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zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Chemie der Ruhr-Universität Bochum
Vorgelegt von Eugen Florin Turcu
Bochum, July 2004
This work was carried out between April 2001 and July 2004 at Lehrstuhl für Analytische Chemie, AG Elektroanalytik & Sensorik under the supervision of Prof. Dr. W. Schuhmann.
Tag der mündlichen Prüfung Referent Korreferent Prüfer
20. Juli 2004 Prof. Dr. W. Schuhmann Prof. Dr. W. S. Sheldrick Prof. Dr. C. Wöll
1. Introduction: Why going smaller?
2. Methods microstructuring 2.1 Lithography 2.1.1. Photolithography 2.1.2. Soft-lithography 2.1.3. Other methods for microstructuring 2.2. Ink-jet printing/lithography – the flow through piezo microdispenser 4 4 6 8 9
3. Tools for probing microstructures 3.1 Scanning Probe Microscopy (SPM) 3.2 Scanning Electrochemical Microscopy (SECM) 3.3 Notes 12 14 25
4. Electrodes for electrochemistry in small volumes 4.1 Integrated working/reference assembly 4.1.1. Preparation of precursor electrode 4.1.2. Chemical deposition of silver onto the body of precursor electrodes 4.1.3. Application of the coaxial Pt-µWE/Ag-RE in SECM 4.2 Miniaturised Ag/AgCl reference electrode 34 39 27 28 30
5. Micropatterning and microelectrochemical characterisation of biological recognition elements 5.1 Enzyme microstructures 5.1.1 About enzymes 5.1.2 Glucose oxidase (GOD) 5.1.3 Patterning of GOD by means of piezo microdispenser 5.1.4 Visualisation of GOD microstructures by SECM 5.2 Defined adhesion/growth of living cells 5.2.1 Introduction 42 44 51 53 62 71 72
Conclusions 18.104.22.168 Conclusions 5.1 DNA microarrays 5.4 Detection of DNA hybridisation in the repelling mode of SECM 5.2.2 What is available so far? 5.2.3 DNA microstructures 22.214.171.124 Detection of DNA hybridisation – What are the options? 5.3.4 Notes 6.3. Experimental 7.3. Acknowledgment 9.5 Conclusions and outlook 5. Curriculum vitae 73 74 79 80 82 87 97 118 124 127 131 136 139 140 157 . References 10.3 Results and discussion 5.3 The repelling mode of SECM – A new and promising assay for imaging DNA microarrays and detecting DNA hybridisation 5.
Albert Schulte. 2004. Electrophoresis. 2003. Wolfgang Schuhmann. Int. Wolfgang Schuhmann. Schmitz. Chem.. “Label-free electrochemical recognition of DNA hybridisation by means of modulation of the feedback current in SECM”. submitted. 43. Gerhard Hartwich. . Mathieu Etienne. Florin Turcu. Gerhard Hartwich. • Anh Nguyen. “High resolution constant distance scanning electrochemical microscopy on immobilised enzyme micropatterns” G. Bioanal. 46-49. • Florin Turcu. Neurosci.. Chem. • Florin Turcu. Oliver J. Solon Thanos. Imaging and Microscopy. 131. Albert Schulte. Jane Hübner. 2004. • Albert Schulte. “Imaging immobilised ss-DNA and detecting hybridisation by means of the repelling mode of scanning electrochemical microscopy (SECM)”.. Ed. Wolfgang Schuhmann. Wolfgang Schuhmann. Siegmar Gäb. David Melchior. Wolfgang Schuhmann. Florin Turcu. Bioelectron. “Analysis of alkyl polyglicosides by capillary electrophoresis with pulsed-amperometric detection”. 141-148. Hans-Willi Kling. “Scanning electrochemical microscopy (SECM) in nanolitre droplets using an integrated working/reference electrode assembly“.T. accepted. 2004. 2004. • Florin Turcu.I. Albert Schulte. Karla Tratsk-Nitz. 5. submitted.PUBLICATIONS • Florin Turcu. “Ink-jet printing for micropattern generation of laminin for neuronal adhesion” J. Angew. 2003. 3482-3485. Anal. Biosens. Methods. Peter Heiduschka.
Authors have found an open field to express their own thoughts and views about all the things they are writing about. Let us consider two of them that are widely spread: “Analytical Chemistry”. my questionnaire moves further to the next and most important question: Why do we need miniaturisation? There are unlimited possibilities to answer.Why going smaller? Micropatterning and microelectrochemical characterisation of biological recognition elements 1. Each book has its main content. What. a clear example for the need of 1 . In particular. This gives the explanation why book’s contents look so different even if they are dealing with the same topic. many theoretical and technical insights. nucleic acids. however. However. how to perform precise measurements and how to get rid of different errors that could appear besides. Before going into details I would like to raise a question: Why do we need microstructures? If you answer: because we need miniaturisation. This thesis is entitled “Micropatterning and microelectrochemical characterisation of biological recognition elements” and may suggest to the reader a survey of all kinds of biological microscopic structures: how to make and how to use them. these were an enzyme. which somehow can be related to this general topic. it is tackling the micropatterning of biological recognition elements. which follows the most important ideas of the subject it is speaking about. most of the books have very general titles. and living cells (in particular neurons). is “Why this one? Does it say enough about the content of that writing?” Well. “History of …” and many others that have something in common: they all speak about something. Furthermore. the Scanning Electrochemical Microscope (SECM) has been chosen as the main tool for exploring the obtained microstructure and for imaging their local (electro)chemical properties with high spatial resolution. should be expected from this piece of work? In fact. Introduction: Why going smaller? A question that often comes in mind when reading the title of a new book or journal article. A piezoelectric microdispenser has been used as microprinting device to create complex enzyme structures on different materials and also was used to prepare oligonucleotide microarrays. Analytical Chemistry explains what an analysis means and how many types we know.
Napier (the inventor of logarithms). Instead of abacus and “Neper’s bones“ all other machines were quite big. Nevertheless. It was characterised by a huge volume and poor computational speed (Image courtesy of the Computer History Museum). just to mention the most famous of them.76 square feet. Something else changed. Eckert the chief engineer of team involved in the construction of ENIAC. Top leading scientists were later involved in the development of these sophisticated machines: Leonardo da Vinci. As Euclid used to finish his (successful) mathematical demonstration: “Quod Erat Demonstrandum” (QED)! a Fig. Pascal built several mechanical machines. named “Pascalines” for helping his father in adding and subtracting large sequences of numbers (he was a tax collector). b 1m2 = 10. Its name is ENIAC (Electronic Numerical Integrator And Computer) (Figure 1). In conclusion. The first representative electronic computer able to carry out general-purpose computations was borna in USA in 1946. miniaturisation means smaller and smarter tools for tomorrow! This is a „sine qua non” condition of all future technological development in order to improve our lives. About 50 years later. Mauchly was the chief consultant and J. 1 ENIAC – an early digital computer. Gottfried Wilhelm von Leibniz. tools that everyone loves when they are small and very efficient in doing lots of work for us. thus. Wilhelm Schickard.Why going smaller? miniaturisation can be found with developing computers. In the 20th century the basics of electronic computing systems were about to be discovered and developed by Alan Turing and John von Neumann. ENIAC required a room of about 10x10 m2! 2 . I suppose it had been made in the smallest format possible at that time. too: the computing speed increased about many hundred thousand times. The long history of mechanical calculators has its roots in the Antiquity when the “abacus” was the computing tool. Just by judging its dimensions and capability one can conclude that it had been a real “Dinosaur” as it occupied a large surface (about 1000 square feetb) for a low computational power (for instance 5000 additions per second). A laptop with more than 2 GHz clock frequency is a common tool these days. the new generation of computers became more compact (less than 1 square feet) due to the achievements with the miniaturisation of their components. J. P. Blaise Pascal.
About a year before. I contacted her and asked for help in elucidating the structure of a polymer. I was wrong. Sweden). Elisabeth Csöregi (Lund University. It was days after this event when I realised Wolfgang is not a place but the name of my future boss!!! Ups! In the next period of time some e-mails were exchanged between Wolfgang and me. however. discuss with me about my request. Dr. 3 . Well. Instead. Biosensors? I never heard about this. some bits of information were displayed on the website of the ELAN-group (Electroanalytik. she mentioned that I could go to Wolfgang in Germany. The entire work presented in the following chapters is based on the concept of miniaturisation. a document that I did not even have at that time. my PhD work is dedicated to micropatterning of biological recognition elements. a novel coaxial microelectrode for advancing microelectrochemical measurements in small electrolyte volumes was developed. However. but I knew a little about chemical sensors! Pdf files of publications describing the topics and research directions in Wolfgang’s group were sent by e-mail. was focusing on the development of a simple strategy to detect hybridisation of nucleic acids on DNA chips. thus I was informed about what I could do in Germany. Having this in my mind. in the Biosensors laboratory at Ruhr University of Bochum. Csöregi went on explaining me I have to send her a CV (curriculum vitae and not a cyclic voltammogram!) and the details of the passport. although a bit late. As mentioned above. The major part of the dissertation. Working on the technology of DNA microarrays was enjoyable to me not only because it is a hot-topic these days but also because I had the opportunity to work on something that was completely new to our lab. What a surprise! I am lucky! I can go abroad and study. I couldn’t read them because the suitable software for opening such files had never been installed on the only computer of the Chemistry Department at North University of Baia Mare. Furthermore. Unfortunately. Biosensors) and gave me a rough idea about my future colleagues and research topic.Why going smaller? Confession: It happened in summer 2000. that I received a phone call from Prof. which I accidentally prepared in 1989. But where is this place “Wolfgang”? I though it must be a small town because I never heard about it! This were my thoughts as E. I thought that she would. particularly.
or one can “write” with a “nano-pencil”. in earlier times. Methods for microstructuring Lithography “As tiny as possible” seems to be the present day trend of science and technology with respect to any kind of device. the microfabrication. I would like to present here. Scaling down structures originated in the field of electronics where devices became smaller in size and the individual components of the integrated electric circuitries had to fit on limited space. that itself means “writing on materials”a. soft lithography. to my opinion the key word “lithography”. exposure and development. The steps involved in the photolithographic processes are: wafer cleaning. Photolithography Photolithography is the process of copying geometric shapes from a mask to the surface of a substrate. hard baking. For instance stamps or printer-like “ink” dispensers occupy an important place among the microstructuring tools. photoresist application. microfabrication procedure has a strong impact in most of the areas of contemporary science and technology and the knowledge and experimental procedures for miniaturisation were transferred from electronics also to chemistry and biochemistry for creating sensors with better performances. Thus. Nowadays. The working principle resembles the formation of the “positive image” from the negatives on to a developed film in photography (Figure 2). The ability to generate patterns of biomolecules on different material surfaces is important for biosensor technology. a Lithography is a combination of the Greek words „lithos“ and „graphein“ with the meaning of stone and write respectively. is in fact sufficient to cover all the existing methods. no matter how the patterned substance reached the substrate. nano-pen lithography.1. 2. such as photolithography. and spotting techniques.Methods for microstructuring 2. one of which being “art of engraving on precious stones”. and fundamental studies in cell biology. the common categorisation will be used. the following part that is dedicated to a succinct presentation of each micropatterning approach. Many ways of microstructuring are derived from common devices used every day. barrier layer formation. 2.1.1. It denoted. 4 . tissue engineering. Although this is the accepted classification of micro/nano-structuring techniques. various items. the underlying method is a lithographical one! However. soft baking. mask alignment. not as a dead point of miniaturisation but as a reliable multi-purpose technique for fabrication of chemical and biological analysis tools. There are several well established ways to pattern biomolecules onto substrates.
the negative image on the film is transferred onto the photo-paper in such a way that the final positive image is larger as the picture captured on the photo-film. the material is called a positive photoresist. In contrast. In photography. which cause the regions exposed to light to be either more or less susceptible to chemical etch. This pattern can then be etched into the underlying wafer by either a wet chemical etch or a plasma etch. Fig. which removes either the exposed (positive photoresist) or the unexposed (negative photoresist) photoresist.Methods for microstructuring The wafer is initially coated by spin-coating with a photoresist. the photoresist material undergoes some light-induced chemical reactions. Light from the source is focussed through some lenses onto a "mask" (reticle). Unlike a slide projector. The ability to project a clear image of a very small feature onto the wafer is limited 5 . no lenses are shown. The resist is finally "developed" by exposing it to the chemical etchant. the stepper does not enlarge the image but actually reduces it in a similar way than sunlight is generating a shadow of a cloud that is smaller than it actual size. When the image is projected onto the wafer. 2 Comparison between the photography (left) and photolithography (right). The stepper functions similarly to a slide projector and creates high-contrast monochromatic images. For simplicity. If the exposed regions become more susceptible to the etch. containing the desired image in order to produce it on the wafer. while it is a negative photoresist if it becomes less susceptible. The substrate then has a patterned polymer coating on its surface. the image grafted on the mask is reduced in size. in the photolithographic processes. The desired pattern is then projected onto for example a wafer in a machine called a stepper.
2. to produce polymeric nanostructures very similar to (or indistinguishable from) those on the surface of the original master. re-replicated using a rigid organic polymer. features of 100-200 nm can be obtained. the stamp is covered with the desired molecules (thiols. tolerates only little variations in the material that can be used (there is not a large selection of available photoresists on one hand. micromoulding in capillaries (MIMIC) and solvent-assisted micromoulding (SAMIM). an photochemically curable polyurethane (PU). With replica moulding. There are some procedures resorting to stamps/moulds made of flexible polymers rather made hard materials. Microcontact printing uses a PDMS stamp (copied from a master that is previously prepared by photolithography) to transfer molecules onto surfaces. high cost due to the necessary sophisticated facilities and technologies. a liquid precursor of PDMS is pressed against a patterned surface with nanometer-sized relief structures.PDMS.2. microtransfer moulding (µTM). enzymes). proteins. Whitesides in which the stamp is made from an elastomeric material namely polydimethylsiloxane .and nano-patterning1. M. The soft rubberlike stamp provides a large-area contact on the molecular scale. not simply adopted for patterning non-planar surfaces. Soft-lithography Alternatives to photolithography were developed in the last 2-3 decades and showed their potential for micro. even on rough or slightly curved surfaces and molecules are transferred directly from the stamp to the surface. "Soft lithography" is a new high resolution patterning technique developed at Harvard by Prof. the cross-linked PDMS is cautiously peeled off the structure perfectly copying the morphology of what was called the original master3. Members of the “soft-lithography” family are microcontact printing (µCP). provides very poor control over the chemistry of patterned surfaces (especially when complex organic functional groups are desired at the patterned surface). The major disadvantages of photolithographical methods are listed below: the smallest achievable dimension is limited by the optical diffraction. The stamp is allowed to dry and afterwards pressed onto the surface to be patterned. nucleic acids. With ultraviolet light.1. and on the other hand common wafers such as glass or ceramics are not that suitable as substrates). G. for example. In a first step. A great advantage of this approach is that it can produce 6 .2. replica moulding (REM).Methods for microstructuring by the wavelength of the light that is used and the aperture of the lense (Rayleigh diffraction). The nanostructures present on the PDMS replica are. After curing. in turn.
In microtransfer moulding4. an amorphous fluoropolymer material Fig. the mould is removed to reveal newly prepared 3D-structures. This technique allows formation of both interconnected and isolated microstructures and is also suitable for building up 3D-structures layer by layer. The softness of the mould leads to mould deformation in the process of patterning or mould preparation and. the dried and clean mould is positioned over the substrate.Methods for microstructuring solid copies with smaller or larger features as the original master! This is achievable because the PDMS copy can be mechanically bent (Figure 3). 7 . The mould is placed on the substrate and a solvent able to corrode the substrate is filled in the capillaries. 3 The soft elastomeric structure can be mechanically bend and thus making possible the preparation of copies with larger (l1) or smaller (l2) features as the original master. An intrinsic problem with any lithography based on an elastomeric mould originates from the material properties of the mould. In contrast. Accordingly. typically for feature sizes smaller than several hundred nanometers. Once the polymer is hardened. With micromoulding in capillaries. Quasi-3D-structures are possibly created. solvent-assisted micromoulding does not deposit a particular polymer or self-assembled monolayer at the surface. the deformation gets worse as the pattern size becomes smaller. of course. but removes material from the substrate in an etching process. The filled mould is then brought into contact with the substrate. To overcome this problem. thiol microstructure are available by this procedure. the recessed regions of a stamp are filled with a solution or pre-polymer and the exceeding liquids are removed away from the crests. In the next step the precursor of the polymer is forced to enter the microscopic channel formed by capillary forces. the mould must be prepared before being used for patterning. When the solvent is completely evaporated. If the mould is filled for instance with a solution of an alkane thiol in ethanol. the mould is removed. to all the above-described soft-lithographic procedures.
normally polymethylmethacrylate (PMMA. such as nickel. • Electroforming: the microcavities generated by the removal of the irradiated plastic material can be filled with metal by electroforming processes. Be is also used at large scale for X-ray transparent windows in nuclear reactors and in radiation detectors. Other methods for microstructuring A promising alternative is the LIGA process that was developed at the Institute of Microstructure technology (IMT) in the early eighties under the leadership of Dr.2 µm are obtainable. Ehrfeld6. by „shadowing”. W.Methods for microstructuring was used as a mould material.3. or alloys. The unique properties of the mould material made it possible to pattern densely populated extremely fine features (80 nm line-width)5. Exposure to radiation modifies the plastic material in such a way that it can be removed with a suitable solvent. such as nickel-cobalt and nickeliron. copper and gold. In this way. and parallel X-radiation. As a matter of fact. high aspect ratio structures with heights of up to 1 mm and a lateral resolution down to 0. • Plastics moulding: Plastics moulding is the key to low-cost mass production by the LIGA process. • Deep X-ray lithography: LIGA requires a highly penetrating. LIGA made possible the mass-production of microcomponents at low-cost. The steps involved are X-ray lithography. The lateral structural information is transferred by illumination of the mask with deep X-ray radiation into a plastics layer. Of note. electroformation and plastics moulding. intense.1. The metal microstructures produced as mentioned above are used as moulding tools for the production of reliable replicas of the primary structure in large quantities and at low cost. Mask-free methods for micropatterning surfaces were established using the tip of scanning probe microscopes to locally modify surfaces down to nm range. The application of X-radiation imposes the use of specific materials for the mask with the „transparent” part made of very thin foil of metals such as titanium or berylliumb. 8 . The way of inducing b Due to its poor electronic structure. its low atomic weight recommends it as moderator for slowing down rapid neutrons in a nuclear reactor and hence promoting the self-sustaining nuclear reaction. typically supplied by a synchrotron. Plexiglas). and the absorbers consisting of a comparatively thick layer of gold. the negative pattern of the plastics structure is generated as a secondary structure out of metals. leaving behind the structure of the unexposed plastic (the „shadowed areas”) as the primary structure. 2.
the flow-through piezo-microdispenser Simple but cost-effective lithographic processes that can be applied even to irregular substrates under ambient conditions have been developed. the SECM tip. Thomas Laurell from the same 9 . May be inspired by the work of Hertz. Ink-jet lithography is a valuable tool providing a non-destructive and localised surface modification technology in which small droplets of ink (or other liquids) are jetted from a small aperture (nozzle) directly to a specified position of the substrate. Fig. The idea of obtaining grey-scale printing was to control the number of droplets deposited in each pixel (spot) so that the amount of ink volume in each pixel was adjusted to create the desired grey tone10. electrochemical: a microelectrode. is used to locally generate a reagent that is able to etch the substrate. 4 “Writing” with a nanoscopic ink-pen that is the AFM tip. The “ink“ molecules that are dissolved in the solution underneath meniscus bind due to their high affinity to the substrate. 2. University of Lund developed about 25 years ago continuous ink-jet techniques that had the ability to modulate the ink-flow characteristics for grey-scale ink-jet printing on different media. Professor Hertz from the Department of Electrical Measurements. DPN7-9. mechanical: an atomic force microscope (AFM) with its sharp tip operated in the contact mode scratches the surface or transfers material from the tip to the surface (dip-pen nanolithography. Ink-jet printing/lithography . Figure 4). optical: the optical fibre probe of a near-field scanning microscope (NSOM) exposes the photoresist at local areas underneath the tip.Methods for microstructuring controlled alteration of substrates by scanning probe lithography (proximal probe lithography) relies on the following interactions: electrical: a scanning tunnelling microscope (STM) tip generates local electrical fields that modify the surface underneath.2.
c Two silicon plates. In contrast to a deposition from a spotting needle. Fig. which expands and contracts upon the excitation with suitable alternating voltages. The inner chamber is then enclosed by the two plates to give a volume of few microlitres down to hundreds of nanolitre. As shown later. obtained by anisotropic etching (one for the nozzle and another for inlet/outlet of solution and membrane). That way the system is able to generate a series of short pulses of pressure each of them ejecting a single droplet with a volume of roughly 100 picolitres. are glued and sealed with waterproof epoxy. In brief. a microdispenser can be used with much more viscous solutions because it is actively forcing the solution out of the reservoir using the piezo-actuator. an enzyme–polymer mixture that certainly is more viscous than pure water had to be micropatterned on a chosen substrate.Methods for microstructuring Department developed a microdispenser11 capable to shoot and deposit picolitre-sized droplets of solutions on substrate surfaces. 5 Schematic representation of the piezo-microdispenser head. The membranec is coupled to a piezoelectric ceramic actor (3x3x2 mm3) and fastened by in a plastic frame (Figure 6). the microdispenser is capable to emit ultra small droplets of solutions of choice based on rapid movements of a flexible silicon membrane. for instance. Alternative voltage pulses applied to a thin silicon membrane eject picoliter droplets of solution (yellow) towards the substrate. 10 . this was helpful when. An obvious advantage of this printing procedure over stamp-based methods is that tiny droplets can be deposited in small cavities12. A schematic representation of a microdispenser is shown in Figure 5.
A pump is connected to the chamber via short silicon tubes. The excess of the filling solution is drained out through the outlet consequently avoiding an exposure of the membrane to overpressure inside the piezo chamber. one droplet is hurled through the nozzle. pyramidal nozzle with an orifice size of 40×40 µm and directed perpendicular towards the target surface where the deposition typically make spots with diameter of about 70 µm. piezo actuator (2). droplets emitted downwards can be delivered with high precision from the nozzle to a chosen area of the opposing substrate. etched silicon chips (3). Certainly. The flow-through microdispenser has proven to be ideal for applications in biochemical and analytical chemistry where small sample volumes and rapid sample handling are key features. 6 Two versions of a piezo-microdispenser head: nozzle (1). 11 . If the droplets are shot in a horizontal plane. Any time when a voltage pulse is applied to the piezo. at least if there is not strong air motion. Fig. they can travel without any change of the trajectory up to several centimeters. A precise movement of the target substrate relative to the microdispenser nozzle during microdispensing can be used to deposit either rows of individual spots or lines and grids from overlapping lines.Methods for microstructuring The droplets are released through the specially designed.
These were accomplished by using not only visible light but also electromagnetic waves with a shorter wavelength such as UV or fascicle of accelerated electrons. 3.16. Up till the 80’s of last century. astronomy and mathematics. Hooke (1667) and A. studied various microscopic objects and published the in “Micrographia”. bacteria. The design of the optics of microscopesb changed their look dramatically over time.Tools for probing microstructures 3. oil. 1703) British scientist known for his work in physics. Nederland 1632 – id. and Z. Scanning-probe microscopy (SPM) refers to a large number of techniques that employ needle-like probes with physically small tip dimension that are mechanically scanned back and forth across a surface. 1590). Janssen. Over the years. 1635 – London. and other tiny creatures in a drop of water and the circulation of blood corpuscles in capillaries. 12 . he made lenses with great curvature. which is just about the thickness of a hair14. 1723. Leeuwenhoek (1697)a. Antony van Leeuwenhoek (Delft. The probe tip can be made sensitive to a variety of surface properties and the measure of distance-dependent interactions between the probe tip and surface is actually used for imaging the interface.) self-educated. microscopes developed into sophisticated instruments with improved magnification and resolution (maximum allowed by Abbe diffraction). Going along with changing the principles of image generation. a Robert Hooke (Freshwater. First scientific observations using microscopes to explore the microcosmos were reported by R.1. built microscopes and was the first to observe yeast. As a matter of fact. b The word microscope comes from Greek language: “micro” = small and “skopein” = look at. visualisation techniques typically generated secondary images arising from the interaction of light with structural parts of objects under study. simply a drop of water. AFM and STM. This is the more “classical” picture of a microscope. these crucial tools. We are used to think about microscopes as a holder that contains a tube having two lenses: the eyepiece (ocular) and an objective (H. Then a new set of techniques appeared that provided images of an object without depending on the diffraction of light. the tubular form of the old-fashioned microscopes turned into new designs. honey or glass fixed on a small round cut in a metallic plate or a tube containing two lenses fixed at its extremities were the plain devices to observe things otherwise invisible to the naked eyes. The most widely used high-resolution imaging tools certainly are the atomic force microscope (AFM) and the scanning tunnelling microscope (STM)15. Tools for probing microstructures Scanning Probe Microscopy (SPM) Microscopes are representing powerful tools for extracting and mapping detailed information about the microstructure of an object13 and helped to unveil secrets of nature that were long time hidden to observers since human eyes are limited to a resolution in the order of 100 µm. At the early stage of development.
This has been demonstrated by employing an AFM for watching biological macromolecules in motion or simply biopolymers such as DNA that are coiled or stretched on a substrate18. STM gives access to imaging the topography of conductors with atomic resolution and has been successful for example even in imaging individual atoms laying on compact surfaces. Recording the deflection of the cantilever as a function of lateral position provides topographical images with up to nanometer resolution. a distance-dependent tunnelling current is flowing and may alter laterally with changes in the tip-to-sample separation. With the STM tip being in extreme proximity to surface and scanned. on the other hand. AFM is taking advantage of force interaction between the sample surface and a sharp tip that is attached to a flexible cantilever. Although. the scanning electron microscope was in fact discovered by M. Table 1 Probe Microscopy Scanning Tunnelling Microscopy Scanning Near-Field Optical Microscopy Atomic Force Microscopy Magnetic Force Microscopy Scanning Electrochemical Microscopy Acronym STM SNOM AFM MFM SECM Appearance year 1982 1985 1987 1987 1989 Notes in-situ technique c Although the first (transmission) electron microscope was presented by M.e. Rohrer were awarded the Nobel Prize for Physics in 1986 and shared the prize with E. SPM is applicable for “in-situ” investigation of samples. the different SPM devices (Table 1) have many technical components in common (i. This and their high spatial resolution made scanning probe microscopes so popular in a relatively short time. the systems that are used for precise micropositioning of the probe tip). Ardenne in 1938. namely G. As it is not restricted to conducting surfaces.17. In general. Indeed. 13 . Ruska who first presented the electron microscope in 1931 in Berlin)c. The first commercial available SEM came up in 1966 (Cambridge Instruments Comp. Ruska in 1931. the working principles behind the instruments differ quite a lot depending on the specific interaction between the tips and sample. With STM.Tools for probing microstructures were invented by the same scientist.19. a voltage is applied between a nanometre-sized metal tip and a conducting surface.). Binnig (he and H. studying the target in its ambient environment is a great advantage because no vacuum is needed as for example with a scanning electron microscope (SEM). AFM is more suitable for biological imaging. Knoll and E.
Tools for probing microstructures
Scanning Ion Conductance Microscopy Scanning Capacitance Microscopy Scanning Chemiluminescence Microscopy Scanning …. Microscopy
SICM SCM SCLM …
Among the numerous SPM devices, only one is based on an important technological advancement in modern electrochemistry: the voltammetric and potentiometric microelectrode. Approximately at the same time, W. Engstrom20 and A. J. Bard21 introduced voltammetric microelectrodes as new type of scanning probes and paved the way for so-called scanning electrochemical microscopy (SECM). Being attached to highprecision positioning devices, the micrometre-sized tip of a disk-shaped microelectrode enabled local electrochemical measurements while being scanned across the sample surface. As will become clear below, the spatial resolution for SECM imaging is somewhat limited and much lower than for AFM/STM. SECM, on the other hand, can more easily provide images of variations in interfacial (electro) chemical activity.
Scanning Electrochemical Microscopy (SECM)
The development of ultramicroelectrodes was one of the most important contributions to electroanalytical chemistry. As their name implies, ultramicroelectrodes are extremely small, with dimensions in the order of micrometers or less. This small size and the electrode characteristics that come with it (low capacitive currents, low IR drop, enhanced mass transfer - see note 1 - and high faradaic current densities) can be exploited in a number of unique applications22. Among them are measurements in highly resistive media, high-speed voltammetry and electroanalysis in very small volumes. The integration of ultramicroelectrodes into a scanned probe instrument led to the invention of scanning electrochemical microscopy which allows observing local electrochemical activity at the liquid/solid interface or at microscopically small object. How does SECM work? A little information about the basic principle of SECM will be introduced because this is necessary to understand the following parts of this work. With SECM, amperometric or potentiometric ultramicroelectrodes with radii, r, in the order of a few µm or even less are employed as electrochemically active scanning probes (SECM tips). In case of an amperometric SECM tip, an electric current will flow if a redox active compound is 14
Tools for probing microstructures
present and the tip polarised at proper potential. Tip interaction with a target surface via an electroactive species results in distance-dependent variation of the tip current. One tool – multiple operational modes The SECM23,d as a whole and self-containing instrument helps in different modes to visualise electrochemically active targets or to modify surfaces exposed to electrolyte solutions. These are the amperometric feedback, the generation/collection, penetration, and the surface modification mode24. In general, the amperometric tip current (iT) is plotted as a function of its horizontal coordinates (X and Y) for imaging superficial electrochemical activity of a sample. In the following, modes I to IV are briefly described and possible applications discussed.
Fig. 7 Feedback mode of SECM: for a given redox reaction Ox→Red carried out at the SECM tip, a steady-state current is recorded in bulk of the solution I=Ilim (A); in the vicinity of an electrically conductive surface the Ox species are regenerated with the consequence of local rise of Ox concentration and hence a higher current recorded at the SECM tip (B); thus an insulating substrate obstructs the diffusion of Ox to the tip, thus lowering the measured faradic current (C). Generation-collection mode of SECM: for instance, surface confined species generate an electroactive compound that is detected at the SECM tip (D).
I. The amperometric feedback mode of SECM Let us imagine a disk-shaped microelectrode (active diameter smaller than about 50 µm) that is immersed in a solution containing a redox species (e.g. ferri- or ferrrocyanide). Shortly after the potential of the electrode is set to a value suitable to oxidise or reduce the
The acronym SECM stands for the instrument itself - scanning electrochemical microscope - as well as for the specific field of probe microscopy that is scanning electrochemical microscopy.
Tools for probing microstructures
electroactive compound, a steady-state faradaic current is observed. The magnitude of this current depends, besides the rate of electron transfer, on the concentration and diffusion coefficiente of the electrochemically active substance and on the diameter of the electroactive disk of the microelectrode. For an unstirred solution, the steady-state current (i∞) (Figure 7A) is purely controlled by diffusional mass transport of the electroactive species and given by the well-known equation:
i∞ = 4 ⋅ n ⋅ F ⋅ D ⋅ c ⋅ a
where n denotes the number of electrons taking part in the electrode process, F the Faraday number (1F = 96484.6 C⋅mol-1), D the diffusion coefficient (cm2⋅s-1)f, c the concentration of electroactive species (mol⋅cm-3), a the radius of the disk-shaped electrode (cm); i∞ is thus measured in ampere (A). Probing a target with SECM clearly requires the microelectrode tip to be brought close to the target surface since otherwise the surface-specific information can not be collected. In the vicinity of a surface, however, the current measured by the SECM tip changes significantly dependent on the tip-to-sample separation and the nature of the approached surface25,26. How? and why? are the two questions to be answered in the following. We should consider two cases: 1. the surface is electrochemically active (electric conductor, enzyme modified). 2. the surface is electrochemically inactive (electric insulator, enzyme-free). 1. An electrochemically active surface: the positive feedback We are bound to a great degree by our own innately human way of thinking and judging phenomena around us which is the common sense! We just simply have learned that some things happen and have a certain course. This helps us to understand new items by comparing the new facts with old schemes in our brain. Quite often, in the physics/chemistry (especially the quantum mechanic, relativistic theory, quantum chemistry…) the common sense deplorably fails! During my PhD, I had the chance to introduce several students to the basics of SECM, and therefore I noticed that typically the answer to the question “What should the current be if the SECM microelectrode is brought close to a conductive surface?” is wrong. The students anticipated that the current will drop to zero when the electrode is approaching a surface; their common sense suggested them
As obtained from the Fick’s first equation, the diffusion coefficient is a linear function of temperature: D = k⋅R⋅T, where k is a constant, R is the gas constant and T is the temperature of solution. f D has typical values ranging within 10-5 – 10-6 cm2⋅s-1 that depends strongly upon the composition of the electrolyte.
In SECM practice. 17 . at least under this experimental conditions. Why? We shall remember that any electroconductive surface will gain a superficial. According to the Nernstg equation. Nobel Prize for Chemistry in 1920. in a supporting electrolyte containing only Fe3+ and no other redox species (especially no Fe2+) is controlled by the electroactive species present in the bulk of electrolyte (see note 2).in 0.Berlin 1941). and thus able to oxidise Fe2+ back to Fe3+ (equation 2): g Walther Nernst (Briesen. Not only is the steady-state current higher close to the surface but the form of CV changes from hysteresis to a perfect S-shape. The potential of a gold surface. negative or positive net charge when it is in contact with a solution of a redox active species. Against the common belief. for instance. 5 mM [Fe(CN)6]3. is expected to be significantly more positive than the E0 of the Fe3+/Fe2+ redox couple (see below). German physicist and chemist with contributions in thermodynamics. the solution contains typically an electroactive species that can undergo reversibly redox transformation. Ag/AgCl 3 M KCl reference electrode. 8 Cyclic voltammograms recorded at a 10 µm Pt microelectrode in bulk (blue line) and at about 15 µm above a gold surface (dark line). A redox compound in solution will electrically charge the inert metallic interface (Pt or Au) to a potential of which polarity and amplitude are controlled by the ratio of the Red/Ox concentrations of the redox species. West Prussia 1864 . 500 mV/s scan rate.1 M phosphate buffer and 3 M NaCl and 0.05 M NaOH.Tools for probing microstructures that the current must decrease irrespective of whether the surface is conductive or not. Fig. the current increases (see Figure 7B and 8). the surface will decrease the mass flow of the reacting species towards the electrode’s disk. the open circuit potential the gold surface.
and represented schematically in Figure 1B.059 V.33 exp − ( L) = 0. E0 is the standard electrode potential measured against a normal hydrogen electrode (NHE) at 25° C. a redox mediator is amperometrically reduced (or oxidised) at the SECM tip with a potential ensuring diffusional control of the tip current. Here. The variation of the positive feedback SECM tip current with tip-to-sample distance is given by equation 327: iT 0. radius of active area ( µm ) A plot of iT versus L is known as approach curve and can be used to calculate and adjust the tip position of a microelectrode of known dimensions. Important to obtain the negative feedback is to position the SECM tip into the nearfield. tip.78 1. the amperometric tip current decreases compared to 18 . the local concentration of Ox in the gap between the microelectrode tip and surface increases compared to bulk concentration and hence the amperometric current measured at the tip increases. This effect is called the positive feedback. due to its polarisation (see above) it will be oxidised (or reduced). and 1 M activity. 3 i∞ L L where L = distance between SECM tip and sample ( µm ) . E0 = +0.generated species simply diffuses into the bulk without undergoing any further reaction. If T = 298 K. a.356 V. 2. n is the number of electron transferred in the redox reaction.Tools for probing microstructures 0 E Fe3+ / Fe 2 + = E Fe3+ / Fe 2 + + RT a Fe3+ lg nF a Fe 2 + eqn.68 + eqn. the constant in front of logarithm is 0. When the tip is brought close to a contacting surface. 2 where E (V) is the actual electrode potential. tipgenerated species has a chance to interact with the charged interface and. however.314 JK-1mol-1). With the SECM tip far above a surface. Due to the lower flux. An electrochemically inactive surface: the negative feedback The surface of insulating target is inert to the redox species and at small tip-to-sample separation mechanically hinders the free diffusion of the reacting species towards the electrode’s tip.07 + 0. In SECM. T is the absolute temperature (K). R is the gas constant (8. Note: Surface-immobilised enzyme can act in the similar way as a bare metallic surface and regenerate redox species that is consumed by the SECM. too. of the two ions. This process is known as “enzyme-mediated positive feedback”28. Consequently.
A sample with alternating conducting and non-conducting areas was made by firmly pressing a finger tip against a chemically deposited layer of silver on glass. image acquisition is achieved by scanning the SECM tip laterally at a user-defined fixed height and simultaneously recording the tip current as a function of position (constant-height feedback mode imaging). and approach curves (I/I∞ vs. For a diffusion-controlled electrochemical reaction at an SECM tip. the tip-to-sample distance has a major impact on the hindrance of diffusion and the extent of negative feedback. This contact led to local removal of silver at areas where the papillae of the skin was touching the silver film. SECM imaging in the negative and positive feedback mode Negative and positive amperometric feedback are highly distance-dependent.51 2. tip-to-sample separation d) permit to place the SECM tip at appropriate working distances within the regime of the nearfield (at a height of about a few times the SECM tip radius or less). This criterion must be achieved in order to allow SECM measurements. the relative faradaic current variation obeys with good accuracy (1. 3. Typically. To give you an idea about SECM imaging in the amperometric feedback mode. The degree of negative feedback also is influenced by the ratio of the total diameter of the microelectrode and the diameter of the active area.2%) equation 4: iT ( L) = i∞ General note: 1 1. Obviously. I would like to present a SECM image of a fingerprint that was scanned with a Pt microdisk electrode in a solution containing 5 mM [Ru(NH3)6]3+ as the active mediator. and a disk-shaped electrode with 1:10 disk to insulation ratio. This observation is regarded as negative feedback. As can be seen from Figure 10. Figure 9 is showing in a schematic the current profiles that one expects when a SECM tip is scanned at a constant height across samples exposing lateral variations in topography and conductivity. 4 A SECM tip is said to be positioned within the working distance when the measured current is experiencing the influence of the conductive or insulating surface.66 exp − L L eqn.40 0. 19 .29 + + 0. the variation in conductivity and/or heterogeneities and variation of the flatness of the sample were nicely revealed.Tools for probing microstructures the value obtained with the tip in bulk (Figure 7C).
Dark areas correspond to low currents and indicate where the finger’s papillae touched the thin layer of chemically deposited silver on glass surface. (B) electrically non-conductive surfaces that have topographic heterogeneities. 20 .Tools for probing microstructures Fig. The imaging was performed with a 10 µm Pt disk electrode in the feedback mode of SECM. Fig. 10 SECM micrograph of a fingerprint.1 M phosphate buffer(pH 6. a topographically homogenous but electrically heterogeneous surface can also be image in the feedback mode (C).7). Scanning solution: 5 mM rutheniumhexamine chloride in 0. 9 Schematic representation of SECM tip reduction current profiles expected when scanning the tip in amperometric feedback over (A) electrically conductive.
The generation . and operated amperometrically. It can also be achieved with SECM tip-generated etching reagents. some semiconductors47 or metals like copper48.Tools for probing microstructures II. corrosion31 of different materials32-34. Of course. a SECM-based fabrication of microstructures of conductive polymers38 such as polypyrrole39 and polyaniline40 and polythiophene41 or polybithiophene42 derivatives by oxidative formation of polymerisable radical-cations from their monomers has been described. biological tissues. solutions with a concentration gradient. Thus. Also. Penetration mode of SECM This mode involves the movement of the SECM tip along a single direction that is perpendicular to the target (Z-axis). 21 . This is achievable.collection mode of SECM As the name suggests. IV. For instance. Hence. for instance bromine45 that act as curving tool and has found application in wet etching of silicon46. inorganic microstructures were created by tip-induced reduction of different metallic cations36.37. III. the generation process can be carried out by the substrate and the products gathered at the microelectrode. it is said the SECM works in the tip-generation/substrate-collection mode (TG/SC). or even soft films are appropriate for being investigated with a tip in the penetration mode of SECM. gels. and the properly polarised substrate collects the products. Opposite to the case above mentioned. Surface modification by means of the SECM The SECM has been successfully used for modifying numerous surfaces35. it involves the production of an electrochemically active species at one place. it generates a concentration profile by consuming a substance available in solution. If the microelectrode is positioned within the working distance. by taking advantage of the SECM tips to generate reagents for directed chemical transformation of the substrate. Micromachining is a controlled alteration of surfaces for creating microstructures. Self-assembled monolayers (SAM) of alkanethiolates patterned on gold43 by means of SECM allowed the local formation of biologically active microstructures containing for instance glucose-oxidase44. which after a short diffusion through solution is immediately detected at another place. any medium that allows the microelectrode to break through without harming it is of interest for this SECM mode. a situation that is described as substrate-generation/tip-collection mode of SECM29 (Figure 7D) Typical examples where the tip is used as a collector are studies of the diffusion of metabolites released from living cells30. on the one hand due to the possibility of accurately moving microelectrodes over a substrate and on the other hand.
on surfaces with larger variations in topography and at surfaces that display both.Tools for probing microstructures The item writing mode of SECM is occasionally accounted in papers regarding surface modification by SECM but it is in fact nothing more than the above mentioned techniques49. 22 . Moreover. and scanned over the structure. The very strict experimental requirements (perfect alignment of the laser source with the mirrors. variation in topography and conductivity. In both cases. led to efforts for improving the “constant-height” methods. a clear interpretation of the measurements would be difficult because changes in the tip current arising from distance variations cannot easily be differentiated from those originating from alterations in surface activity. depending upon the scanning direction. The introduction of the “constant-distance” SECM employing an optical detection scheme for shear forces between the electrode tip and the surface permitted the operation of probe tips with electroactive diameters below a micrometer and simultaneous imaging52. since the tip-to-sample distance has to be in the orders of few tip radii of the UME (within the nearfield) it is obvious that with a decreasing electrode size scanning in this mode becomes hard51. photodiode detector and tapered capillary of the ultramicroelectrode) required with the optical read out of the damping of shear forces. A non-optical shear force-based distance control was established by replacing the laser beam and the light-sensitive diode with a piezo receiver54. In the case of a tilt surface. The integrated shear force-sensitive and computer-controlled feedback loop of this mode forces the SECM tip to follow the contours of the surface during the entire time of scanning and imaging. This not only allows simultaneous acquisition of the electrochemical tip response.50. Constant distance mode of SECM Most often SECM experiments are performed with the SECM tip kept at “constant-height” above the substrate. Constant height mode is only applicable when changes of the sample height or the overall surface tilt do not exceed the tip-to-sample distance. there are some inherent limitations especially on tilted samples. a particular substrate has a rough surface with gaps or heights of sizes not far from the total diameter of the scanning tip. it may collide with the surface protuberances.53. For instance. However. the electrode tip could either crash on the surface or retreat from the working distance. the sample topography but also effectively prevents against tip crash even with smallest SECM tips. If the tip is positioned in the working distance.
Glass pulled capillaries constituted the body and the insulation of the electrodes. or potentiometric monitoring of anions (chlorine)63. In addition to diskshaped microelectrodes.60 electrodes have gained recognition and found applications in SECM.and nano-electrodes with active diameters of 1-25 µm55.62.56. insulated by anodic electrophoretic deposition of paint (EDP)65. SECM tips were also manufactured from pulled capillaries filled with ion-selective ionophors64. Besides potentiometric. Carbon fibre microelectrodes (CFMEs). 23 . A remarkable electrode for simultaneous electrochemical. New electrode materials allowed direct measurement of pH61. Drawback Spatial resolution of SECM is primarily determined by the size of the SECM tip and for that reason not on atomic scale as for instance with scanning tunnelling microscopy (STM) and atomic force microscopy (AFM).58 or hemispherical59. most of the SECM tips were fabricated with noble metals or graphite in disk-shaped micro. optical and topographical images was obtained from an optical fiber surrounded by a gold ring electrode and an electrophoretic insulating sheath68. However. the electrochemical nature of the scanning probe offers an exceptional chemical selectivity and SECM therefore serves as an excellent tool for studying interfacial (electro)chemical properties and reactions.Tools for probing microstructures SECM measuring tips At the beginning. ring57.66 were used as SECM-tips for low-noise recordings of neurotransmitters release67.
Unlike the Faraday cage that is useful anytime when recording low amperometric currents. 24 . X and Y motors move the sample according to the given scanning parameters: speed. distance between data points and time for data acquisition. 11 Scheme of representing the scanning electrochemical microscope (left) and photo of the SECM used in this work (right). Fig. The microelectrode is positioned close to the sample with the Z stepper motor. Scale bar on the right image represents 35 cm. When the working distance is achieved. the vibration-free platform is useless if the SECM tip’s height is not controlled by shear-forces.Tools for probing microstructures Home build SECM set-up All SECM experiments gathered in this volume were carried out with the following set-up (Figure 11).
In the in the case of the macroelectrode a number of unit-volumes are emptied (9 white squares in this example) and 13 units are in vicinity to supply the mass transfer (blue squares).44 and the microelectrode 5. Notes Short after being introduced to preparation of microelectrodes by Dr. Fig. by diffusion.3. why it is believed the mass transfer towards a microelectrode is enhanced compared to a macroelectrode! I felt something is missing to me. something like a qualitative representation of the diffusional processes that take place around a macro. because as big are the resulting numbers as efficient is the mass transfer! For the above example. where the macroelectrode has 1. 25 . This model is valid for spherical geometries as well. E. Once the first species were consumed.and microelectrode the surface. Let us name the as unit volume (orange square).Tools for probing microstructures 3. whereas. I think is possible and didactically useful to compare the ratios of the blue square to white square. it becomes clear why the last mentioned is better in enhancing the diffusion of reactive species in direction of electrode surface. a Qualitative model explaining why the mass transfer of active species towards an electrode surface is more efficient for microelectrodes as compared to macroelectrodes. Bonsen. is much higher for a microelectrode as compared to a macroelectrode (Figure a). Quite a while after that moment I have got in mind a clear picture of a reasonable explanation showing why “the mass transfer is improved”. fresh material is pushed towards the electrode by forces derived from the chemical potential of the present species. 1. the volume of electrolyte of which content of redox species is fully emptied after the potential step was applied to the electrode. without resolving mathematical equations. The mass transfer is improved because the resource of the fresh material that is carried towards the electrode. in the case of the microelectrode one white square has only 5 blue squares around. (2001) I had difficulties in understanding.
3 M NaCl at a scan rate of 100 mV/s. 0. Fig. This means that the species in high excess (usually in bulk) are controlling the potential of the substrate while the traces of the partner species are thermodynamically instable (Figure b). so-called open circuit potential (OCP) of noble metal . for instance the reduction of ferricyanide. Thus. If an electrochemical in relation is carried out at the tip of a SECM that is positioned in the working distance.Tools for probing microstructures 2.1 M phosphate buffer. the potential of the surface is polarised at approximately +400 mV versus the Ag/AgCl 3M KCl reference electrode (potential measurement at zero current. b The stability potential of the redox couple ferro/ferricyanide. These domains are revealed in a cyclic voltammogram performed with a 10 µm Pt disk electrode in a solution of 5 mM ferricyanide. These two compounds are partners of a redox couple. the minute quantity of ferrocyanide will be quickly consumed at the conductive substrate and this before being able to affect the perturb the OCP of the metal – solution interface. Ag/AgCl 3M KCl reference electrode.aqueous solution. The OCP is hence a critical number for it determines what species can exist at a certain potential (circa 300 mV in the mentioned case).reference electrode couple). 26 . An electrolyte containing [Fe(CN)6]4lowers the open circuit potential to about -200 mV. Example: a portion of a noble metal is immersed in [Fe(CN)6]3.
Electrodes for electrochemistry in small volumes 4.66.1. respectively. … but the reference? When aiming on applying precisely positionable microelectrodes for measurements in ultra-small electrolyte volumes it is crucially important that the total diameter of their sensing tips including the insulating material is sufficiently small. Electrodes for electrochemistry in small volumes 4. The motivation to work on the development of such electrode originated from an observation that a colleague of mine complained against the difficulty to operate a three-electrode configuration in the tiny vials of a microtitre plate (Figure 12). Fig. 12 Working electrode small. Integrated working/reference assembly The information about SECM probes given before dealt with conventional type of diskshaped microelectrodes. However.69.72. Microelectrodes fulfilling this prerequisite have been constructed either by sealing carbon fibres or thin metal wires into tapered tips of pulled glass micropipettes56. 27 . an electrode will be described that is well suitable for electrochemical measurements in small volumes of electrolytes and especially for SECM applications in nanoliter droplets. and chemical vapourdeposited quartz73. these needle-type electrodes have to be jointly used with a reference electrode (RE) to accomplish at least a 2-electrode configuration as required for voltammetry.70 or by coating carbon fibres with a thin layer of an electrochemically depositable polymer 65. In the following.
4. which can occur when the tips of two separated mobile electrodes are brought closely together and moved autonomously from each other.D. the preparation of the proposed electrode system is difficult and needs a lot of hands-on experience to reach a sufficient success rate. Concentric layers of metal were deposited on the insulation of microelectrodes by sputtering76.D. Furthermore. Preparation of precursor electrode Disk-shaped Pt microelectrodes served as precursors for the fabrication of coaxial Pt/Ag electrode assemblies.1. the tapered tips of the double-barrelled microelectrodes are fragile and can easily break during operation. which 28 . However. L 100 mm). Pt wires were sealed into the tips of tapered glass capillaries and then silver was sputtered on the glass surface to form a Ag/AgCl reference electrode.1. The reference system was separated from the analyte solution with an agar salt bridge that was placed in the tip opening.Electrodes for electrochemistry in small volumes Clearly. I.75 mm. This probe design obviously had an advantage for measurements in extremely small volumes since a combined µWE/RE assembly is more easily to position. A carbon fibre was sealed with epoxy into one of the two tapered channels of pulled theta glass capillaries while the other kept a macroscopic Ag/AgCl wire in a chloride solution. Polishing the tip of metal-coated microelectrodes would lead to the exposure of the active disk that is concentrically surrounded by the insulation and an outer ring of metal and some used this approach for the fabrication of a coaxial microelectrode designed for in vivo measurements of nitric oxide75. only a single micro-positioning device and avoids the risk of collision. They were constructed from 10 µm-diameter Pt wires that were tightly sealed into the tapered ends of pulled borosilicate glass capillaries (O. needs not as much space.5 mm. 1. a technically easier alternative for the construction of dual probe tips is offered by depositing a metal onto the outer surface of the (glass) insulation of disk-shaped microelectrodes. 0. Recently a miniaturised dual electrochemical probe for voltammetry and SECM in nl droplets was introduced74. In contrast to the well established method of back-connecting the electrodic material to external copper wire with bi-component silver epoxy glue. galvanostatic techniques) of non-wetting substrates can be also achieved with the scanning droplet cell method82 where the substrate under investigation must act as the working electrode. a completion of such a 2-electrode assembly in highly restricted space is a challenging task. chemical vapour or electroless deposition (also used for preparation of substrates in microcontact printing77) were also used to establish an active electrical shield for reducing the stray capacitances and improving the microelectrode noise78-81. In principle. Note: spatial resolved surface analysis (potentiostatic.
the glue-wires contact is usually lost) 5. However. B). The ohmic resistivity of the Zn-to-Pt connection was found to be low and the electrode typically worked reliable. 29 . the wires are stiff enough not to bend when adding the copper wire and there was a good contact. zinc – tin is strongly binds against glass (because of the instant wetting resin) connection is fragile (once the glass capillary breaks. and 0. The melting solder entrapped the Zn particles and the wires ensuring good electrical contact between all components. which after placement was melted by careful heating in an coiled filament at high currents. In this case. D). no efficient control during electrode preparation 4. 1 hour) 3. 1 µm.5 mm) were attached to each other by filling the capillaries with a small amount of Zn powder and crushed tin solder.3 µm). therefore these electrodes can successfully replace the epoxy back-connected electrodes. expensive 5. To overcome this problem. no oven necessary (ready made electrode) 3. gloves) 2. no single zinc particle is wasted 2. The Pt and a Cu wire (Ø 0. inexpensive This method was applied first for thicker platinum wires without using zinc.Electrodes for electrochemistry in small volumes is awkward an alternative and easy method was developed. most part of the glue is wasted (it remains onto the preparation dish. the narrow space between the copper wire and the capillary tip was filled with zinc powder (Figure 13 C. for thin platinum wires this method is not suitable because the delicate wire can break or turn away as the copper is inserted and hence no electrical contact is established (see Figure 13 A. requires a oven for fastening the two components (800 C. glue – platinum/copper wires Zinc powder 1. Tabel 2 Bi-component silver epoxy glue 1. Table 2 outlines the advantages of zinc-based electrodes over epoxy ones. The ohmic response does not change over a wide range of scan rates. every preparation step is under control 4. Smooth Pt micro disks were exposed by polishing the tip at 90° on emery paper (grade 320 to 2000) and then on a polishing cloth wetted with alumina suspension (particle sizes: 3 µm.
1. The deposits typically had a morphology reflecting to some extent the irregularities of the abraded surface of the underlying glass and were mechanically sufficiently stable not to be damaged by handling of the electrodes and performing electrochemical 30 . the chemically deposited silver appeared as a uniform coating of white colour. 2 ml of 5% AgNO3 solution were then treated with 10% NaOH solution until no more dark-coloured precipitate (Ag2O) was formed upon addition. taken out and immediately dried with the handheld dryer. D). here.2. As can be seen from the Figure 14. To enhance the rate of Ag deposition. For further Ag deposition. The Ag2O was converted into soluble [Ag(NH3)2]OH by slowly adding 28% NH4OH until the solution became clear and colourless. the microelectrodes were rinsed with distilled water before heating (Figure 14). Obviously. this treatment increased the roughness of the substrate herewith offering a better physical support for anchoring the Ag deposit to the glass through microscopic fissures in its surface. B). wiped with soft tissue paper and rinsed with a stream of acetone in order to remove the grinding dust. the body of the Pt microelectrode was preheated with a heat gun. 13 Only solder alone can not connect the copper lead to the thin Pt wire (A. Zinc microparticles can establish an intimate contact between the external copper lead and the the glass-embedded platinum wire (C. Tollens’ reaction and formation of metallic silver was induced by adding a few ml of a reducing agent. Chemical deposition of silver onto the body of precursor electrode Well-adhering layers of Ag were accessible only when the glass insulation of Pt microelectrodes was rubbed with emery paper (grade 320). In a small tube. it is 10 % glucose.Electrodes for electrochemistry in small volumes Fig. For uniform and compact Ag layers the treatment had to be repeated multiple times. 4. and then quickly immersed into the freshly prepared silvering bath for only a few seconds.
31 . However. rinsing with pure water (3). The following experiments were performed with an electrode identical to the one in Figure 18. However. the electrode surface can be covered with ordinary nail varnish. a current density of 0.6 A/dm2 was applied to form well-adhering and stable Ag deposits.Electrodes for electrochemistry in small volumes measurements. Optionally. 14 The transformation of the precursor electrode (left) into the silver coated electrode (right) requires three steps: blowing hot air on the precursor (1). Fig. tips with diameters down to about 50 µm were obtained by successively polishing them at an angle in order to reduce the thickness of the glass insulation at the apex and then upright to expose the Pt disk. Reproducibly. the total tip diameter was about 200 µm giving a RG value (dtotal/dPt disk) of about 20. respectively. Tips of Ag-coated microelectrodes were carefully polished on emery paper (grade 2000) and polishing cloth soaked with alumina to re-establish a clean electroactive Pt disk. In the particular case shown. At room temperature.5-0. to enhance the stability of the silver layer and extend its lifetime. thicker layers of silver could be deposited on pre-coated microelectrodes by galvanic Ag deposition from a thiosulphate-based silvering bath (pH 10) (Figure 16). Finally. A top view of the tip of a polished Pt/Ag electrode assembly is shown in the scanning electron micrograph of Figure 17. a flat tip geometry was obtained with the 10-µm-diameter Pt wire well centred in the glass insulation and the thin layer of chemically deposited Ag. a copper wire was firmly attached to the silver layer using a heat shrinking tube (Figure 15). dipping into the Ag+ solution (2).
Fig. 32 . 15 The external connection of the coaxial silver layer.Electrodes for electrochemistry in small volumes Fig. 16 The thickness and morphology of the chemically deposited silver layer can be adjusted by electroplating of silver in an electrolysis cell with a silver anode.
Fig. 33 .17 Scanning electron micrograph of the Ag-chemically coated electrode.Electrodes for electrochemistry in small volumes Fig. 18 The completed coaxial working/reference electrode.
In both cases. even without chloridisation. 100 mV/s. Figure 19 compares two cyclic voltammograms of 5 mM [Ru(NH3)6]Cl3 that were recorded in a single well of a 384-well microtitre plate (Figure 20) at a coaxial PtµWE/Ag-RE electrode assembly with either the Ag layer of the assembly as internal pseudo reference (A) or a miniaturised Ag/AgCl 3 M KCl system external reference electrode (B). 19 Cyclic voltammograms recorded vs. 5 mM [Ru(NH3)6]Cl3 in 0. The half-wave potential (E1/2) for the reduction of Ru3+ measured vs.1 M phosphate buffer. However. the internal pseudo reference was shifted by about -60 mV as compared with the external reference electrode. 4. the inner reference (A) and an external Ag/AgCl reference electrode (B). For instance. the CVs displayed the expected sigmoidal shape (steady state) that is characteristic for microelectrodes.3. Applications of the coaxial Pt-µWE/Ag-RE in SECM It was thought that this electrode could also be turned to the development of a robust and easy to use (micro-)electrode arrangement supporting straightforward SECM imaging in nanoliter volumes of solutions. the shape of the CVs did not change notably over time indicating that the coaxial Ag electrode is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability of the coaxial Pt-µWE/ AgRE electrode assembly for SECM applications.Electrodes for electrochemistry in small volumes In order to verify ability of this electrode assembly to perform electrochemical measurements a series of experiments were designed to address this problem. 34 . Fig. it has been attached to the Z-positioning element of the SECM and used as a scanning probe with integrated reference electrode in electrolytes containing 5 mM [Ru(NH3)6]Cl3.1.
in control measurements. tip approach curves were recorded in both configurations on insulating (glass) and conducting (gold) surfaces. In contrast. The resulting negative (positive) feedback curves were properly overlapping and in good accordance with the curves from theoretical calculations (not shown). the external Ag/AgCl reference electrode. This clearly demonstrates that a recycling of tip-induced Fe2+ at the conducting outer ring of silver does not disturb significantly the positive feedback interaction between the Pt-µWE and a conductive sample surface. since the distance between the Ag ring and the Pt-µWE is about 100 µm which is far too large to be within the regime of the electrochemical feedback of a 10 µm diameter microelectrode.Electrodes for electrochemistry in small volumes Fig. For the given electrode geometry this was expected. In a large-volume electrochemical cell. spacing: 25 µm) (Figure 22). vs. approach curves were performed on gold using either an ordinary Pt disk microelectrode or a Pt-µWE/ Ag-RE electrode assembly with the ring of silver as SECM tips. The Pt-µWE. As can be seen in Figure 21. 20 Electrochemistry performed with the integrated Pt working / Ag reference electrode assembly in a single well of a 384-well microtitre plate. was operated in the amperometric feedback mode and scanned at constant height. SECM test trials were then carried out in small droplets (< 1µl) of the mediator solution that was placed on top of an array of four Pt band microelectrodes (length: 1 mm. Furthermore. it was kept at -350 mV vs. the obtained positive feedback curves were almost perfectly lying on top of each other. width: 25 µm. the integrated coaxial Ag-RE or. 35 . To induce diffusion-limited reduction of the dissolved redox mediator. an alteration of the feedback behaviour cannot be excluded when reducing the total tip diameter of PtµWE/Ag-RE assemblies below about 20-30 µm.
1 M KCl. 21 Approach curves that were recorded on a gold surface with (∆) the tip of a coaxial 10 µm-diameter Pt-µWE/Ag-RE electrode assembly and (O) a 10 µm diameter Pt disk microelectrode of similar RG value and with no silver deposited on the insulation. tip approach curves were used to position the tip of the Pt/Ag electrode assembly in close proximity to the sample surface. scan speed: 1 µm/s. 22 Schematic of the SECM experiment designed to scan with the coaxial electrode in a 500 nl solution and of the Pt array band used as a substrate. Typically. a working distance was chosen at 36 . Fig.Electrodes for electrochemistry in small volumes Fig. electrolyte: 5 mM [Ru(NH3)6]Cl3/0. As usual.
Fig. ultimately increasing the SECM tip currents in due course of imaging. However. the experiment will take considerable time and hence. 5 mM [Ru(NH3)6]Cl3) in 0. Figure 23 shows a representative 3-dimensional feedback image of the Pt microstructure that was acquired with the coaxial Pt-µWE/Ag-RE electrode assembly in a droplet of 500 nl volume of mediator solution.1 M phosphate buffer. a strategy to prevent solution 37 . the amperometric tip current changed virtually by a factor of two. with low scan speeds for SECM imaging. 23 3-D SECM image acquired by operating a coaxial Pt-µWE/Ag-RE as dual electrode scanning probe in the amperometric feedback mode on a Pt band microarray. imaging the Pt band microstructure provided the first evidence about the operability of the coaxial SECM tip arrangement for measurements in small volumes. The Pt bands are clearly visible with negative feedback observed over insulating areas in the neighbourhood of the Pt bands and positive feedback due to the regeneration of consumed mediator molecules above the electrochemically active Pt bands. Within the one hour of the experiment. Especially.Electrodes for electrochemistry in small volumes which the amperometric tip current dropped off to about 50 % of the value measured in bulk solution. Accordingly. This effect is confirmed by Figure 26A showing a set of fifty line-scans that were obtained on the Pt band microarray one after the other at scan speeds of about 5 µm/s. we observed that solution evaporation became a critical parameter for image acquisition due to rapid changes in the volume of the nl droplets. scan speed: 5 µm/s. a raise in mediator concentration took place. Successfully.
adding glycerine to the electrolyte to lower the water vapour pressure. A layer of paraffin oil serves as a protection against solution evaporation and the O-ring keeps the electrolyte in the proper place.Electrodes for electrochemistry in small volumes evaporation is of key importance when aiming for long-term SECM measurements in microvolumes. As shown in the schematic representation in Figure 24 and 25. Various methods have been proposed previously to stabilise small sample volumes. Fig. among them. Here. a large-diameter O-ring of appropriate thickness was used to keep the paraffin oil in place and hinder it to spread that thin that the droplet would be exposed to air and hence start to fade away. 38 . 24 Schematic representation of SECM in nanoliter droplet. regulating the humidity in their environment by maintaining a water-saturated atmosphere in specially designed closed chambers. or covering the solution with mineral oil8385 . the tiny droplets of ruthenium solution were protected with a reasonably thick layer of paraffin oil against evaporation.
the Pt-µWE was first placed into the droplet before adding the paraffin oil to the cell to avoid a contamination of the surface of the tip electrode with the oil. Figure 26B is a clear proof that paraffin oil indeed offered an effective diffusion barrier for water molecules and did not permit nl-sized droplets to evaporate and change their volume significantly throughout SECM imaging.2. a reference electrode had to be made. This experimental design is simple. Miniaturised Ag/AgCl reference electrode SECM experiments performed in a conventional electrochemical cell required the use of a pseudo-Ag reference electrode considering the bulky Ag/AgCl 3M KCl reference electrode available on the market. Certainly. 25 Photographs showing the aqueous droplet over the four Pt band. Again. 4. It turned out that a pseudo Ag/AgCl reference can not be used in oxidising media (such as ferricyanide solutions). For this reason. fifty line-scans were taken successively on the Pt band microarray at a scan speed of 5 µm/s but at this time taking advantage of the aforementioned strategy of evaporation protection. all covered by paraffin oil (left) and the immersed electrode positioned at the working distance over the Pt array (right).Electrodes for electrochemistry in small volumes 500 nl of electrolyte Pt band array Fig. a Pasteur pipette was used as specified (Figure 27) together with: 39 . and primarily was chosen to gain maximal spatial freedom for tip movements in X and Y direction. which is stable and small enough to fit in the available holder of the SECM tip. This was clearly indicating that the droplet volume and concentration of the mediator could be kept reasonably stable for the duration of the experiment when using coverage with paraffin oil. the current values of the first and fiftieth scans differed from each other by only 3-4 %. To build a trustworthy Ag/AgCl 3M KCl reference electrode. because the electrode potential is greatly affected. Although the line scans were not fully superimposed. a prerequisite for successful carrying out long-term SECM measurements in nl volumes.
Electrodes for electrochemistry in small volumes a ceramic frit with a diameter about 1mm mm.1 M phosphate buffer solution with a coaxial Pt-µWE/Ag-RE as scanning probe. The chloridisation of the silver wire was carried out in a typical solution of 3M KCl and 0.1M HCl in water (1:1 volumes) with a Pt coil as counter electrode. A B Fig. 40 . 3-4 cm of silver wire. A similar reference electrode was chosen as reference electrode for all experiments described in the next chapters. The shift of the electrode potential measured versus a commercial Ag/AgCl electrode is within few mV.(3M KCl) inside the capillary is high enough keep the potential constant and to sustain low currents for long time in a two electrodes set-up as usually used. The short cylindrical frit (about 2 mm length) is sealed into the narrowed part of the Pasteur pipette with the flame of a portable piezo-torch. shrinking tube. 50 line scans in (A) were recorded with the droplet exposed to air while the ones in (B) were obtained using a layer of paraffin oil to protect the droplet against solution evaporation. A relative thick and adherent layer of AgCl is obtained in two potential steps: 1 min at 5 V and then 10 min at 15 V. The reserve of Cl. 26 Droplet SECM carried out on a Pt band microarray in 500 nl droplets of a 5 mM [Ru(NH3)6 ]Cl3 in 0.
This operation can be done easily at anytime. The syringe shows the way of refilling the capillary with fresh KCl. glass cutter and the cutting line (5). complete electrode (6). frit (4). Pasteur pipette (1). storing tube (2).Electrodes for electrochemistry in small volumes Fig. 41 . electrode (3). 27 Simple. four-steps procedure for manufacturing of a Ag/AgCl 3M KCl reference electrode.
will require microfabrication methods that effectively assemble sensitive biological components (e. One example that illustrates our approach is the use of a copolymer of ethylene and vinylacetate (Vinnapas® EP16 W1) and glucose oxidase (GOD) for the assembly of micropatterned enzymes. Thus. SEM (Scanning Electron Microscope) and SECM micrographs have demonstrated that micro-deposition allows enzyme assembly to be performed with high spatial selectivity while the deposition conditions are sufficiently mild to retain the molecular structure of the enzyme (the activity of trapped enzyme had been high enough to produce detectable amounts of hydrogen peroxide). 1 Wacker Polymer Systems GmbH & Co. KG. The size and large number of different biosensors necessary to perform a complex analysis is a hurdle for small amounts of samples (Figure 28).1. The actual technology of micropatterning Biological Recognition Elements (BRE) such as photolithography.g. Enzyme Microstructures A biosensor is intended to be specific to a unique substrate (analyte). The work described throughout this subchapter was focussing on the application of polymers as structural materials used to entrap enzymes as recognition element. The EP16 offers thin-film properties that allow it to be locally deposited onto different surfaces in response to an applied voltage on a piezo microdispenser. piezo-microdispensing permits the creation of multi-analyte arrays with densely packed biosensors by immobilising BREs onto different substrates and preserving the activity of these molecules. and viable cells) onto suitable substrates. wine etc. Burghausen. This marriage. selfassembled monolayers. however. proteins. real samples (physiological fluids. an array is a promising candidate for becoming an ideal tool for detecting a large spectrum of analytes with a miniscule device. Once the enzyme and the polymer are mixed. However. enzymes.) are usually made up of multiple components and monitoring all of them would require a bundle of biosensors. Micropatterning and microelectrochemical characterisation of biological recognition elements 5. The successful marriage of biology and microelectrochemistry will result in improved biosensing devices that can have wide applications. waste waters. Germany 42 . nucleic acids. soft-lithography.Enzyme microstructures 5. the enzyme can be locally assembled onto a given surface.
43 .Enzyme microstructures Fig. 28 Schematic showing the power of miniaturisation in reducing the number of individual sensors and laboratory space.
5.1.1. About enzymes
Due to its unique 3-dimensional structure, each enzyme is specific to its substrate or to structurally similar compounds. Enzymes and substrates need proper circumstances to interact with each other. In the second half of the XIXth century, E. Buchnera had
investigated the alcoholic fermentation of sugars and proved the yeast cells are not necessary to ferment glucose, fructose or maltose. A juicy extract of brewer’s yeast is able to perform this task. His belief was that a protein, named by him zymase, must be responsible for this process. Inspite of XIXth century dynamism, lack of adequate information and minimal equipments made the separation of enzymes from cells and the structural analysis complicated and thus explaining why Buchner had doubts in considering the zymase as a member of enzyme family. It was a fact that “there are
important differences between fermenting action and the action of ordinary enzymes. The latter is solely hydrolytic and can be imitated by the simplest chemical means86”.
Well, he was wrong but this example intended to briefly give a view of the problems and ideas that scientists had encountered about 100 years ago working on enzymes. The biological processes that occur within all living organisms are chemical reactions, and enzymes regulate most. Without enzymes, many of these reactions would not take place at a perceptible rate. Enzymes are remarkable catalysts! Not only do they effectively accelerate the rate of the reaction, but they also limit the potential side reactions so that the yield of a reaction is essentially 100%. The overall energy of the reactant interaction with the active site can be determined from the equilibrium constants of the reactant-enzyme complexes [ES]. The equilibrium constants are usually in the order of 102 – 1010 M which corresponds to free energies of interaction on the order of -12 to -60 kJ/mol. As can be noticed, these energies are approximately 10 to 15 times lower than those associated with covalent bonding. Enzymes catalyse all aspects of cell metabolism. This includes the digestion of food, in which large nutrient molecules (such as proteins, carbohydrates, and fats) are broken down into smaller molecules; the conservation and transformation of chemical energy; and the construction of cellular macromolecules from smaller precursors. Many inherited human diseases, such as phenylketonuria87 (PKU - a genetic disorder that results in deficiency of an organism to convert phenylalanine to tyrosine), albinism (a genetic circumstance which
Eduard Buchner (Munich, Germany 1860 – 1917, Munich) won the Nobel Prise in 1907 for “his biochemical researches and his discovery of cell-free fermentation” (see: http://www.nobel.se/chemistry/laureates/ 1907/buchner-bio.html)
results in a lack of melanin pigment in the eyes, skin and hair of both animals and humans) and many others, result from a deficiency of a particular enzyme. Enzymes have valuable industrial applications, the fermenting of wine, leavening of bread, curdling of cheese, or brewing of beer have been practiced from earliest times, but not until the 19th century were these reactions understood to be the result of the catalytic activity of enzymes. Medical applications of enzymes make the metabolism products monitoring easier and less stressful to the patients (one example to be mentioned is the biosensor for glucose). In the early stages of the studies on fermentation, the notions “enzyme” and “ferment” coexisted88. Latin and Greek inspired innumerable scientists over time when looking for a name to be given to a new substance, living creature or phenomenon. And this is exactly what happened to enzymes: enzumos is a Greek word meaning “yeast” and fermentum i.e. a Latin word having the same meaning. Monitoring health-relevant molecules in body fluids became accomplishable in a simple manner due to the high (sometimes absolute) specificity of enzymes that make them perfect tools able to function in complex environment such as blood.
Enzyme classification A close view on the functional structure of enzymes shows proteins are not sole components but often accompanied by other groups like FADH2, NADH, porphyrins. Some enzymes made of pure proteins are able to attach and convert the substrate into product. However there are a large number of enzymes that use special non-proteic units to establish a catalytic reaction. This is the basis of an enzyme classification (Figure 29) as briefly depicted below: 1. Holoproteinsb - are enzymes entirely built from aminoacids 2. Proteides - contain beside a protein (apoenzyme) that is inactive but specific to a substrate, a chemical part that undergoes enzyme specific reaction namely cofactorc. Upon attachment type, the cofactor is called a prosthetic group, if binds covalently and is called coenzyme if binds loosely (see note 1).
From Greek “holos” that means “whole” The cofactor apart from enzyme is able to catalyse the reaction of numerous substrates.
Fig. 29 Enzyme classification. Enzymes only made of proteins are called holoproteides whereas enzyme containing proteins and other compounds are named as proteides. An apoenzyme is inactive as long as the cofactor is apart. The cofactor is covalently or loosely linked to the apoenzyme and thus it is named prosthetic group or coenzyme respectively.
Enzyme coding “Enzyme Committee” (EC)89 that is a subdivision of “Nomenclature Committee of the International Union of Biochemistry and Molecular Biology” (NC-IUBMB) established the enzyme nomenclature. The large variety of enzymes determined EC to assign each enzyme a code made up of four numbers (say A.B.C.D.). “A” indicates one of the 6 major classes (see note 2). “B” and “C” codes the affiliation to a sub- and a sub-subclass of enzymes. “D” is just a numerator that shows the place of enzyme in the list with enzymes of the same sub-subclass. According to EC nomenclature, one can refer to glucose oxidase as EC 126.96.36.199. Commonly, the glucose oxidase is known as GOD. This name will be used in the following parts of this work. Enzyme activity Enzyme activity means enzyme ability to transform a substrate into a product. Either a product or a reagent (even the substrate) is experimentally detected in order to measure, under controlled conditions, the enzyme activity. It is influenced by many factors (substrate, solvent, temperature, inhibitors, ionic strength, pH and cofactor). Two kinds of disturbing factors of enzyme activity: 1) specific to each individual enzyme as substrate, cofactor, and inhibitor. For most of enzymes, some substrates can react with but at different rates. This justification is valid for the cofactor and inhibitor too; 2) factors that control the protein conformation as solvent, ionic strength, pH and temperature affects the protein backbone shape. Temperature influences both the kinetic and the protein 46
These activities are typically measured in terms of the activity unit (U).“ It uses minutes instead of seconds as its unit of time. The old unit (U) was generally nameless for approximately 30 years and only occasionally called “international unit. Rackerd and he meant that detailed studies on how enzyme catalyses the conversion of one substance to another is generally waste of time until the enzyme has been purified from other substances that make up a crude cell extract. from mitochondria. An international unit (I. Another unit. the ATP synthase in 1960. d Efraim Racker (1913 Neu-Sandez. The mixture of thousands of different proteins released from a disrupted cell that typically contains several that direct other rearrangements of the starting material and the product of the particular enzyme’s action. nano. Kilo Novo.moles) converted by a certain enzyme quantity within a unit time. Of a practical interest is the specific activity defined as the number of units corresponding to 1 milligram of enzyme preparation (U/mg). “Don’t waste clean thinking on dirty enzymes” said E. The enzyme activity has to be measured at the optimal temperature of the given enzyme (see note 3)! Dissolved oxygen must be taken into account for the enzymes that use it during the substrate transformation. which is not keeping with SI or with the usual way of expressing rate constants in chemical kinetics. which are based on physical changes for instance lowering viscosity. which is used but did not receive widespread acceptance is the katal (kat) even though recommended by International System of Units (SI). One katal will catalyse the transformation of 1 mol of substrate per second. Poland – 1991 Syracuse. These units are gradually falling into disuse. New York. Only when one has purified the enzyme to the point that no other enzymes can be detected can one feel assured that a single type of enzyme molecule determines the conversion of substrate to product and does nothing more90.) corresponds to a unit time of 1 minute.or pico. Parameters that are more relevant are the activity of the enzyme preparation.About enzymes conformation. Enzyme kinetics Enzyme catalysed reactions are basically chemical reactions and a kinetic study is meaningful when aiming to understand the reaction mechanism or calculating the yields. as its purity is often low and not to mention that parts of it might be in an inactive state. 47 . which is defined as the amount of substrate (micro-.U. USA) American scientist that isolated. and supposedly better understood by industry. Enzyme activity related definitions The amount of enzyme that is involved in a biochemical reaction is difficult to determine in terms of grams of enzyme. Sometimes non-standard activity units are used such as Soxhet. Anson (AU – Anson unit).
k 2 -1 constant of activated complex break-up. Symbols have the following meanings: S – substrate. k – constant of activated complex consumption. v = k⋅S ⋅ v=k (first order reaction). the rate of the enzymatic reaction depends linearly on the amount of enzyme added and on the amount of substrate as well. the enzyme undergoes no net change. it must actually have a mechanism with steps that involve the enzyme reaction. [S ] << K M → vP = k 2 [E0 ][S ] KM 3. Conclusion: although the net reaction is simply S P. [S ] = K M → vP = v P max 2 Nowadays. Michaelis and M. for high substrate concentrations S. P – product. From the above listed experimental data and assuming that the formation and breakdown of the [SE] has reached a value that is no more changing during reaction (steady-state approximation). 5 Accordingly. E stands for the enzyme and S for the substrate. Menten. Particular cases: 1. for low substrate concentrations S. k 1 – constant of activated complex formation. the reaction rate is proportional to S. the reaction rate is independent on S. Herein. They proposed the following reaction scheme: S+E k1 k-1 [SE] k2 P+E In the conversion of S to product P. b. [SE] – activated complex. both more general and more specific models of enzyme kinetic exist for the enzymes that do not follow the mechanism described by Michaelis-Menten equation. Enzyme kinetics obeys Michaelis-Menten equation (L.About enzymes Experiments show that: a. (zero order reaction). E – enzyme. e 48 . [S ] >> K M → vP = vP max = k 2 [E0 ] 2. than the following equation can be derived (equation 5): vP = k2 [E0 ][S ] K M + [S ] Eq. 1913)e.
49 . three approaches towards the synthesis of such non-natural catalyst that mimic real enzymes are presented.. Enzymes are isolated and purified from plants or animals and due to abundance of other biomolecules this process is complex and time consuming. Inorganic An artificial enzyme analogue of Nafion/lead-ruthenium oxide pyrochlore (Py) chemically modified electrode (NPyCME) was synthesized by in situ precipitation through blocking of Nafion’s hydrophilic zones91. - KM gives quick information about enzyme affinity to the substrate (the lower the value of KM. A great advantage could be given by possibility of on-demand straightforward synthesis of enzyme-like materials. This issue already raised lot of attention in the last decades and achievements till date are encouraging.About enzymes The third equation can be used to define KM as substrate concentration at which the reaction rate is half of the maximum rate. i. Briefly. is true as long as the enzyme concentration is small compared to the concentration of the substrate. Artificial enzymes Surprisingly. the optimal pH and temperature must be set. when determining KM. KM (normally expressed in mM) is a short hand of: Note: - k −1 + k 2 and therefore has no dimension. k1 - KM indicates the required substrate concentration to achieving half of the maximum reaction rate. k1 the fundamental characteristic of the Michaelis-Menten mechanism is a transition from first-order to zero-order kinetics near a critical substrate concentration that is KM. - - the steady-state approximation. - it is useful to refer to k −1 as KS (the dissociation constant of [ES]). the stronger is the binding between substrate and enzyme and hence more product is obtainable in a given time). it has been possible to design assemblies of inorganic or organic molecules that have remarkable catalytic properties and high specificity to substrate. [SE] = ct. for the majority of enzymatic systems k2 is small compared to k-1 and thus KM ≅ KS.e.
to fasten) is. it should be a transition state analogue of the aimed enzymatic reaction). subsequently. an imprint containing the functional groups in a certain spatial orientation remains. So. how is this accomplished? We shall imagine one is aiming to generate a catalyst for a reaction having a transition state [TS]∗. In brief. the hapten is then transformed into an antigen by covalent binding of a polypeptide (usually bovine serum albumin. the following steps are required: 1. a cross-liked polymer is structured around a molecule that acts as a template (for best performances of the artificial enzyme. BSA). a substance resembling the structure of the transition state is synthesised (known as hapten/haptenef. The polymer has to have a high degree of cross-linking in order to preserve the shape of the template. such polymers are obtainable with little effort for industrial production. Moreover. the newly formed antigen ([TS]∗-BSA) is injected in small portions in a mouse. 3.About enzymes Organic The plausible translation of the principle of enzymatic catalysis to artificial catalysts has attracted also organic/macromolecular scientists since the 60’. Biochemical It is well known that antibodies are protein complexes used by the immune system to identify and neutralise foreign entities (called antigens) that accidentally enter a living organism. 50 . 2. This feature lead to the idea of creating an artificial antibody (also: abzyme. After removal of the template. catalytic antibody or catmab – from catalytic monoclonal antibodies). 4. formally. an incomplete antigen that can stimulate the antibody production only in combination with a particular protein. The monomers are bearing functional groups that can later interact with the template through covalent or non-covalent binding.94. f Hapten (from the Greek word “haptein” . the antibody is isolated from the serum of the mouse and used as a catalyst. Each antibody is binding only a single type of antigens in a similar way as an enzyme binds its substrate. One should not forget that the exclusive properties of enzyme rely on their polymeric structure! Synthetic polymeric molecules are attractive chiefly because of their chemical and physical properties (can withstand heat and chemical attacks)92. Otherwise the cavity will shrink and the substrate will not fit in. In principle. The first abzymes were reported in 1986 and since then various types were described93.
The GOD activity is colorimetrically detected by the increase in absorbance at 460 nm resulting from the oxidation of o-dianisidine (highly toxic compound which can cause skin irritation and sensitisation) through a peroxidase coupled system. It is present in any solution unless it is purposely removed (physically or chemically) and re-oxidise GOD back to its active structure with concomitant formation of hydrogen peroxide: GOD-FADH2 + O2 GOD-FAD + H2O2 The dissolved molecular oxygen is a co-reagent in this case. A glucose biosensor with improved electrode kinetics is calling for controllable concentration of the co-reagent and high diffusion towards the core of GOD. An orange colour is generated in this two steps reaction. Since the GOD´s discovery as an “antibiotic”. As inhibitors Ag+. However. Although specific to β-glucose. glucose oxidase consists of two identical polypeptide chain subunits each of them containing one Fe atom and one FAD (Figure a). but optimal at pH 5. pH 6. The KM is about 33 mM and the turn-over number is approximately 2.Glucose oxidase 5. FAD is a member of the adenilic acid derivates that play a key role in biochemical processes as cofactors. It works in a wide range of pH. it is employed to determine total glucose because as a result of the consumption of β-glucose.form from the equilibrium is converted to the β. 51 . One unit of glucose oxidase causes the oxidation of 1 µmol o-dianisidine per minute at 25oC. (shown subsequently to be due to peroxide formation) there has been a wide interest in GOD chiefly because of its value in glucose determination.form by mutarotation.1. Hg2+ are the most important. 1 FAD is the acronym of Flavine-Adenine Dinucleotide. it is not any longer able to transform more glucose. Cu2+.3x104 molsubstrate /second ⋅ molenzyme. The redox moiety of GOD changes its redox state from oxidised to reduced as glucose (reduced form) turns into gluconolactone (oxidised form).5. Structurally. the α.2 Glucose oxidase (GOD) It is a flavoenzyme1 that catalyse the following reaction: β-D-Glucose + GOD-FAD δ-D-gluconolactone + GOD-FADH2 Once a GOD molecule had converted a single glucose molecule into gluconolactone. GOD can transform an unlimited number of glucose molecules by the supportive action of dissolved molecular oxygen. New developments in glucose monitoring of blood are aiming at finding a replacement for the natural co-reagent with electrochemically active complexes of transition metals such as osmium or ruthenium.
2 52 . 30 The molecular structure of FAD. causing the black mould on certain types of fruit and vegetables. are stable for years when stored cold. The redox active parts are coloured in magenta and located on the isoalloxazine ring (blue. it is incompatible with strong oxidising agents and is better to keep it away from moisture. makes it affordable by many laboratories (104 units costs 20 € or 106 costs 900 €. Besides these. Aspergillus niger is a fungus. However. FAD could be seen as being made up of riboflavin residue (dark blue) and an ADP (adenosine diphosphate. Dry preparations having a yellowish colour. on the right side) of FAD. On the left is shown how the riboflavin residue undergoes redox reaction. shown in red). About FAD FAD’s (Flavin Adenine Dinucleotide) molecular structure and redox behaviour are shown in Figure a. ADP residue N O O O O H2 P O C N N NH2 N H3C H3C 2H+ / 2eR H3C N N O Riboflavin (Vitamine B2) residue O H3C H FADH 2 N H O N FAD oxidate state R N N O + 2H+ / + 2eH N N O H N N O H O OH P OH OH OH CH2 CHOH CHOH CHOH CH2 H3C H3C N N reduced state Fig. from Sigma Product Catalogue for Germany 2002-2003). Only glucose oxidase isolated from Aspergillus niger2 was used in the present experiments.Glucose oxidase Glucose oxidase is perfect to work with as a trial-enzyme because of its high stability in solution. described in chapter 5. the low cost. non-pathogenic to humans. R denotes the ADP-[CH2-(CHOH)3-CH2)].
SECM. microdispensers). the reader should be acquainted with the enzyme micropatterning techniques available up to date and their advantages/disadvantages.Patterning GOD by means of piezo microdispenser 5. urea and other substances. the enzyme concentration was not optimised. that the enzymes are preserving their activity after being shot through the microdispenser nuzzle (withstanding the shear forces and elevated pressure). The spatial resolution of such micro/nano structures is determined by precision in controlling the distance between two neighbouring spots. hands-on devices can not compete with the highly precise micropositioning stepper motor or piezo elements for creating biosensor architectures with high spatial resolution.3 Patterning GOD by means of piezo microdispenser 5. two ways of placing biological recognition elements onto a surface are available so far: Active placement Active placement refers to the procedure of delivering small droplets of biomolecule directly at the point of interest (capillary. simple tools coordinated by hand (capillaries)95 or sophisticated machinery controlled by computers (laser confocal microscope96. self assembled monolayers of thiols). Gaspar98). Before proceeding to present specific details of this work. The affinity of surface is tuned with an appropriate functionality to facilitate the specific binding of the biomolecules (for instance. Therefore. or any other compound of interest in water. increasing attention has been given to the formation of localised biomolecule microstructures in the last decade. food or physiological fluids. by means of the substrate generation – tip collection mode of SECM. Excellent 3-dimensional SECM images of different surface confined enzymes were achieved.3. Since biosensors are wished to become simply tools for home-monitoring of glucose.1.1. Indirect methods These methods require a chemical pre-modification of the surface aiming at the localised immobilisation of biomolecules. Aim The main goal of the effort is directed to enzyme micropatterning and to demonstrate the possibility of constructing a complex self-containing biosensor array for multiple applications: monitoring of drugs. S. the polymer matrix used to entrap the enzymes at a certain surface had not the optimal concentration. Conversely. pollutants. 53 . It was already shown (M. Nevertheless. Mosbach. However.97. Consequently.1. microdispenser) are used to create basic (spots) or complex (lines or grids) microstructures.
it becomes clear why a local electrochemical probe must be placed close to the enzymatic patches in order to be able to measure the enzyme activity/substrate concentration through the redox active products. the enzymatic reaction is slowed down because this product is (a weak) inhibitor of GOD. this minute quantity represents an apex for the given amount of GOD and reaction time. A large disk-shaped electrode is not appropriate for investigating micrometric structures (because it could a The dynamic range is the ratio of the maximum value versus the lowest detectable value of a specific parameter.25 x 10-16 mol of GOD will generate roughly 40 million product molecules per second (and of course will consume the same number of glucose molecules). I sought to further develop the complexity of the enzymatic patterns and to study the possibility to increase the dynamic rangea of the microsensor arrays. as D-glucono-1. and therefore.Patterning GOD by means of piezo microdispenser Following their foot steps. is measured in B (Bel) or dB (decibel). Several factors merge their contributions to make the reaction sluggish: a. a spot of GOD preparation pasted at a surface by means of piezo microdispenser contains 6. with the enzyme free in a large volume of solution. the diffusion of the substrate towards the enzyme molecules is hindered by the film and the surface on which the enzyme lies. The available product is even less if the enzyme is immobilised into a thin film of polymer. enzyme is not free to accommodate its position with regard to the incoming substrate (as would happen in bulk where it can rotate and translate in any direction). Now.5-lactone is formed and is accumulating in the film. 54 . c. 6. one also decreases the amount of product that can be produced within a given time. Note An enzyme that is interacting with the substrate in bulk will generate a certain amount of product. If these molecules diffuse away into a volume of 2 ml (of the electrochemical cell used in SECM measurements). Taking into account these facts. one increases the difficulty in analysing the product. Typically. b.25 x 10-16 mol of GOD (for 1 mg/ml GOD in the spotting mixture and a droplet of 100 pl). If the same quantity of enzyme is by any way grafted to a surface. As already mentioned. in a logarithmic scale: B=lg(value1/value2). when one reduces the dimensions of the catalyst patch. In ideal case. Typically. then there is an analytical difficulty in measuring the product in the resulting ≅ 2 x 10-10 M solution. then the speed of the product formation is smaller as in bulk.
1. The yellowmilky solution did still contain polymer aggregates with an overall size that could block the 55 . it was possible to overcome the drying of the enzymatic mixture at the nozzle. Obviously. We shall find in chapter 3 and 4 a detailed description of the microelectrodes properties/manufacturing. 31 Schematic of the control set-up of the microdispenser. 5. General consideration about GOD microdispensing Home-made microdispensing set-up The schematic view of a home-made microdispenser control set-up is depicted in Figure 31. A home-made electronic board combined the carrier signal (from the wave generator) with the power applied to the piezo actuator.5 cm2 (chip).2. The trigger signal generated in the computer was send to the wave generator where the power output of the power supply was modulated in order to produce on-demand-release of droplets. Repairing/cleaning the dispenser is not easy and successful any time. Fig.Patterning GOD by means of piezo microdispenser record signal from multiple enzyme microspots at the same time) and hence microelectrodes were preferred (active diameter 10 µm). the next chip was positioned in the shooting direction of the microdispenser.5 x 0. 0. In this way.3. The surface chosen for patterning glucose oxidase was gold sputtered on silicon wafer. Such plates were cut in rectangular pieces with appropriate dimensions. The rely card made possible the control of two stepper motors via a single motor-card. Once a chip was spotted. This unwanted clogging had to be avoided because normally it compromised the chips by shooting droplets out of the line. at a distance of 1 cm from the head of the microdispenser. Improved adherence of the gold layer to the silicon support was typically attained by using an intermediate layer of titanium between Au and silicon. A plastic holder allowed the placement of 5 chips in a row. the microelectrode is moved across the enzymatic microarray with a micropositioning system (see chapter 2). The mixture containing the enzyme was prepared by adding 1 mg GOD and 2 mg EP16 in 1 ml tri-distilled water.
Patterning GOD by means of piezo microdispenser
microdispenser. Such big particles have to be removed prior to shooting. It was noticed that a filter unit of 5 micrometers pore-diameter retained not only large amounts of the polymer but even enzyme. Alternatively, centrifugation of the enzyme-polymer mixture was found to be a better choice. Centrifugation for half a minute at a speed of 700 rotation/min (for a normal lab centrifuge) was enough to settle the large aggregates. The clear supernatant had been carefully sucked into a 1 ml syringe and further used to fill the chamber of the microdispenser. Although the droplets are visible to naked-eye, while shooting they have to be well illuminated with a fiber optic light from a side in order to see if they are ejected from the microdispenser. Once a droplet touches the surface of the gold it appears as a glittery spot for 1-2 seconds till it gets dried. The polymer matrix is hydrophilic and thus is capturing a lot of water around. In this case the GOD is rather mobile (swimming between polymer chains) and could dissolve away from the polymer if not allowed to cure for 20-24 hours before being further used. This GOD preparation had enough activity for months even after keeping it at room temperature. However, to avoid a probable loss of enzyme from the polymeric matrix all enzyme micropatterns herein presented were cured 24 hours. 188.8.131.52. Simple GOD microstructures A PC in combination with Windows software programmed in Microsoft Visual Basic 3.0 (Microsoft, Unterschleißheim, Germany) was used for the control of all system parameters
Fig. 32 Snapshot of the software interface used for micropatterning with the piezo microdispenser.
Patterning GOD by means of piezo microdispenser
(like: number of droplets per spot, distance between and the number of lines; number of spots per line), and also for data acquisition. A snapshot of the user-friendly interface of the mentioned software is shown in Figure 32. With this automated device one could simply create a variety of microstructures of GOD captured in the Vinnapas EP16® polymer dispersion. The SEM micrographs shows, arrays of spots (Figure 33A) with different enzyme concentration or even grids (Figure 33B) with variable load of enzyme were achieved. The polymer film is thinner in the central part than at the rims (see Figure 33C) where apparent heights of 1-2 µm were measured for with the electron-beam parallel with the surface of the microstructure (the apparent heights was used because the enzyme-polymer film was not metalised by sputter-coating prior to imaging). Figure 33D gives a closer look at the nanoscale structure of the sputter coated gold. As plainly visible, the gold surface is not smooth but full of cracks and gaps. It is possible that these features have a certain role in stabilising the polymeric layer.
Fig. 33 SEM micrographs of spots and grid of piezo-dispenser patterned GOD containing polymer. The dispensing mixture contains 1 mg/ml enzyme and 2 mg/ml EP16 W (A, B, C). Number of droplets / spot is increasing from the upper right corner to the lower left corner of the image (A, B). Close-up of a single spot (C). Gold sputtered surface appears as broken up. This feature could explain why EP16 adheres so well to this gilded surface (D). Scale bar represent: 200 µm (A, B), 20 µm (C) and 1 µm (D).
Patterning GOD by means of piezo microdispenser
Gold is a material that has a certain affinity to many compounds, especially to organic functional groups. Is it possible to immobilise glucose-oxidase on a gold surface without resorting to any polymers? To address this question, two aqueous solutions were prepared one containing only polymer (2 mg/ml) and other only enzyme (1 mg/ml). The idea was to dispense the droplets from each preparation as spots and then to compare the morphology of the corresponding films. A well adhering and rather homogenous covering was obtained with the polymer suspension in water (Figure 33A). Surprisingly, the enzyme alone formed solitary accumulations and hence indicating low natural affinity for gold (Figure 34B)
Fig. 34 SEM images corresponding to 2 mg/ml pure polymer spot (A) and pure 1 mg/ml GOD spot (B). Scale bars are identical and represent 20 µm. Both were aqueous solutions.
A versatile tool for micropatterning biological molecules is the microdispenser, it offers a straightforward procedure to create complex geometrical structures automatically, or semiautomatically or to generate as already mentioned, enzyme gradients. For this, multiple droplets having identical composition are directed towards the same area of the target surface. Now, precautions must be taken owing the fact that 1) the overall enzyme activity is not increasingly proportional to the number of droplets/spot and 2) the diameter of the spots is directly proportional to the number of droplets/spot; these two issues will be discussed in more detail.
Two approaches could be used to change the biological recognition element spot concentration. A negative answer could suggest a misuse of the enzyme. A series of SEM micrographs shows the spots with variable diameters obtained by increasing the number of droplets deposited on each target place. but the disadvantage is the necessity to refill the dispenser every time and for each concentration. For the same composition of the enzyme solution (1 mg glucose oxidase and 2 mg polymer per ml of tri-distilled water). the target place can be shot multiple times and the advantage is that the complex arrays can be created without refilling the microdispenser. which is not affected by n). 3 n Diameter (µm) 1 75.Patterning GOD by means of piezo microdispenser Discussion Enzyme exposure to the substrate and its relation to diameter/number of droplets In order to optimise the glucose biosensor array. the diameter of the spots increases with the number of droplets (say n) as depicted bellow (Table 3): Tab. a larger volume will produce a larger diameter spot and considering a constant film height one can calculate the ratio of the droplet/spot radius.7 2 97. Let us assign the following parameters for droplet and spot (which is in fact a disk) (Table 4): Tab. firstly. Is the enzyme activity increases with n? (It is about activity of a spot as active entity not about the specific enzyme activity. Secondly.0 3 133. or in other words that is useless to try to increase spots activity in this way. it can simply be achieved by increasing its concentration in the shooting solution.3 Note: the numbers on this table are valid only for the mixture composition described above. the enzyme concentration in the spots was varied. 4 Droplet V R R' h volume of a single droplet radius of a single droplet - Spot (Disk) volume of a single spot radius of a single spot height of the spot 59 . Theoretically.
73 . However.41 1. 35 The droplet and its corresponding spot (disk). Fig. Consequently the active substance in the mixture is less exposed to the solution in the case of a double droplet as for single one (Figure 36). Tab. For a triple volume the resultant ratio is 3 = 1. a larger droplet will splash and spread more around the impact area as compared to a small one. the film is a bit thicker as for a single droplet of the same solution. and hence the possibility to find a relation between the droplet volume and the corresponding spot radius (Figure 35). These are theoretical values and will be used as a reference to calculate the deviation of the real spot diameters and to explain why the “spots activity” is not proportionally increasing with parameter n ' (Table 5).Patterning GOD by means of piezo microdispenser The volume is the only parameter that has equal values for both droplet and spot.28 1. ' R2 V2 = ' R1 V1 1/ 2 n = 2 n 1 1/ 2 The ratio of the volumes is equal to the ratio of the number of droplets and this explains the second term of the upper equation. Rn is the radius of a spot made of n droplets. For instance. Radius of the disk is a function of the droplet volume. Indeed. Similar ideas lead to an equation describing the relation of any two spot radii when their volume ratio is known. if the volume is doubled then the radius ratio is 2 = 1.76 Remarks about film ticker as single drop expected thickness 1.41 .73 R3' R1' Remarks presented here are roughly valid for bigger droplets. 5 Theoretical ' R2 R1' Experimental 1. for a double sized droplet. 60 .
Light coloured molecules stand for “hidden” enzyme. (A) thin film exposes it better as a tick film (B).Patterning GOD by means of piezo microdispenser This behaviour will decrease the response signal of a biosensor (an amperometric current in this case) because of the “hidden” enzyme in the thick film. Vinnapas EP 16 polymer dispersion had a good behaviour in terms of permeability for water and glucose. Fig. 36 The exposure of the enzyme in dependence from polymer film thickness. A well permeable polymer film usually is enough to make the active compound reachable from the bulk of the substrate solution. 61 .
4 Visualisation of GOD microstructures by SECM An indication that surface immobilisation of GOD is possible with the use of a piezomicrodispenser is only half the way showing that the construction of a biosensor could be done in an easy and flexible manner by ink-jet printing. two lines were spaced at 500 µm to each other. 37 Optical photographs of GOD/EP16 polymer microstructured on gold surface.Visualisation of GOD microstructures by SECM 5. Metallic wires (Pt 10 µm and Ag 500 µm) were used as scale bars. Thus. Typically.1. 62 . or 10 µm Pt) (Figure 37). Fig. Integrated scale bars along the length were represented by metallic wires with known diameter (either 500 µm Ag. The SECM was the chosen instrument for demonstrating a set of quantitative and qualitative measurements of micropatterns of glucose oxidase. The spotting solution contained 1 mg/ml GOD and 2 mg/ml EP16. It is important to demonstrate that this approach would preserve the natural and main important feature of an enzyme: its catalytic properties. it is better to inspect them with the aid of a light-microscope to check for possible errors (such as unwanted satellite droplets or discontinuities in the pattern). Although such an arrangement of enzyme/polymer microstructures can still be observed with the naked eye. each new droplet was shot out of the nozzle along the X axis with a displacement of 50 µm (for an average spot diameter of 75 µm). The freshly dispensed microstructures were allowed to cure overnight at ambient temperature. To obtain continuous lines. Microlines with variable enzyme content The microdispenser chamber was carefully filled with a mixture of GOD (1 mg/ml) and Vinnapas® EP16 polymer dispersion (2 mg/ml). before being studied by SECM. it was proceeded to probe the activity of the surface confined glucose oxidase by measuring the released hydrogen peroxide from a GOD microstructure in the presence of glucose.
1 M phosphate buffer pH 6. the working microelectrode is too far from the chip to detect the minute concentration of anything released from the enzyme preparation. Subsequently. or a cell screwed from the top. The ultramicroelectrode (UME) is brought manually to about 1 mm above a clean area of the gold chip and in the vicinity of the enzyme pattern. Fig. The refinement of the height of electrode was typically achieved in the feedback mode of SECM. namely +600 mV vs. The incoming glucose is converted at the enzyme microstructure into gluconolactone and hydrogen peroxide.Visualisation of GOD microstructures by SECM To facilitate the SECM measurements these enzyme modified gold chips had to be first placed into an electrochemical cell (EC cell). A solution of glucose in 0. it could be a simple Petri dish. As the glucose and GOD are reacting. the two electrodes (a 10 µm Pt disk-shaped ultramicroelectrode and an Ag/AgCl pseudoreference electrode) necessary to perform electrochemical measurements are positioned inside the EC cell. the reference electrode. Now. the solution of the mediator was removed and the chip was thoroughly rinsed with pure water. if the scanning ultramicroelectrode of the SECM is kept at constant potential. the products (gluconolactone and H2O2) start to diffuse away into the bulk of the solution. 63 . the oxidation of hydrogen peroxide occurring at the Pt disk can be monitored (Figure 38). For this experiment.1 M phosphate buffer was then poured into the cell over the GOD micropattern. the approach was done in a 5 mM solution of [Ru(NH3)6]3+ in 0. In this position. Once the chip (5x5 mm2) is in place. until the tip-current increased with 30% of the bulk value. a small cup. In this particular case. which is subsequently detected at the Pt disk electrode.7. 38 Schematic showing the underlying idea of the measurement of released hydrogen peroxide in the generation-collection mode of SECM.
7 – red. and 20 – for dark blue. the maximal current response in each case (A. A 25 µm Pt disk electrode as SECM probe was scanned over the three lines of the microstructures at different substrate concentrations (Figure 39). The produced microstructure consisted of three parallel lines with 500 µm spacing. C) is proportional to the glucose concentration in bulk. Glucose concentration were (mM) 5 – yellow. Therefore. 10 – cyan. Fig. The enzyme concentration was varied through varying the number of droplets deposited per target spot. Line A was prepared by dispensing 1 droplet/spot whereas lines B and C were prepared with 2 and respectively 3 droplet/spot. in the amperometric recording three current peaks with roughly equal heights can be noticed. It turned out that the enzyme was used in excess compared to the amount of available glucose.1 M phosphate buffer pH 6. The spotting mixture included 1 mg/ml GOD and 2 mg/ml polymer Vinnapas® EP16 in 0. Accordingly. Calibration curves obtained in the generation-collection mode of SECM Optimal concentration of enzyme in a microstructure cannot merely be calculated because the KM value changes when the enzyme is immobilised onto a film at a surface.1 M phosphate buffer. 64 . 25 µm Pt SECM tip.7. Supporting electrolyte 0.Visualisation of GOD microstructures by SECM The obtained amperometric current is proportional to the concentration of the substrate if the enzyme is not in the saturation regime. For this reason an uncomplicated enzyme micropattern was designed and investigated to find the favourable concentration of GOD. the number increased from line 1 (A) to line 3 (C). the entire microstructure is screened and the currents corresponding to specific locations within the studied area are recorded by a computer. B. Because of the motion of the tip. 30% current increase. 39 Line-scans over a GOD microstructure obtained in generationcollection mode of SECM. Nevertheless.
The spotting solution contained 0. with the constant-height mode the tip crash is at high risk on tilted or rough surfaces.1 mg/ml enzyme and 2 mg/ml polymer dispersion. This mode of scanning has the advantage of being fast and straightforward. the constant-height mode of SECM has certain limits on heterogeneous surfaces displaying variations in both. especially when decreasing the size of the SECM tip for imaging 65 . enzymatic microstructures were imaged only with the SECM tip moved at constant height above the surface.1 mg/ml and 2 mg/ml Vinnapas® EP16 in the dispensing solution. conductivity along with topography. to obtain calibration curves. (A) bird-view of the grid.1 M phosphate buffer. Thus. calibration curves. Glucose concentration was 100 mM. Figure 40 shows the SECM (A – bird view and B – three dimensional) images of this grid. a micro-grid with increasing concentration of GOD from top to bottom and left to right was scanned again with a 25 µm Pt tip in the generation-collection mode of SECM. On these samples. 25 µm Pt SECM tip. However. changes in the tip current arising from distance variations for principal reasons cannot easily be differentiated from ones originating from alterations in conductivity.Visualisation of GOD microstructures by SECM These plots are. in fact. Imaging achieved in the generation-collection mode of SECM. Note: if the enzyme is working in the saturation range. (B) three-dimensional representation of the grid. In this case. Furthermore. 40 SECM images of a microgrid with increasing GOD concentration from top to bottom and from left to right. then the kinetic of the GOD enzymatic reaction depends upon the diffusional transfer of O2 from outside gas atmosphere. High-resolution constant-distance SECM on immobilized enzyme micropatterns Up to this point. A GOD concentration of 0. 30% current increase. white areas indicate higher concentration of hydrogen peroxide. the enzyme concentration should not be raised to more than 1 mg/ml because the analytical signal (amperometric current from the oxidation of H2O2) is not improved. elevated areas stand for increased enzyme concentration. In addition. Fig. resulted in a saturation of the enzyme. it can be performed with standard Pt-glass microelectrodes. Supporting electrolyte 0. and high concentration of glucose (100 mM) in the bulk.
This is the heart of shear-force constant distance mode of SECM. In the non-optical shear force based distance control54. at its resonance frequency. 41 Schematic representation of the set-up as used for constantdistance mode SECM with an integrated non-optical (piezoelectric) detection of the distance-dependent shear forces. two piezoelectric plates are glued to the upper part of the highly flexible. Hence. Fig. a mode in which the scanning tip follows the profile of the sample at constant distance. 66 . a change in the vibration properties of the tip appears and is noticed as a shift in vibration amplitude and phase. is an oscillating measuring tip that is forced to vibrate. The upper plate vibrates the SECM tip at its resonance frequency while the lower serves as a piezoelectric detector of the amplitude of tip oscillation and frequency. This does not only allow simultaneous acquisition of the electrochemical tip response and the sample topography but also effectively prevents against tip crash even with smallest SECM tips.Visualisation of GOD microstructures by SECM at higher resolution. On the contrary. The foundation of the constant-distance SECM. This frequency comes from superposition of the hydrodynamic friction force and the excitation mechanic force. constant-distance SECM offers the advantage of a accurate control of the tip-to-sample distance since the integrated shear force-sensitive and computer-controlled feedback loop of this mode forces the SECM tip to follow the contours of the surface during the entire time of scanning/imaging. respectively. in the electrolyte solution. Any object that happens to be close to (under or aside) the vibrating tip will increase the friction because it stops the solvent molecules oscillating around the probe tip. needle-like ultramicroelectrodes just above the region where the pulled capillary starts to get thinner (Figure 41).
shear forces start to appear in close proximity of the surface. for example at 70 . most of the Fig. The morphology of the spot appears non-homogeneous and displays small local variations in the density of the enzyme/polymer film. Figure 42A presents a typical scanning electron micrograph (SEM) of a 70-µm-diameter glucose oxidase/Vinnapas® EP16 spot on glass. The unpolished tip was used because it offers much smaller total tip dimension as a polished. disk-shaped nanoelectrode. which leads to a damping of the tip vibration and a phase shift. In this way.80 % of the unaffected value with the tip far above the surface. the tip-to-sample distance is usually automatically stopped at a user-defined degree of damping of the vibration amplitude. both effectively registered by connecting the detecting piezoelectric plate to a dualphase analogue lock-in amplifier. The diameter of the spot is about 76 µm. A first experiment showed how efficient this technique is in guiding the vibrating microelectrode across a rough surface without colliding the tip with the protuberances on the surface. Obviously. 67 . the tip can be scanned in X and Y directions. The positive or negative shift in the vibration amplitude and phase as recorded by the lock-in amplifier provides an output signal for the Z-axis stepper motor that can move the electrode up or down unless they reach the user-defined set-point value resulted from the electrode approach curve. computer and Z positioning system makes up the computer-assisted feedback loop that accurately controls the tip-to-sample distance all through the scanning experiment. A microscopic spot of an enzyme/polymer mixture was dispensed on a glass surfaces and visualised in air using the unpolished tips of a pulled Pt-nanoelectrode as scanning probe. 42 Scanning electron micrograph (A) and shear-force topography image (B) showing a nonhomogeneous distribution of an enzyme/polymer mixture inside the circular area of a microscopic spot of glucose oxidase/ Vinnapas® EP 16. Continuous communication between the lock-in-amplifier. Once the tip is in the working-distance.Visualisation of GOD microstructures by SECM During the approach of the SECM tip towards the sample.
which is responsible for the shear force contact while the current resolution is related to the size of the electro active area of the tip. over these polymeric microstructures and to detect variations in the samples topography and the amperometric current image of the mentioned arrangement of lines. a quartz glass-insulated Pt-disk nanoelectrode had to be used for the visualisation of the prepared enzyme-containing polymer microstructures. Such a nanoelectrode can be made by simultaneously pulling a quartz glass capillary together with an inserted platinum wire using a laser-based micropipette puller56. The enzyme was entrapped only in the middle line (2 mg/ml polymer and 1 mg/ml GOD) whereas the outer lines contained pure polymer (2 mg/ml). Figure 42B illustrates the topography of the same spot as imaged using the constant-distance mode of SECM with an integrated piezoelectric detection for shear forces. SECM 68 . With this arrangement.Visualisation of GOD microstructures by SECM two compounds deposits upon drying in a thin rim at the edge of the spot. The topographic resolution of constant-distance SECM depends on size the sharpest part at the very end of the probe tips. a polymer micropattern consisting of three lines (70 µm in width each) made of Vinnapas® EP16 and with glucose oxidase was microstructured by means of the piezo microdispenser. the application of the non-optical shear-force constant distance SECM was then supposed to be focused on the simultaneous imaging of topography and enzymatic activity of a polymer-enzyme micropreparation in aqueous solution (Figure 43). Careful grinding the tips of the pulled capillaries on polishing pads leads to the exposure of active Pt-disks with diameters of far below one micrometer. local micrometer-sized topographical variations are clearly visible in the shear-force image underlining unequivocally the ability of the shear-force based constant-distance mode of SECM to resolve surface topography at high resolution. To visualise the activity of the enzyme microspot. To practically verify the above mentioned assumptions. Thus it should be possible to scan with the vibrating tip of the SECM positioned within the near field. With the encouraging output achieved in air. As with SEM. high-resolution constantdistance SECM imaging of the topography and localized glucose oxidase activity was performed in solutions containing 50 mM glucose. A disk-shaped Pt nanoelectrode with tip diameters of about 500 nm was used as scanning probe for the measurements. In order to fulfil this requisite the following strategy was considered: a number of polymer (P) and polymer-enzyme (P&E) microstructures (such as lines) patterned onto a surface should present both topographic and chemical activity features when immersed in a solution containing the substrate of the given enzyme.
an Ag/AgCl pseudo reference electrode. This was expected as only the middle line contained active enzyme and thus. a product of the enzymatic reaction that takes place within the polymer matrix (red line). while the stable feedback loop guaranteed a constant tip-to-sample distance of about 100 . To allow the local detection of enzymatically generated H2O2 in the generator/collector mode the SECM tip was poised to a potential of 600 mV vs.1 . Although the topographical resolution with disk-shaped Pt nanoelectrodes was found to be not as good as with the tapered tips of unpolished electrodes. the local production of H2O2 is limited to the area covered by that line. On the other hand. line scans of topography (blue) and amperometric SECM tip current (red) were both simultaneously acquired by scanning across the enzyme/polymer linemicrostructure (Figure 44B). The shift of the resonance frequency of the vibrating tip due to the shear forces is used to obtain the topographic image of the studied microstructure (blue line). an increase in the amperometric tip current was observed only just above the middle polymer line. 69 . Fig. small lateral variations in the topography of the enzyme/polymer structure are still visible.Visualisation of GOD microstructures by SECM imaging in the constant-distance mode of operation was carried out at scanning speeds of 0. 43 Trajectory of a SECM tip scanned over a 3-dimensional microstructure made of a polymer (P) in which the central part contains a biological recognition element such an enzyme (E).200 nm. for instance. As clearly visible in Figure 44A. Polarisation of the SECM tip at proper potential could be used to achieve an image of the electrochemical activity of the surface by detecting.1 µm s-1 for X and Y displacements.
Measurements performed in the constant-distance mode of SECM with a 500 nm Pt-disk nanoelectrode. With such achievements. and with GOD immobilised only in the central line (B). SECM is widening its application field to objects with nanoscopic surface features and (electro) chemical inhomogeneities. 44 (A) Line scans of the topography (blue) and amperometric SECM tip current (red) simultaneously acquired in solution containing 50 mM glucose by scanning across a polymer microstructure consisting of three lines of Vinnapas® EP16. for instance. Conclusion The combination of nanometre-sized SECM tips with the non-optical shear force positioning allowed simultaneous imaging of the topography and of the local chemical activity of. enzyme-containing polymer microstructures with high spatial resolution.Visualisation of GOD microstructures by SECM Fig. Scale bar: 100 µm. 70 .
who prepared the neurons. at the Ruhr University of Bochum. Editor: Rainer Waser. the special buffers and also incubated neurons over the laminin microstructures (for details see chapter 6 “Experimental”). Münster. University Eye Hospital Münster. A close-up of the microdispenser and positioning system is shown in Figure 46. 2003. and that it is a complementary part of the work directed to the growing of neurons on artificial microstructures – it was done in cooperation with Dr. “Neuroelectronic Interfacing: semiconductor chip with ion channel. P. Indeed. Fromherz. According to his confession. a 71 . holders. Hearing all this I was thinking that if this would have been realised by now. power supplies and obviously lots of connectors. I realised how complicated it is to fulfil this taskb. The microdispensing work was mostly done at the Eye Hospital. the idea of developing such devices came up in an unexpected way.Defined adhesion/growth of living cells 5. Wiley-VCH Berlin. Getting irritated while using a personal computer (a Macintosh) about 20 years ago. b This is a considerable technical challenge because an ionic current in the cell has to interact with the electronic current in the silicon chip and between these two parts (cell–chip) is gap of several tents of nm that blocks the current flow. I would have wrote my PhD thesis faster and enjoyed the springtime! Ok. I travelled several times to Münster together with a heap of equipment (computer. the so-called neuroelectronic interfacing. wave generator. Defined adhesion/growth of living cells In the beginning of the year 2004. I had an opportunity to listen to a lecture by Professor P. Heiduschka from the Department of Experimental Ophthalmology. plugs and replacements for the microdispenser) (Figure 45). P. He is the director of Max Plank Institute for Biochemistry in Martinsried (near Munich) and world-wide known as the initiator of the research towards binding the neurons to electronic devices. Actually. and brain”. nerve cells. this was only a minor thought.2. the electrical interfacing of semiconductors and neurons necessitate micropatterning techniques to control neuronal networks. Fromherz. the task was to prepare microstructures of laminin on different substrates for growing chicken embryo neurons. microdispenser. Nanoelectronics and Information Technology. For this. In the framework of an ELMINOS project. 781-810. All the following results are shared with Karla Tratsk-Nitz. Artificial networks of living neurons are supposed to facilitate the understanding of the properties and functions of neurons. Sooner or later they will be grown in-purpose in damaged tissues of the human/animal body in order to repair locally the connection of the nerve cells. he dreamt to replace the keyboard with an electronic implant in the brain that could possibly transfer all the wished commands directly to computera.
Defined adhesion/growth of living cells Fig. (D) piezo microdispenser and positioning system. (B) rely card. 45 Microdispenser set-up as mounted and used at the Eye Hospital. (E) microdispenser leads.1. (C) cold-light lamp. (B) reservoir of the spotting mixture.2. Introduction Study of properties and function of neurons belongs to the major research fields in life sciences. Fig. 46 Close-up of the active area: (A) the microdispenser head. The aim of the present study was to check whether laminin patterns can be created by the microdispensing technique and if such patterns are suitable for patterned adhesion of neurons. (E) laminar flow bench. Münster. (D) sample positioning stepper motors. Connections between neurons are established by synapses between axons and 72 . 5. (C) power supplies. (A) wave generator.
multi-domain protein122 with many binding cites for cellular receptors123. wells or similar structures101-105 or spatially dissolved chemical modification of the surface106. They include modifications of surface topography by grooves. which can be achieved by microcontact printing (stamping)108-112.or pico„In vitro“ experiments/processes are taking place out of a living organism (in a test tube. In order to study the properties of neuronal network (signal propagation. From the Latin word “vitreum” – glass / vitreus – made of glass. and a patterned attachment of two different cell types could be achieved after coupling an RGD peptide to quinone groups121. and adhesion and growth promoting properties of laminin could be achieved by adsorptive deposition of a polylysine layer prior to adsorption of laminin130. where contacts between neurons can easily be traced99.107. If microstructured electrodes are present on the substrate.124.2. photolithography99. Many of the methods of patterned surface modification are restricted to certain material properties of the substrate and/or require expensive equipment and complicated procedures. we choose the so-called ink-jet printing11.100. Electroactive structures were also created by microcontact printing. for instance). such investigation might be difficult owing the complicated structure of naturally developed neuronal networks. Different approaches have been tried to achieve defined adhesion of neurons on artificial substrates. This method has been known by commercial printers for several years.2. they can be coated selectively as it has been demonstrated for adhesion of neuroblastoma cells on platinum stripes coated with laminin-derived peptides119 and axonal outgrowth on laminin-coated platinum stripes120. c 73 . which should be suitable for a variety of substrate materials. we used a piezoceramic actuated dispenser developed at the Lund Institute of Technology (for details see chapter 2). Otherwise. Laminin has been used for many years as substratum for in vitro cultivation of neurons127-129. It plays a crucial role in the development and maturation of the nervous system125. For this study.132 and microcontact printing133-135. 5. they should be grown in vitroc in a defined pattern. What is available so far? A spectrum of different technologies for the directed growth of living cells is available. An alternative method having the ability to create well defined and reproducible surface microstructures at the micron level is using an excimer laser beam to micro-sculpture a polymeric film118. Laminin is a large.126. or neurotransmitters release). Initially developed for the handling of very small volumes of nano. On the search for a simple and inexpensive method. Normally they occur in living organisms that means “in vivo”. There are also reports of patterned deposition of laminin by photolithography131.Defined adhesion/growth of living cells dendrites.114-116 or plasma polymerisation117.
Therefore. for MALDI-TOF MS136. To circumvent any contamination of the equipment. The result is shown in Figure 46A. all instrumentation and materials involved in this project were sterilised prior use with a 70% aqueous solution of ethanol. positioning system. It can be seen that the liquid in the lines contracts. At the very beginning of this common project. Note: Special circumstances as handling living cells requires a sterile environment. continuous lines with one droplet/spot were made. In addition. no impurities could enter the space where the microstructures are obtained.3. A constant stream of filtered and sterilised air was continuously flowed outwards the hood. Accordingly. Although the contraction is still visible. the idea was to check whether the aqueous solution of laminin is proper for use in this form or it needs a support. with a high variability regarding the geometrical shape of the pattern. e.137. a mixture of laminin and Vinnapas® was used. single irregular dots are formed. The successful growth of neurons depends partially on the degree of sterility of gloves and equipment. 74 . the microdispenser can also be utilised for the creation of substance patterns on substrates.g. Moreover. and glass cover slips) were placed into a laminar flow bench after being disinfected. 5. this mixture of laminin and Vinnapas® was used in the experiments with the neurons. Results and discussion In the further described experiments with neurons. This behaviour can be attributed to the properties of laminin that possesses strong intermolecular interactions. continuous lines could be created now (Figure 46B). with a width of 100 µm and a distance of usually 500 µm. In order to obtain continuous lines on the glass substrate. as a result.Defined adhesion/growth of living cells litres in microscaled analytical devices. prior to use. the microdispensing set-up (micropump. This has been demonstrated already by the creation of microstructures glucose oxidase and other enzymes on gold substrates98. The solution shot onto the substrates contained 2-4 mg/ml Vinnapas® and 20 µg/ml of laminin diluted in Hank’s balanced salt solution.2. one has to wear rubber gloves all the time and has to clean them with alcoholic solution as often as one retracts the hands from the hood. Therefore. they were. several substrate materials can be used. centrifuged (10·g) for 30 seconds and the agglomerates settled down at the bottom of an Eppendorf tube. Since agglomerates of the polymer could clog the microdispenser nozzle.
Fig. there are also some fluorescent spots visible outside the lines.Defined adhesion/growth of living cells As the next step. 47 Staining of laminin–Vinnapas lines using the anti® laminin antibody and Cy2 -conjugated secondary antibody. if the laminin molecules entrapped in the polymer are still accessible to surface binding molecules so that neurons could bind to them. it was checked. It could be seen. 75 . by fluorescent microscopy. because there is still enough laminin present on the lines. Scale bar: 200 µm. Fig. Vinnapas® lines with and without laminin were created and stained with an antilaminin antibody. that the antibody was really bound to the lines containing laminin in a dot-like manner (Figure 47). Nevertheless. it should have no significant effect on the patterned adhesion of neurons. ® As depicted in the above images. 46 Comparison of lines shot on glass slides with: (A) laminin solution and ® (B) a mixture of laminin and Vinnapas . This could mean that some laminin molecules might have been washed out of the polymer matrix during the long procedure of immunochemical staining. whereas no staining can be seen if the anti-laminin antibody or laminin were omitted. For this purpose.
76 . Immunocytochemical staining was performed with an antineurofilament antibody. Neuronal adhesion is not restricted solely to the lines. Examples of the behaviour of neurons on glass cover slips are shown in Figures 48. In order to verify that adhered cells are neurons. A suspension of neurons was applied onto the substrates with the lines. To obtain this fluorescent image. so that they do not become part of a neural network developing on the lines. It is obvious that adhesion of neurons follows the laminin-Vinnapas® lines. b) 1 hour incubation with α-laminin antibody that previously dissolved in BSA phosphate buffer. d) 1 hour incubation with the fluorescent tagged antibody dissolved in 1% BSA phosphate buffer. neurons adhering outside the lines are not able to outgrow neurites. For this experiment a solution containing 4 mg polymer and 14 mg laminin/ml was used. a new set of experiments were carried out in Bochum this time.Defined adhesion/growth of living cells Moreover. ® lines on Coverage of lines with neurons is not perfect at some places indicating some inhomogeneities in the composition of the polymer lines. 48 Examples of neurons cultured on laminin–Vinnapas glass cover slips. c) three-times gently rinsing with phosphate buffer. as several cells were found also outside the lines. The microstructures were subjected to the following incubating and rinsing steps: a) 1 hour incubation in 1% BSA phosphate buffer. Fig. The vast majority of cells were found to be stained indicating that neurons were attached to the laminin-Vinnapas® lines (Figure 49). a Vinnapas®-laminin mixture was patterned onto glass cover slips.
They could provide sites for non-specific cell adhesion. h) drying and preserving in a cold.e. silicon and glassy carbon. possibly by non-specific inclusion of the fluorescent dye DiI into the polymer matrix.Defined adhesion/growth of living cells e) rinsing with phosphate buffer. g) rinsing with phosphate buffer. where lines were made only with Vinnapas® i. ® Steps d) and g) have to be carried out in the absence of intense light. 49 Neurons on a laminin–Vinnapas line on a glass cover slip. The cells are visualised by means of immunocytochemically staining with an anti-neurofilament anti-body. if laminin was present in the Vinnapas® (Figure 50B). Small and faint fluorescent dots were seen on the polymer lines on all opaque substrates. Figure 50 shows the result of a negative control performed on glassy carbon substrate. No adhesion of neurons occurs on Vinnapas® lines that are lacking laminin (Figure 50A). dark place. f) 30 minutes immersion in 5% glutaraldehyde. Glassy carbon is a material which possesses a variety of heterogeneities in its structure. It was also checked whether laminin-Vinnapas® lines were suitable for neuronal adhesion on gold. Fig. without laminin. 77 . it was found that there were several cells on the glassy carbon surface outside the lines. Indeed. A good adhesion of neurons onto the polymer lines on glassy carbon could be observed.
whereas no cells could be found on the polymer lines (Figure 51A). A completely unexpected finding was observed on gold substrates. The cluster-like gathering of the neurons indicates that pure glassy carbon surface is not appropriate for neuronal adhesion. thus providing additional potential adhesion sites for the neurons. The reason for this behaviour could be leaching of laminin out of the polymer lines and subsequent local adsorption of laminin at the glassy carbon surface. Although the lines seemed to be preserved. At the moment. Nevertheless. The whole surface was populated more or less densely with neurons. ® 78 . The broken lines indicate position and width of the polymer lines. we cannot explain this behaviour. Due to small number of adhering cells. 50 Behaviour of neurons on: (A) Vinnapas lines and (B) laminin– ® Vinnapas lines shot onto glassy carbon. and further experiments should be performed. On silicon substrates. Both scale bars represent 100 µm. individual cells and their connections can easily be traced. The broken lines indicate position and width of the polymer lines. Both scale bars represent 50 µm.Defined adhesion/growth of living cells Fig. only a very weak adhesion of neurons could be found at all (Figure 51B). 51 Behaviour of neurons on laminin–Vinnapas lines shot onto: (A) gold and (B) silicon substrates. ® More cells adhere non-specifically outside the lines. Fig. they exhibited normal neuronal appearance and a good growth of neurites. only few neurons adhered to them. some of them forming dense clusters. The bright dots of fluorescent dye visible on the lines represent most probably stained cellular debris attached to the lines.
These findings open the route for the generation of complex small neuron arrays and for the electrochemical investigation of the obtained neuron matrix.2. It is not yet understood why neurons adhered to the pure gold surface instead of the polymer lines.4. though to a different extent depending on the substrate.Defined adhesion/growth of living cells 5. Vinnapas® turned out to be a useful polymer matrix for laminin in order to achieve adhesion of neurons and even outgrowth of neurites. It swells in an aqueous environment. It may be that sub-micrometric features of the surfaces can promote better the cell adhesion and growth on smooth supports rather than rough ones. which contained the adhesion-supporting laminin. Conclusions It could be demonstrated that the adhesion of the neurons followed the prepared micropatterns. exhibiting hydrogel-like properties. Primary embryonic neurons are able to attach to lines made from a mixture of laminin and Vinnapas®. 79 .
scientists around the world were seeking to contribute with their best to the new challenge. It is a currently developing technology that is reshaping molecular biology. The problems to be solved had been different in the last decades and it became time to take advantage of the enormous amount of information encoded in DNA and used it to get better tools for detecting and curing genetic diseases. space era and not to mention the most recent one: the century of genetic engineering. and cytosine (C) (Figure 52) could not stay apart of this phenomenon. New improvements in life care must pay attention to the detection of genetic vectors as DNA strands which can give a great deal of information about the incipient phase of a disease development. The information storage is achieved by a genetic code made up of sequences of the A. DNA microstructures Many metaphoric names have been given to the XXth century. the application of highly selective genetic tools in a parallel manner will play a significant role to perform multiple analyses Fig. 80 . DNA represents a cellular library with the complete information required to form complex organisms. inherited or acquired. of electronics. DNA chips or microarrays represent the foundation of the DNAbased high-throughput analysis. In this context. T. The century of speed. 52 The four nitrogenous bases present in the structure of DNA. thymine (T). In this century. With every new idea and great discovery. The scientific and technological revolution have created numerous new research fields and the achievement rates were so high that the century itself was named after key words specific to the new found disciplines.DNA microstructures 5. Each period of mankind history had its particular implements or methods to identify and to cure diseases. guanine (G). at once.3. the “super-molecule” encrypting the secrets of life within its long chain made of four basic units adenine (A). G and C units. Some ideas about important aspects of DNA microarray are introduced in the following. Deoxyribonucleic acid (DNA).
bases of one strand are bound by H-bonds with bases of the other strand. the long DNA moleculesb are found within entities called genes that are defined as the physical and functional units of heredity. G. The size of the genome is usually measured in terms of base pairs (bp) and largely varies from as little as 50 thousand (Phage λa) to billions (human genome has about 3 billions bp). Stretching the DNA molecule from a single human cell. A chromosome is the self-replicating genetic material during cell division process. 81 . The nuclear DNA that is the physical support of this information is called genome. the genes are coupled in an array of genes that are known as chromosomes. T. A and T are hold together by two H-bonds. The four organic bases are attached to the side of the chain (Figure 53). The unique arrangement of the four constituents. Three secondary structures were identified by X-ray crystallography for double stranded DNA: A-DNA and B-DNA are two geometries of the standard right-handed double helices. C). the nucleotides (A.DNA microstructures The DNA Any cell of an organism contains in form of DNA molecules the complete set of genetic information necessary for building a fully functional new similar organism. Phage λ specifically infects E. An ester bound between a phosphate group and the hydroxyl of deoxyribose generates a nucleotide. a b A phage (or bacteriophage) is a virus that contaminates only bacteria. thus A pairs with T. coli. Condensation of a purine or pyrimidine base with deoxyribose produces a nucleoside. In a cell nucleus. in a gene ensures the distinctive features of individual organisms.8 meter. while Z-DNA is the left-handed double-helical structure that is only stable at high concentrations of NaCl or MgCl2. Two single DNA strands are able to bind to each other forming double stranded DNA obeying the base-pairing rules of Watson and Crick: • • • Fig. B1-B3 are nitrogenous bases. To a higher level of organisation. and C with G. DNA as biopolymer The structure of a DNA polymer consists of repeating chain sugar (β-D-deoxyribose) residues linked by phosphate units. purine bases bind pyrimidine bases. 53 A fragment of DNA. leads to a DNA string as long as 1. while G and C are hold by three Hbonds (Figure 54).
140 in 1989. E. Fig. 54 Formation of hydrogen bonds (red doted lines) between adenine-thymine (AT) and cytosine-guanine (CG) as postulated by Watson-Crick base-pairing rules. His team thought this new tool would be of value for the analysis of complex protein mixtures deriving from recombinant DNA technologies. Roger Ekins was the first who described a “microspot multianalyte immunoassay”139.DNA microstructures 5. 55 Example of microarrays and their possible applications. Southern had the idea of using nucleic acids molecules to interrogate other nucleic acid molecules attached to a solid support141. 82 . Nevertheless. A microarray is named according to the reagent that is confined at the surface rather than to the analytes they aim to detect. Figure 55 is exemplifying different types of microarrays together with possible applications142. DNA microarrays A microarray138 is a spatially ordered and miniaturised arrangement of a large number of surface immobilised reagents. Since 1995.1. the term “microarray” got slowly in wide spread use. Fig. Typically.3. the spot sizes are less than 200 µm in diameter.
1999. This nomenclature will be used throughout the chapter about DNA microarray which also are frequently called DNA chips. 83 . 1) a "probe" is the tethered nucleic acid with known sequence. This is a prerequisite to gain profit from the mountain of information resulting from the completion of the Human Genome Project146-149 (HGP. According to the nomenclature recommended by Bette Phimister (Nature Genetics Supplements.DNA microstructures It is common use to call the surface-immobilised reagent the probe and the analyte in the sample the targetc (Figure 56). 56 Hybridisation at a DNA microarray (chip). It should be mentioned that the name GeneChip® is owned by Affymetrix Incorporation and refers to their high density. Fig. Due to the large number of different probes. DNA targets. The underlying principle is the same for all microarrays. the term “gene chip” is often used as a general terminology within the microarray technology. whereas a "target" is the free nucleic acid sample whose identity/abundance is being detected. the microarray can identify thousands of DNA fragments simultaneously which offers the chance to perform genetic analysis on a huge scale. from the bulk of the solution. Typical applications of DNA microarrays are150-153: c In the literature one can find two confusing nomenclature systems for referring to hybridisation partners. but both commonly used "probes" and "targets". bind to their complementary DNA probes (match) but do not hybridise to the non-complementary ones (mismatch). DNA microarrays exploit the preferential binding of complementary single-stranded nucleic acid sequences. oligonucleotide-based DNA array. formally started in 1990) which otherwise would be of no use but simply a collection of data. The unknown sample (target) is hybridised to the ordered array of immobilised DNA strands whose sequence is known (probes)143-145. 21. However. no matter how they are made.
Hopes The beginning of the era of molecular detection of cancer started last year. 84 . 5. f The term cDNA denotes a complementary DNA that is a single-stranded DNA molecule complementary in base sequence to a RNA strand. comparison of gene expression in different populations of cells (for instance healthy versus diseased cells. silicon.1. Researchers are convinced that they understand gene expression patterns now well enough to use them in life-altering treatment decisions. nucleic acid microarrays are currently dominating the field. e The expression (transcription) level of a gene is the amount of its corresponding mRNA present in the cell. Thus. but most used are glass. 5. Owing to the fact that DNA probes can be easily synthesised “in situ”160. g Oligonucleotide is a DNA molecule usually composed of 25 or fewer nucleotides. The immobilisation of artificial155 or natural deoxyribonucleic acid on solid supports is a crucial step for any application in the field of DNA microarrays. determination of the expression level (abundance) of a genee. 3. sometimes even 80-mers are called oligonucleotides maybe because they look small compared to a typical cDNA that has 500-5000 bases. identification of the sequence and gene mutation (such as Single Nucleotide Polymorphism – SNPd). Unlike DNA. there is no chemical d SNP is a DNA sequence variation that occurs when a single nucleotide in the genome sequence is changed. or the evolution of gene expression during the embryonic development.1. plastic or gold-covered slides.3. About 2 of every 3 SNPs involve the replacement of C with T. drug discovery (pharmacogenomics) which is aiming to find correlation between therapeutic responses to drugs and the genetic profiles of patients. the first ever clinical study to assign patients to a standard or aggressive therapy based on a gene scan is expected to lead to the implementation of DNA microarrays as routine clinical tools in hospitals within the next years154. when a team of cancer specialists recruited a group of women for clinical test for breast cancer investigations. The preparation of DNA microarrays Various materials can be used for grafting DNA strands.DNA microstructures 1. 2. disease diagnosis).161. toxicological research (toxicogenomics) which is aiming to find the correlations between toxic responses to poisons and changes in the genetic profiles of the organisms exposed to such pollutants. It determines the efficacy of the hybridisation and influences the signal strength for the detection. 4. Thousands of spots of natural or synthesised156 single-stranded DNA probes in the form of cDNAf / oligonucleotidesg (see note 5) are fabricated by highspeed robotics using contact or non-contact printing methods157-159.
“In-situ” synthesis of a library of oligonucleotides consists of stepwise building a number of different DNA strands from its particular nucleotides by using ink-jet printing162 or photolithographic methods similar to those used in the semiconductor industry/silicon technology (Affymetrix. G. CombiMatrix is applying Lab-on-a-Chip technology for in-situ simultaneous synthesis of thousands of DNA probes. reagents that facilitate the in-situ synthesis of DNA fragments. 85 . However. Thiol-modified DNA was patterned with an atomic force microscope (AFM) on a resistcovered gold surface with line widths as small as 15 nm164. This process is repeated until the desired DNA chip is ready (4-6). T. Deprotected DNA probes react with one of the protected monomers (A. The protected probes of a DNA film are rendered ready for chemical reaction only in the areas exposed to ultraviolet light (1-2). A remark about the DNA spot diameter: photolithographic-based in-situ synthesis can produce extremely tiny spots with diameters down to 2 µm which is much smaller than the 100 µm obtainable by ink-jet spotting. A great advantages of this approach is the highly parallel synthesis of hundred of thousands molecules since it saves time and reduces costs. via an electrochemical reaction. Individual addressable microelectrodes of an array locally generate. Fig. Figure 57)163.DNA microstructures possibility yet to build proteins at a microarray surface and thus protein molecules must be delivered to the surface for the formation of protein chips. 57 Affymetrix is fabricating the high-density probes GeneChip® through a combination of photolithography and combinatorial chemistry. or C) and increase the length of the addressed probes (3). patterning with an AFM tip is not a largely accepted method for DNA microarrays preparation due to the low production rates.
Texas. 58 Schematic of a spotting machine (arrayer).165. accurately and quickly access to high density DNA microarrays. Chemistry Nobel Prize Laureate in 1984. Merrifield (FortWorth. Fig. A robotic platform transfers the DNA sample from the wells of a microplate to the slide in the form of a spot under careful control of the temperature and humidity. Evidently. Fast and precise delivery of the DNA at the substrate surface is typically achieved by means of a spotting machine or arrayer (Figure 58). h R. the DNA probes must be modified at one end with an appropriate functional group (thiol166 or amino167) for establishing a covalent bond between the probe and chip surface. USA 1921).DNA microstructures The most commonly used technology is based on the localised immobilisation of complete DNA strands that are prepared by solid phase synthesish. 59 Examples of split-pins. In this case. an automation of the DNA chip fabrication gives more reproducibly. 86 . B. Fig. washing and drying stations are cleaning and drying the pens in between spotting steps. The precise delivery of the substance at the chip surface occurs by contacting the spotting pen with the chip in a computer-assisted process. The spotting solutions are kept in microtitre plates. „for his development of methodology for chemical synthesis on a solid matrix“. A key element of an arrayer is the spotting pen that is shown schematically in Figure 59 (see note 6). (2) Slot ended at the upper part with a larger cut used for storing the larger quantities of spotting solution. (1) Normal slot filled with the spotting solution.
3. price and complexity of fluorescencebased DNA detection systems as well as the dependence of the method on the long-term stability of the fluorescent dyes are major drawbacks.1. Figure 60).2. If. sophisticated instrumentation is required for reading out the weak light signals emitted by the DNA labelled spots169. If the DNA targets to be identified are part of a complex mixture (such as blood or tissue extract). As a consequence of the Watson and Crick base pairing rules (see note 7) the kind of interaction between the DNA probe and target is the formation of the double helix. the probes are labelled one has to take advantage of a quenching of fluorescence. A concise review of the established recognition methods of DNA hybridisation is provided in the following. is known for its excellent sensitivity and reliability and turned into the standard for the detection of hybridisation. however. the fundamental scheme that researchers are looking for is a method to discriminate between a certain single stranded DNA (ss-DNA) and its corresponding double strand (ds-DNA) when both of them are possibly present at the microarray surface. 5. They are discussed considering their working principle (optical. Detection of DNA hybridisation is performed in multiple ways but optical. but did not show anything about the way a DNA microarray could be used. Laser scanning fluorescence microscopy using for example fluorophor-labelled target DNA. Detection of DNA hybridisation – What are the options? The above discussion on DNA microarrays intended to give a brief description of this revolutionary tool. If the targets are tagged then the corresponding spots on the chip are not appearing fluorescent unless the DNA probes are hybridised. a process known as hybridisation of DNA. Accordingly.2. In general. 87 . Following the hybridisation with the unlabeled targets. mass sensitive or electrochemical methods) or in concordance with the features of the attached active groups/reporters (label and label-free methods). mass sensitive and electrochemical techniques are dominating. This is only observed if the labelled probe strands are flattened on the surface of a metal.DNA microstructures 5. thinking about a widespread application the size. Nevertheless. the probes will stretch upwards placing the label far above surface and thus increasing the detected fluorescence (TIFI168 – target induced fluorescence increase. Optical detection Either the DNA targets or the probes are functionalised with a fluorescent dye. the compatibility/match of the purines and pyrimidines would guarantee that preferentially the wished targets will be measured.3.
DNA microstructures Fig. This simple principle is used to monitor hybridisation of nucleic acids by measuring the frequency shift that follows the formation of the double strand170-172. Such methods are not only label-free but also able to detect in real-time DNA hybridisation and hence can provide kinetic information. 88 . If the power used to vibrate the quartz is kept constant. with a mass sensitivity being 2500 times higher than for a 20 MHz quartz micro balance173.2. A quartz crystal is forced to oscillate at its own resonance frequency (5 to 20 MHz).3. Another integrated biosensor technology based on thin-film bulk acoustic resonators has been introduced. As the above. The sensor has been proved to be suitable to detect proteins as well as DNA molecules. The QCM response is directly related to mass variations and thus easy to interpret (see note 8). the oscillation frequency will changes upon mass addition to its surface. the detection principle of these sensors is label-free and relies purely on a resonance frequency shift caused by mass loading of an acoustic resonator. 5. Mass sensitive detection A widespread and methodically investigated representative of this sensor type is the quartz crystal micro balance (QCM).2. A limitation of this mechanical approach to detect hybridisation is the difficulty to use it for large number of samples (parallelisation) in comparative genome studies with the goal to identify hundred or thousands DNA fragments in relatively short time. 60 Target induced fluorescence increase (TIFI) detection of hybridisation is based on the fact that the fluorescence of the label of a single stranded DNA probe is much lower as compared to the fluorescence signal of the hybridised probe.
however. As emphasised in a series of recently published review articles174-179 electrochemical DNA chips can be much simpler in instrumentation and are easier to miniaturise.2. 2003) and Motorola’s eSensor™ 180 is a good sign that an EC-based technology indeed has the potential to offer relatively cheap.DNA microstructures 5.3.and nanofabrication. are strongly supporting long-range electron transfer184-186 through the duplex (π way) towards conductive carriers. 89 . for instance. signalling probe. In view of that. other as used to binding the capture probe. In general. a remarkable number of electrochemical (EC) hybridisation assays have been proposed as practical alternatives to optical readouts. Hill and co-workers made use of this observation for establishing sensitive (see note 9) schemes for the electrocatalytic detection of hybridisation and mismatch recognition189.3. Barton.182. a redox or fluorescent label is transferred at the chip surface where is afterwards detected and thus giving a proof for the hybridisation. Figure 61 gives a typical example of a sandwich-like DNA chip where generally three DNA fragments are involved: a capture probe that is confined at the chip surface and binds the target. the recent appearance of commercially available systems such as Toshiba’s Genelyzer™ (Toshiba. since the fabrication of electrochemical devices actually is well compatible to micro. another DNA fragment. Stacked arrays of aromatic heterocyclic base pairs in the core of immobilised ds-DNA. electrochemical detection of base pairing benefits from differences in the intrinsic electrical properties of single (ss) and double (ds) stranded nucleic acids183 and/or employs easily oxidisable or reducible hybridisation indicators and redox labels. With this complicated stepwise procedure. that is worse in the presence of disruptive mismatches and not observed with ss-DNA187. Such sandwich-like hybridisation assay are used for determining hybridisation or even point mutations via electrochemical redox signals arising from ferrocene-tagged signalling probes (AMBERi)181. i AMBER is the acronym for amperometric bioelectronic reporter. easy-to use and portable analytical platforms for high-throughput DNAbased diagnostics. an effect. is labelled and complementary to a part of the target.190. Electrochemical detection of hybridisation The complexity of fluorescence-based detection systems as well as the limited dissemination of mass sensitive techniques is hindering the development of independent medical points-of-care devices based on DNA microarrays.188. In fact.
ruthenium or cobalt199. such as hemin. (Motorola eSensor™).DNA microstructures Fig. Methylene blue (MB) has good voltammetric behaviour and an appropriate structure for binding to ds-DNA.200 complexes have found applications as electroactive intercalators too. It had been shown that the interaction between DNA and some transition metal complexes changes from electrostatic to intercalative with increasing ionic strength193. were also used as intercalators197. Threading intercalators are derivatives of polyaromatic systems bearing an electroactive label on the side arms (naphthalene diimide194. 90 . Osmium198. MB could be employed as signalling tag for ds-detection196. This approach is simple on the one hand and on the other hand is highly selective. K > 106 M-1 > hydrophobic interaction. A self assembled monolayer containing molecular wires facilitate the current flow. and thus allowing the redox label to exchange electrons with the electrically polarise chip. Thus. Natural compounds. Electrode surface modification with functionalised multi-walled nanotubes (MWNT-COOH) turned to be a suitable intermediate for DNA immobilisation with j An intercalator is a type of DNA ligand that inserts or intercalate between adjacent base pairs of ds-DNA. 61 Sandwich-like assay for detection of hybridisation.192. K >105 M -1 > electrostatic interaction.and ds-DNA (the binding affinities of metal complexes with DNA are generally in the following order: intercalation. The signalling DNA probe is immobilised at the chip surface via the target. hence offering good discrimination rates between ss. A capture DNA probe bind first the non-labelled target (1).195). They intersperse within the ds-DNA thus allowing EC detection of DNA on arrays (Figure 62). K > 103 M -1)191. There is a category of compounds known as intercalatorsj that are appreciated by the DNA biosensor community because they opened a new route for the detection of ds-DNA. but they are toxic and their synthesis is time consuming.
It has numerous applications in display devices (such as flat panels.213. 91 . The intercalator has a polyaromatic core with labels linked to its side chains.DNA microstructures Fig. Intrinsic electroconductive properties of the DNA double helix allows electrons to be wired from the label towards chip or vice versa. Other approaches for transduction of hybridisation take advantage of differences in the binding affinities of dissolved redox active metal complexes for ss-DNA and ds-DNA202-204. In this case. lead to notable improvement of signal-noise (S/N) ratio when gold nanoparticle probes and silver enhancement are used to detect hybridisation214. it is used to prepare transparent conductive coatings by electron-beam evaporation or sputtering. high melting point: ~1900° C. The water oxidation can be overcome by a suitable modification of the DNA chip surface. the redox label is detectable at the electrically polarised chip surface only if hybridisation took place. field emission). 62 Schematic showing the application of intercalators for detecting DNA hybridisation. for instance with indium tin oxide (ITO)k. improved rates of the electron transfer between the electrode and daunomycin as redox intercalator201. It has been reported that modifications of the chip/electrode surface with polyelectrolytes such as poly(allylamine hydrochloride) and poly(styrenesulfonate) and above-mentioned ITO. One should mention that target-modified gold nanoparticles have another advantage because they k Indium tin oxide (ITO) is in fact indium oxide doped with tin oxide (In203:Sn02). the oxidation of guanine (Figure 63) and adenine moieties205-208 (only these two are used because the redox potential is suitable) or amplification strategies with metal nanoparticles209-212. photovoltaic devices and heat reflecting mirrors. the high potentials involved could hydrolyse the water. When guanine bases are oxidised at the surface of the microarray itself.
An example is the enzyme-amplified hybridisation test from Heller’s group. November and Eppendorf Instrumente. 63 Oxidation of guanine bases of single stranded probe DNA can be achieved with tris(2. Fig. the target or oligonucleotides that are used as autonomous signalling probes. which enables detection of duplex formation between surfaceanchored target strands and enzyme-tagged signalling probes by amperometrically monitoring the product of the enzymes action as it is exposed to the substrate218-222.DNA microstructures induce instability in the duplex. It is sensitive but relies on immobilising both the targets and the probes at the surface of magnetically and electrochemically active microspheres217. Instead of using dissolved redox active hybridisation indicators. is employing enzymes in 92 . This significantly alters the melting profile of the hybridised DNA. allowing single base mismatch to be perceived215. Hybridised DNA has the guanine bases hidden inside the core of the double helix. Silver nanoparticles labelled with DNA probes and Anodic Stripping Voltammetry (ASV) of Ag+ were used as ds-DNA detection assay216.2’-bipyridyl) ruthenium(III) electrogenerated at the surface of the DNA chip. Nevertheless. Fraunhofer Gesellschaft. these approaches require extra amounts of noble metals and supplementary chemical steps for enhancement of signal. A merge project of several German companies. A technique for detecting DNA hybridisation has been reported that is using “electroactive beads”. namely Infineon Technologies. Siemens. marker molecules can also be covalently bound to strands of the DNA probe.
the ds-DNA can wire electrons along its π-way. Even if multiple probes labelled with the same light sensitive complex are simultaneously present at the chip surface. In brief. Redox active compounds released by an enzymelabelled target are consequently cycled between the interdigitated electrodes E1 and E2 while the corresponding current is monitored. It becomes clear that a microelectrode array is not necessary. whereas in the case of hybridisation. two neighbouring microelectrodes E1 and E2 are polarised at opposite potentials. LADER). An interesting technique based on light-induced electron transfer from a DNA probe label to the chip has been developed by FRIZ Biochem (Light Addressable Direct Electrical Readout. An electron donor/acceptor complex label of a DNA probe is selectively illuminated with ultraviolet light. They cycle the redox active compounds released by the enzyme-labelled target upon hybridisation enabling the detection and quantitation of hybridisation amperometrically (Figure 64). Fig. In addition. If the probe bearing the label is single stranded.DNA microstructures detection of DNA hybridisation in an interdigitated array of gold electrodes223. no current is measured. this method permits the particular DNA probes to be identified by directing the UV beam in the right spot. a high sensitivity is achieved due to the ability of the complex to amplify the current by pushing up to 106 charges/second in the measuring electrical circuit (a mediator in bulk ensures that the 93 . 64 Microelectrode array for amperometric detection and quantitation of hybridisation.
Thus. This method requires two chronocoulometric measurements. The redox active labels are then kept relatively close to the conducting chip surface and detectable by means of a potential step that is applied to the chip (working electrode). Therefore. uses short strands (4 bases only) of redox-labelled DNA as reporter to probe the statue of the surface immobilised DNA. The charge transport through the electrical circuit due to the electrochemical transformation of the label is recorded as a fast decaying charge-time curve. one prior and another subsequent to hybridisation. the integral of the charge-time curve is smaller as in the first case with the difference being the indication of hybridisation. a chronocoulometric procedure. FRIZ Biochem. A similar experiment is then performed after hybridisation of the probes with their complementary targets. 94 .DNA microstructures electron donor/acceptor complex is permanently brought back to initial state subsequent to UV-label interaction) (Figure 65). Fig. From the same company. Displacement of the short labelled reporter DNA by the longer targets is reducing the concentration of redox labels in the vicinity of the chip surface (Figure 66). 65 LADER resorts to the use of light induced electron donors/acceptors to pump charges up/down only along π-way. This detection scheme does not call for target labelling as many of the other electrochemical assays but still requires the use of labelled signalling DNA. Firstly. Electrically Detected Displacement Assay (EDDA). ss-DNA generates no current while the dsDNA can wires an amplified number of charges due to the regeneration of the label by the electrochemically active mediator in the bulk of solution. the immobilised DNA probes are hybridised with a number of the labelled reporter.
derived from C-E curves). Fig. In a potential step experiment the replacement of signalling probes with targets is detected as a significant drop of the charge integral.and ds-DNA228. Recently. for example. Janata and co-workers proposed a new approach for simple and direct electrochemical detection of a hybridisation. Contrary. presented a disposable electrochemical biosensors based on ds-DNA that was immobilised on the surface of a screen-printed graphite electrode (SPGE). which is based on the electrostatic modulation of the flux of chloride ions through a polypyrrole film into which the DNA probes are entrapped229. the transport of Cl. 66 With the electrically detected displacement assay (EDDA). short signalling strands of DNA are tagged with a redox active compound and used as a reporter. electrochemical impedance spectroscopy225-227 (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dE/dt on the potential at constant current) are also used to electrochemically study the interaction of different redox labels with ss. Voltammetry was employed to investigate the electrode surface and changes in the DNA redox properties (oxidation of guanine base) were monitored in order to study the interactions between DNA and the 95 .ions is controlled by the status of the DNA probes with higher values of diffusion observed in case of ss-DNA (Figure 67). studies were reported on using electrochemical DNA biosensor as a screening tool for environmental pollution monitoring230. neither the capture probe nor target is labelled. The reporter that is hybridised with probe strands is displaced when the chip is exposed to the target.DNA microstructures Capacitance measurements224 (dependence of the differential capacitance C of the electrode double layer on potential E. Interestingly. Lucarelli231 and co-workers.
However. Other methods Besides the above mentioned schemes for detecting the hybridisation of DNA. 5. 96 . 5.3. hybridisation detection is still an active field of ongoing research and development. a series of papers suggest surface plasmon resonance232 (SPR) as a label-free and/or real-time hybridisation assay.2.2. Fig.and dsDNA. The last decade witnessed a remarkable development of SPR use in biomedical applications233 and it seems that DNA chip technology is on its way to taking advantage of this reliable and sensitive technique234.DNA microstructures analytes. 67 Modulation of Cl.3. Although yet not specifically addressed to the detection of hybridisation.4. the principles behind the strategy may be helpful as well to discriminate between ss.fluxes by polypyrrole (PPy) film for label-free detection of DNA hybridisation.5 Concluding remarks A number of schemes for detecting hybridisation events on the surface of DNA microarrays have been developed ranging from optical readouts using a sophisticated and expensive instrumentation to rather simple electrochemical assays.
3. The silver staining procedure formed locally conductive regions at which the SECM tip current was amplified due to the appearance of positive feedback (Figure 69). A silver-enhanced SECM imaging of DNA hybridisation was demonstrated236. This complex can oxidise guanine bases while itself is reduced to the initial state. 68 Detection of DNA hybridisation via SECM tip generated [Ru(BiPy)3] (III). Fig.3. This ruthenium complex is a strong oxidising agent and able to oxidise guanines. DNA duplex regions were successfully visualised by SECM using ferrocenyl naphthalene diimide as intercalating hybridisation indicator237.3. calf thymus DNA) immobilized onto aldehyde-modified glass substrates was achieved in the generation/collection mode of SECM through the oxidation of guanine residues by tipgenerated [Ru(bpy)3]3+-molecules235 (Figure 68). the visualisation of oligonucleotides and polynucleotides (poly[G]. Compared to non-hybridised DNA spots. The repelling mode of SECM: A new and promising assay for imaging DNA microarrays and detecting DNA hybridisation 5. The oxidation current of [Ru(BiPy)3] (II) is thus higher above the ds-DNA as compared to areas carrying ss-DNA. In addition.1.3.DNA microstructures 5. SECM and DNA microarrays Recently. 97 . an increased current is observed over an area with hybridised DNA due to the larger number of guanine bases present in the double strands. The related increase in the measured tip current made hybridised spots visible. Capture probes attached to insulating glass slides were hybridised with biotinylated targets and only regions where sequence-specific hybridisation had occurred could be developed by the adsorption of streptavidin-gold nanoparticles followed by electroless silver particle deposition.
The idea of using a negatively charged redox compound to probe the status of DNA strands immobilised at a chip surface appeared very exciting and was thought to offer a straightforward alternative to the present hybridisation detection assays especially to those based on electrochemistry.3.3. In a further step. 5. As illustrated in Figure 70. This coulomb interaction and modulation of diffusional mass 98 . but also could be used as an unconventional approach for inspecting the quality of spotted nucleic acids microarrays. Imaging and detection principle At pH values above about 5. Often miniaturisation that is essential for the fabrication of individual medical point-of-cares instruments is difficult to achieve.DNA microstructures Fig.3.towards DNA-modified regions of a surface will therefore be effectively hindered due to electrostatic repulsion. This detection scheme turned out to be not only a tool for detecting hybridisation. Aim The abovementioned methods that were designed to address a key issue of present genetic research. have inherent drawbacks: they are either complicated due to a high number of steps necessary for delivering the final result. the detection of hybridisation on DNA chips. gold nanoparticles are bound to the double strand via streptavidin – biotin interactions and than a silver reduction is carried out over the gold nanoparticles layer unless a compact film of silver is obtained (3). the diffusion of an anionic species An.2).2.3. A ruthenium complex mediator is recycled only above the hybridised area where the silver layer could be produced. the phosphate groups of nucleic acids are likely to be deprotonated. 69 Detecting hybridisation with feedback mode SECM. or they need of sophisticated machinery. 5. A biotinylated target is hybridised to the DNA probes (1.3.
DNA microstructures transport provided the basis for establishing a novel label-free electrochemical detection of hybridisation. and a steady-state current value is measured.can be reduced to [Fe(CN)6]4-. However. Additional negative charge is introduced by hybridising the probe strands with a complementary target. For ss-DNA.towards a DNA-modified surface.is hindered above surfaces carrying DNA and virtually unaffected in the DNA-free areas. [Fe(CN)6]3. 70 Schematic representation of the influence of coulomb interaction on the diffusion of An. the diffusion of tip-generated [Fe(CN)6]4. close to the chip surface. the tip-generated [Fe(CN)6]4. This electrochemical recycling is increasing the tip current compared to the value in bulk (positive feedback of SECM). a cathodic tip current arises from the diffusion limited reduction of ferricyanide ions at the tip electrode.(Iss) is higher than for the ds-DNA (Ids) due to the increase in negative charges through formation of aggregates between the probe and unlabeled target. For a given density of DNA probes.and the phosphate groups at the backbone of the immobilised DNA strands hinders the diffusion of the anionic species to the underlying surface. At a properly polarised SECM tip. the flux of An. Fig. Electrostatic repulsion between An. number of bases per individual strand. ionic strength of the 99 . Hybridisation thus becomes detectable thanks to an enhancement of the effect of coulomb interaction on the diffusional flux of electroactive species. a DNA chip with the recognition element spotted in a regular pattern on a conducting surface is electrochemically highly heterogeneous. Because the phosphate groups of nucleic acids and a negatively charged mediator are presenting a repelling force to each other. Far above a conducting DNA chip surface (Au).is diffusing towards the Au surface and an oxidised back to [Fe(CN)6]3-. Due to the influence of repulsion.
to the surface is mainly determined by the ss/ds status of DNA strands within the probe spots.3. Munich. Moving the SECM tip at fixed height above an oligonucleotide spot gives reason for a sudden drop in the SECM tip current since here tip-generated [Fe(CN)6]4. Germany. The determination of the relevant fluxes of anions can be achieved by subjecting DNA chips to local electrochemical measurements.5 x 7. the DNA chips contained DNA spots 100 . experiments have been carried out on DNA microarrays of synthetic 20-mer oligonucleotides. Typically.-species being higher in case of ss-DNA. these spatially resolved measurements can be achieved by performing SECM in the amperometric feedback mode.4. They were supplied by FRIZ Biochem GmbH. Immobilisation of the 20mers was accomplished using the selfassembly of the 3'-thiol modified strands that were spotted from a 250 mM phosphate buffer aqueous solution on Au sputtered glass surfaces (2.3. Oligonucleotides and the substrate To prove the abovementioned hypothesis right. transfer rates for Au-induced recycling diminished in turn leading to a decreased redox amplification by the positive feedback effect.molecules are repelled. electrolyte solution and concentration of the mediator.DNA microstructures Fig. As shown in Figure 71.5 cm2) with a professional microarrayer (see Materials and Methods). 71 Schematic of the principle of DNA visualisation in the repelling mode of SECM. 5. the electrostatic interaction and corresponding modulation of the diffusional transport of [Fe(CN)6]4. with fluxes of the [Fe(CN)6]4.
Freshly prepared DNA chips were post-assembled with 1 mM propanethiol in deionised water. Fig. overnight. Fluorescent tags were necessary to provide comparative fluorescence measurements of the hybridisation events. The base sequences are shown in Figure 72. For this reason. As can be noticed from this figure. Fig. This increased hybridisation efficiency and helped to avoid unspecific adsorption of target strand. the two DNA probe strands include all the four bases.DNA microstructures of two different 20-mer sequences. The layout of the DNA chip is depicted in Figure 73A. an intermediate layer of chromium or titanium is applied on the glass before gold is sputtered (Figure 73B). and at room temperature. but its adherence to glass slides is poor. 73A The layout of the DNA chip. 101 . A thin film of gold is necessary for the specific anchoring of the thiol-modified DNA probes to the chip. denoted as Oligo8 and Friz12 (O8 and F12 respectively). 72 The base sequence of the probes and targets used for spotting/hybridisation.
hence. In order to facilitate the positioning of an SECM tip near to a DNA microstructure and to shorten the scanning time of the DNA chip by choosing only a particular area of the chip. with the help of these momentary marks. a computer-assisted precise electrochemical approach of the electrode is performed to position the electrode tip well within the feedback distance. The SECM tip is positioned over the DNA microarray in two steps. There are two types of marks: temporary and permanent. The DNA-free areas are not noticeably changed by this treatment owing the hydrophobic properties of the thiol-modified surface. 73B The sandwich structure of the DNA chip. Permanent marks – are fine scratches engraved with a scalpel on the gold surface. they are obtained by rinsing the chip with some drops of 1M NaCl PBS and air drying. First. the chip was marked further with permanent marks. are not visible with naked eyes. 2. the crystals are dissolved when the electrolyte is filled in the electrochemical cell. Subsequently.DNA microstructures Fig. For both. DNA spots consist of a monomolecular film of DNA molecules and. However. the reference electrode is attached to its holder and the electrolyte is filled into the cell. Marking the DNA chip 1. Although they are meaningful for the positioning of the electrode. Then. Figure 74 indicates these marks on the chip. electrochemical approach curve and line scan 102 . Temporary marks – are polycrystalline deposits of potassium phosphates and NaCl grown at the hydrophilic DNA modified surface of the chip. a series of marks were stained or engraved at the surface of the chip. the tip of the microelectrode is manually placed close to the “L”-shaped scratch (point 2 in Figure 74) and roughly 1 mm above the surface.
3. the SECM tip is polarised at 0 mV versus the Ag/AgCl 3 M KCl reference electrode in order to reduce ferricyanide under diffusion-limited conditions. Fig. The width was 28.DNA microstructures measurements. exact start point (4). Four screws pressed down the O-ring onto the chip surface in order to make a water-tight seal. the SECM feedback remained to be positive.3. a cell as shown schematically in Figure 75 was made of Plexiglas. Conventional electrochemical cell The rectangular DNA chips were too large to be fixed in the standard electrochemical cells available in the laboratory. Accordingly. The inner cylindrical hole was 15 mm in diameter but had a larger opening at the bottom to allow an O-ring to be placed. the length 55 and height 15 mm. the electrochemical cell and the microarray could be driven by computer-controlled stepper 103 . The electrochemical cell and set-up for measurements on DNA microarrays Numerous electrochemical cells were and could be designed for SECM applications. 1. 74 Marks on the DNA chip as coordinates for the positioning and imaging. a tip-to-sample distance of 10-15 µm corresponded to an increase in the amperometric tip response of about 50%. The electrochemical cell with the DNA microarray fixed to the bottom was mounted on the two-axis translation stage of the SECM.5. Of course. horizontal line (3). Though the gold surface was in most of the cases post-assembled with propane-thiol. longer alkane-thiols (C6 or C12) would gradually turn the feedback to negative as the access of the anions to the gold surface is hindered. approximate start point of the scan (2). 5. For the chosen 10 µm diameter platinum disk microelectrode. vertical line (1). but here are presented those that have been used in the experiments of this PhD work. Thus.
75 Drawing of a typical SECM screwed-from-the-top electrochemical cell. 2. The advantage of this simple arrangement is that it tip placement and positioning is easier for the user since no walls of a chamber are hindering the optical observation of the microelectrode and the sample. Furthermore. Fig. Fig. 76 Simplified electrochemical cell for SECM measurements. O-ring (2).DNA microstructures motors in X. the body of the cell is made of Plexiglas (1). Vacuum fat was used to keep the O-ring in place and obtain a water-tight seal to the chip surface. substrate (3). lower volumes of electrolyte are needed which could be an advantage when one is aiming on monitoring the hybridisation of the 104 . Tip approach (Z-movement) was achieved with a third stepper motor that was mounted perpendicular to the ones moving the cell and kept the Pt disk microelectrode that was used as SECM tip. a simplified cell was constructed with little efforts and only an O-ring defining the electrolyte volume (see Figure 76). Simplified cell In addition to the conventional electrochemical cell.625 µm per half step. Y direction with a nominal resolution of 0. translation stage (4).
105 . The solvent evaporation is considerably reduced if it is ensured that the surface of the solution is not having a convex curvature but plain or concave (Figure 77).DNA microstructures probe strands with their complementary targets in real time while the duplex formation takes place. 77 The curvature of the surface of the electrolyte in the simplified electrochemical cell has a high impact on the evaporation rate of the solvent: convex (A) > plain (B) > concave (C). Since the chip has to be exposed to a certain concentration of targets. a lower volume would help to save on the expensive synthetic target oligonucleotides. Fig.
78 The first SECM scan over a ss-DNA microarray indicated the position of some DNA spots (lower parts of the curve). The SECM tip was polarised at +400 mV vs. experiments were carried out in the presence of a negatively and positively charged mediator. 5 mM [Fe(CN)6]4in 0. Indeed.1 M phosphate buffer and 1 M NaCl. In Figure 79. Proof of principle In order to prove that electrostatic repulsion is responsible for the creation of contrast between the DNA-modified and bare chip surface. The latter should not experience a repelling force from the phosphate groups of the immobilised ss-DNA and the feedback response of the SECM tip thus not be disturbed. see below). scan rate 10 µm/s. Obviously this was a remarkable result because it demonstrated that the underlying idea of the proposed new detection protocol was right: the downwards peaks as visible in this SECM image are a visualisation of the DNA spots. Ag/AgCl 3 M KCl in order to oxidise the mediator. It is a single line scan carried out in 5 mM ferrocyanide in 1M PBS with a 10 µm Pt disk electrode scanned at 5 µm/s.3. When larger areas of a DNA chip were scanned.DNA microstructures 5. Imaging ss-DNA in the repelling mode of SECM The preliminary experiments were performed in a ferrocyanide solution in 1M PBS. For this reason. colour bird-view or 3-D plots could be obtained by converting the raw data into the desired image files (here not shown. the upper curve displays a 106 . Fig. the DNA spots appeared in the SECM images as areas displaying a sudden drop of the anodic tip current.6.3. repelling forces between the negatively charged mediator and the phosphate groups of the DNA strands on surface seemed to have a strong impact on the amperometrically feedback current of the SECM. Figure 78 represents a SECM image of a DNA microarray that was recorded at the very beginning of this project.
The current to the left and right are indicative of the unbiased positive feedback.5) was used as the mediator and the SECM tip was polarised to 0 mV vs. As expected.5 s waiting time before data acquisition is performed. Ag/AgCl/3 M KCl. The drop of the tip current almost completely disappeared when substituting [Fe(CN)6]3. distance between two measuring points in X direction: 5-10 µm. and inset) which most probably is due to a pure steric hindrance of diffusion. 25 µm distance between two neighbouring X-line scans). SECM measurements and the evaporation of solvent A typical DNA microstructure had 24 spot within a 1200 µm x 1500 µm square.1 M potassium phoshate/3 M sodium chloride. 79 Line scans acquired by scanning the tip of a 10 µm diameter Pt microelectrode at a fixed height of 10 µm across a spot of single stranded oligonucleotide. backward scan 500 µm/s. Applying the typical parameters for imaging in the SECM feedback mode (scan speed: forward scan 5-10 µm/s. With [Ru(NH3)6]3+. 10 to 20 hours are needed to complete a full image of the DNA array.by [Ru(NH3)6]3+. only a negligible decrease in the SECM tip response was observed (Figure 79. 0. Negatively charged [Fe(CN)6]3.DNA microstructures Fig. 107 . Positively charged [Ru(NH3)6]3+ (5 mM in 0.5) was the mediator and the SECM tip was polarised to –400 mV vs. lower curve.(5 mM in 0.solution during a complete move over an individual oligonucleotide spot. pH 6. pH 6.1 M potassium phoshate/3 M sodium chloride. representative amperometric recording taken in a [Fe(CN)6]3. the DNA strands drastically lowered the current values above the DNA spot. Ag/AgCl/3 M KCl. The different behaviour of the two mediators gave evidence that really an electrostatic repelling force is responsible for a DNA-induced modulation of the tip response. This was as expected and verifies the reduced rates of redox recycling due to electrostatically hindered diffusion of tip generated species.
1 M phosphate buffer and 3 M NaCl.DNA microstructures Fig. 5 mM ferricyanide in 0. From this it was assumed that solution evaporation at the given experimental conditions did not influence the experiments on the DNA chips notably. cyclic voltammograms recorded at the beginning and the end of the acquisition of a full SECM image of the 24-spot DNA microarray did not display significant differences in the diffusion-controlled amperometric currents (see Figure 80). evaporation of water from the measuring buffer was expected to lead to a raise in the concentration of the mediator. scan rate 100 mV/s. On the other hand. Image acquisition took 16 h. 80 CVs measured before (black) and after (red) recording of a complete SECM image of a DNA chip in the imaging solution. and hence to an increase of the faradic current at the SECM tip. However. A practical and straightforward method for lowering the influence of non-specifically adsorbed ss-DNAs is to post-assembly the DNA chip with alkane-thiols directly after spotting the DNA probes. Non-specific adsorption Imperfections in the arraying process may lead to inhomogeneities in the deposition of the DNA spotting solution and the formation of areas with a certain degree of non-specific adsorbed DNA strands that surround the desired DNA spots. to displace loosely bound (physisorbed) DNA probe strands. Amperometric current increase due to the loss of solvent is ignorable. At this time scale. the presence of a dense alkane-thiol monolayer is reducing the contrast between the background signal (DNA free 108 . As can be clearly seen in the Figure 81. in a dynamic equilibrium. the gloom accompanying the ss-DNA 20-mer spots (A) is totally vanished for a chip subjected to a post-assembly with hydroxy-propanethiol (B) since alkane-thiol molecules are able.
That a ring-like deposition of the biological recognition element takes place when spotting a DNAcontaining solution becomes evident from the SECM image shown in Figure 84. A strong jet of buffer directed towards the chip surface can be used as well. Removal of DNA strands that are physically adsorbed at the chip surface can also be achieved by soaking the chip in saline buffer for few hours (Figure 82A. see note 10) is contributing to a heterogeneity in the distribution of the DNA strands within an individual spot. Fig. Spot size: 100 µm in length. 5 mM ferrocyanide in 0.1 M phosphate buffer and 1 M NaCl. The higher density of probe strands at the edge of the spot leads to an enhanced repelling which is visible in the profile of this image.1 M phosphate buffer and 1 M NaCl. Furthermore.DNA microstructures surface) and DNA spots and thus decreases the sensitivity of the SECM imaging in the repelling mode of SECM. B). 109 . without fearing that the shear forces caused by the liquid flow could damage the self-assembled film! Fig. The doughnut effect results from a capillary flow of the bulk of a drying droplet outwards the edge that can carry any dispersed material to the margin (see Figure 83). 81 The effect of thiol post-assembly on the non-specific ss-DNA adsorption: without post-assembly (A) and with hydroxyl-propanethiol (B). 82 The effect of saline buffer soaking on the non-specific adsorption: no buffer soaking (A) and with soaking (B) in 0. the so-called edge effect (“doughnut” effect.
84 The ring-like deposit (RLD) of a ss-DNA spot (30 µM probe concentration) visualised in the repelling mode of SECM. such phenomena have to be considered when aiming at quantitative measurements of ss. Certainly. The microdispenser as an alternative tool for the preparation of DNA microarrays Most of the experiments for the evaluation of the repelling mode of SECM as a tool to detect hybridisation on DNA microarrays were carried out on commercially available chips that were supplied by FRIZ Biochem. Germany. 83 Formation of a ring-like-deposition (RLD) Fig. The repelling mode of SECM is very sensitive to variations in the density of probes.or ds-DNA. and thus could be an excellent tool for inspecting the quality of the spotting procedure.DNA microstructures Fig. 5 mM ferricyanide in 0.1 M phosphate buffer and 1 M NaCl. These microarrays were prepared 110 . Munich.
the chip was rinsed with 1 M NaCl phosphate buffer and used for scanning in the repelling mode of SECM. Fig. the spotting solutions contained 3’-thiolated adenine 20-mer in 250 mM phosphate buffer (pH 6. 111 . Typically.1 M phosphate buffer with 5 mM ferricyanide as mediator. Longer contact between the gold-covered glass slide and the corrosive mixture could peel off the gold film. a piezo microdispenser (GeSIM mbh. the gold surface has been immersed 5 minutes in Piranha mixture and than rinsed thoroughly with tri-distilled water. Due to the fact that the resolution in X and Y direction was not the same (10 µm distance between two data point along X-axis and 25 µm along Y-axis). Figure 85) was used to fabricate in-house DNA microarrays. Although the functioning of this particular piezomicrodispenser is similar to the one presented in Chapter 2 and 5.DNA microstructures with a pin-spotting instrument. Alternatively to this routinely used method of patterning DNA chips.7). After 4 hours of curing time in which the thiolated DNA strands were chemisorbed onto the gold surface. Germany. The piezo-dispenser head is shown in the inset. Before spotting. 85 Ink-jet set-up used for micropatterning spots 230 µm diameter of DNA microarrays on gold surfaces. there are little differences between them in the electronic part of the set-ups This commercially available dispenser is capable to deposit spots about 230 µm in diameter. Figure 86 displays a home-made DNA array in which the probe spots are still covered with the spotting solution. especially if the metal layer underneath is exposed and attacked. the circular spots of patterned DNA probes were imaged in the SECM micrograph as squares. The imaging of the single stranded DNA probes was carried out in 3 M NaCl in 0.
it appeared in the course of all experiments involving these entities that one type of DNA (F12) blocked more the diffusion of ferricyanide ions than the other (O8). 112 . 86 Example of a home-made DNA microarray. If would be possible to prove beyond any doubts that strands having the same number of nucleotides but different sequence are distinguishable by the repelling mode of SECM. 5 mM ferricyanide in 0. this could lead to a powerful. Figure 87 undoubtedly visualises this significant difference between the two oligonucleotides.DNA microstructures Fig. 87 Repelling mode of SECM is a method possibly sensitive to the sequence of the oligonucleotides immobilised at the chip. Fig. Note: no shadows around the spots and no heterogeneities within the spots are observed! Sequence specificity Two oligonucleotides with equal number of bases but different sequences should offer the same repelling force when interacting with negatively charged redox species. DNA probe concentrations were 100 (first line) and 30 µM (second line) for both F12 and O8. The image on the right is a SECM micrograph of a small area of the same microarray.1 M phosphate buffer and 3 M NaCl. White spots represent the DNA modified regions of the Au surface (low feedback current). The photographic image to the left displays a freshly prepared microarray where the droplets of solution are not yet dried. Although only two different DNA strands (O8 and F12) were studied. cheap and uncomplicated assay for comparative genomic studies. Two 20-mers (F12 and O8) oligonucleotides with different sequence can be clearly distinguishable in these SECM micrographs.
3. Besides. For this reason the following results will be discussed in terms of the concentration of the spotting solutions that were used to produce the studied DNA chips. there were four parameters found during the investigations that were considered critical and sufficient for the proper study of DNA hybridisation.DNA microstructures 5. Any quantitative model wished to describe completely the interactions between the DNAs or other polyelectrolytes and ions in solution call for an accurate description of the potential of the electric field around the charged backbones.7. Figure 88 illustrates in a series of representative SECM line scans and images that the modulation of the SECM tip current due to repulsion between the mediator and probe strands is well correlating with the concentration of spotting solution.3. Although not exactly known. almost no effect was observed and the spots were almost invisible for the repelling mode of SECM. The modified Poisson-Boltzmann (MPB) equations together with the Booth’s theory of water dielectric saturation and an experimental dependence of water dielectric constant on ionic concentrations are normally used to calculate the mean electrostatic potential and ionic distributions around a DNA-like highly charged cylindrical polyion240. with probe concentrations ranging from 100 µM to 1 µM. however. Variations in surface density of the probes were accomplished by spotting equal volumes of probe solutions. Of course. many factors could be identified as important. With the lowest concentration (1 µM). but accidentally (effect of tip-tosample distance) or in purpose. the contrast (defined as the difference of the signals above a probe spot and the neighbouring surface) was significantly improved and better SECM images of the single stranded oligonucleotide were achieved at higher probe concentrations. in a set of experiments a number of oligonucleotide spots that were different from each other only in the density of the immobilised probes were examined. Effect of DNA probe concentration At first. Factors affecting the imaging quality in the repelling mode of SECM Imaging of ss-DNAs in the repelling mode of SECM is influenced by a set of parameters. the surface density was expected to be correlated to the concentration of the probe in the spotting solution. electrostatic attraction between charged cylindrical polyelectrolytes in aqueous medium can be induced by multivalent counterions239. These achievements are presented in the following. This was seen as an indication that the strands in fact 113 . The conformation of a single flexible polyelectrolyte molecule with a hydrophobic backbone in aqueous solution is effected by the interplay of the short-range intramolecular attraction and the long-range Coulomb repulsion238. Typically.
molecules to approach the Au surface and undergo redox recycling. This.3 (for the bird-views). oligonucleotide strands in dots spotted from the highest concentrated solution (100 µM) effectively blocked the diffusion of [Fe(CN)6]4. Nevertheless. However. pH 5. however.in 1 M NaCl/0. from the observed saturation of the influence of electrostatic interaction on the SECM signals at higher concentration it became obvious that choosing a suitable superficial density of probes is a prerequisite when aiming on the imaging of DNA strands and detecting hybridisation. pH 6. Fig. This suggested that above a certain density the electrical field produced by closely arranged probe strands is well protecting the spot region against passage of free-diffusing anions.1 M phosphate buffer.towards the gold surface and offered good contrast.1 M phosphate buffer.7 (for the line scans) or 5 mM [Fe(CN)6]3. In contrast. Scan speed 10 µm/s.DNA microstructures were so loosely arranged that their anionic phosphate groups could not really hinder [Fe(CN)6]4. 114 . 88 Representative SECM line scans (top) and bird-view SECM images (bottom) obtained by imaging 120 µm diameter spots of a 20 base oligonucleotide at concentration ranging from 1 to 100 µM by means of the repelling mode of SECM. Oligonucleotide spots were detectable and could be successfully visualised with the repelling mode of SECM in a wide range of concentrations. Measuring solutions: 5 mM ferricyanide in 3 M NaCl/0. This is confirmed by a qualitative model.couple reaches saturation at a critical probe concentration. SECM tip: 10 µm Pt disk microelectrode. explains why the impact of coulomb repulsion on the diffusion of the [Fe(CN)6]3-/[Fe(CN)6]4. which is schematically depicted in Figure 89. the “30-µM” spots nearly equally influenced the tip current.
The observed feedback current would then become independent from the number of negative charges localised within the spot thus preventing to distinguish between ss.and hybridised DNA probes at higher concentration. In this case. Effect of mediator concentration The influence of the redox mediator concentration on the performance of the repelling mode of SECM for imaging single-stranded nucleic acids was investigated in electrolyte solutions containing either 5 mM or 50 mM [Fe(CN)6]3-. 89 Qualitative model representing schematically the coulomb interaction between ss-DNA and a negatively charged redox species indicated by the arrows. individual probe strands can be considered as thin negatively charged rods that. even after duplex formation. Fig. the diffusion-limited current at a disk-shaped microelectrode is proportional to the bulk concentration of the redox species according to the equation i = 4⋅ n ⋅ F ⋅ D ⋅ r ⋅ c 115 . anionic species will be repelled by coulomb interaction and hence will not find access to the surface. On the other hand.DNA microstructures Due to the presence of anionic phosphate groups in their backbones. the distance between two adjacent strands should not be too large to allow for a significant modulation of the diffusional flux of the redox species to the underlying Au surface. Obviously. Within r1. Since. in first approximation. the entry for anions within the probe spot would be fully blocked. this assumption leads to the consideration that probe strands should not be arranged too close to each other to avoid overlapping of their electric fields. The circles represent the “forbidden” area at which anions do not find access to the chip surface.concentration. A selected spot of probe oligonucleotide was sequentially imaged in these electrolytes with the electrochemical cell being rinsed many times with phosphate buffer and the scanning solution of choice to ensure accuracy of the [Fe(CN)6]3. form a cylindrical electric field with a radius r1 around themselves.
90 Influence of the mediator concentration on the visualisation of ss-DNA in the repelling mode of SECM. As shown in Figure 90. the ratios of tip currents obtained in the vicinity of an imaged spot and just above it were apparently not dependent on the concentration of the chosen mediator. the contrast for imaging oligonucleotides in the repelling mode can not be enhanced by varying this parameter.1 M phosphate buffer. also the current that is observed due to redox recycling at the same tip-to-sample distances is proportionally higher in solutions of the higher mediator concentration because the feedback current is directly related to the number of tip-generated species available. SECM tip: 10 µm Pt disk microelectrode. a 10 fold increase in the concentration of the redox mediator is leading to a 10 fold increase in the amperometric tip current in bulk solution.7. Oligonucleotide spots were therefore subjected to repelling-mode SECM measurements in solutions with significantly different total ion concentrations. however. Since the extension of the electric double layer is changing with the ionic strength of a surrounding solution. the repelling force between phosphate groups at immobilised DNA strands and free-diffusing negatively charged redox species should be strongly affected by the composition of the electrolyte. The line scans presented in 116 . Effect of ionic strength It is well-known that two charged particles are only facing electrostatic interaction when their individual electric double layers start to overlap. F = Faraday constant. and r = radius of the electroactive electrode surface. 5 mM (top curve) or 50 mM (bottom curve) [Fe(CN)6]3. Hence. D = diffusion coefficient. scan speed 10 µm/s. As a matter of fact. c = bulk concentration of the redox active compound.in 3 M NaCl/0.DNA microstructures with n = number of transferred electron. Fig. probe concentration: 10 µM. pH 5.
A compromise must be found to ensure on the one hand good SECM imaging and on the other hand to provide a suitable environment for the stabilisation of doublestranded DNA after hybridisation has occurred. Regarding the model. the better was the contrast (Figure 92).DNA microstructures Figure 91 demonstrate that the contrast of SECM imaging indeed was enhanced when lowering the ionic strength of the measuring solution from 3 to 0.in phosphate buffer of the given ionic strength. the closer the SECM tip was scanned across the DNA microstructure.g. SECM tip: 10 µm Pt disk microelectrode. variations of the tip-to-sample distance due to e. In fact. For large scan lengths. Apparently.02 M (left) or 3 M (right). However.02 M. a surface tilt could easily disturb the current response or even lead to tip crash.at the tip electrode. the probability for loosing the redox species through lateral diffusion is lower at a smaller spacing between the microelectrode tip and chip surface. precaution must be taken when applying SECM in constant-height mode. Worth mentioning that the enhancement in contrast achieved in solutions of low ionic strength is of little benefit for the detection of hybridisation since the aggregates of probe and target strands are not stable under this condition. respectively. Fig. 91 Effect of the ionic strength of the electrolyte solution on the visualisation of ss-DNA in the repelling mode of SECM. Probe concentration: 10 µM. As expected. Line scans were obtained at an ionic strength of 0. scan speed 10 µm/s. 117 . 5 mM [Fe(CN)6]3. The reason for this observation is an improved collection of tip-generated [Fe(CN)6]4at the chip surface and recycled [Fe(CN)6]3. the extension of the electrical double layer expands when lowering the ionic strength. this corresponds to an increase of r1 and thus to a reduction in the surface area available for redox recycling. even though it is meaningful to scan in close proximity. Effect of tip-to-sample distance One and the same oligonucleotide spot was imaged sequentially in the repelling mode with the SECM tip positioned at working distances of 6 and 15 µm.
It was an outcome of the first experiments on detection of DNA hybridisation in the repelling mode of SECM that the way the hybridisation and imaging were carrying out was not optimal. merely the visualisation of single stranded oligonucleotides in the repelling mode of SECM was demonstrated. the difficulties described above were plainly solved in an extremely easy manner. Several questions can arise from such a situation: Are observed differences in the current peaks of the ss-DNA and ds-DNA induced by the hybridisation or are variations in the probe density responsible for the effect? Is the SECM tip positioned at the same height over the hybridised and control chips? Little variations possibly could have an impact on the obtained results? Well. Detection of DNA hybridisation in the repelling mode of SECM So far. Being aware of the multiple factors that are affecting the imaging of ss-DNA. With this approach. 92 Influence of the tip-to-sample distance on the visualisation of ss-DNA in the repelling mode of SECM. SECM tip: 10 µm Pt disk microelectrode.4.in 3 M NaCl/0. Nevertheless. the investigations were proceeded to study the influence of the probe density on the recognition of hybridisation. For instance.7. scan speed 10 µm/s. a few µl droplet of the target solution were placed over all the DNA spots of the microarray and covered with a glass cover slip which basically meant that hybridisation could not be selectively performed at a given area of the chip. pH 5. 5 mM [Fe(CN)6]3. Line scans were obtained at a tip to sample distance of 6 µm (left) or 15 µm (right). 5. the major challenge of work was to develop a straightforward electrochemical approach for detecting the hybridisation of DNA in a truly label-free manner.3. namely. probe concentration: 10 µM.DNA microstructures Fig. the spots of one DNA microarray was divided in two sub-areas by means of the 118 . the DNA chip subjected to hybridisation and the one designed to be the control chip were not one and the same.1 M phosphate buffer.
the control and hybridisation experiments can be performed on the same chip. had been exposed to the hybridisation solution containing the complementary target and the other to target-free hybridisation solution (control). Ag/AgCl 3 M KCl and positioned at about 15 µm above the chip surface) that was scanned across two otherwise identical microstructures one of which. Hence. a “blind” hybridisation had to be carried out only on the control area.DNA microstructures vertical scratch (see Figure 93). In this way. while soaking the chip in saline buffer. The vertical scratch defining the control and hybridisation regions of the chip was found helpful because it is acting as a barrier stopping the solution wetting the forbidden area at the opposite side (Figure 94). The status of individual DNA spots was worked out by measuring and comparing the current response of the SECM tip (a 10 µm Pt disk polarised at 0 mV vs. This ensures that the probe spots should be of fairly identical properties and that the tip-to-sample distance above the ss. the control spots and hybridised areas must be equal times in contact with buffer solutions. 93 Layout of the DNA chip after hybridisation on the right side with DNA target (A’) complementary only for one type of probe (A).and ds-DNA spots are not varying too much since they are only a fraction of 1 mm apart. It had been shown in the previous pages that a considerable number of DNA probe strands is washed off. 119 . whereas the hybridisation solution should not touch the control region. Fig. To be able to perceive the hybridisation event. however.
5. Except for the lowest probe concentration.1 1 0. SECM measurement with 5 mM ferricyanide in 3 M PBS. This could result in unwanted effects on the amperometric feedback behaviour of the chip. the gold surface will be gradually covered with impurities from solutions. 2. chip rinsing with 3 M PBS. 3 µM.01 Real hybridisation 2 0. This closed system helped to minimise the evaporation of the 5 µl droplet solution of target DNA. 5 µM. 2 hours hybridisation on the right side (in the hybridisation cell). and 1 µM probe concentration is shown in Figure 95 and 96. Otherwise. probe strands should not be too close to each other to avoid that the sphere of 120 . the DNA spots could be well visualised but a visible change in the tip current upon hybridisation was only observed at the 10 µM DNA probe. that otherwise will dry out before the completion of the hybridisation. scratch.DNA microstructures A typical hybridisation protocol consists of a series of steps: 1. binding of the target strands is increasing the net charge and enhancing the electric field within the probe spot. 10 µM. it is recommended to filter all buffer solutions through filter units with a pore size of 5 µm. 3. A Petri dish was used as the hybridisation cell (Figure 94) in which the DNA chip and a small plate filled with the same buffer as used for hybridisation were placed. 4. In theory. Accordingly. Owing to the affinity of gold for many compounds. disturb the electrochemical approach of the electrode and lead to noisy SECM images. Figure 97). the “forbidden” area for free-diffusing redox-active anions is expanded upon hybridisation (r1→r2.5): Blind hybridisation Target (µM) Buffer (M) NaCl (M) SDS (%) 0. 2 hours blind hybridisation on the left side (in the hybridisation cell). For hybridisation detection. A synopsis of the results of a typical hybridisation experiment obtained for 100 µM.01 Before and after hybridisation the chips were rinsed with 3 M PBS.1 1 0. Hybridisation experiments were performed at room temperature with the following solutions prepared in phosphate buffer (pH 6.
DNA microstructures Fig. which are expected to have a significant impact on the possibility to detect DNA hybridisation with repelling-mode SECM. a good current contrast between probe spots and hybridised spots can be obtained. In that case. 121 . By means of proper selection of a set of parameters as can be clearly seen in Figure 95. the separation between two single strands needs to be approximately 2⋅r2 (or ε) for optimal detection of base pairing (see note 11). In principle. making it difficult for the hybridisation of target to have an additional effect and become detectable. their electrical fields overlap. already for single strands the flux of anions towards the chip surface would be blocked to a high extent. 94 The DNA hybridisation cell was a simple Petri dish in which the DNA chip and small water container were placed.
in 3 M NaCl/0. Fig. 95 Detection of hybridisation on an oligonucleotide microarray by means of repelling mode of SECM.1 M phosphate buffer. The current traces to the left correspond to nonhybridised strands (control) whereas the traces to the right were acquired on hybridised spots. Single line scans were carried out over pairs of DNA spots. Scan speed 10 µm/s. SECM tip: 10 µm Pt disk microelectrode.DNA microstructures Fig. 5 mM [Fe(CN)6]3.7. 122 . Probe concentration in spotting solution: 1 to 100 µM. pH 5. 96 A close-up of the 10 µM ss-DNA and ds-DNA amperometric current peaks as measured at the scanning tip in the repelling mode of SECM after hybridisation with the complementary target.
Interestingly. in a few minutes. The current observed above the control region (not exposed to any dissolved DNA) was much higher due to positive feedback than in the region which was exposed to target DNA. 123 . This observation shows that the surface density of DNA probes will change when the chip is dehybridised in NaOH. which in principle restrains the number of hybridisation-dehybridisation cycles of a certain microarray. Thus.01% in the hybridisation solution are of great help since the target could not be any longer observed by SECM at the gold plane. It should be mentioned that SDS is not efficient at very high target concentration (10-20 µM) and it is impossible to overcome unwanted physical adsorption of targets by increasing the SDS concentration because its solubility in saline buffers is rather low. Moreover. an exposure of a gold surface to a solution containing DNA molecules is altering its feedback behaviour that way that it appears less conductive. Typical SDS concentrations of 0. wash off the excessive target. Upon duplex formation the forbidden area increases with radii changing from r1 (rss) to r2 (rds). to the right). Non-specific adsorption of target during hybridisation process DNA has a high affinity to gold and therefore is able to forms stable films non-specifically adsorbed targets on the substrate. 2 M NaOH solutions can. In order to diminish the amount of non-specifically adsorbed targets and enhance the detection of double helix. as a side effect.DNA microstructures Fig. loosely bound DNA probes are removed as well (Figure 98. it is recommended to use detergent additives such as sodium dodecylsulphate (SDS). This is confirmed by the observations shown in Figure 98 (to the left). 97 Qualitative model representing schematically the coulomb interaction between dsDNA and negatively charged redox species An-.
The coulomb interaction is modulating the diffusion properties of the electroactive species in the vicinity of probe strands and thus had a strong impact on the transport of the negatively charged species towards the conducting chip surface in DNA-modified regions. for example. the ionic strength of solution and the tip-to-sample distance are influencing the capability of so-called repelling mode SECM to visualise DNA while the concentration of the chosen mediator has not effect on the contrast of imaging. 98 Non-specifically adsorbed DNA target on a hybridised DNA chip after hybridisation with solution lacking SDS (left).DNA microstructures Fig. A device could. However.3. The method is based on the electrostatic repulsion between the phosphate groups of DNA and free-diffusing [Fe(CN)6]4.ions and is straightforward and reliable. after a short 2 M NaOH treatment (right). The density of the capture probe.5. the obtained results promote. Using SECM in the amperometric feedback mode. significant decreases in positive feedback currents are observed above spots of nucleic acids due to the local appearance of repulsion between the active components of the assay. 124 . Miniaturisation of the detection set-up Verification of the developed detection scheme was performed by local electrochemical measurements in a sophisticated SECM set-up that obviously is not suitable for common use in medical diagnostics. in principle. 5. consist of an array of immobilised probes in a base plate and an array of individually addressable Pt microelectrodes in a cover plate able of being revolved upon a lateral hinge as suggested in Figure 99 (similar Pt microelectrode arrays have already found many applications especially in biosensors). The same chip. Conclusions and outlook A truly label-free electrochemical method for visualising immobilised nucleic acids and their hybridisation on microarrays was developed. the design of a simplified electrochemical device that could be produced using microfabrication technology.
For instance. preliminary results in “imaging” DNA chips by means of the appearance of salt deposits are 125 . Fig. the relation between the concentration of DNA probes and size of the salt deposit was not as precise as would be needed for detection of DNA hybridisation. Apparently.or dsDNAs? Theoretically. However. During those operations. Hybridisation could not be yet detected. Could such phenomena be used for specific imaging of immobilised ss. the microelectrodes and DNA spots could be arranged on top of each other and kept at proper working distance. The preliminary experiments showed that the amount of salts trapped at DNA-modified areas is proportional to the concentration of the saline buffer (Figure 100) and that high density spots grow bigger deposits as small ones. instrument-free detection of DNA hybridisation The preparation of the DNA chips for hybridisation and SECM measurements required rinsing-steps for removing unwanted compounds off the chip surface. to a certain extent (Figure 101). saline buffers were used before marking the DNA chip. Label-free. 99 Miniaturised clamping device for reading out DNA hybridisation from a multielectrode array. as many counterions would be necessary to compensate their electrical charge. special circumstances may lead to crystalline deposits of which dimensions are correlated with the electrical charge of the polyanions because as many deprotonated phosphate groups are present at a spot. it was noticed that salt deposits are formed on the surface of the chip exactly in the positions of the DNA spots.DNA microstructures Using specially designed spacer and alignment elements. The difference in the response of an individual Pt microelectrode before and after exposure to a sample would evaluate the status of the opposite capture probe and indicate hybridisation in case the complementary target had been present in the sample solution.
Fig. 100 First step to a label-free instrument-free detection of hybridisation? The concentration of the rinsing buffer determines the thickens of the salt deposit at the ss-DNA spots! The yellow rectangular is pointing out a printing error. Fig. Future work has to be directed towards finding the appropriate conditions (substances.DNA microstructures encouraging because the differences between DNA probes of various surface densities are clearly noticeable within the salt patterns (Figure 100). solvents) for inducing specific crystal/deposit formation around DNA spots. 126 . 101 Another suggestive example of dependence between the size of the salt deposit and concentration of ss-DNA probes for two 20-mers with different sequence (F12 and O8).
growth. Laminin is a major component of basement membranes of living cells. The discovery was first presented in 1985. Laminin a Kary B. one great example being Polymerase Chain Reactiona (PCR) would not become possible without enzymes that normally function at elevated temperatures. Hence. 4. C-O. EC 6 Ligases Ligases are enzymes that catalyse the joining of two molecules with concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate. Owing these functions it is used as a thin coating on tissue/cells-culture surfaces or as a soluble additive to culture medium. Aspergillus niger. 3. Directed growth of living cells 4. EC 2 Transferases Are enzymes transferring a group from one compound (generally regarded as donor) to another (acceptor). including neurite outgrowth. Optimal working temperature of an enzyme is to be associated with the conditions the source of enzyme is living under. and differentiation. a bacteria living in hot springs. 5. EC 1 Oxidoreductases To this class belong all enzymes catalysing oxido-reductions. 3. 127 . EC 4 Lyases These enzymes cleave C-C. USA) American scientist that received the Nobel Prize for „his invention of the polymerase chain reaction (PCR) method” in 1993. Special application in biochemistry.4 Notes Enzyme microstructures 1. 2. The common name is “dehydrogenase” but “oxidase” is used when oxygen is an electron acceptor. The six major classes of enzymes according to EC: 1. C-N and other bonds by other means than by hydrolysis or oxidation. The optimal growth temperature of this specific organism is 35-37 ° so that any enzyme obtained from this C fungus best works at 35-37 °C. 6. migration. needs a hot medium (about 72 ° to C) work properly. EC 5 Isomerases They catalyse changes within one molecule. It has numerous biological activities including promotion of cell adhesion. EC 3 Hydrolases These enzymes catalyse the hydrolysis of various bonds. The term “holoenzyme” denotes an apoenzyme linked to its corresponding cofactor. its DNA-polymerase is suitable to using in PCR for mass-copying of DNA excluding cloning techniques.Micropatterning and microelectrochemical characterisation of biological recognition elements 5. the fungus used to produce citric acid and gluconic acid in industry is a accessible source of glucose oxidase. DNA-polymerase that is isolated from Thermus aquaticus. Mullis (1944. 2.
ii. Although. The pins of the spotting machines are made of stainless steel. Pauling and R. organic and biological chemistry were intersected in order to elucidate a mysterious process: DNA replication. iv. To obtain cDNAs libraries. hybridisation at the surface of the DNA microarray. Corey. teams of foremost scientist as L. Simultaneously. In either case. or "split" pins. titanium or other metals that have sufficient resistant against corrosion and certain hardness to minimise the blunting of the sharp tips. distinct from the normal bacterial genome) and then transferred in Escherichia coli where the plasmid is replicated many times. the cDNA is extracted and purified from the bacterial content. the cDNA is cloned into a plasmid (an extra-chromosomal circular DNA molecules. A preferred method for deposition of very small (1-2 nanolitre) quantities of DNA-probe solution onto the slide involves the of use pins. The cDNA are used as probes in DNA microarrays. S. which pick up much more sample at a time and are capable of a multi-dispensing mode of operation. Several scientists were at the same time in run for elucidating the structure of nucleic acids in the early 1950s 241 . this RNA could be used for grafting it onto a microarray as probes. In these circumstances. and cell differentiation. ongoing research programmes in the physical. 7. Complementary DNA is synthesized in laboratory from a messenger RNA template (mRNA – messenger RNA). To understand better why this is important for the hybridisation experiments. B. Franklin and R. Wilkins et all242. mRNA is prone to being destroyed especially by RNA-digesting enzymes that normally are everywhere where our fingers touched a surface. E. DNA microstructures 5. the mRNA is translated into proteins by ribosome. chromium. Electrical Discharge Machining (EDM) is normally used to cut the fine slots in the tip of the pins. To prevent the loss of mRNA. At last. promote cell attachment. extraction of mRNA and the reverse transcription: genes which code for protein are transcribed into messenger RNA’s in the cell nucleus.Micropatterning and microelectrochemical characterisation of biological recognition elements has been shown in culture to stimulate neurite outgrowth. i. which dispense just once per sample collection action. it is preferred to reverse-transcribe the mRNA into more stable DNA that is the complementary cDNA. scanning the hybridised microarray. this is not practical. Gosling understood the DNA 128 . chemotaxis. let us follow the steps of a virtual experiment for the study of a comparative gene expression. These may be "solid" pins. 6. and R. fluorescent labelling of cDNA’s. vital for the mass production of DNA microarrays. v. After being released in the cytoplasm. iii. choosing the cell population: two different kind of cells of an organism are selected. sample is transferred passively by means of surface tension as the tip gently touches the slide surface.
244 even though she was very close to get it245. Gooding. München. 8. page of interest is 1152). 255-257. The two DNA chains are held together by interactions between the nitrogenous bases on opposite strands. D. The famous DNA Base Pairing Rules. I find this is a misused technical detail because such limits were achieved only with very long DNA chains. Piper Verlag GmbH. the novel DNA structure even suggested a possible copying mechanism for the genetic material247. J. For instance. Wolke “Was Einstein seinem Friseur erzählte”. 2001. Consequently. Watson and F. A is the surface of the exposed quartz crystal. It is frequent that scientist are reporting extremely low detection limits of ds-DNA. Pauling build a wrong model of the double helix while R. who suggested a model explaining that: DNA has two anti-parallel and complementary strands in a double-helix arrangement246.3 ×10 6 ×ν 0 × A where ∆ν denotes the shift of the frequency corresponding to ∆m. iii. namely 550 amol (J. there are 2H-bonds holding A and T together and 3H-bond for C and G. ii. in the above mentioned case was used a 1497 bases DNA strand and if one takes into account that there are the same number of reporters (guanines) on each strand. 14. 9. ν0 is the resonant frequency of the bare quartz surface. a mass as small as 10-11 g is measurable by QCM248. a ring is observed at the margins. The edge (doughnut) effect is a ring-like deposit (RLD)249 which results from a capillary flow of the bulk of a drying droplet outwards the edge that can carry any dispersed material to the margin. Purines base pair with pyrimidines. A pairs with T and vice versa while C pairs with G and vice versa. “Electrochemical DNA hybridisation biosensors”. 2002. frequency shifts of 10-2 or even lower can be detected. are : i. Franklin did not bother at all about the possible arrangements of the DNA constituents243. C. 129 . H. the ring is less visible. For a given working-frequency of 10-15 MHz. iv. Sugar makes the liquid viscous and the outwards flow is slowed down. For a funny explanation please read the book of Robert L. Electroanalysis. Consequently. this is equivalent to 1497 of individual and simple species. These interactions are called base pairing. Example: a coffee drop falls on the table and by splashing and drying out creates a brownish disk. It was J. After the complete drying of the drop. Furthermore.Micropatterning and microelectrochemical characterisation of biological recognition elements molecule is a helix. then one should agree the human being is also able to see with naked eyes single molecules: a big mono-crystal of diamond is a single molecule. The relation between the frequency-shift of a quartz crystal microbalance and the change in the mass of the film attached to the crystal is given the next equation: ∆m ∆ν = − 2. Note: this ring will not be noticed if the coffee contains sugar. adenine (A) and guanine (G) are purines and cytosine (C) and thymine (T) are pyrimidines. C and G base pair is stronger as A and T. If such ideas are accepted. because of the additional hydrogen bond. or? 10. Crick.
and finding the solution could be of great deal of help for mass production of DNA microarrays. the problem is solvable. Despite the fact that describing the overall electrostatic interaction between involved parts is not a challenge. or where the DNA’s charges are distributed not along a straight -road but coiled as function of the ionic strength of the solution are only two examples of significant obstacles not easy surmountable. 130 . At some point in elaborating this qualitative model of DNA-DNA interaction. the problem could not be solved because of turning an theoretical equation valid in vacuum condition to complex environment where the electrical permittivity of solution differs in bulk and around the polyelectrolyte strands.Micropatterning and microelectrochemical characterisation of biological recognition elements 11. when knowing the optimal surface density could save expensive probes! It may worth finding the ε distance only if the repelling-like detection techniques will raise enough attention and have a market among the already commercialised DNA detection kits. I sought to establish an equation between R1 and the optimal separation distance ε for a given set of DNAs (target and probes) and negative charged redox mediator. Nevertheless.
Experimental Chemicals and Materials for the preparation of microelectrodes Solutions were prepared with triply-distilled water. Four different substrates were used for the experiments: glass cover slips. KG. Germany. and 10% Glucose. Bochum). 20 g/L Na2B4O7 ⋅10H2O (borax) and 20 g/L Na2SO4 ⋅10H2O. Chemicals and Materials for the preparation of enzyme microstructures Glucose oxidase. Germany). Tollens’ reagent for the electroless deposition of silver consisted of a mixture of 5% AgNO3. Paraffin (mineral) oil was from Sigma (EC number 232-455-8). 131 . Polishing cloth (red) was from Heraeus Kulzer.3.3-tetramethylindocarbocyanine perchlorate) was from Molecular Probes Europe.. Filter units FP 30/5.000 units/g solid. Pt wire was from Goodfellow/Germany and glass capillaries from glass capillaries Hilgenberg/Germany. Germany. 1 µm and 0. Chemicals and Materials used in the experiments regarding the defined adhesion/growth of living cells Laminin from the mouse Engelbreth–Holm–Swarm sarcoma was from Boehringer Mannheim.3 µm was from Leco Co. Chemicals were purchased from Sigma Aldrich (Deisenhofen.Experimental 6.1 M KCl as supporting electrolyte. USA.1 M KH2P04/ K2HP04 buffer or 0. Dassel. pure silicon samples coated with a 130 nm gold layer (Chemistry Department at the Ruhr University. Burghausen. 10% NaOH.3. 28-30% NH4OH. Louis. The Ag electroplating bath consisted of 40 g/L Ag2SO4.. pure silicon samples provided by the Institute of Thin Film and Ion Technology.. 3 µm. Wehrheim. 380 g/L Na2S2O3 ⋅5H2O.0 CN (black rim) were purchased from Schleicher & Schuell. Solutions of glucose oxidase and Vinnapas® EP16 W were prepared with triplydistilled water. type X-S from Aspergilus niger activity of 119. Germany. DiI was applied as fluorescent dye (1. Zinc powder was from Fisher Chemicals and soldering wire from Conrad/Germany. Germany. St. Leiden. Alumina polishing suspension. Lakeview Ave. USA. Cyclic voltammetry and SECM were performed in solutions containing 5 mM [Ru(NH3)6]Cl3 and either 0. The Netherlands.1-dioctadecyl-3. was purchased from Sigma Chemical Co. The polymer dispersion Vinnapas® EP16 W was kindly provided by Wacker Polymer Systems GmbH & Co.
Darmstadt. Fürstenwalde. and glassy carbon samples (Hochtemperatur-Werkstoffe GmbH. Germany. The personal computer for controlling the microdispensing procedure used software programmed in Microsoft Visual Basic 3. Instrumentation Basic components of the SECM set-up Experiments were carried out with a home-built SECM in a one-compartment cell in twoelectrode configuration.. Neurones were prepared from embryonic chicken forebrain under sterile conditions. Germany. Germany). miniaturised Ag/AgCl/3M KCl reference electrode. Germany. was used.Experimental Research Centre Jülich. Germany. Chemicals and Materials for the preparation of DNA microstructures KH2PO4. Germany provided oligonucleotide microstructures (DNA chips) along with the complementary targets. type 33120A. Microspotter for DNA microarray fabrication For spotting a microarrayer from Cartesian Technologies. Deisenhofen. Germany. K2HPO4⋅3H2O. Microdispenser set-up The microdispenser was operated together with a wave generator from Hewlett Packard. Unterschleißheim/Germany). Electrochemical measurements were carried out with a low132 . High current power supply (32 V and 24 A) was from Statron Elektronik. Stepper motors for precice movements of the substrate had a resolution of 0. Stepper motors with a resolution of 0. Tamm. Inc.0 (Microsoft. The personal computer used software programmed in Microsoft Visual Basic 3. Staufen. FRIZ Biochem. Faraday cage was home build and the low-noise VA10 amplifier from npi electronics GmbH. Inc. USA equipped with a printhead (ChipMaker™) and microspotting pins (120 µm diameter) from Telechem International. Potentials were measured against the chemically deposited Ag coating or an independent. Germany and SDS (sodium dodecylsulphate) from Merck. Thierhaupten.625 µm per half step and were from Owis. K3[Fe(CN)6] and [Ru(NH3)6]Cl3 were from Sigma. Munich. Unterschleißheim/Germany). NaCl.625 µm per half step were from Owis. Germany. Staufen.0 (Microsoft.
Note: in the case of Pt wires with diameter bigger as 50 µm. Experiment protocols Preparation of glass Pt-disk microelectrodes Borosilicate glass capillaries (O.D. 1 µm. Other instrumentation A fluorescence microscope (Axiophot. Piezo microdispenser with 700 nl droplet size. The Cu wire was fastened at the upper part of the capillary with shrinking tube I order to avoid breaking the contact between Cu and Pt.e. Platinum wires with diameters of 10 µm were tightly sealed into these tapered ends of pulled capillaries. which after placement was melted by careful heating with the coiled filament at 9 A.3 µm). AgSO4) and for this reason the solution has to be kept in the ultrasonic bath for few minutes. 1. One should know that the colour of the solution 133 . process carried out with a classical capillary puller. shortens the embedding time of Pt in the molten glass tip. The back connection between the Pt wire and copper lead was established by filling the capillaries with Zn powder (10 µm particles) unless the protruding Pt is fully covered and then with crushed tin solder. Germany) was used for the observation of stained neuronal cells. This reduces the risk of getting air bubbles between the Pt wire and glass insulation.Experimental noise VA10 amplifier from npi electronics GmbH. Silver electroplating The Ag electroplating bath consisted of 40 g/L Ag2SO4. Germany.75 mm. Tamm.D. and 0. The compounds are not all easily dissolved in water (i. 20 g/L Na2B4O7· 10H2O (borax) and 20 g/L Na2SO4· 10H2O in tri-distilled water. Germany. the Zn powder is non necessary but only the solder. The particular coiled filament in this case used a current of 17 A to heat glass tube. was from GeSIM mbh. 0. Smooth Pt micro disks were exposed by carefully polishing the tip at 90° on emery paper (grade 320 to 2000) and then on a polishing cloth wetted with alumina suspension (particle sizes: 3 µm. The open end of the capillary is connected to a water vacuum pump all along the heating process. 380 g/L Na2S2O3 · 5H2O. L 100 mm) were tapered down to form a closed sharp tip. and additionally.5 mm. Zeiss. I.
Bonhoeffer. “Selective growth of rat Schwann cells in neuron. at 37 0C.6 A/dm2 is the ideal. I.4. the brain was dissociated by incubation in 1 mg/ml trypsin in HBSS for 10 min. G. pH 7. After decapitation. and fixed with a solution of 4% paraformaldehyde in PBS overnight at 4 0C. and cells and neurites can be visualised with a fluorescence microscope2. 7. a current density of 0. The serum-free S4-medium was prepared starting from Dulbecco’s modified Eagle’s medium (Gibco BRL Life Technologies. Dissociated cells were prepared from the forebrain of the chicken embryo. After a second washing step by centrifugation. After 2 days of cultivation in the S4 medium in an incubator at 37 0C and with 5% CO2. Tennekoon. In order to reduce DNA-mediated aggregation of cells. K.Experimental is dark green or even black even if all substances used to prepare the mixture are colourless. do not waste it! At room temperature. 100 µl solution of DNAse were added. “A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue”. 1987. 134 . however. the cells were re-suspended in the so-called S4-medium.. the cultured neurones were washed shortly with phosphate buffer saline (PBS). 1-9. fertilised chicken eggs were incubated at 37. and cell suspensions were given onto the substrates. The samples were washed again with PBS. The cell suspension was washed by centrifugation at 550 × g for 6 min in HBSS containing trypsin inhibitor solution (1 mg/ml) and subsequently incubated with trypsin inhibitor (3 min. The lipophilic dye is incorporated very quickly into the membranes of cells and neurites. J. M. So. J. G. Scotland) according to the procedure described literature1 with the modification that a higher glucose concentration was applied. 2 P.and serum-free primary culture”. the solution must be stirred to enhance the mass transport towards the cathode. McKhann. at 37 0C). The cells located on opaque substrates were stained for 5 s in a solution of DiI in 70% ethanol. 697-713. high current are allowed to flow through he electrolysis cell. Neurosci. Defined adhesion/growth of living cells Neurons preparation For the preparation of prepare the neurons. Thanos. Godement.5-0. Vanselow. 1987. F. The appearance of dendrites is a sign that the electrochemical reaction is kinetically limited and thus if.7 0 C and 60% humidity for 8–10 days. 1 L. Cells on glass cover slips could be inspected with usual phase contrast microscopy. Development. Needham. A lower current will take too long time for preparing a reasonable thick layer of silver whereas a higher current leads to formation of Ag dendrites. S. 101.
135 . Antibody against neurofilament (Sigma) was added in a dilution of 1:400 in 1% BSA in PBS overnight at 4 0C. the cell cultures were inspected with a fluorescence microscope.1 M phosphate buffer was prepared from 0.7). neuronal cultures were washed and fixed as described above. The same procedure was applied for the immunochemical staining of the polymer lines on glass cover slips. an antilaminin antibody (Sigma) was used for the detection of laminin.7). Then.1 M phosphate buffer a solution of 1M NaCl was prepared for hybridisation purposes (pH 6. Measuring buffer With the o. Dehybridisation solution The dehybridisation solution contained only 2M NaOH in NaOH. SECM measurements were performed in a phosphate buffer containing 3 M NaCl and 5 mM ferricyanide (pH 5.5). DNA microstructures Phosphate buffers The 0. West Grove. After three washing steps with PBS. PA) was added in a dilution of 1:200 in 1% BSA in PBS for 1 h.05M KH2PO4 and 0. they were washed with PBS three times in order to remove the paraformaldehyde and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. The samples were washed again three times with PBS. and a secondary antibody labelled with Cy2® (Jackson Immuno Research Laboratories.05 K2HPO4· 3H2O in one litter of tri-distilled water (pH 6. This solution should be used only fresh and filtrated through at least 5 µm filter unit prior to use. In order to check whether laminin being entrapped in the polymer could be recognised.Experimental Immunochemistry For immunochemistry.
simple. some of them being defined from the very beginning. it was of great help to have a modular spotting set-up that could be adapted to the special conditions (clean rooms. for instance. that was not that familiar to me. ask first of all. reliable. Multi-analyte sensors will offer a great deal of help in monitoring metabolites or pollutants. When it was planned to work on the directed growth of neurons. we sought to add study the conditions in which an enzyme base microarray could be used to monitor glucose concentration. especially of those based on nucleic acids. for manufacturing of the microstructures by means of specific tools. The output was very encouraging giving that the cells clearly followed our microstructures. is mostly directed towards the invention of a sensitive. This study involved the SECM. in this case enzymatic reactions. are strongly tuned to lab experiments but have also had to do with development of theoretical aspects of microstructures. Münster.Conclusions 7. and especially the label-free approaches are given much attention these days because they fulfil all the requirements for developing individual medical point-of-care devices. it turned out that even patterning laminin is a challenge due to its structural fragility. a piezomicrodispenser in this particular case. others coming up during the course of this work. flow benches) of that laboratory. and thus. and their possible applications. which are key elements of the chemical/biochemical sensor technologies. I sought to make use of one of the intrinsic property of 136 . These results open a new route to. Although. miniaturised device for the detection of DNA hybridisation on DNA chips. Special care had been taken to avoid laminin getting damaged and to minimise contaminations of the instrumentation and chips and to preserve the vitality of neurons. owing its abilities to probe electrochemically the course of. development of neuron networks useful in studying cell growth or for coupling microelectronics to living organisms. the experiments were performed in the laboratory of Eye Hospital. Investigations of microstructures of various components of living cells. and cost-effective. It turned out that calibration curves for glucose could be obtained in a broad domain of concentrations by varying the content of enzyme within the microstructures. Conclusions The aims of this PhD thesis. The tremendous advance of microarrays. It was shown that with the ink-jet printing technique. Along with long-ago established fluorescence detection of hybridisation. the electrochemical methods. The microdispenser has found another interesting application in the field of biosensor microarrays. complex geometries are microstructured in a straightforward manner. With background knowledge in SECM.
Two or three electrodes can be easily fit in a single vial of microtitre plate due to the novel electrode assembly developed in the framework of this PhD. to establish a method for a label-free electrochemical procedure to visualise surface confined DNAs strand and to detect their hybridisation. the superficial concentration of DNA probes must have a special value as resulting from a qualitative model described in this work. Thus it can be prepared in-house in laboratories lacking sophisticated instruments for coating by sputtering or vapour deposition of metals. and to gain practical experience in manipulating specific instrumentation. A great advantage of this coaxial Pt working/Ag reference electrode comes from its easy manufacture with common instruments. by shortly mentioning some of the significant results of this PhD it was sought to outline the alternative methods or improvements that could be of use in analytical chemistry. this can be produced with available material in any laboratory. 137 . Its fabrication. developed at some points in the course of my PhD time. dedicated to the detection of hybridisation explains the difficulties that had been overcome before it could be proved that it is possible to distinguish the double stranded DNA signal from those belonging to the single stranded DNA. it was noticed that in order to improve the sensitivity of this technique. Personal note Thanks to the variety of fields this PhD covered over the last few years. Investigating a number of DNA microarrays in the newly introduced repelling mode of SECM. However. theories related to that part of science regarded as electrochemistry. in this group. possible applications and advantages as well as disadvantages were described.Conclusions DNA. Part of the DNA chapter. for developing my own ideas and checking whether there are valid or not. In conclusion. the different research topics I was involved in matched nicely with each other and thus allowed me to unify the results within a broad title such as “Micropatterning and microelectrochemical characterisation of biological recognition elements”. Microelectrochemistry in small volumes is needed when the amount of available analyte is reduced or for combinatorial electrochemistry in micro. the main factors that have an essential impact on the quality of this mode of DNA imaging were revealed. I have found a proper atmosphere. namely the negative electrical charges of the DNA backbones. I had the opportunity to learn/understand concepts. a miniaturised Ag/AgCl x M reference electrode preparation was presented. and as the above mentioned electrode system. Moreover.or nano-titre plates. Additionally.
the detection of DNA hybridisation should end up with a miniaturised and trustworthy device based either on electrostatic repulsion between an anion and the deprotonated phosphate groups.Conclusions There are several items I would like to continue in one way or another. Manufacturing of integrated electrode assemblies is a funny and relaxing habit that could deliver more useful SECM tips or just simple miniaturised electrodes. I hope I will find a chance to go on with these and other scientific plans! 138 . For instance. I believe that the micropatterning of biomolecules can still be further improved and performed with common instrument in a reliable and reproducible way.from ds-DNA. or based on the DNA-induced salt crystallisation (DISC) at the DNA spots that theoretically could differentiate ss.
patience and understanding. Peter Heiduschka for the successful collaboration on neuron growth on micropatterned surfaces. he also guided me in writing this thesis. Thomas Erichsen for his useful software and daily computer troubleshooting. 139 . Dr. Kalathur Ravi for making the English readable and for the pleasant teamwork in the lab. Düsseldorf. Dr. Dr. Wolfgang Schuhmann for his guidance along electrochemistry. Dr. Dr. constant encouragement. Acknowledgment I would like to thank all those who have been generous with their time and helped me by any mean over the last three years. October 2002. I am in particular thankful to: My esteemed supervisor Prof. Gerhard Hartwich for providing DNA chips and knowledge about DNA microarrays.Acknowledgment 8. Rolf Neuser for the great SEM images and his friendliness. Dr. Gerhard Hartwich and Wolfgang Schuhmann. Albert Schulte for the sleepless nights in the lab and his helping from the very beginning and all along my PhD time.
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Notes chapter 5
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FAMILY NAME FIRST NAME ADDRESS
TURCU EUGEN FLORIN
Markstrasse 329 44801 Bochum, Germany
0049-234-3384897 (private) 0049-234-3226202 (work)
E-mail DATE OF BIRTH PLACE OF BIRTH NATIONALITY MARITAL STATUS
firstname.lastname@example.org 1973.10.18 Baia Mare, Romania Romanian Married
Florin Turcu ACADEMIC DEGREE • Diploma in Physics and Chemistry, from the Faculty of Science, North University of Baia Mare, Romania; Diploma thesis: “Active protection against corrosion“, July 1997.
EDUCATION • Since 01.04.2001, PhD student with Prof. Dr. Wolfgang Schuhmann, Lehrstuhl für Analytische Chemie, Abteilung Elektroanalytik und Sensorik, Ruhr Universität Bochum. The topics of the PhD thesis include the creation of enzyme microstructures and micropatterns of living cells, as well as the development of a new, truly label-free electrochemical detection of DNA hybridisation. • 1999 – 2000 Research assistant at Department of Chemistry, North University of Baia Mare; Chairman and organizer of the Chemistry Club that was designed for participants from the Faculty of Science and the local high schools. Also, I taught Chemistry at my high school. • 1997 - 1999 Technical assistant at Chemistry Department, North University of Baia Mare. • 1992 – 1997 Studies in Physics and Chemistry at the North University of Baia Mare, Romania. Graduation: July 1997.
RESEARCH EXPERIENCE • Basic knowledge about (micro-) electrochemistry. • Scanning electrochemical microscopy (SECM) and its application for the visualisation of variety of samples (enzyme microstructures, DNA spots, patterns of conductive polymers).
• Fabrication of micropatterns of living (neuronal) cells. Ruhr Universität Bochum. 11th-17th July 2003. ADDITIONAL QUALIFICATIONS • Soft Skills I & II: “Communicating with Audience & Giving Presentations” at Graduate School of Chemistry and Biochemistry. • Four-probe conductivity measurements. • Fabrication and characterisation of enzyme microstructures. • Microelectrode fabrication. German (good in understanding) Bochum.07.Florin Turcu • DNA microarrays and electrochemical-based label-free detection of DNA hybridisation. • • Fluent in spoken English and good in written English.2004 159 . characterisation and application. 05.
Gerhard Hartwich. • Anh Nguyen. Albert “Imaging by Schulte. 2003. Siegmar Gäb. Angew. 141-148. Chem. Anal.07. 46-49. recognition Wolfgang of DNA Schuhmann. Jane Hübner. David Melchior. “High resolution constant distance scanning electrochemical microscopy on immobilised enzyme micropatterns” G. “Analysis of alkyl polyglicosides by capillary electrophoresis with pulsed-amperometric detection”. “Scanning electrochemical microscopy (SECM) in nanolitre droplets using an integrated working/reference electrode assembly“. 2004.. Bioelectron. 5. 131. • Florin Turcu. in press. “Ink-jet printing for micropattern generation of laminin for neuronal adhesion” Journal of Neuroscience Methods. 2004. 3564-3567. Oliver J.. 05. Mathieu Etienne. Albert Schulte. 2003. Gerhard Hartwich.T. Florin Turcu. Schmitz. Wolfgang Schuhmann. • Florin Turcu. and of Wolfgang detecting scanning Schuhmann. Solon Thanos. 43. Electrophoresis. • Albert Schulte. Chem. Bochum.. 3482-3485.Florin Turcu PUBLICATIONS • Florin Turcu.. Angew. Karla Tratsk-Nitz.I. Ed. Florin Turcu. Albert “Label-free Schulte. Imaging and Microscopy.2004 160 . Wolfgang Schuhmann. 116. • Florin Turcu. Int. Chem. Biosens. hybridisation immobilised of the ss-DNA means repelling mode electrochemical microscopy (SECM)”. submitted. 2004. 2004. Hans-Willi Kling. submitted. electrochemical hybridisation by means of modulation of the feedback current in SECM”. Wolfgang Schuhmann Peter Heiduschka. Bioanal.
Albert Schulte. „Label-free electrochemical detection of hybridisation events on DNA chips”. Schmitz. Albert Schulte. 14-17th of September. Gerhard Hartwich. Mathieu Etienne. Oral presentation. Poster presentation.Florin Turcu CONFERENCES Florin Turcu. Herbert Wieder. Dublin. Wolfgang Schuhmann. Florin Turcu. Austria. Gerhard Hartwich. Albert Schulte. Poster presentation. Siegmar Gäb. Book of Abstracts V13. Austria. Wolfgang Schuhmann. Florin Turcu. „Coaxial referenceworking electrode assembly for electrochemistry in small volumes“ ELACH-6 Conference. Albert Schulte. Polo Scientifico di Sesto Fiorentino. Germany. Book of Abstracts p. „High-resolution shear-force dependent constant-distance Bochum. Anh Nguyen Minh Nguyet. Florin Turcu. Wolfgang Schuhmann. 25-28th October 2003. 20-22nd November 2003. 05. Poster presentation. Vienna. P166. Salzburg. 4th EMBL PhD Student International Symposium. Dublin City University. Florin Turcu. Ireland. Heidelberg. Oral presentation. 30. 8-12th February 2004. Gerhard Hartwich. Wolfgang Schuhmann.2004 161 . Jane Hübner. Ingrid Fritsch. University of Florence. Florin Turcu. Oliver J. Wolfgang Schuhmann.07. 2003. Albert Schulte. 11-12th June 2004. Thomas Kratzmüller. „Ink-jet dispensing of DNA microarrays and label-free electrochemical detection of DNA- hybridisation events“ Workshop on new trends in nucleic acid based biosensors. Dominik Schäfer. “Repelling mode of SECM: a new approach for visualising DNA microarrays and detecting hybridisation” The 3rd Workshop on Scanning Electrochemical Microscopy (SECM). “Optimisation of the analysis of alkyl polyglycosides by MEKC-PAD” 17th International Symposium on Microscale Separation and Capillary Electrophoresis.
BioSensorSymposion (BSS). 30. Germany. Düsseldorf. Frischmann. 15-20 September. Thomas Kratzmüller. Book of Abstracts p. ISE 2002. “Formation of chemically active microstructures as a basis for novel miniaturised analytical devices”. W. Albert Schulte. G.07. “Label-freie electrochemische Detektion von Oligonukleotid-Hybridisierung mittels electrochemischer Rastermikroskopie (SECM)”. Wolfgang Schuhmann. “Defined adhesion and growth of neurones on microstructured polymer patterns made by ink-jet printing”. 7-11th September 2003. Düsseldorf. K. Thanos. Tagungsbuch p. New York. Peter Heiduschka. 53rd Annual Meeting of the International Society of Electrochemistry. Florin Turcu. Oral Presentation.Florin Turcu mode SECM“ 226th ACS National Meeting. Germany. Florin Turcu. 3. 26 März 2003. 187. Oral Präsentation. Book of Abstracts p. ISE 2002. Florin Turcu. Tratsk-Nitz. Wolfgang Schuhmann. Poster presentation. Oral Präsentation. Düsseldorf. 9th International Conference on Electroanalysis. Wolfgang Schuhmann. INCOMSondersymposium / 74. High Resolution SECM imaging of complex enzyme microstructures“. 28. S. Wolfgang Schuhmann. USA. Mathieu Etienne. 05. 76. April 2003 Potsdam. Bernardo Ballesteros Katemann. Schuhmann. Bochum. P. “Lokale Detektion von Oligonukleotidspots mittels elektrochemischer Rastermikroskopie (SECM)”. Florin Turcu. Thomas Erichsen. AGEF-Seminar. Hartwich. Germany. Herbert Wieder. Marcus Mosbach. März / 1. Mathieu Etienne. Germany. 53rd Annual Meeting of the International Society of Electrochemistry. 15-20 September. Florin Turcu.2004 162 .
Book of Abstracts vol. Romania. Düsseldorf.07. Cracow. pp. Poland. Calimanesti-Caciulata. Poster presentation.22 in the Abstracts Book. The XXVth Chemistry and Chemical Engineering National Conference. 05. Poster Präsentation. Romania. Florin Turcu. Wolfgang Schuhmann. Karla Tratsk. Book of Abstracts pp.2004 Bochum. Kyoto.2004 163 . 6-8th of October 1999. Bertrand Ngounou.07. 15-17 May 2002. elements “Non-manual as a immobilisation for the of biological of prerequisite preparation miniaturised biosensor systems”. Bucharest. 16-18th of October 1997. Florin Turcu „Study on the chemical separation of silver from silvercopper electrotechnical alloy”. Poster presentation PS 3. Germany. 26 April 2002. Thomas Erichsen. Peter Heiduschka. Japan. Oral presentation O 46 in the Book of Abstracts.Florin Turcu 9-13 June 2002. 266. “Polymere Mikrostrukturen als Basis zur lokalisierten Zelladhäsion“. Chemistry and Chemical Engineering National Conference. Florin Turcu. ELMINOS. Albert Schulte. 1. Florin Turcu „Polymer receptor for a methanol chemical sensor“. Poster presentation P2-3. 85-86. Bochum. Wolfgang recognition Schuhmann. The Seventh World Congress on Biosensors. 05. Mathieu Etienne.
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