XIV RENCONTRE TRANSFRONTALIERE CAPTEURS ET BIOCAPTEURS

XIV TROBADA TRANSFRONTERERA

SOBRE SENSORS I BIOSENSORS
BANYULS SUR MER 2009 24-25 septembre /setembre 2009

Programme
Thursday, September 24th Sensor and biosensor development 14:00 Registration 14:45 15:00 Welcome 15:00 15:15 Aptamer selection against Ochratoxine A for the development of electrochemical aptasensor. L. Barthelmebs, J. Jonca, A. Hayat, B. Prieto-Simon, T. Noguer 15:15 15:30 An electrochemical biosensor design for protein detection based on carbon nanotubes and aptamers. P. Kara, M. Guix, A. Escosura-Muniz, M. Maltez, M. Ozsoz, A. Merkoi 15:30 15:45 Surface IR for chemical characterisation and direct label-free target detection by biosensors. S. Boujday, C. Mthivier, C.M. Pradier 15:45 16:00 Microfabrication of u-FFE device integrating a multichannel optical detection system for the continuous monitoring of separation process. P. Couceiro, J. Alonso Chamarro 16:00 16:30 Coffee break 16:30 16:45 Electrodeposition rationalization of gold nanoparticles for cysteine electrooxidation. M. Combrouze, K. Fajerwerg, B. Chaudret, M. Comtat

16 :45 17:00 Gold nanostructured composite sensor for the electrochemical genosensing of thiolated double-tagged PCR products. P. R. Brasil de Oliveira, A. Lermo, S. Campoy, H. Yamanaka, J. Barb, S. Alegret, M. Isabel Pividori 17:00 - 17:15 Thermo-sensitive liposomes via Curvature tuned preparation method for application in bioassays and drug delivery. R. Gen, D. Murphy, M. Ortiz, C.K. OSullivan 17:15 - 17:30 Investigation of electrochemical responses of different sized gold nanoparticles using screen-printed carbon electrodes C. Parolo, M. Maltez-da Costa, A. de la Escosura, A. Ambrosi, A. Merkoi 17:30 19:30 Poster session and degustation of wines from the Banyuls area 20:30 Dinner at the restaurant Les Elmes Friday, September 25th Medical and environmental applications 9:30 9:45 Structural and electrochemical properties of a multi-walled carbon nanotubes tyrosinase matrix for phenol detection. M. Guix, M. Roldn, G. Alarcon, B. Prez, A. Ambrosi, A. Merkoi 9:45 10:00 Artificial Neural Networks for the selective bio-detection of organophosphate and carbamate insecticides. G. Istambouli, A. Rhouati, M. Cortina-Puig, J.L. Marty, T. Noguer 10:00 10:15 Electrochemical Biosensors for the Determination of the Antioxidant Capacity. M. Cortina-Puig, C. Calas-Blanchard, J.-L. Marty

10:15 - 10:30 Magneto Immunoseparation of Pathogenic Bacteria and Electrochemical Magneto Genosensing of the Double-Tagged Amplicon. S. Libana, A. Lermo, S. Campoy, J. Barb, S. Alegret, M. I. Pividori 10:30 11:15 Coffee break 11:15 11:30 Development of algal biosensors for water quality monitoring of lagon ecosystems and coastal waters. C. Durrieu, L. Volatier, H. Guedri, T. Baussant 11:30 11:45 Interferences in heavy metals detection using screen-printed voltammetric electrodes with interest for seawater quality control. M. Cadevall, G. Aragay, A. Merkoi 11:45 12:00 Integrative sampling systems for the monitoring of dissolved marine toxins. G. Gimnez, O. Henry, C. Brissard, P. de la Iglesia, M. Fernndez-Tejedor, J. Diogne, C.K. OSullivan, M. Camps 12:00 12:15 Automatic microfluidic system for the electrochemical detection of cancer proteins. C. Kellner, A. Fragoso, C.K. OSullivan 12:15 12:30 Electrochemical genosensor For Coeliac Disease Predisposition Diagnosis. H. Joda, D. Cournane, V. Beni, C. K. OSullivan and I. Katakis 12:30 12:45 Development of electrochemical biosensors and solid-phase amplification methods for the detection of human papillomavirus genes. L. Civit, A. Fragoso, C.K. OSullivan 12:45 13:00 Meeting closure 13:00 15:00 Lunch at the Marine Station

Posters
1. Carbon nanotube based biosensor for flow through monitoring of phenol. G. Alarcon, M. Guix, A. Ambrosi, M.T. Ramirez Silva, M. Palomar Pardave, A. Merkoi. 2. Biosensors based on polyphenoloxidase enzymes for the determination of phenolic compounds. M. Alcaraz, J. Tejerina, S. Hernndez, S. Fabiano, M. I. Pividori, S. Alegret. 3. Heavy metal screen-printed electrodes integration in an in-situ voltammetric probe. G. Aragay, A. Puig-Font, M. Medina, A. Merkoi and M. Malizia, P. Moscetta, L. Sanfilippo, P. Moscetta 4. Monolithic capillary electrophoresis system with integrated reservoirs, separation electrodes and amperometric detection on LTCC technology. E. Arasa-Puig, M. Puyol, J. Alonso Chamarro 5. Bulk reference electrode based on LTCC technology for electrochemical sensors. E. Arasa-Puig, J. Alonso Chamarro 6. Yessotoxin determination by phosphodiesterase inhibition-based colorimetric and electrochemical assays. M. Camps, P. de la Iglesia, M. Mola, G. Gimnez, M. Fernndez-Tejedor, J. Diogne 7. ALARMTOX: assays and biosensors for the detection of biotoxins from aquatic media H. Eixarch, P. de la Iglesia; J. Diogne; M. Fernndez-Tejedor, C. Flores, V. Guibert, V. Dumont, J. Caixach, J.-L. Marty, M. Camps 8. Ceramic microfluidic devices as a powerful tool for the simple synthesis of gold nanoparticles S. Gomez-de Pedro, M. Puyol, J. Alonso 9. Direct electrical detection of point mutations at Multi walled carbon nanotube modified electrodes. P. Kara , M. Guix, A. Escosura-Muniz, M. Ozsoz, A Merkoi

10. Gliadin immunosensing in gluten-free products for food safety of celiac patients. T. Laube, S. Alegret and M. I. Pividori 11. A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis in dairy farms by electrochemical geno sensing. S. Fabiano, A. Soutullo, S. Hernndez, M. I. Garca, A. Lermo, S. Libana, S. Campoy, S. Alegret, M.I. Pividori 12. Rapid detection of food pathogens by electrochemical magneto immunosensing. S. Libana, A. Lermo, S. Campoy, J. Barb, S. Alegret, M. I. Pividori 13. Microanalyzer for Enzymatic Reactions with an Integrated Thermal Preconditioning Step. C.S. Martnez-Cisneros, N. Ibez-Garca and J. Alonso 14. Miniaturized Amperometric Analyzer for Free-Chlorine Determination. C.S. Martnez-Cisneros, Z.M. da Rocha, R. Oliv-Monllau, M. Ferreira, A.C.Seabra, M.R. Gngora-Rubio, and J. Alonso. 15. PDMS based lab on a chip with electrochemical detection usign screen printed electrodes. M. Medina Snchez, S. Marn Mancebo, M. Guix, G. Alarcn, A. Ambrosi, A. Merkoi, D. A. Garzn 16. Preparation of aptamers against lupine potential allergens. P. Nadal Polo, V. Cengiz Ozalp, N. Canela, I. Katakis, C. K. OSullivan 17. DF508 Cystic fibrosis Determination Electrochemically. H. Nasef, V. Beni and C. K. OSullivan via Methylene blue

18. On line monitoring of low chlorine concentration using an optimized rigid composite material based on multiwall carbon nanotubes R. Oliv-Monllau, J. Bartroli, M. Baeza and F. Cspedes 19. Ultramicroelectrode arrays: a promissing analytical tool for (bio)sensing J. Orozco, C. Fernndez-Snchez and C.Jimnez-Jorquera

20. Enzymatic biosensor for L-lactate analysis in wine based on the incorporation of lactate oxidase by the phase inversion technique in a polysulfone/multi-wall carbon-nanotube onto screen-printed electrodes S. Prez, S.Snchez, E. Fbregas 21. Towards aptamer mediated detection of coeliac disease toxic High Molecular Weight Glutenins. A. Pinto, M. Svobodova, M.C. Bermudo, P. Nadal Polo, C.V. Ozalp, I.D. Katakis, C.K. OSullivan 22. High-sensitive flow-immunosensing system for okadaic acid assessment B. Prieto-Simn, J.-L. Marty, I. Karubea and H. Saikia 23. Electrochemical sensors for in situ monitoring in oceanic systems. A. Ruffien-Ciszak, D. Thouron, V. Garon, P. Gros and M. Comtat 24. Enhancement of Response of Graphite Epoxy Composite Electrodes Modified with Metal Nanoparticles Synthesized by Intermatrix Synthesis Technique. P. Ruiz, M. Muoz, J. Macans and D.N. Muraviev 25. Selection of aptamer against Prostate Specific Antigen for biosensor application. M. Svobodov, I. Katakis and C. K. OSullivan 26. Aliquoting Structures based on capillary force valves for construction of centrifugal microfluidic platforms O. Ymbern, C.S. Martnez-Cisneros, E.Soria, V. Catalan, J. Alonso. the

Abstract list

ORAL COMMUNICATIONS

Surface IR for chemical characterisation and direct label-free target detection by biosensors Souhir Boujday(1,2)*, Christophe Mthivier(1,2), Claire-Marie Pradier(1,2) (1) UPMC Univ Paris 6, UMR CNRS 7609, Laboratoire de Ractivit de Surface, F75005 Paris, France (2) CNRS, UMR 7609, Laboratoire de Ractivit de Surface, F75005 Paris, France Tel: +33 1 44 27 60 01, Fax: +33 1 44 27 60 33, email: souhir.boujday@upmc.fr, UMR 7609 UPMC-CNRS, case 178, 4, Place Jussieu 75252 Paris CEDEX 05 France Direct label-free detection is a growing challenge in biosensing applications. We recently observed the efficiency of surface IR spectroscopy (PM-RAIRS) as transduction technique, allowing target detection without any labeling or revealing step. The studied immunosensors were elaborated by forming amine or acid terminated thiol self assembled monolayers on gold surfaces. The antibodies were then immobilized using a binding protein that could be protein A or G or a secondary monoclonal antibody depending on the target and on antibodies availability. It is now well known that controlling immunosensor elaboration is crucial to build up an efficient specific system. Surface IR spectroscopy enables structural and quantitative chemical characterisation of each elementary elaboration step including affinity constant calculation. We were able to correlate the collected data to the efficiency of the sensing layer.1 Surface IR based immunosensors were also applied to the detection of real target such as the pathogenic bacterial Staphylococcus aureus,2 the herbicideatrazine,3andthe atmospheric pollutant benzo-apyrene4 and its sensitivity was comparable, in some cases higher, to that attained with a more conventional ELISA method. We propose via the above cited examples, to give a synthetic review of the numerous advantages of the Surface IR Spectroscopy in biosensing applications, simultaneously as a powerful characterisation tool during elaboration and as a competitive direct label-free transduction technique.
2927

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amide I 1655 cm-1

Increasing protein amount

Increasing BaP amount

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amide II 1550 cm-1

7 M 5 M 3 M 1 M

2300

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1300

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1 S. Boujday et al., J. Phys. Chem. B, 112(21) (2008), 6708-6715. 2 S. Boujday et al., Microchim. Acta 163 (3-4) (2008), 203-209. 3 M. Salmain et al., Anal. Biochem. (2008), 373(1), 61-70. 4 S. Boujday et al., Talanta (2008), doi:10.1016/j.talanta.2008.10.064

Aptamer selection against Ochratoxine A for the development of electrochemical aptasensor. BARTHELMEBS* Lise, JONCA Justyna, HAYAT Aktar, PRIETO-SIMON Beatriz and NOGUER Thierry. IMAGES (EA 4218). Universit de Perpignan, 52 avenue Paul Alduy, 66 860, Perpignan Cedex. *Tel : 04 68 66 22 56, Fax : 04 68 66 22 23, Mail : barthelm@univ-perp.fr In recent year, artificial DNA/RNA sequences, named aptamers have appeared as promising recognition molecules for analytical applications. Aptamers have comparable affinities as antibodies for target analytes and they present many advantages, such as possibility of chemical synthesis, low cost, high temperature resistance DNA or RNA aptamers are produced using the SELEX process, a technique for screening very large combinatorial libraries of oligonucleotides by an iterative process of in vitro selection and amplification. Since aptamers could be used as alternative candidates to antibodies and enzymes as biorecognition elements in biosensors, the aim of our work is to initiate the development of aptasensors in our laboratory using a well-known target molecule : Ochratoxin A (OTA). We have firstly established the implementation of the SELEX process in order to produce highly selective and sensitive DNA aptamers against OTA. These aptamers have been selected for their ability to bind to OTA, from a library of initially about 1x1018 randomised ssDNA molecules. The SELEX process is characterised by the repetition of successive steps consisting of selection (binding, partition and elution), amplification and conditioning. After 15 rounds of SELEX, the resulting DNA pool from OTA selection has been cloned and sequenced. DNA aptamers have been tested for their affinity with OTA. Those having the best affinity towards OTA have been characterised and produced by chemical synthesis. Once ending this first step, two indirect competitive enzyme-linked assay strategies have been investigated for the development of OTA electrochemical sensor using modified aptamer. In the first configuration, OTA-BSA conjugate has been immobilised on the graphite surface of screen printed electrodes. Biotinylated aptamer and streptavidin-HRP conjugate were used for the detection of OTA. In the second configuration, amino aptamer have been attached onto self-assembled monolayers (SAMs) on gold surface electrode and the detection was performed using OTA-HRP conjugate. The main objective of this work is to provide important insight into the role of aptamer as new receptors in biosensors, in order to produce in the futur several aptamers against other toxines and bacteria and to develop new aptasensors.

Interferences in heavy metals detection using screen-printed voltammetric electrodes with interest for seawater quality control M.Cadevall (1, 2), G. Aragay (1, 2), A. Merkoi (1, 2, 3) (1) Nanobioelectronics and Biosensors Group, Institut Catal de Nanotecnologia (2) Departament de Qumica, Universitat Autnoma de Barcelona Bellaterra, Catalonia, Spain (3) ICREA, Barcelona, Spain E-mail: miquel.cadevall@campus.uab.es, arben.merkoci.icn@uab.es Anodic stripping voltammetry is an interesting technique for the determination of heavy metals, i.e. cadmium, lead and copper, in seawater with interest for environmental studies. The simultaneous quantitative analysis of these elements using screen-printed electrodes and voltammetric stripping analysis in a flow system use to suffer from interferences between heavy metals. The detection limits as well as the linear ranges for each metal have been studied firstly and the results obtained including the stability of the response during measuring in a flow through spiked seawater sample will be shown. Experimental design method as an interesting tool to determine the effects of interferences between heavy metals is also used. By applying this method the response ranges without interferences for each studied metal will be given reducing in this way the number of measurements. The obtained results are with interest for further applications of the developed screen-printed sensors as low-cost screening devices for automatic control of heavy metals pollution.

Development of electrochemical biosensors and solid-phase amplification methods for the detection of human papillomavirus genes Laia Civit1, Alex Fragoso1, Ciara K. OSullivan1,2
1

Nanobiotechnology and Bioanalysis Group, Departament dEnginyeria Quimica, Universitat Rovira i Virgili, 43007 Tarragona, Spain. 2 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010 Barcelona, Spain

The human papillomavirus (HPV) is one of the common sexually transmitted infections, affecting the skin and mucous membranes. There are more than 40 HPV types that can infect the genital areas of men and women. HPV has been detected in virtually all invasive cervical cancers and has been confirmed as the major cause of this cancer. It is believed that most cervical cancers develop when various aggressive genetic HPV strains activate certain oncogenes (cancer-causing genes). Oncogenes called E6 and E7 are particularly important because they interfere with certain protective proteins, such as p53 and pRb, respectively. Under normal conditions, these proteins limit cell growth. Therefore, once they are blocked, cell growth can run rampant, leading to tumour development and cancer [1, 2]. Since HPV cannot be cultured efficiently and the clinical performance of serological assays is poor, diagnosis of HPV infection is almost entirely based on molecular tools. Various techniques based on molecular detection of HPV DNA are exploited based on the extraction of DNA from clinical samples followed by PCR amplification and detection of the amplicons. However, detection of viruses by PCR is complicated by their high mutation rates. Another, less explored, possibility for HPV detection is the use of electrochemical biosensors with amperometric (labeled) and/or impedimetric (label-free) detection. Electrochemical biosensors have the advantage of being inexpensive, sensitive, and suitable for mass manufacturing, also offering the possibility of miniaturization [3]. In this work we present the development of electrochemical DNA sensors based on the immobilization of DNA fragments on electrodes for the recognition of sequences related to subtypes of HPV and the development of an electrochemical real-time PCR technique. The surface chemistry of the various assay formats will be optimized for sensitivity, stability and occurrence of non-specific interactions. Preliminary results for the development of electrochemical DNA sensors and study of an optimum surface chemistry for the electrochemical real-time PCR are presented. Bibliography: [1] X. Liu, A. Clements, K. Zhao and R. Marmorstein, Structure of the Human Papillomavirus E7 oncoprotein and its mechanism for inactivation of the retinoblastoma tumor suppressor, J Biol Chem, 2006, 281, 578586. [2] N. Munoz, F.X. Bosch and S. de Sanjose et al., Epidemiologic classification of Human Papillomavirus types associated with cervical cancer, N Engl J Med, 2003, 348, 518527. [3] Daniela DellAtti, Michele Zavaglia, Sara Tombelli, Gloria Bertacca, Andrea O. Cavazzana, Generoso Bevilacqua, Maria Minunni and Marco Mascini Development of combined DNA-based piezoelectric biosensors for the simultaneous detection and genotyping of high risk Human Papilloma Virus strains, Clinica Chimica Acta, 2007, 83, 140-146

Microfabrication of -FFE device integrating a multichannel optical detection system for the continuous monitoring of separation process
Pedro Couceiro, J. Alonso Chamarro Grup de Sensors i Biosensors, Departament de Qumica, Universitat Autnoma de Barcelona, Edifici C-Nort, 08193 Bellaterra, Spain Phone: +34935811836 julian.alonso@uab.es

Free Flow Electrophoresis (FFE) is a continuous separation technique, in which a narrow sample stream is continuously inserted in a separation chamber filled with running buffer. By the application of an electric field perpendicular to the buffer flow stream direction, the charged species in the sample, are deflected to different angles being separated according to there electrophoretic mobility.

We present a -FFE hybrid devices fabricated using green ceramic tapes and glass, in which the electric field is generated by electrostatic induction across an insulating wall(1). This configuration eliminates bubble formation associated with electrolysis, a major problem in -FFE devices. The FFE device was microfabricated based on Low Temperature Co-Fired Ceramics (LTCC) technology
(2) (3)

, and using a stamp-and-stick technique

. This low-temperature

adhesive bonding method involves the stamping of one substrate ( LTCC), containing its set of microfabricated features, onto a glass substrate spin-coated with PDMS adhesive. This operation serves to transfer a thin layer of adhesive selectively onto protruding surfaces of the device substrate which, after removal from the glass substrate, is aligned and bonded (i.e., sticking) to a second glass substrate. The use of LTCC-Glass hybrid devices, allow for rapid, low cost, 3D microfabrication offered by the LTCC technology, with the transparency of glass to integrate multiple optical detection flow cells to continuously monitor the separation channels.

References:
(1) D. Janasek, M. Schilling, A. Manz , J. Franzke, Lab Chip, 2006, 6, 710713 (2) M.R Gongora-Rubio,P.Espinoza-Vallejos,L.Sola-Laguna,J.J.Santiago Aviles. Overviews of low temperature co-fired ceramics tape technology for meso system (MsST), Sens. Actuators A: Phys. 89 (2001) 222-241 (3) S. Carroll, M. M. Crain, J. F. Naber, R. S. Keynton, K. M. Walsh and R. P. Baldwin, Lab Chip, 2008, 8, 1564 - 1569

Development of algal biosensors for water quality monitoring of lagon ecosystems and coastal waters C. Durrieu* (a), L. Volatier (a), H. Guedri (a), T. Baussant (b) (a) Universit de Lyon, ENTPE, rue Maurice Audin, 69518 Vaulx-en-Velin, France (b) International Research Institute of Stavanger (IRIS)-Biomiljo, Mekjarvik 12, 4070 Randaberg, Norway

The harmful effects of toxic chemicals on natural ecosystems has led to an increasing demand for early-warning systems to detect those toxicants at very low concentration levels. Lagoons ecosystems and coastal water receive large volumes of pollutants from rivers, these ecosystems need continuous monitoring to control quality of water. Biosensors can respond to this requirement, as they can provide fast, sensitive and cost-effective measurements which can be operated for on-site monitoring. In the study presented hereafter, algal whole cell biosensors based on metabolic perturbation of immobilized algal cells in the presence of toxicants have been developed Three marine algal strains were used for the biosensor design : Phaeodactylum tricornutum, Dunaliella tertiolecta and Ostreococcus tauri. These algae were immobilized through physical entrapment on a quartz microfiber placed in front of an optical fiber bundle as optical transducer or by cross linking reactions with bovine serum albumin before transfer on to the sensitive area of a micro conductometric electrode for detection. Tests were carried out with solutions containing metal cations (Cd2+, Zn2+, Pb2+) or Diuron and glyphosat, tested as pesticides. Results show toxic responses on photosynthetic and enzymatic (alkaline phosphatase and esterase) activities with limit detection at 0.1ppb level. These encouraging findings permit to consider these biosensors as competitive tools to more traditional techniques used in toxicity assessment of saline water.

Electrodeposition rationalization of gold nanoparticles for cysteine electrooxidation Magali Combrouze1, Katia Fajerwerg1, Bruno Chaudret1, Maurice Comtat2 Laboratoire de Chimie de Coordination UPR CNRS 8241, Universit de ToulouseUniversit Paul Sabatier 2 Laboratoire de Gnie Chimique UMR 5503, Universit de Toulouse-Universit Paul Sabatier katia.fajerwerg@lcc-toulouse.fr Gold nanoparticles (AuNPs) have drawn remarkable interest in the past few years because of potential applications in microelectronics, optical devices, catalysis, electroanalytical and bioanalytical chemistry [1-2]. There are many methods to prepare AuNPs including chemical reduction of gold salts, photolysis, radiolysis, organometallic chemistry, etc [3-6]. Among them, electrochemical deposition is a rapid procedure for the synthesis of AuNPs on electron conducting surfaces [7]. The present work deals with electrochemical deposition of AuNPs on glassy carbon electrodes (GCE) from aqueous solutions of HAuCl4,3H2O and KNO3 used as support electrolyte. The first goal is to show that the use of four electrochemical methods: - steady state voltammetry on a rotating disk electrode, - cyclic voltammetry, - constant potential chronoamperometry on a static electrode - constant potential chronoamperometry on a rotating disk electrode enable to determine the diffusion coefficient of AuCl4-, cathodic electron transfer () and the heterogeneous rate transfer constant (k0) and to better understand the coupling between mass transfer of electroactive species, nucleation and growth of AuNPs. The second goal allows us to demonstrate the reductive role of L-cysteine on AuCl4- and to predict the rate of reduction. One of the main reasons that AuNPs deposited on GCE show properties different from those of a bulk gold electrode is the size effect: atoms at the surface have lower coordination numbers and are therefore less stable than bulk atoms. The smaller a particle, the larger the fraction of atoms at the surface, and thus the more reactive is the surface, which makes nanosized materials of great interest as substrates for analytical and bioanalytical applications. 1. M.Daniel, D. Astruc, Chem.Rev. 104 (2004) 293 2. J.M. Pingarrn, P. Yez-Sedeo, A. Gonzlez-Corts, Electrochim. Acta 53 (2008) 5848 3. Z. Ma, H. Han, Colloids and Surfaces A : Physicochem. Eng. Aspects 317 (2008) 229 4. E. Katz, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 6042 5. M. Mirdamadi-Esfahani, M. Mostafavi , B. Keita, L. Nadjo, P. Kooyman, A. Etcheberry, M. Imperor, H. Remit (2008) 41/2 98. 6. S. Gomez, K. Philippot, V. Collire, B. Chaudret, F. Senocq, P. Lecante, Chem. Commun., (2000) 1945 7. M. Oyama, S.-Y. Yamaguchi, J. Zhang, Anal. Sci. 25 (2009) 249
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Thermo-sensitive liposomes via Curvature tuned preparation method for application in bioassays and drug delivery Rkan Gen1, Deirdre Murphy3, Mayreli Ortiz1, Ciara OSullivan1,2 Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira I Virgili, Av. Pasos Catalans, 26, 43007, Tarragona, Spain. 2 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010, Barcelona, Spain. 3 Department of Biochemistry, University College of Cork, Cork, Ireland. e-mail: mayreli.ortiz@fundacio.urv.cat, ciara.osullivan@urv.cat
1

Having one hydrophilic head group and one-two hydrophobic tails, liposomes have the capacity to encapsulate huge number of water-soluble substances in their aquatic core, as well as oil-soluble substances among the lipid bilayer. Being a natural product and having the properties which facilitates the surface-modification of them, liposomes have seen increasing interest as carrier and encapsulation material in several research areas such as pharmaceutics, cosmetics, and biosensor technologies, and as model systems in cell biology (cell membrane physiology, function and cell trafficking). Moreover, sensitivity of the liposomes can be tuned via tailor designing of their lipid formulation. There are a plethora of reports on the design of liposomes which are sensitive/ resistant to environmental changes, such as pH sensitive, thermo-stable/sensitive, light or magnetic field sensitive liposomes, properties that can be exploited to design carrier systems responding to their environment. Liposomes are very attractive vesicle, however, they also have drawbacks, such as long preparation methods with additional post-homogenization steps (over 20 hours) leading to time-consuming processes with low monodispersity in size; low stability, toxicity of the organic solvents (most commonly chloroform and methanol) used in preparation and problems in scale-up, resulting in limitations for the success and commercialization of liposome products. Thus, the development of a more rapid preparation method with increased homogeneity, size control, and high entrapment of encapsulated materials is a challenging topic. In the work reported here, an ultra-rapid, one-step Small Unilamellar Liposome (SUVs) preparation method is described, obtaining highly homogeneous and extremely stable liposome populations using both charged and neutral lipids. Moreover, there is no pre/post step requirement of the method and the liposomes can be prepared in less than 1 hour. The method has been demonstrated with a known thermo-sensitive liposome formulation for the encapsulation of the horseradish peroxidase (HRP) enzyme. Thermo-sensivity and the size of the liposomes were controllable by varying the lipid ratio. Encapsulation efficiency, temperature controlled enzyme release and stability of these liposomes was characterized using TEM imaging and spectrophotometric methods. The developed liposomes demonstrate high stability and homogeneity in size that could be applied in several research approaches, such as bioassays, drug delivery and gene therapy.

Integrative sampling systems for the monitoring of dissolved marine toxins Gimnez G1,2; Henry O3; Brissard C4; de la Iglesia P1,2; Fernndez-Tejedor M1,2; Diogne J1,2; OSullivan CK3; Camps M1,2 IRTA, Ctra. Poble Nou, km. 5.5, 43540 Sant Carles de la Rpita, Spain 2 Xarxa de Recerca en Aqicultura 3 Nanobiotechnology & Bioanalysis Group, Universitat Rovira i Virgili, Av. Pasos Catalans, 26, 43007Tarragona, Spain 4 Universit de Perpignan, 52 Av. Paul Alduy, 66860 Perpignan Cedex, France gemma.gimenez@irta.cat Some species of marine microalgae naturally produce highly toxic compounds that can be accumulated by filter-feeding organisms, such as mussels, and reach human through the food chain. Programs for seafood safety monitoring are performed worldwide in order to prevent intoxications due to seafood consumption, mainly in bivalve production areas. Research efforts focused on the improvement of monitoring programs in any of their facets are constant; among them, toxins concentrations in seawater are an interesting data for prevention actions, as they have been suggested as an alarm earlier than microalgae concentrations for predicting the presence of high levels of toxins in mollusk flesh. Lipophilic toxins embrace a wide range of compounds, causative of different types of intoxications (e.g. okadaic acid and derivatives, causative of the diarrheic shellfish poisoning (DSP) syndrome; yessotoxins; cyclic imines, etc.). The presence of some of them in seawater has already been successfully monitored using passive sampling systems based on resin (HP20-DIAION)-filled sachets. For example, the presence of dissolved spirolides has been detected for the first time in the Catalan coast by using these devices (Mallat et al., 2007). Domoic acid (DA) and its derivatives are responsible for the amnesic shellfish poisoning (ASP) syndrome. Their hydrophilicity prevents their sampling by resins. Consequently, other strategies for their monitoring have been envisaged. In this direction, polymeric matrices from 2-(trifluoromethyl)acrylic acid (TFMAA, computationally modelled; Piletska et al. 2008) or 4-vinyl pyridine (VyP, molecularly imprinted; Nemoto et al. 2007) monomers have been synthesised and their capability of adsorbing DA both in acidified buffer and non-acidified f/2 media (culture media = seawater + nutrients) has been evaluated in a cartridge format, demonstrating the retention of the total amount of spiked DA. Afterwards, polymers have been immersed in f/2 media spiked with DA for 1 week as prior step before immersion into cultures. Finally, polymers have been immersed in cultures of Pseudo-nitszchia calliantha, a DA producer, in order to check the polymers performance in conditions closer to the final goal (seawater sampling). Work is in progress to evaluate the applicability of polymers as passive sampling supports for DA sampling in the sea. References: Mallat, E.; Krock, B.; Fernndez-Tejedor, M.; Caillaud, A.; Caete, E.; Elandaloussi, L. M.; Franco, J.; Cembella, A.; Diogne, J. (2007) First approach towards the implementation of passive sampling adsorption devices for the identification of lipophilic toxins in the coastal embayments of the Ebro Delta. Sixth International Conference on Molluscan Shellfish Safety, pp. 336 342.
1

Nemoto, K.; Kubo, T.; Nomachi, M.; Sano, T.; Matsumoto, T.; Hosoya, K.; Hattori, T.; Kaya, K. (2007) Simple and effective 3D recognition of domoic acid using a molecularly imprinted polymer. Journal of the American Chemistry Society, 129: 13626 13632. Piletska, E. V.; Navarro Villoslada, F.; Chianella, I.; Bossi, A.; Karim, K.; Whitcombe, M. J.; Piletsky, S.; Doucette, G. J.; Ramsdell, J. S. (2008) Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid. Analytica Chimica Acta, 610: 35 43.

Structural and electrochemical properties of a multi-walled carbon nanotubes tyrosinase matrix for phenol detection M Guix , M Roldn , G Alarcon , B. Prez , A Ambrosi and A Merkoi
3 1,2 2 1,3 1 1 1,2,4

1 2

Nanobioelectronics & Biosensors Group, Catalan Insitute of Nanotechnology, Spain;

4

Autonomous University of Barcelona, Spain; Autonomous Metropolitan University, Mexico; ICREA, Barcelona, Catalonia, Spain E-mail: mguix@cin2.es

Electrochemical biosensors represent an interesting tool for the detection of phenolic compounds . An alternative biosensor design for phenolic compounds based on screen-printed carbon electrodes (SPE) modified with multi wall carbon nanotubes (MWCNT) and tyrosinase (Tyr) has been developed and the results obtained will be shown in this work. A novel visualization methodology, based on the use of immuno-fluorescence and Confocal Scanner Laser Microscopy (CSLM), was adopted to quantify and visualize the immobilization of tyrosinase enzyme over MWCNT-modified screen-printed electrodes. This technique is extensively used in the charcterization of live strucutres , but also to the characterization of new materials . CSLM resulted an extremely powerful technique, which allowed a clear visualization of the distribution of the enzyme within both MWCNT and carbon electrode layers with an addition morphological data useful for a better understanding of the interaction between biomolecules and electrode materials. Other techniques, such as Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), were also used to determine the differences between the two different materials. Electrochemical analytical performances of the biosensor were investigated in order to found the optimal fabrication design, but also to study the enzyme stability over the time. The adsorption technique used to modify the SPEs with MWCNTs and Tyr resulted promising in terms of cost, simplicity and analytical performances. A detection limit of 1.35M and a sensitivity of 47.4 AmM-1 within a linear range of response from 2.5 to 75M phenol have been obtained. The developed biosensors show very good performance for use as disposable devices while being saved in a freezer (-18C) during a period of up to 2 months. 1 Gorton, L. Electroanalysis, 1995, 7, 2345. 2 Kim M. A. and Lee W. Y. Anal. Chim Acta, 2003, 479 (2): 143-150 3 Carralero V., Mena M. L., Gonzalez-Corts A., Y ez-Sedeo P. and Pingarrn J. M. Biosens. Bioelectron, 2006, 22: 730-736 4 Zhang T., Tian B., Kong J., Yang P. and Liu B. Anal. Chim. Acta, 2003, 489 (2): 199-206 5 Kam N. W. S, Jessop T. C., Wender P.A., Dai H. J. Am. Chem. Soc., 2004,126 (22), 6850-6851 6 Cheng J., Fernando K. A. S., Veca L. M., Sun Y. P., Lamond A. I., Lam Y. W. and Cheng S. H. ACS Nano, 2008, 2(10), 2085-2094 7 Sun G., Fang H., Cheng C., Lu P., Zhang K., Walker A.V., Taylor J. S. A. and Wooley K. L. ACS Nano, 2009, 3 (3), 673-681 8 Rao A. K. and Creager S. E. Analytica Chimica Acta 622, 2008, 1-10 Keywords: Phenol detection, Screen-printed electrodes, Confocal microscopy, carbon nanotubes
7-8 5-6 1-4

Electrochemical genosensor For Coeliac Disease Predisposition Diagnosis Hamdi Joda1, Deirdre Cournane2, Valerio Beni2, Ciara K. OSullivan2, 3 and Ioanis Katakis1 1 Bioengineering and Bioelectrochemistry Group, 2 Nanobiotechnology & Bioanalysis Group Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Pasos Catalans, 26, 43007 Tarragona, Spain 3 Institucio Catalana de Recerca i Estudis Avanats, Passeig Lluis Companys 23, 08010, Barcelona, Spain *Corresponding Authors: ioanis.katakis@urv.cat & ciara.osullivan@urv.cat

Coeliac disease (CD) is a small intestinal inflammation, affecting genetically susceptible individuals, triggered by intake of gluten protein presents in certain cereals [1]. Prevalence of CD in Europe and United States is about 1% in the general population [2]. Most of CD cases remain undiagnosed for many years because of highly variable clinical spectrum and atypical presentation. The gold standard test for CD diagnosis is the smallintestinal biopsy, an invasive and expensive test [3]. Strong relation between CD and Human Leukocyte Antigens (HLA) has been proven; 95% of patients carry HLA DQ2 genotype while the remaining carries the DQ8 [4, 5]. Patients negative to both genes are unlikely to develop CD. Normally HLA typing is performed by serological methods, DNA-based methods including sequence-specific primer or sequence-specific oligonucleotide and sequencebased typing. These methods are rather laborious, time consuming and expensive. Electrochemical genosensors are low cost, easy to use, rapid, suitable for miniaturization and easy to integrate in more complex systems devices. The aim of this work is the development of an electrochemical genosensor for CD associated HLA typing. The genosensor is built up by self assembly immobilization of HLA allele specific DNA probe onto gold surface. Surface chemistry, in terms of probe concentration, backfiller nature and immobilization approach has been evaluated. Target hybridization was detected by amperometry based on the monitoring of the catalytic response of an enzyme label. The developed surface allowed limit of detection in the order of low nM concentration (ca. 1 nM). Attempt to improve the detection limit will be performed by the use of DNA modified liposomes with encapsulated enzymes. Moreover, some preliminary results on the selective detection (typing) of HLA alleles are presented. Probes for the selective detection of DQA1*0201, DQA1*03, DQA1*05 and DQB1*02 alleles have been designed and tested. Clearly probes for DQA1*0201 and DQA1*03 alleles proved good discrimination ability, having a responses of unspecific sequences below 40% of the specific one. Further optimization of probes for DQA1*05 and DQB1*02 alleles will be performed in term of probe design. Additional improvement of allele discrimination will be attempted by optimization of the stringency conditions. Reduction of assay time will be targeted by hybridization at controlled temperature or by developing pre-incubation strategies. Finally simultaneous detection of alleles, from real sample and from multiplexed PCR will be performed.

References [1] Marsh MN.. QJM. (1995), 88(1), 9. [2] Dube C, Rostom A, et al. Gastroenterology (2005), 128:S57. [3]Hill ID, Dirks MH, Liptak GS, et al. J Ped. Gastroenterol Nutr. 2005;40:1-19. [4] Sollid LM. Annu Rev Immunol (2000), 18(5):53. [5] S. Guandalini, P. Gupta. Clin. Applied Immunol. Rev. 2 (2002) 293305.

Artificial Neural Networks for the selective bio-detection of organophosphate and carbamate insecticides

G. Istambouli, A. Rhouati, M. Cortina-Puig, J.-L Marty and T. Noguer* Universit de Perpignan Via Domitia, IMAGES EA4218, 52 Av. Paul Alduy, 66860 Perpignan Cedex, France noguer@univ-perp.fr

The extensive use of insecticides in modern agriculture has raised serious public concern regarding the environment and food safety, and considerable efforts have been devoted to the development of highly sensitive detection methods. Biosensors have been described for many years as being good candidates as substitutive or complementary tools to conventional methods, as they can provide real-time qualitative information about the composition of a sample with minimum preparation. A special interest has been paid to biosensors for carbamate and organophosphorus insecticides, due to the fact that these compounds strongly inhibit cholinesterase enzymes. In the last years, the sensitivity of these devices has been considerably improved by using highly sensitive genetically-modified enzymes. However, a drawback of acetylcholinesterase-based biosensors is related to their poor selectivity, as the target enzyme can be unequally inhibited by various classes of chemicals. To overcome this problem, we proposed to use as complementary enzyme a phosphotriesterase (PTE), which is able to specifically hydrolyse some organophosphate compounds but has no activity towards carbamates. This work presents the use of different Artificial Neural Networks for the selective detection of organophosphate and carbamate insecticides using two different acetylcholinesterases (wild type from electric-eel & genetically-modified from Drosophila melanogaster), in association with PTE. The detection is made either using the enzyme in solution or immobilized on the surface of a screen-printed electrode.

An electrochemical biosensor design for protein detection based on carbon nanotubes and aptamers

1,2

,1

1,

,1,3

P. Kara
1

, M. Guix , A. Escosura - Muniz M.Maltez M. Ozsoz , and A Merkoi

Nanobioelectronics & Biosensors Group, Cat. Inst. of Nanotechnology, Bellaterra, Catalonia, Spain
2

Ege University Faculty of Pharmacy, Analytical Chemistry. Department, Turkey 3 ICREA, Barcelona, Catalonia, Spain E-mails: arben.merkoci.icn@uab.es ; pinar.kara@ege.edu.tr

Aptamers are short single-stranded DNA or RNA sequences which have high specific affinity in binding protein targets. Aptamer based studies represent a novel modern and a strategic approach in bioanalytical analysis for specific protein detection. Due to their high stability, chemical simplicity, and easy applicability, they can be used as alternative captures to antibodies. Thereby, aptamer applications hold a great promise for biosensing of protein, and for developing protein arrays. Current studies based on electrochemical methods represent a new approach for molecular recognition of aptamer protein interaction. Various formats based on single-stranded aptamer sequences immobilized onto different kinds of trasnducers and the use of electroactive labels such as methylene blue or ferrocene have been reported. A label-free bioelectronic detection of aptamer thrombin interaction based on electrochemical impedance spectrometry (EIS) technique will be reported. The 5amino linked aptamer sequence was immobilized onto multiwalled carbon nanotube modified screen printed electrode surfaces. The binding of thrombin to aptamer sequence was monitored by EIS transduction of the R in the presence
3-/4ct

of 5mM [Fe(CN) ]
6

. This study represents an alternative electrochemical biosensor for the detection

of proteins with interest for future applications.

Keywords Aptamer, thrombin, electrochemical impedance spectrometry, screen-printed electrodes, carbon nanotubes

Automatic microfluidic system for the electrochemical detection of cancer proteins Christian Kellner,1 Alex Fragoso,1 Ciara OSullivan1,2 Nanobiotechnology and Bioanalysis Group, Departament dEnginyeria Quimica, Universitat Rovira i Virgili, 43007 Tarragona, Spain. 2 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010 Barcelona, Spain
1

This paper reports the development of a prototype microfluidic device with electrochemical detection for the detection of cancer markers. The system includes an automatic pump module and a smart valve module, which are connected over tubes to the electrochemical detection chip, including the targets on a 2D-electrode array. The syringe has 1 input/output and a maximum volume of 2500 l. The valve system has 1 input and 6 outputs. Over this setup air is pumped into reservoirs and fluids are flowed through channels to a calibration and a sample chamber. These two automatic devices are controlled with a software written for the fluidic actions. The program includes all washing steps, the injection steps of sample, calibrator, secondary antibody and the substrate inclusive incubation times. The electrochemical detection is carried out by connecting the system to a portable potentiostat which is controlled with an independent software. The developed automatic microfluidic system was used for the detection of carcinoembryionic antigen (CEA) in real serum samples obtained from breast cancer patients.

Gold nanostructured composite sensor for the electrochemical genosensing of thiolated double-tagged PCR products Paulo R. Brasil de Oliveira2, Anabel Lermo1, Susana Campoy3, Hideko Yamanaka2, Jordi Barb3, Salvador Alegret1, Mara Isabel Pividori1* Grup de Sensors i Biosensors, Departament de Qumica, Universitat Autnoma de Barcelona, Bellaterra, Spain 2 Universidade Estadual Paulista, Instituto de Qumica, Laboratrio de Electroanalitica, Campus de Araraquara, Sao Paulo, Brazil 3 Unitat de Microbiologia, Departament de Gentica i de Microbiologia, Universitat Autnoma de Barcelona, Bellaterra, Spain *Contact: Isabel.Pividori@uab.cat
1

The use of nucleic acids as biorecognition element represents an exciting interdisciplinary area of research in converging nano-bio-info-cogno technologies. The oriented and improved immobilization of single-stranded DNA to solid substrates, followed by hybridization and detection of this event has gained importance over the past decade, due to its use as genomic detection tool in DNA chips, DNA biosensors, lab-on-a-chips based on micro fluidic techniques and self-assembled molecular electronic circuits. Because of the strong affinity of thiols to metal surfaces, sulfur chemistry is widely employed when attaching DNA, especially to continuous gold films or, instead, to gold nanoparticles-modified electrodes, to increase the electroactive surface. In this work, a novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double-tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. This assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomol range. Rigid conducting gold nanocomposite represents an interesting material for the improved and oriented immobilization of biomolecules with excellent transducing features for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors [1] .

[1]

Oliveira Marques, P.R.B.; Lermo, A.; Campoy, S.; Yamanaka, H.; Barb, J.; Alegret, S.; Pividori, M.I. Anal. Chem., 81 (2009) 1332-1339.

Magneto Immunoseparation of Pathogenic Bacteria and Electrochemical Magneto Genosensing of the Double-Tagged Amplicon Susana Libana1, Anabel Lermo1, Susana Campoy2, Jordi Barb2, Salvador Alegret1, Maria Isabel Pividori1* 1 Grup de Sensors & Biosensors, Unitat de Qumica Analtica Edifici Cn, Universitat Autnoma de Barcelona, 08193, Bellaterra, Barcelona. 2 Unitat de Microbiologia, Departament de Gentica i Microbiologia E-mail: Isabel.Pividori@uab.cat The control of food quality has become of growing interest for both consumer and food industry since the increasing incidence of food poisoning is a significant public health concern for customers worldwide. While additives were at one time a major concern, nowadays microbiological issues are the greatest. Among food pathogens, Salmonella enteritidis has been the source in the last decade of many outbreaks, while Salmonella typhimurium and other antibiotic-resistant salmonellae have also recently become a concern. A rapid and sensitive method for the detection of food pathogenic bacteria is reported. In this approach, the bacteria are captured and preconcentrated from food samples with magnetic beads by immunological reaction with the specific antibody against Salmonella. After the lysis of the captured bacteria, further amplification of the genetic material by PCR with a double-tagging set of primers is performed to confirm the identity of the bacteria. Both steps are rapid alternatives to the time-consuming classical selective enrichment and biochemical/serological tests. The double-tagged amplicon is then detected by electrochemical magneto genosensing. The IMS/double-tagging PCR/m-GEC electrochemical genosensing approach is used for the first time for the sensitive detection of Salmonella artificially inoculated into skim milk samples. A limit of detection of 1 CFU mL-1 was obtained in 3.5 h without any pretreatment, in LB broth and in milk diluted 1/10 in LB. If the skim milk is preenriched for 6 h, the method is able to feasibly detect as low as 0.04 CFU mL-1 (1 CFU in 25 g of milk) with a signal-to-background ratio of 20. Moreover, the method is able to clearly distinguish between pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods as well as with PCR strategies.

Investigation of electrochemical responses of different sized gold nanoparticles using screen-printed carbon electrodes C. Parolo1, M. Maltez-da Costa1, A. de la Escosura1,2, A. Ambrosi1, A. Merkoi1,3
1

Nanobioelectronics & Biosensors Group, Institut Catal de Nanotecnologia, Barcelona, Spain 2 Instituto de Nanociencia de Aragn, Universidad de Zaragoza, Zaragoza, Spain 3 ICREA, Barcelona, Spain E-mail: arben.merkoci.icn@uab.es Abstract: Gold nanoparticles (AuNPs) have been used for analytical and biomedical purposes for many years. Rapid and simple chemical synthesis, a narrow size distribution and efficient coating by thiols or other bioligands has enabled gold nanoparticles to be used as transducers for several biorecognition binding applications. Electrochemical properties of AuNPs have been already exploited to design highly sensitive analytical systems applied to DNA [1] and protein [2] detection. Electrochemical methods used in combination with AuNP tracers represent an interesting approach to develop modern point-of-care (POC) diagnostic devices as well as rapid medical testing systems, due to the high sensitivity, low cost, user-friendly and portable dimensions of the instrumentations. In this work we present a deeper investigation on the electrochemical properties of AuNPs in relation to their size, concentration and surface modifications, using direct voltammetric (DPV) and electrocatalytic techniques with screen-printed carbon electrodes as novel transducer platform for AuNP detection. AuNPs with sizes ranging from 2 to 100 nm have been preliminarily characterized by means of transmission electron microscopy (TEM), z-potential measurements, UVVisible absorption and Inductively Coupled Plasma Mass Spectrometry (ICPMS). Modifications of the AuNP surfaces have been performed immobilizing DNA and protein molecules. Preliminary results regarding the effect of the size, the concentration and the surface modifying molecule over the electrochemical responses toward AuNPs with interest for several biosensor technologies applications will be presented. Acknowledgments MAT2008-03079/NAN and NANOBIOMED (CONSOLIDER-INGENIO 2010, CSD2006-00012) from MEC (Madrid) and VALTEC08-1-0007 from CIDEM (Barcelona) are acknowledged. Keywords Screen-printed electrode, electrochemical detection, gold nanoparticles, biosensing. References [1] M.T. Castaeda, A. Merkoi, M. Pumera, S. Alegret, Biosens.Bioelectron. 22 (2007) 1961. [2] A. Ambrosi, M.T. Castaeda, A.J. Killard, M.R. Smyth, S. Alegret, A. Merkoi, Anal. Chem. 79 (2007) 5232.

Electrochemical Biosensors for the Determination of the Antioxidant Capacity M. Cortina-Puig, C. Calas-Blanchard, J.-L. Marty BIOMEM group, Universit de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France E-mail: mcortina12@gmail.com Reactive oxygen species (ROS), naturally generated during the metabolism, can damage biological structures such as proteins, lipids or DNA. Inside the human body, the antioxidant defensive system prevents their effect but sometimes these natural defences are overwhelmed by an excessive generation of ROS and a situation of oxidative stress occurs. In this case, cellular and extracellular macromolecules can suffer oxidative damage, causing tissue injury1, 2. Antioxidants are synthetic or natural substances that prevent or delay the oxidative damage by scavenging the free radicals. The determination of free radicals and antioxidants has been widely investigated in the food technology. Traditional techniques such as spectrophotometry, fluorescence, and gas or liquid chromatography are being replaced by other innovating technologies3. In this direction, electrochemical biosensors are promising tools since they exhibit advantages such as minimal sample preparation, selectivity, sensitivity, reproducibility, relatively low cost, rapid time of response and simple use for continuous on-site analysis. In food science, research aims to detect and quantify the ability of some compounds to scavenge free radicals. In this sense, two different kinds of biosensors are reported in the antioxidant domain. On the one hand, several amperometric biosensors for the detection of mono and polyphenols (the main antioxidant compounds in food) have been developed on the basis of enzymes such as tyrosinase, laccase or peroxidase4. These configurations allow the evaluation of the usually named total phenol content. On the other hand, biosensors for the assessment of the antioxidant capacity are based on the free radical scavenging ability. In order to assess the antioxidant capacity of a substance based on the measurement of the superoxide radical (O2-) concentration, two main types of biosensors have been developed, using cytochrome c (cyt c) or superoxide dismutase (SOD) enzyme5. (1) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine; Oxford University Press: New York, USA, 2007. (2) Halliwell, B.; Aruoma, O. I. FEBS Lett. 1991, 281, 9-19. (3) Prior, R. L.; Wu, X.; Schaich, K. J. Agric. Food Chem. 2005, 53, 4290-4302. (4) Mello, L. D.; Kubota, L. T. Food Chem. 2002, 77, 237-256. (5) Prieto-Simn, B.; Cortina, M.; Camps, M.; Calas-Blanchard, C. Sens. Actuators, B 2008, 129, 459-466.

Abstract list

POSTERS

Carbon nanotube based biosensor for flow through monitoring of phenol

1,3 1,2 1 3 4

G. Alarcon , M. Guix , A. Ambrosi , M.T. Ramirez Silva , M. Palomar Pardave , A. 1,2,5* Merkoi (1)Nanobioelectronics & Biosensors Group, Catalan Institute of Nanotechnology Campus UAB, 08193 Bellaterra, Barcelona, Catalonia, Spain. (2) Chemistry Department, Autonomous University of Barcelona, Campus UAB, 08193 Bellaterra, Barcelona, Catalonia, Spain. (3) Chemistry Department, Autonomous Metropolitan University Unit Iztapalapa, 02200 Mexico City, Mexico (4) Chemistry Department, Autonomous Metropolitan University Unit Azcapotzalco, 02200 Mexico City, Mexico (5)ICREA, Barcelona, Catalonia, Spain *arben.merkoci.icn@uab.es

A biosensor for phenol detection based on a screen-printed electrode modified with tyrosinase, multiwall carbon nanotubes and glutaraldehyde is prepared and applied in a FIA system. Several parameters such as the working potential, pH of the measuring solution, biosensors response time, detection limit, linear range of response and sensitivity are studied. The obtained detection
-6

limit for phenol was 0.14 10 M. The biosensor shows a stable response (RSD 15%) within a 15 days working period at room temperature . The developed biosensor is being applied for phenol detection in seawater samples and seems to be a promising alternative for automatic control of sea waters contamination. The developed detection system can be extended to other enzyme biosensors with interest for several other applications.

Biosensors based on polyphenoloxidase enzymes for the determination of phenolic compounds Mirta Alcaraz1, Jimena Tejerina1, Silvia Hernndez 1, Silvia Fabiano 1, Mara Isabel Pividori2, Salvador Alegret2
1

laboratorio de sensores y biosensores, facultad de bioqumica y ciencias biolgicas, universidad nacional del litoral, argentina. 2 grup de sensors i biosensors, departament de qumica, universitat autnoma de barcelona, bellaterra, espaa Contact: sfabiano@fbcb.unl.edu.ar

The determination of phenol derivates is very important in food, medical, environmental ground and surface water. Some of them may cause the danger to the health an environmental pollution because of their inherent toxicity. Recently, a large number of efforts have been made for the simple and effective determination of phenolic compounds. The amperometric biosensors have been applied for this because of the advantages such as good selectivity, low cost, and easy automation. Amperometry is a method to measure the electric current that flows as a result of reactions generated at the electrode. in the present work we describe a biosensor based on metalloenzymes (tyrosinase and laccase) immobilized on the surface of graphiteepoxy composite (gec) electrodes. This composite presents physical, chemical and electric properties that make it appropriate for the electrochemical sensing. Amperometry were the techniques chosen to obtain the analytical response. Nafion, a perfluorinated anionic polyelectrolyte was used for the fabrication of enzyme-modified electrodes due to ease of use and good electrical conductivity. In addition, nafion films have high chemical stability, good biocompatibility and the ability to resist interferences from anions and biological macromolecules. In order to optimize the functioning of biosensor, different operational conditions were assayed as regards pH, ionic strength, applied potential, etc. This methodology was very useful for a simple and effective way to develop biosensors for several phenolic compounds determination Perspective in this work is to evaluate environmental real samples with this sensor.

Heavy metal screen-printed electrodes integration in an in-situ voltammetric probe

1,2 5 1 5 1,3 5 1,2,4

G. Aragay , A. Puig-Font , M. Medina , A. Merkoi

*
6

and M. Malizia , P. Moscetta , L. Sanfilippo , P. Moscetta

1

Nanobioelectronics and Biosensors Group, Institut Catal de Nanotecnologia

2

Departament de Qumica, Universitat Autnoma de Barcelona Bellaterra, Catalonia, Spain

3

On leave from Universidad Nacional de Colombia, Bogot

4

ICREA, Barcelona, Spain

5 6

SYSTEA S.p.A., Italy

Sysmedia S.r.l., Italy *E-mail: gemma.aragay@uab.cat , arben.merkoci.icn@uab.es

The successful integration of heavy metal screen-printed sensors (SPE) into the in situ voltammetric probe so as to achieve the required analytical performance is strongly dependent to the flow electrochemical cell and the whole flow through and electronic setup of the system. A new flow through cell that better fits to the developed SPEs ensuring a tight close as well as an easy electrode replacement system has been designed and all the corresponding electrochemical parameters including those of the liquid flow are accordingly optimized. The whole system probe that will ensure the smooth running of all the analytical steps including its hardware is shown. The application of the developed SPEs using the developed in situ probe system represents the final step so as to achieve the objective of WARMER project for voltammetric detection of heavy metals. The hydraulic circuit, the voltammetric flow cell and the hardware (voltammetric measuring unit) have been integrated into a single system. Some preliminary studies related to the theoretical models of the designed flow cells including some simulations regarding the effect of the liquid flow rate upon the pressure homogeneity/distribution inside the bulk of the electrolytic cell are shown. The whole analytical parameters optimizations (deposition time, conditioning time, conditioning potential, frequency and step potential) are presented in detail. The standard addition method has been studied and the number of heavy metal standard additions (including the concentrations) has been accordingly selected. The analytical performance of the final voltammetric measuring system (automatic analyzer) based on the use of SPE as detectors and the flow cell is still in process at our laboratories and the final results will be shown later.

Monolithic capillary electrophoresis system with integrated reservoirs, separation electrodes and amperometric detection on LTCC technology

E. Arasa-Puig, M. Puyol, J. Alonso Chamarro Analytical Chemistry Department, GSB, Universitat Autnoma de Barcelona, Spain e-mail: julian.alonso@uab.es

The miniaturization of conventional analytical instrumentation has been in the eye of researchers during the last decades and mainly since the concept of -TAS appeared. It was then, when the integration of some stages of the analytical procedure such as the sample introduction and pre-treatment, chemical reaction, separation and detection were feasible. CE is one of the possible analytical techniques developed at micro-scale as it requires simple instrumentation like electrodes to apply the electrical field to drive flow and thus, avoiding the coupling of pumps and valves. However, the mostly employed detection methods LIF (LaserInduced Florescence) and MS (Mass Spectroscopy), based on a complex instrumentation, eclipse this feature. In this sense, the integration of electrochemical methods is attractive due to the small physical dimensions required for CE (Capillary Electrophoresis) systems and their compatibility with microfabrication techniques. The LTCC (Low Temperature Co-fired Ceramics) technology is capable of integrating detectors (electrochemical, optical), micro channels and electronic circuits in the same device. A fully integrated LTCC microfluidic system for CE based on amperometric detection (Figure 1) has been constructed. The separation potential can be applied by means of silver screenprinted electrodes (c) in contact with the integrated buffer (a) and sample reservoirs (b) and a grounded electrode (g) positioned in an integrated amperometric cell placed at the end of the separation channel (white lines). An embedded Pt foil as the auxiliary electrode (h) and a silver screen-printed pad as the reference electrode (f) with a working electrode based (e) on an endchannel platinum wire inserted onchip constitute the amperometric detection system. The behavior of the CE microsystem has been evaluated with a mixture of dopamine and catechol solutions as model compounds. The obtained results show they can be well resolved due to the little dimensions of the capillary and the position of the working electrode, and demonstrate the viability of the easy integration of all the components to perform a CE procedure, from the sample uptake until the detection, in a single device by using this technology.

Figure 1. Photography of the CE device based on the LTCC technology: a) buffer reservoirs, b)sample reservoir, c) one of the separation electrodes, d) reservoir, e) working electrode, f)reference electrode, g) ground electrode, h) counter electrode. White lines represent the inner injection and separation channels.

Bulk reference electrode based on LTCC technology for electrochemical sensors

E. Arasa-Puig, J. Alonso Chamarro Analytical Chemistry Department, GSB, Universitat Autnoma de Barcelona, Spain e-mail: julian.alonso@uab.es Advances in micro-fabricated electrochemical sensor on silicon, plastic or ceramics substrates, call for the development of a reference electrode (RE) system that can be integrated readily with other working electrodes on the same chip. The Low Temperature Co-fired Ceramics (LTCC) technology is a miniaturization technique capable of integrating detectors (electrochemical, optic), micro channels and electronic circuits in the same device. Extracting benefits of these characteristics, in this work we propose the construction of a miniaturized bulk reference electrode based on LTCC technology for electrochemical sensors. The LTCC-RE consists (Figure 1) on a liquid junction with a protective layer of polyurethane which separates the
Capacitor

Ptsheet Filling solution inlet andoutlet

Filling solution inlet andoutlet Capacitor

internal filling from de test electrolyte. The filling solution is in contact with an Ag screen-printed path which acts as Ag/AgCl reference electrode. In order to improve the performance, the
Liquid junction 5.5x1.6x0.2mm Liquid junction Connection to external setup Ptsheet Connection to external setup

Electrical screenprinted contact (capacitor/Pt)

Reference electrode

device integrates a Pt sheet which diminishes the impedance and

together with a capacitor filters the high frequencies of electrical current.

Figure1.Photosandschemeofdeviceconstruction.

The proposed electrodes with a filling solution KCl 0.1M exhibit a negligible potential drift and stable potential during at least four days in different test solutions without any handling of the device. solution. The results obtained in cyclic voltammetry and in potentiometry are comparable with those obtained with a commercial reference electrode. Device fabrication is reproducible, being the deposition of protective layer in the liquid junction the most crucial point. Stable potentials are also obtained when K2SO4 0.1M is used as filling

Yessotoxin determination by phosphodiesterase inhibition-based colorimetric and electrochemical assays.

Camps M; de la Iglesia P; Mola M; Gimnez G; Fernndez-Tejedor M; Diogne J IRTA, Ctra. Poble Nou, km. 5.5, 43540 Sant Carles de la Rpita, Spain monica.campas@irta.cat

Yessotoxins are marine toxins with a polycyclic ether structure produced by dinoflagellates, which accumulate in shellfish. Although they are not diarrheic toxins and there is no evidence that they are toxic to humans, they are the main cause of false positives in the mouse bioassay (MBA) for lipophilic diarrheic shellfish poisoning (DSP) toxins. The purpose of this work has been the development of functional assays for yessotoxin detection, based on the interaction between this toxin and the phosphodiesterase enzyme, and the measurement of the enzymatic activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates have been tested in order to find those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that have provided with the highest absorbance values and density currents have been p-nitrophenyl phenylphosphonate and -naphthyl phosphate, respectively. Afterwards, several electrochemical techniques have been evaluated, differential pulse voltammetry (DPV) showing the highest sensitivities and the most reproducible measurements. An inhibitory effect of yessotoxin on the phosphodiesterase activity has been observed in both the colorimetric and the electrochemical assays. Limits of detection (LOD = 20% inhibition) of 1.1 and 0.7 M have been attained by colorimetry and electrochemistry, respectively. The assays have been applied to the analysis of yessotoxin production by Protoceratium reticulatum cultures and results have been compared with LC-MS/MS analysis. Current research is focused on the removal of matrix effects for proper quantification and the study of the inhibitor effect of several yessotoxin analogues. With the aim of developing the electrochemical biosensor, phosphodiesterase has been immobilised on graphite electrodes by entrapment into photopolymers, achieving 100% immobilisation yields. In order to improve the limits of detection, other immobilisation techniques that do not create diffusion barriers are being studied.

ALARMTOX: assays and biosensors for the detection of biotoxins from aquatic media Eixarch H1; de la Iglesia P1; Diogne J1; Fernndez-Tejedor M1; Flores C2; Guibert A3; Dumont V3; Caixach, J2; Marty J-L4; Camps M1 IRTA, Ctra. Poble Nou, km. 5.5, 43540 Sant Carles de la Rpita, Spain 2 IDAEA, CSIC, C. Jordi Girona, 18-26, 08034 Barcelona, Spain 3 CRITT-BioIndustries, INSA 135, Av. de Rangueil, 31077 Toulouse Cedex, France 4 BIOMEM IMAGES, UPVD, 52 Av. Paul Alduy, 66860 Perpignan Cedex, France monica.campas@irta.cat The presence of microalgae species able to produce toxins in the aquatic environments has a negative influence in several areas, such as food safety, health, environment and socioeconomy. Contamination of shellfish, drinkable water and nutritional algae or direct exposure to biotoxins may cause general illnesses and diseases both in humans and animals. Moreover, closure of coastal shellfish production areas due to the presence of toxins slows progress in aquaculture. Similarly, commercialisation of drinkable water and development of agriculture and cattle farming in continental areas can be affected by the presence of biotoxins. Contamination of shellfish and recreational areas also has implications for tourism sector. The ALARMTOX project is focused on the development and validation of assays and biosensors for the detection of biotoxins from aquatic media, with the aim of establishing new technologies that will guarantee the quality of continental waters as well as fishery and aquaculture products for human consumption. These new technologies should be more specific, more sensitive, faster and less expensive than the currently used analytical methods, for instance the mouse bioassay that, despite being a useful surveillance tool for consumer protection, presents ethical and selectivity limitations. In this project, CRITT-BioIndustries-INSA is in charge of the production of genetically engineered protein phosphatases sensitive to microcystins and okadaic acid and derivatives. BIOMEM-IMAGES-UPVD and IRTA are responsible for the development of colorimetric assays and electrochemical biosensors. IDAEA-CSIC and IRTA will validate the assays and biosensors for microcystins and okadaic acid, respectively, by comparison with LC-MS/MS analysis. Besides, IRTA is coordinating the project. Several public institutions and private enterprises are collaborating by providing the project with water, microalgae and shellfish samples from a great variety of ecosystems. ALARMTOX is a project co-financed by the European Union through the Interreg IV B SUDOE (South-West Europe) Territorial Cooperation Programme. It is integrated into the first priority of the Programme: Promotion of innovation and constitution of stable cooperation networks in technological matters. The presented opinions only compromise the partners and, consequently, in no way represent the opinion of the Territorial Cooperation Programmes management bodies. www.alarmtox.net
1

Ceramic microfluidic devices as a powerful tool for the simple synthesis of gold nanoparticles Sara Gomez-de Pedro, Mar Puyol, Juli n Alonso Sensors and Biosensors Group, Department of Chemistry, Universitat Autonoma de Barcelona, Bellaterra, Spain Sara.gomez@uab.cat Microfluidic reactors offer certain advantages over conventional chemical processes for the synthesis of gold nanoparticles thus being a powerful tool for this purpose. Nanoparticles are usually prepared by means of batch processes, where some steps such us the addition of reagents, injection volume, rate of stirring, local temperature and concentration fluctuations are difficult to control, obtaining as a result particles with rather broad size distributions. Microfluidic systems allow varying in a rapid, controlled and precise way most of experimental variables, leading to more uniform particles. Few microfluidic devices have been proposed for this purpose and they are based on the most common materials employed for miniaturization (glass, silicon or polymers). However, most of these methods require a postfabrication sealing of the microchannels that can produce leaks at the joints and show poor chemical and thermal inertness. Ceramic tapes based on the LTCC (Low-Temperature Co-fired Ceramics) technology, not only solve problems related to the lack of sealing but also bear high temperatures or extreme conditions, at which the synthesis of some types of nanoparticles take place. All this with a rapid prototyping and without the need of using sophisticated facilities. This significantly reduces the costs and the production time. Moreover, its multilayer approach allows the design of 3D structures, where mechanic, electronic and fluidic components can be integrated in a single device. Thus, a careful and accurate control of the synthesis can be performed by the introduction of passive mixers or by the monolithic integration of temperature control systems based on resistor/thermistor couples. Furthermore, other steps for the stabilization or modification of the nanoparticles can be easily carried out by the addition of more channels in the same microfluidic system. The LTCC technology is a potential tool for the development of analytical microsystems devoted to biomedical applications and integrating as well as optical as electrochemical transducers.

Fig. a) Ceramic microfluidic device, consisting of a 40L passive mixer and four inlets for reagents. b) Example of the gold nanoparticles synthesized in a ceramic microfluidic device.

Direct electrical detection of point mutations at Multi walled carbon nanotube modified electrodes
1,2 ,1, 1 2 ,1,3

P. Kara
1

, M. Guix , A. Escosura - Muniz , M. Ozsoz , A Merkoi

Nanobioelectronics & Biosensors Group, Catalan Insitute of Nanotechnology, Bellaterra, Catalonia, Spain;
2

Ege University Faculty of Pharmacy, Analytical Chemistry. Department, Turkey 3 ICREA, Barcelona, Catalonia, Spain E-mails: arben.merkoci.icn@uab.es ; pinar.kara@ege.edu.tr

Electrochemical hybridization biosensors hold a great promise in viral, bacterial identification, single nucleotide polymorphisms, mutation detections and genomic sequencing. They may greatly reduce the assay time and simplify the protocol. Such fast on-site monitoring schemes are required for quick preventive action and early diagnosis. The appearance of guanine signal after hybridization with inosine substitute probes and target sequences eliminates the external labels and shortens the assay time. The oxidation signals of guanine have been employed to detect hybridization. Such use of intrinsic signals of DNA is nowadays preferred because it reduces the assay time. A label free electrochemical genosensor based on point mutation related to Factor II gene detection is developed in this study. The genosensor relies on voltammetric transduction of guanine oxidation at multi-walled carbon nanotubes modified screen printed electrode (MWCNT-SPE) surfaces. The physical adsorption is used to modify the SPEs with MWCNTs followed by SNP detection based on guanine signal. The electrochemical transduction of guanine oxidation is accomplished after hybridization with a 45 mer target sequence with a detection limit of 0.17 nM.

Keywords DNA hybridization, point mutation, guanine oxidation, screen-printed electrodes, carbon nanotubes

Gliadin immunosensing in gluten-free products for food safety of celiac patients Tamara Laube, Salvador Alegret and M Isabel Pividori* Grup de Sensors i Biosensors, Universitat Autnoma de Barcelona Campus UAB, Edifici Cn, 08193, Bellaterra, Spain. E-mail: Isabel.Pividori@uab.cat

Celiac disease, also known as gluten-sensitive enteropathy, is an autoimmune disorder that affects approximately 1% of the population in Northern Europe and North America. The only treatment that is known untill now is a lifelong avoidance of the cereal protein gluten in the diet. Due to this fact, to increase food safety for celiac patients, gluten and celiac disease have been included in food regulations and labelling in order to prevent harmful effects of gluten-containing food or food components in celiac patients. Moreover, the US Food and Drug Administration (FDA) defined in 2008 that foods labelled with the term gluten-free, may not exceed a content of 20 ppm. With this aim, it is essential to have a reliable method of analysis to control the contents of glutenfree foods and there is still a need for an assay that permits the easy, rapid and on site testing of incoming raw materials as well as the contamination monitoring during the industrial processing. Electrochemical biosensors offer an exciting alternative for the descentralized analysis without professional supervision, keeping in mind some of their advantageous properties, like simple manipulation, high sample throghput, rapidity of analysis and low cost, which contrast with the more conventional analytical instrumentation used for food analysis. The present work is focused on the development of immunosensing strategies for the detection of gluten in food, based in optical and electrochemical detection (through a magneto-ELISA system and magneto-sensor, respectively). In both cases, the immunological reaction was performed on magnetic beads as a solid support, to which the antigen, in this case gliadin (toxic protein fraction of gluten), was covalently immovilized to the tosyl-activated surfaces. The biorecognition strategy is based on a competitive assay, which can be direct or indirect, using an anti-gliadin antibodyperoxidase (HRP) conjugate as the enzymatic label, or a primary antibody without tracer and a secondary antibody conjugated to the same enzyme, respectively. The subsequent detection is then achieved through a suitable substrate and mediator for the HRP enzyme. Excellent LODs (5,13x10-3 ppm) were achieved with the electrochemical immunosensor, according to the requirements for gluten-free products. These novel strategies could be of great promise for rapid, simple, cost-effective and on-site analysis of food samples.

A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis in dairy farms by electrochemical geno sensing S. Fabiano3, A. Soutullo4, S. Hernndez3, M. I. Garca4 A. Lermo1, S. Libana1, S. Campoy2, S. Alegret1, M.I. Pividori1* Grup de Sensors i Biosensors, Unitat de Qumica Analtica, Unitat de Microbiologia, Departament de Gentica i Microbiologia, Universitat Autnoma de Barcelona, Bellaterra, Spain 2 Laboratorio de Sensores y Biosensores, Facultad de Bioqumica y Ciencias Biolgicas, Universidad Nacional del Litoral, Argentine 4 Laboratorio de Diagnstico e Investigaciones Agropecuarias, Ministerio de Agricultura, Ganadera, Industria y Comercio de Santa Fe, Argentine *Contact: Isabel.Pividori@uab.cat
2 1

Tuberculosis in humans and other mammals is usually caused by Mycobacterium tuberculosis or Mycobacterium bovis. While M. tuberculosis is the single greatest cause of infectious disease in humans worldwide, M. bovis affects the largest number of animals throughout the world. In humans, the global prevalence of tuberculosis infection is about one-third of the worlds population, a number which is expected to grow steadily [1] . M. bovis causes a disease known as bovine tuberculosis which can be easily transmitted between farm animals. The disease due to M. bovis is also an important zoonosis mainly associated not only with labour risk in dairy farms, but also with the consumption of contaminated dairy products with the bacilli. As such, the isolation of M. bovis from milk samples of storage tanks, not-well pasteurized milk, and milk samples from tuberculin non-reactive cattle was reported [2] . The conventional methods for tuberculosis diagnostic (sputum microscopy, chest radiography, histopathology, tuberculin test, and culturing methods) are time consuming and lack of specificity and sensitivity [3] . In this work a very sensitive assay for the rapid screening-out of raw milk stored in bulk tanks contaminated with TBC based on electrochemical genosensing is proposed as an alternative to time-consuming methods. The features of this approach are discussed and compared with classical methods as well as with inter-laboratory PCR assay.
Bloom, B. R., Murray, C. J. L. Science, 257 (1992) 10551064. Pardo, R.B., Langoni, H., Mendoa L.J.P. and Chi, K.D. Braz. J. Vet. Res. Anim. Sci., 38 (2001) 284287. [3] Tiwari R. P., Hattikudur N. S., Bharmal R. N., Kartikeyan S., Deshmukh N. M., Bisen P. S. Tuberculosis 87, (2007) 193201.
[2] [1]

Rapid detection of food pathogens by electrochemical magneto immunosensing Susana Libana1, Anabel Lermo1, Susana Campoy2, Jordi Barb2, Salvador Alegret1, Maria Isabel Pividori1* Grup de Sensors & Biosensors, Unitat de Qumica Analtica Edifici Cn, Universitat Autnoma de Barcelona, 08193, Bellaterra, Barcelona. 2 Unitat de Microbiologia, Departament de Gentica i Microbiologia E-mail: Isabel.Pividori@uab.cat Many factors have contributed to recent food emergencies, such as the increasingly complexity of the food production chain because of mass production. One of the most effective ways for the food sector to protect public health is to base their food management programs on Hazard Analysis and Critical Control Point (HACCP). Biosensing devices can be considered as ideal tools to be implemented in HACCP programs, for being used as an alarm' to rapidly detect the risk of contamination by food pathogens in a rapid, inexpensive and sensitive manner and in a wide variety of food matrixes. The novel procedure presented here is suitable for the rapid and sensitive on-site analysis of Salmonella in HACCP. In this approach, the bacteria are captured and preconcentrated from milk samples with magnetic beads by immunological reaction with the specific antibody against Salmonella. A second polyclonal antibody labeled with peroxidase is used for the electrochemical detection. Then, the modified magnetic beads are easily captured by a magneto sensor made of graphiteepoxy composite (m-GEC) which is also used as the transducer for the electrochemical detection. The magnetic immunoseparation and the detection with a second specific antibody were able to effectively replace the selective plating. As such, the time of the assay was considerably reduced from 3-5 days to 50 min without any pretreatment. A limit of detection of 5103 cfu mL-1 and 7,5103 cfu mL-1 in LB and in milk diluted 1/10 in LB broth, respectively was obtained. With a preenrichment of 6 h of the skimmed-milk, the method was able to detect 1,4 cfu mL-1, while if it was preenriched for 8 hours, 0,108 cfu mL-1 (2,7 cfu in 25 g of skimmed-milk sampled in 5 portion of 5 g each) were detected, according to the legislation. Moreover, the method is able to clearly distinguish between food pathogenic bacteria such as Salmonella and E. coli. The features of this approach are discussed and compared with classical culture methods.
1

Microanalyzer for Enzymatic Reactions with an Integrated Thermal Preconditioning Step

C.S. Martnez-Cisneros, N. Ibez-Garca and J. Alonso
Grup de Sensors i Biosensors. Departament de Qumica. Universitat Autnoma de Barcelona. Catalunya. Spain. e-mail: cynthia.martinez@uab.es

The continuous need of highly integrated analyzers able to perform all the steps associated to the classical analytical process has produced an increasing interest in the miniaturization field. The integration of pretreatment steps, such as termostatization, results very useful when the analysis involve the use of biologic agents that improve their reaction rate at certain temperatures. Trying to full fit this need, in this work, a miniaturized analyzer that includes a
2 1 3 4 A T R

monolithic thermal preconditioning step is presented. The device is based in the LTCC technology and integrates an embedded pair sensor/actuator for its termostatization. The system performance was evaluated using a LED-photodiode based optical detection system for the determination of the alkaline phosphatase activity and its inhibitors, by measuring the products obtained from the

Figure 1: Microanalyzer for enzyme determinations. T: thermistor; R: resistor; A: LED; B: photodetector; 1,2: reagent inlets; 3: detection cell; 4: outlet.

enzymatic reaction. For this purpose, the device included a detection cell (see figure 1). Since the microanalyzer operation was regulated by means of a microcontroller, experiments using different and precise steady state temperatures could be carried out in order to determine the optimum value. The alkaline phosphatase activity was evaluated at five different temperatures (environmental temperature 20C, 30C, 35C, 37C and 40C). Significant improvements in the sensibility of the system were observed while temperature increased until the enzyme denaturing temperature was reached.

Miniaturized Amperometric Analyzer for Free-Chlorine Determination

C.S. Martnez-Cisneros1 Z.M. da Rocha2, R. Oliv-Monllau1, M. Ferreira2, A.C.Seabra2, M.R. Gngora-Rubio3, and J. Alonso1.
1

Grup de Sensors i Biosensors. Universitat Autnoma de Barcelona. Catalunya. Spain.

2

Laboratorio de Sistemas Integrveis. Universidade de So Paulo. Brasil.

3

Instituto de Pesquisas Tecnolgicas de So Paulo (IPT). Brasil e-mail: cynthia.martinez@uab.es

Currently, dedicated devices aimed at solving specific analytical problems are needed. In these cases, low-volume production processes are a better alternative to mass production technologies such as silicon and glass. Following this approach, in this work, the Green Tape technology was applied to the development of a complete amperometric microanalyzer (see figure 1) . The device includes, monolithically, the fluidics, a three-electrode configuration electrolytic cell and the associated
d
1

a b c

electronics, all in a unique device. To increase the system reliability and to avoid its dependence on the lifetime of the working electrode, it was integrated in an exchangeable configuration. Using this approach, any metallic or conductive material able to be used as electrode, i.e.,

Figure 1: miniaturizad amperometirc analyzer. a: reagent inlets; b: reference solution inlet; c: outlet; d: working electrode.

graphite-epoxy composites, can be readily integrated to full fit any application. In this work, the microanalyzer was applied to the determination of free chlorine in swimming pool water. Results were compared with those obtained using the standard colorimetric (N, N-diethyl-p-phenylenediamine, or DPD) method observing any significant difference between them.
1

C.S. Martnez-Cisneros, Z.M. da Rocha, M. Ferreira, A.C. Seabra, M.R. Gongora-Rubio, A Novel Monolithic Continuous Flow

Microanalyzer with Amperometric Detection Based on the Green Tape Technology. Anal. Chem. Submitted.

PDMS based lab on a chip with electrochemical detection usign screen printed electrodes

Mariana Medina Snchez1,2,3, Sergio Marn Mancebo1, Maria Guix1, Georgina Alarcn1, Adriano Ambrosi1, Arben Merkoi1,4, Diego A. Garzn3
1

Nanobioelectronics & Biosensors Group, Institut Catal de Nanotecnologia. Barcelona, Catalonia, Spain
2

Universidad de San Buenaventura. Bogot, Colombia

3

Universidad Nacional de Colombia.

4

ICREA, Barcelona, Catalonia, Spain

The miniaturization and integration in microfluidic systems including nanostructurated electrodes have been shown to be of a great interest for applications in several fields that include clinical analysis, security & safety, environmental monitoring and other industrial applications. An integrated lab-on-a-chip (LOC) system based on PDMS including the electronic connections is presented and its performance is compared with conventional micro systems. The optimization of the fabrication process (design of the mask, photolitrography, molding and sealing with the substrate) will be also shown. Different integrations alternatives between the microfluidic unit and electronic (high potential for the electrophoresis and electrochemical detection electrodes) are presented. The electrodes have been designed & fabricated by using screen printing technology. The developed LOC will be of a low cost and compatible to mass production technologies. It allows the easy modification and other further integrations with interest for several analytical applications. Finally some preliminary electrochemical results that show advances of the developed electrochemical detection system will be shown.

Preparation of aptamers against lupine potential allergens Pedro Nadal Polo1, Veli Cengiz Ozalp2, Nuria Canela3, Ioanis Katakis1, Ciara K. OSullivan1,4 Nanobiotechology and Bioanalysis Group, Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Pasos Catalans 26, Tarragona 43007, Spain. 2 Department of Biochemistry and Molecular Biology, University of southern Denmark, 5230, Odense, Denmark. 3 Unitat de Biologia Molecular, Servei de Recursos Cientfics i Tcnics, Universitat Rovira i Virgili, Paisos Catalans 18, 43007, Tarragona, Spain. 4 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010 Barcelona, Spain. pedro.nadal@urv.cat
1

Aptamers are artificial nucleic acid ligands isolated from complex libraries by an iterative process of adsorption, recovery and reamplification in a process coined SELEX, which permits the identification of unique oligonucleotides from very large populations of random sequence oligomers that bind to the target molecule with very high affinity and specificity. Aptamers are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors. Despite the improvement and increased automation of aptamer selection, a large proportion of the reports for analytical applications use the same aptamers (e.g. the thrombin-binding aptamer). Lupine has recently been added to the list of substances requiring mandatory advisory labelling on foodstuffs sold in the European Union, thus all products containing even trace amounts of lupine must be labelled correctly since December 2008 [1]. This is a response to the increasing number of severe cases of lupine allergies reported during the last decade [2]. The aim of the work reported here is (i) the elucidation of a protocol for the isolation of each of the lupin subunits - , , , - conglutins and subsequently (ii) the development of aptamers against each of these lupine proteins and (iii) their use for the detection of these substances in clinical or food analysis.[3]

References: 1. 2. OSullivan, C.K., Aptasensors the future of biosensing? Anal Bioanal Chem 2002. 372: p. 44-48. Authority, E.F.S., Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the evaluation of lupine for labelling purposes. The EFSA journal, 2005. 302: p. 1-11. Switzerland, C.L.U., Detection of lupine in food commodities. 2007.

3.

DF508 Cystic fibrosis Determination via Methylene blue Electrochemically Hany Nasef1, Valerio Beni1and Ciara K. OSullivan*1,2
1

Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Pasos Catalans, 26, 43007 Tarragona, Spain 2 Institucio Catalana de Recerca i Estudis Avanats, Passeig Lluis Companys 23, 08010, Barcelona, Spain

Cystic fibrosis CF is caused by a mutation in a gene called the cystic fibrosis transmembrane conductance regulator (CFTR) and represents one of the most common life-shortening, childhood-onset inherited diseases. The most common mutation of cystic fibrosis is DF508 which represents 70 per cent of mutations in the white populations of Britain and US and 50 per cent in southern European populations. The following work is investigating the discrimination of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt) sequences via Methylene blue Indicator. The sensor surface was prepared by the formation of self assembly monolayer (SAM) of 21 bp Mutant (or wild type) complementary probe via S-Au bond on a polycrystalline gold electrode. The determination step was achieved after hybridisation with the complementary target (mutant amplicon, 83 bp or wild type amplicon, 85bp). The signal increasing in MB voltammograms was a function of the amplicons hybridisation efficiency on the electrode surface. About 150% difference in the signal was recorded as a discrimination factor between mutant and wild type. The assay was quantitative and linear in the range of 10 100 nM DF508 mutant target exhibiting limit of detection (LOD) of 2.64 nM.

On line monitoring of low chlorine concentration using an optimized rigid composite material based on multiwall carbon nanotubes Rosa Oliv-Monllau, Jordi Bartroli, Mireia Baeza and Francisco Cspedes Grup de Sensors i Biosensors, Departament de Qumica Analtica, Facultat de Cincies, Edifici C-Nord, Universitat Autnoma de Barcelona, 08193 Bellaterra, Spain. rosa.olive@uab.cat

The development of composites based on a conductive phase dispersed in a polymeric matrix has led to important advances in the analytical electrochemistry field, particularly in sensor devices development. They present important advantages respect to others pure materials, such as low background current since the electrode capacitance is strongly influence by the exposed carbon and facile surface renewal, as well as an efficient mass transport of the electroactives species due to the radial diffusion to the spaced carbon particles. Furthermore, they present low cost and easy malleability. From the point of the analytical view, their electrochemical behavior is strongly influenced by the carbon loading. This parameter was studied and optimized for a composite based on resin epoxy and multiwall carbon nanotubes, in a previous work [1]. The simplicity of these materials makes them suitable for their integration in a flow system. Hereby, we have incorporated this optimized composite electrode in a flow cell, for the monitoring of low free chlorine concentration in real water samples coming from a swimming pool. Figure 1 presents the experiment set-up.

Valve

Pump

Mix Coil Flux Cell Reference electrode

Figure 1 Developed flow system for the monitoring of low free chlorine concentration 1.Oliv_Monllau R., Esplandiu M.J., Bartrol J., Baeza M., Cspedes F. New approach for systematic characterization and optimization of conducting composites materials. Carbon (submitted)

Ultramicroelectrode arrays: a promissing analytical tool for (bio)sensing Jahir Orozco , Csar Fernndez-Snchez and Cecilia Jimnez-Jorquera Instituto de Microelectrnica de Barcelona IMB-CNM, (CSIC). Campus UAB, 08193 Bellaterra, Barcelona, Spain. *Corresponding author: Jahir Orozco tel. +34 935947700, Jahir.orozco@imb-cnm.csic.es Many alternative sensors based on noble metals (i.e. Au, Pt, etc), or carbon based materials (i.e. Glassy Carbon, Carbon Paste and Carbon Nanotubes composites) has been extensively explored in the field of analytical chemistry. However, the current trend towards the miniaturisation of chemical sensors and automation of analytical systems has led to the application of utramicroelectrode arrays (UMEAs) as feasible electrical transducer approaches in numerous applications [1]. The term ultramicroelectrode is reserved for electrodes with at least one dimension smaller than 25 m. Comparing with conventional electrodes, ultramicroelectrodes exhibit a series of advantages such as an enhanced mass transport, due to dominance of radial diffusion at the electrode-solution interface, which gives rise to steady-state currents, as well as decreased charging currents and consequently, low ohmic drop, which allow working in high resistivity media [2]. The use of Ultramicroelectrede arrays for in-field monitoring of environmental parameters offers numerous advantages. As solid state microsensors, they can be mounted in small and compact probes designed for in-field measurements [3, 4] or integrated in flow systems for on-line monitoring [5]. Such systems permit automatic sampling and calibration, sample treatment and sensor conditioning. They can be installed near the sampling place (i.e. rivers, lakes, industrial wastes) and can also accomplish some of the requirements for in-field monitoring like low power consumption, high autonomy and robustness. In that way it is possible to obtain information in real time and minimize variations in the sample composition due to transportation or storage. This work illustrates the use of UMEAs in a couple of environmental applications. On one hand, it is shown the development of a high sensitive device based on gold nanoparticles(GNP)-modified UMEAs for quantification of copper in extracts of soil samples. Modification of the UMEAs with GNP is a simple procedure that permits to improve the performance of the device, -a three-fold increase of sensitivity (1.3 nCM-1) and a five-fold higher linear concentration range (0-10 M) compared with bare UMEAs. Quantification of copper in soil samples was successfully performed and results were according to those obtained from the ICP-AES standard method. On the other hand, as a proof of concept, anchorage of an HRP enzyme on the GNP-modified UMEA surface through self assembled monolayers of DTSP was successfully performed and interrogated for the detection of phenolic compounds in aqueous solutions. The performance of the prepared electrodes was evaluated by amperometry in a pH 7.4, 0.05 M PBS background solution. It was noteworthy a dramatic increase in response of the HRP/DTSP/GNP-modified UMEA respect to the bare UMEAs. Values of the corresponding calibration parameters in the concentration range of linear response showed the higher sensitivity of the HRP/DTSP/GNP-modified UMEA (228.6 Acm2 mM-1), which is 3 times higher respect to that attained with the HRP/DTSP/UMEA. Overall, this work shows not only an alternative easy-to-use novel miniaturized device for the rapid and reliable determination of copper in soil samples, but also the potential use of such platforms in the development of biosensors for environmental applications.
References [1] J. Orozco, C. Fernandez-Sanchez, C. Jimenez-Jorquera, Environmental Science & Technology, 42 (2008) 4877. [2] J. Orozco, G. Suarez, C. Fernandez-Sanchez, C. McNeil, C. Jimenez-Jorquera, Electrochimica Acta, 53 (2007) 729. [3] J. Orozco, A. Baldi, R. Baena, A. Cadarso, A. Bratov, C. Jimenez, Measurement Science & Technology, 18 (2007) 935. [4] J. Orozco, A. Baldi, P.L. Martin, A. Bratov, C. Jimenez, Analytica Chimica Acta, 579 (2006) 95. [5] R. Olive-Monllau, J. Orozco, C. Fernandez-Sanchez, M. Baeza, J. Bartroli, C. Jimenez-Jorquera, F. Cespedes, Talanta, 77 (2009) 1739.

Enzymatic biosensor for L-lactate analysis in wine based on the incorporation of lactate oxidase by the phase inversion technique in a polysulfone/multi-wall carbon-nanotube onto screen-printed electrodes
Sandra Prez, Samuel Snchez, Esteve Fbregas Grup de Sensors i Biosensors,Universitat Autnoma de Barcelona, Facultat de Cincies, Campus de Bellaterra ,Barcelona, Spain Sandra.Perez@uab.es

Blood lactate is a clinically valuable diagnostic indicador. However, the importance of lactate is not limited to the medical sector. D- and L-lactic acid are found in many foods and beverages. For instance, the wine industry, the course of malolactic fermentation is monitored by following the falling level of L-malic acid, and the increasing level of L-lactic acid. [1] Amperometric biosensors for L-lactate have been constructed based on the immobilization of the L-Lactate oxidase (LOD) and the Cobalt Pthalocyanine (CoPC) as mediator into a Polysulfone/MWCNT membrane. LOD incorporation has been possible by the phase inversion technique. This method has been used recently for the immobilization of enzymes, proteins, hormones and antibodies into polymers [2, 3]. A thin film of polymer solution (in DMF) is deposited onto screen-printed commercial electrodes and then immersed into an aqueous solution (non solvent) that contains LOD. Thus, an exchange of the solvent by the non solvent causes the membrane coagulation. LOD catalyzes the conversion of lactate to pyruvate and hydrogen peroxide, according to the following reactions:
L-lactate + LODox pyruvate + LODred LODred +O2 LODox +H2O2 (1) (2)

Measurements are carried out in a phosphate buffer solution (PBS), and hydrogen peroxide is followed electrochemically by its electrocatalytic oxidation mediated by CoPC enzyme.
0,25 0,20 0,15

I (A)

0,10 0,05 0,00

y = 2048.7x + 0,0031 2 R =0,9998

0,0 2,0x10-54,0x10-56,0x10-58,0x10-51,0x10-4

[lactate] (M)

Fig.1 The current was monitored amperometrically at 0.350 V vs. S.C.E. and this method showed a linear range of the l-lactate from 110-5 to 110-4 M.

References:
[1] Nikolaus, N., Strehlitz, B., Microchim Acta, 160, (2008), 15-55. [2] Snchez, S., Roldn, M., Prez, S., Fbregas, E., Anal. Chem., 80(17), (2008), 6508-6514. [3] Snchez, S., Fbregas, E., Biosens.Bioelectron., 22(6), (2007), 965-972.

Towards aptamer mediated detection of coeliac disease toxic High Molecular Weight Glutenins A.Pinto1, M.Svobodova1, M.C. Bermudo1, P. Nadal Polo1, C.V.Ozalp2, I.D. Katakis1, C.K.OSullivan1,3 Nanobiotechology and Bioanalysis Group, Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Pasos Catalans 26, Tarragona 43007, Spain. 2 Department of Biochemistry and Molecular Biology, University of southern Denmark, 5230, Odense, Denmark 3 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010 Barcelona, Spain alessandro.pinto@urv.cat
1

High Molecular Weight Glutenins (HMW glutenins) are proteins of the wheat gluten. Recent in vivo studies have demonstrated the toxicity of these proteins to sufferers of coeliac disease 1 highlighting the importance of detecting these proteins in gluten-free foodtuffs. The objective of the work reported here is the selection of an aptamer against HMW glutenins and the subsequent use of these aptamers in fluorescent and electrochemical molecular aptamer beacons and aptasensors. Aptamers are nucleic acid receptors of high affinity and selectivity towards the target against which they are selected from a random library containing 1015 members exploiting the Systematic Evolution system by EXponential enrichment (SELEX) 2 , which is basically a screening method that mimics a Darwinian process. Aptamers have several advantages over other chemical and natural receptors due to their stability and versatility 3 , and can be exploited in assay formats not amenable to antibodies where PCR amplification is utilised to achieve detection of ultra low levels of analyte 4 .

Van de Wal Y et al. Glutenin is involved in the gluten-driven mucosal T cell response. Eur J Immun 1999;29 2 Ellington,A.D, .Szostac,J.W, In vitro selection of RNA molecules that bind specific ligands Nature, 1990. 3 T. Mairal, et al., Aptamers: molecular tools for analytical applications, Anal. Bioanal. Chem., 2008 4 A.Pinto et al. Real-Time apta-PCR for 20000-fold improvement in detection limit, Molecular Biosystem, 2009.

HIGH-SENSITIVE FLOW-IMMUNOSENSING SYSTEM FOR OKADAIC ACID ASSESSMENT

a

B. Prieto-Simn,a,b,c J.-L. Marty,b I. Karubea and H. Saikia

School of Bionics, Tokyo University of Technology, Katayanagi Institute 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan b IMAGES, Universit de Perpignan Via Domitia, 52 Avenue Paul Alduy 66860 Perpignan cedex France c Nanobioenginyeria, IBEC, C/ Baldiri Reixac 10-12, 08028 Barcelona bprieto@ibec.pcb.ub.es

Okadaic acid (OA) is a lipophilic phycotoxin that causes human health concern. It is a secondary metabolite produced by Dinophysis and Prorocentrum dinoflagellates that can be ingested by bivalves, such as mussels, oysters or scallops. The ingestion of bivalves contaminated with OA can cause gastrointestinal problems in humans. The European Union fixes at 160 g of OA equivalentskg-1 the allowed limit in bivalve molluscs and recommends the mouse bioassay (MBA) or alternatively liquid chromatography with fluorescence (LC-FLD) or mass spectrometry (LC-MS), enzyme inhibition assays or immunoassays. Due to the quite common episodes of shellfish contamination with OA and the ethical controversy related to the use of MBA and the expensive equipment and high-skilled personnel requirements of LC, there is an evident need of alternative detection techniques fulfilling the requirements of high sensitivity and accuracy and simplicity. Several biosensors for OA assessment have been shown as emerging screening tools. Enzymatic biosensors have been developed based on the inhibition that OA causes to protein phospatase PP2A. Nevertheless, immunosensors have been the commonest biosensors developed for OA assessment, likely due to the high affinity interactions between antigens and antibodies. Chemiluminescence, electrochemistry, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) have been used as detection methods for those immunosensors. Our aim was to develop a high-sensitive flow-immunosensing system based on kinetic exclusion measurements for the detection of the theoretical lowest limit of detection of OA enabled by the affinity of the anti-OA antibody (IC50= 0.05gL-1 in the assay solution). This highly sensitive automated system allows rapid and reliable OA quantification, with no significant matrix effects for the analysis of spiked mussel and scallop samples. Results were compared with those obtained using a Surface Plasmon resonance (SPR) assay based on the same immunostrategy. Although the SPR assay enabled the detection of the toxin in mussel samples at the limit set by the European Union with good percentages of recovery, matrix effects could not be neglected. Performance features such as high sensitivity and accuracy, low limits of detection and simplicity of the analysis protocol, shows the biosensing-systems based on kinetic exclusion measurements for toxin detection in shellfish samples as highly-performing tools for rapid and continuous screening.

Electrochemical sensors for in situ monitoring in oceanic systems

a a a b b

Audrey Ruffien-Ciszak , Danile Thouron , Vronique Garon , Pierre Gros and Maurice Comtat
a

Laboratoire dEtudes en Gophysique et Ocanographie Spatiales, UMR CNRS 5566,18 Avenue

b

Edouard Belin, 31401 Toulouse Cedex 9, France. Laboratoire de Gnie Chimique, UMR CNRS 5503, Universit Paul Sabatier,118 route de Narbonne, 31062 Toulouse Cedex, France.

The implementation of observatories for biogeochemical studies is a major challenge in oceanography. This will allow to clarify mid and long term biological processes and progress in our understanding of the ocean role in climate evolution. This long term monitoring in marine environments requires an in situ miniaturized autonomous instrumentation with long lifetime, high precision, low detection limit, fast response time and good reproducibility. Electrochemistry is a well adapted method for extreme conditions found in the ocean such as high pressure and high temperature gradient. It provides promising reagentless methods to go further in miniaturization, decrease in response time and energy requirements. At LEGOS, electrochemical sensors for silicate and phosphate are in development. Although silicate and phosphate are not electrochemically active species, their determination is possible by different electrochemical methods based on the detection of silicomolybdic or phosphomolybdic complexes formed in acidic media by the reaction with molybdenum salts (Carpenter and al., 1997). Voltammetric detection of silicates was shown to be feasible within the range of concentrations found in the ocean (between 0.3 and 160 M) in about 6 minutes. Cyclic voltammograms present two reduction and two oxidation peaks giving four values of the concentration and therefore increasing the precision. Then, chronoamperometry is performed on an electrode held at a constant potential. A complete reagentless method with a precision of 2.6 % is described based on the simultaneous formation of the molybdenum salt and protons in a divided electrochemical cell. This latter method is very useful for developing a reagentless sensor suitable for long term in situ deployments on oceanic biogeochemical observatories (Lacombe and al., 2007, 2008). These methods are being adapted to determine phosphate concentrations over the concentration range found in the open and coastal oceans, i.e. 0.5 to 4 M. These developments are part of two ongoing projects: the RTRA STAE MAISOE project within Midi Pyrnes and the european FP7 SENSEnet International Training Marie Curie network.

References Carpenter N., Hodgson A.W.E., and Pletcher D., 1997, Microelectrode procedures for the determination of silicate and phosphate in waters-fundamental studies. Electroanalysis, 9, 1311-1317. Lacombe M., Garon V., Comtat M., Oriol L., Sudre J., Thouron D., Le Bris N., and Provost C., 2007, Silicate determination in sea water: toward a reagentless electrochemical method, Marine Chemistry, 106, 489-497. Lacombe M., Garon V., Thouron D., Le Bris N. and Comtat M., 2008, A new electrochemical reagentless method for silicate measurement in seawater, Talanta, 77, 744-750.

Enhancement of Response of Graphite Epoxy Composite Electrodes Modified with Metal Nanoparticles Synthesized by Intermatrix Synthesis Technique. P.Ruiza, M. Muoza, J. Macansa and D.N. Muravieva
a

Unitat de Qumica Analtica, Departament de Qumica, Universitat Autnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Metal Nanoparticles (MNPs) have attracted great interest within the last years due to their special properties making them suitable for a wide range of applications. One of these applications deals with the use of Polymer Stabilized MNPs (PSMNPs) and nanocomposite membranes on their base in electrochemical applications such as sensors and biosensors. In this case one can substantially enhance both the mass transfer parameters and, as the result, the response time of these devices. In this context the development of the novel techniques applicable for the synthesis of PSMNPs is of particular importance. One of such techniques is the intermatrix synthesis (IMS). The IMS allows to synthesize MNPs in stabilizing matrix of functional polymer (e.g., sulfonated polyetherether ketone, SPEEK), which acts both as a sort of a nanoreactor and as the stabilizing medium for MNPs [1-4]. In this presentation we report the IMS as a way to modify Grafite Epoxy Electrodes (GECE) [5]. Various methodologies have been studied for modifying these sensors when using both ex situ (synthesis of MNPs in a SPEEK-membrane followed by its dissolution and deposition on the surface of GECE) or in situ (by carrying out the IMS directly on the GECE surface inside pre-deposited SPEEK-membrane) methods. It has been show that the different ways of the synthesis of PSMNPs result in the different distribution of MNPs inside the membrane located on the surface of the electrode. This in turn, has been demonstrated to affect not only the sensitivity but also the response time of the sensor along with some other parameters of the system, such as the MNPs composition and their size.
[1] D.N. Muraviev, Contrib. Sci, 2005, 3(1), 19. [2] J. Macanas, J. Parrondo, M. Muoz, S. Alegret, F. Mijangos, D.N. Muraviev, Phys. Stat. Sol. (a), 2007, 204, 1699-1705. [3] D.N. Muraviev, J. Macans, P. Ruiz, M. Muoz, Phys. Stat. Sol (a), 2008, 205(6), 1460-1464. [4] D.N.Muraviev, P. Ruiz, M. Muoz, J. Macans, Pure Appl. Chem., 80(11), 2425-2437 (2008). [5] F.Cespedes, E. Martinez-Fabregas, S. Alegret, Trends Anal. Chem. 15, 296 (1996).

Selection of aptamer against Prostate Specific Antigen for biosensor application. Markta Svobodov1, Ioanis Katakis1 and Ciara K. OSullivan1,2
1

Nanobiotechology and Bioanalysis Group, Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Pasos Catalans 26, Tarragona 43007, Spain. 2 Instituci Catalana de Recerca i Estudis Avanats, Passeig Llus Companys 23, 08010 Barcelona, Spain marketa.svobodova@urv.cat

Aptamers are artificial nucleic acid ligands isolated from complex libraries by an interative process of adsorption, recovery and reamplification, in a process coined SELEX that permits the identification of unique DNA molecules from very large populations of random sequences. Aptamers are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors. Prostate specific antigen (PSA) is a protein produced by the cells of the prostate gland and is commonly used as a marker for diagnosis and monitoring of prostate cancer. DNA sequences binding to PSA have been selected in vitro by selection and amplification process. These aptamer candidates were selected for their ability to bind to PSA using magnetic beads methodology from a 91-mer library of initially about 1x1016 different ssDNA molecules. The SELEX process was carried out until the tenth round and after this round, the selected oligonucleotides were cloned and sequenced. Sequence analysis and alignments were performed obtaining preliminary results. Selected sequenced aptamer clones were tested for their individual binding characteristic to PSA.

Aliquoting Structures based on capillary force valves for the construction of centrifugal microfluidic platforms
O. Ymbern1, C.S. Martnez-Cisneros1, E.Soria2, V. Catalan2, J. Alonso1.
1

Grup de Sensors i Biosensors. Universitat Autnoma de Barcelona. Spain. 2 LABAQUA, S.A. (Alicante) Contact address: oriol.ymbern@uab.es

It is well known the growing interest and high demand for the construction of devices featuring the integration of the total sequence of lab processes to perform chemical analysis. The benefits Lab-on-a-chip or TAS (micro total analysis systems) include portability, reduction of sample and reagents consumption, as well as the integration on a single chip of different stages of the analytical process reducing the user-interference and human error in experimental operation. In this way, centrifugal microfluidic platforms are of particular interest. The use of centrifugal force is a simple and inexpensive way to handle liquids in microfluidic systems due to there is no need the use of pumps or actuators for driving the liquids. Moreover, centrifugal propulsion allows the integration of valving and separating steps, and also enables a high throughput of many tests by highly parallel and automated liquid handling. This work is focused on the construction of a disposable centrifugal microfluidic platform to be used for analytical applications where the fluidic operations such as valving, sample metering, and aliquoting steps are implemented in. Concretely this work is centred in the study of different microfluidic structures in order to handle defined and reproducible sample volumes. The precision of the metered volumes into the microfluidic channels and reservoirs is defined by a system of capillary force valves. The liquid handling is based on the combination of centrifugal and capillary forces, prevailed by radial position, channel geometry and dimension, hydrophobicity and surface characteristics of the substrate material. These structures are proposed as an aliquoting system implemented in a disposable platform for a multiple and independent assays into the same device.

Figure 1. A) CAD design of different microfluidic systems. B) Rotating disk. C) Defined volumes during centrifugation process.

Different polymer materials such as polycarbonate (PC) and cycloolefin copolymer (COC) were applied on the construction of these devices, and different microfluidic structures were fabricated and evaluated. Micromilling technique was applied to construct microfluidic devices and different sealing techniques were applied based on polymer substrate material.

Last Name Alarcon Pinto De la Escosura Alonso Aragay Arasa Puig Armengol Torrella Baeza Barthelmebs Bazin Boujday Cadevall Camps Christian Christophe Civit Pitarch COMPERE Comtat Cortina-Puig Couceiro Durrieu Fajerwerg Figueras Garibo Gen Gimnez Gmez Gros Guix Joda Kara Laube Le Bris Lermo Libana Girona Maltez Marin Martinez Marty Medina Moscetta Nadal Nasef Oliv Monllau Parolo Perez Pividori Gurgo Prieto-Simn Puig Puyol Ruffien Ruiz Sanfilippo Sesal

First Name Georgina Alessandro Alfredo Julian Gemma Eva Pau Mireia Lise ingrid Souhir Miquel Mnica Kellner Cline Laia Chantal Maurice Montserrat Pedro Claude Katia Cristina Diana Rukan Gemma Sara Pierre Maria Hamdi Pinar Tamara Nadine Anabel Susana Marisa Sergio Cynthia Jean Louis Mariana Pompeo Pedro Hany Rosa Claudio Sandra Isabel Beatriz Anna Mar Audrey Patricia Luca N. Cenk

Email galarcon@cin2.es alessandro.pinto@estudiants.urv.cat alfredo.escosura.icn@uab.es cynthia770@gmail.com sandra.domene.icn@uab.es eva.arasa@uab.es pauete@hotmail.com mariadelmar.baeza@uab.cat barthelm@univ-perp.fr ingrid.bazin@ema.fr souhir.boujday@upmc.fr kd_miquel@yahoo.es monica.campas@irta.cat christian_kellner84@web.de celine_chris@hotmail.com laia.civit@urv.cat chantal.compere@ifremer.fr comtat@chimie.ups-tlse.fr mcortina12@gmail.com oriol.ymbern@gmail.com claude.durrieu@entpe.fr katia.fajerwerg@lcc-toulouse.fr cristina.figuerasp@campus.uab.cat diana.garibo@irta.cat rukan.genc@urv.cat gemma.gimenez@irta.cat sara.gomez@uab.cat gros@chimie.ups-tlse.fr mguix@cin2.es hamdi.joda@urv.cat pinar.kara@ege.edu.tr tamara.laube@uab.cat lebris@obs-banyuls.fr anaisabel.lermo@uab.es susana.liebana@uab.cat marisa.maltez.icn@uab.es sergio.marin@uab.es cynthia.martinez@uab.es jlmarty@univ-perp.fr marianamedinas@yahoo.es pompeo.moscetta@systea.it pedro.nadal@urv.cat hinassif@yahoo.co.uk rosa.olive@uab.cat claudio.parolo@gmail.com Sandra.Perez@uab.cat Isabel.Pividori@uab.cat uakala@hotmail.com anna.puig.icn@uab.es mariadelmar.puyol@uab.es audrey.ruffien@legos.obs-mip.fr Patricia.Ruiz.Nicolas@uab.cat luca.sanfilippo@systea.it csesal@marmara.edu.tr

Institute Institut Catal de Nanotecnologia Universitat Rovira i Virgili Institut Catal de Nanotecnologia Universidad Autonoma de Barcelona Institut Catal de Nanotecnologia Universitat Autnoma de Barcelona Universitat Autnoma de Barcelona Universitat Autnoma de Barcelona Images Ecole des mines d'ales Laboratoire de Ractivit de Surface Institut Catal de Nanotecnologia IRTA Universitat Rovira i Virgili LAAS CNRS Universitat Rovira i Virgili IFREMER LGC UMR 5503 Universit de Perpignan Universitat Autnoma de Barcelona Universit de Lyon ENTPE LCC UPR 8241-UPS Universitat Autnoma de Barcelona IRTA Universitat Rovira i Virgili IRTA Grup de Sensors i Biosensors LGC UMR CNRS/INP/UPS 5503 Institut Catal de Nanotecnologia Universitat Rovira i Virgili Institut Catal de Nanotecnologia Universitat Autnoma de Barcelona UPMC Universitat Autnoma de Barcelona Universitat Autnoma de Barcelona Institut Catal de Nanotecnologia Institut Catal de Nanotecnologia Universidad Autonoma de Barcelona UPVD Institut Catal de Nanotecnologia SYSTEA SpA Universitat Rovira i Virgili Universitat Rovira i Virgili Universitat Autnoma de Barcelona Institut Catal de Nanotecnologia Universitat Autonoma de Bracelona Universitat Autnoma de Barcelona Grup de Nanobioenginyeria, IBC Institut Catal de Nanotecnologia Universitat Autnoma de Barcelona LEGOS - UMR CNRS 5566 Universidad Autnoma de Barcelona SYSTEA SpA Marmara University

City Bellaterra (Spain) Tarragona (Spain) Bellaterra (Spain) Barcelona (Spain) Bellaterra (Spain) Bellaterra (Spain) Bellaterra (Spain) Bellaterra (Espanya) Perpignan cedex (France) Ales (France) Paris (France) Bellaterra (Spain) Sant Carles de la Rpita (Spain) Tarragona (Spain) Toulouse (France) Tarragona (Spain) Plouzane (France) Toulouse (France) Perpignan (France) Cerdanyola del Valls (Spain) Vaulx-en-Velin (France) Toulouse (France) Bellaterra (Spain) Sant Carles de la Rpita (Spain) Tarragona (Spain) Sant Carles de la Rpita (Espagne) Bellaterra (Spain) Toulouse (France) Bellaterra (Spain) Tarragona (Spain) Bellaterra (Spain) Bellaterra (Spain) Banyuls (France) Bellaterra (Spain) Cerdanyola del Valles (Spain) Bellaterra (Spain) Bellaterra (Spain) Barcelona (Spain) Perpignan (France) Bellaterra (Spain) Anagni (Italy) Tarragona (Spain) Tarragona (Spain) Bellaterra (Spain) Bellaterra (Spain) Barcelona (Spain) Bellaterra (Spain) Barcelona (Spain) Bellaterra (Spain) Bellaterra (Spain) Toulouse (France) Barcelona (Spain) Anagni (Italy) Istanbul (Turkey)

Svobodova Vuillemin Wallner Ymbern

Marketa Renaud Daniela Oriol

marketa.svobodova@urv.cat renaud.vuillemin@obs-banyuls.fr daniela.wallner@estudiants.urv.cat oriol.ymbern@uab.es

Universitat Rovira i Virgili UPMC Universitat Rovira i Virgili Grup de Sensors i Biosensors - UAB

Tarragona (Spain) Banyuls (France) Tarragona (Spain) Cerdanyola del Valls (Spain)