Fluorescent biosensors of protein function.

VanEngelenburg SB, Palmer AE. Source
UCB 215, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.

Abstract
Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.

High-performance electrochemical biosensor for the detection of total cholesterol.
Ahmadalinezhad A, Chen A. Source
Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B5E1, Canada.

Abstract
We report on a highly sensitive electrochemical biosensor for the determination of total cholesterol. The novel biosensor was fabricated by co-immobilizing three enzymes, cholesterol oxidase (ChO(x)), cholesterol esterase (ChE) and horseradish peroxidase (HRP), on nanoporous gold networks directly grown on a titanium substrate (Ti/NPAu/ChO(x)-HRP-ChE). The morphology and composition of the fabricated nanoporous gold were characterized by scanning electron microscopy (SEM), energy-dispersive Xray spectroscopy (EDS) and X-ray diffraction spectroscopy (XRD). The electrochemical behaviour of the Ti/NPAu/ChO(x)-HRP-ChE biosensor was studied using cyclic voltammetry (CV), showing that the developed biosensorpossessed high selectivity and high sensitivity (29.33 μA mM ⁻¹ cm⁻²). The apparent Michaelis-Menten constant, K(M)(app) of this biosensor was very low (0.64 mM), originating from the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range up to 300 mg dL⁻¹ in a physiological condition (pH 7.4), which makes it very promising for the clinical determination of cholesterol. The fabricated biosensor was further tested using real food samples margarine, butter and fish oil, showing that the biosensor has the potential to be used as a facile cholesteroldetection tool in food and supplement quality control.

Recent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules

Eiji Nakata, FongFong Liew, Shun Nakano and Takashi Morii Institute of Advanced Energy, Kyoto University Kyoto, Japan 1. Introduction The creation of novel molecular tools for detection and monitoring of the transitional concentration and localization changes of biologically important molecules, such as biomacromolecules, signaling small molecules and biologically important ions, is a great challenge in the field of chemical biology. Therefore, much attention has been devoted by chemists and biologists to develop sensing tools that allow real-time tracking of the molecules of interests in vivo or in vitro. (Thevenot, D. R. et al., 2001; Jelinek, R. et al., 2004; Borisov, S. M. et al., 2008) Among them, the fluorescent biosensor, which is defined as the sensor that converts a molecular recognition event to a measurable fluorescent signal change, has recently emerged as a powerful tool for the following reasons. (Hellinga, H.W. et al., 1998; Johnsson, N. et al., 2007; Johnsson, K., 2009; Wang, H. et al., 2009; Liu, J. et al., 2009) Biomacromolecular receptors, such as nucleic acids (DNA or RNA) or proteins, have superior characteristics as the recognition platform because they play crucial roles in numerous biological processes to mediate and regulate a range of strict recognition and chemical reactions within cells. As for the tools for the transducer, the fluorescence detection has the superior physical properties, such as high sensitivity, excellent spatial resolution, good tissue penetration and low cost for the detection system, in contrast to the other detection method including optical, electrical, electrochemical, thermal, magnetic detections. Thus, transducing the molecular recognition events with the fluorescence signals is very appealing and has been one of the most widely adapted methods. (Giepmans, B.N. et al., 2006; Rao, J. et al., 2007) The rational design strategies of fluorescent biosensors have not been matured as generally considered by the researchers in the biological field. A simple strategy to construct a biosensor with tailored characteristics would be to conjugate a

recognition module with a signal transducer unit, although there is no simple methodology to conjugate the recognition module and the transducer unit to afford a usable fluorescent biosensor. Here we focus to overview the progress in the design strategy of fluorescent biosensors, such as the auto-fluorescent protein-based biosensor, protein-based biosensor covalently modified with synthetic fluorophores and signaling aptamers. www.intechopen.com124 Biosensors – Emerging Materials and Applications 2. Auto-fluorescent proteins (AFPs) based biosensors Auto-fluorescent proteins (AFPs) such as green fluorescent protein (GFP) from jellyfish (Shimomura, O. et al., 1962) are widely used as noninvasive fluorescent markers for gene expression, protein localization, and intracellular protein targeting (Chalfie, M. et al., 1994; Lippincott-Schwartz, J. et al., 2001). The application of AFPs is not limited to the fluorescent markers. Various kinds of AFP-based biosensors have recently been developed by fusion of reporter proteins or mutation of AFPs for imaging and sensing important molecules and key events in living cell. ( Zhang, J. et al. 2002; Zhang, J. et al. 2007; Mank, M. et al., 2008; VanEngelenburg, S. B. et al. 2008; Lawrence, D. S. et al. 2007; Ozawa, T. 2006; Prinz, A. et al. 2008) The advantage of AFP-based biosensor is that it can be endogenously expressed in cells or tissues simply by transfection of the plasmid DNA encoding it. This approach is a noninvasive method and therefore avoids damage to the cell. Because AFPs based biosensor can be produced automatically, the influence of dilution due to vital activity, such as cell growth and division, is minimal. Moreover, it is possible to control the localization of biosensors to the sites of interest within cell by introducing a certain organelle-specific targeting signal. These biosensors have been powerful tool for in vivo applications. 2.1 Single AFP based biosensor In the case of biosensors based on a single AFP, analyte binding events affect directly or indirectly to fluorescent properties or formation, respectively, of the chromophore moiety of

A. L. T. whose fluorescent properties are directly affected by the interaction between a target molecule and a chromophore moiety in AFP. biosensors specific for Mercury (II) ion (Chapleau. 2006. R. E. Llopis. Indeed. The cpAFP is a regenerated AFP variant. R. cpAFP variants that detect Ca2+ (Nakai. in which the original N. G.AFP. R. et al. To exploit such intrinsic properties of AFPs. et al.and C termini are connected with a flexible peptide linker to regenerate novel N and C termini at specific positions. Another design strategy of a single AFP based biosensor relies on circularly permutated AFP (cpAFP). Miesenbock. In general. 2000. pH sensitive AFP variants have been developed. 2001). S. W. et al. a conformational change of the receptor associated with the ligand-binding event results a formation of the AFP chromophore. Souslova. that is. J. et al. D. C. V. 1998. 1999. Matsuyama. 1999) A number of cpAFPs with novel termini retained their fluorescence even when a foreign receptor was inserted at the termini. et al. et al. et al. et al.R. 2008) and Zinc (II) ion (Barondeau. et al. et al. (Baird. et al. G. 2007) and an inositol . the fluorescence of most of AFP variants is affected reversibly by moderate acidification of the chromophore. 2002) have been created. T. 2000) The fluorescence of AFP becomes sensitive to other signals by the introduction of specific mutation in close proximity to the chromophore or within the barrel structure. P. Jayaraman. M. et al. Dooley. 1999. S. S. cGMP (Nausch. 2008). (Wachter. 2004). In this manner. 2001. Wachter. J. M. The design of analyte-sensitive sensors utilizes AFP variants. The former is classified as analyte-sensitive sensors and the latter as conformationsensitive sensors. H2O2 (Belousov. The receptor function of the sensor was directly integrated into the chromophore by alteration of the chemical nature around the chromophore. S. Baird. M. Nagai. et al. 1998. which is classified as a conformation-sensitive sensor. et al. 2007. Zn2+ (Mizuno. et al. G. T. V. et al. et al. 2000) Mutants of YFPs showing pH sensitivity bind to halide ion selectively and the binding of anion leads to fluorescence quenching due to the induced pKa shift. (Kneen. 1998.

Kerppola T. like circular permutation and insertion of recognition domains as described in the previous section.5tetrakisphosphate.3. R. AFP sensors in the absence of targets often reveal unavoidable background fluorescence.www. The original N and C termini of GFP were linked with a short peptide linker (orange).5)P4 by the excitation of each distinct absorption band. K. Interestingly. et al.3.3.J. 2009). Schematic illustration shows a fluorescent biosensor for Ins(1. Therefore. Y.4. 2006 ) Regan and co-workers first demonstrated that a split GFP displayed a quite low . R. However.4.2 Split AFP based biosensor It is considered that the formation of a AFPs chromophore requires a properly folded and an intact structure. Ins(1. 2009) (Figure 1). R. and retained ligand affinity and selectivity of the original PH domain.intechopen. Morii and coworkers developed a cpAFP-based sensor for D-myo-inositol-1.( Shyu. many experimental data indicate that slight structural modifications of AFPs. et al. 2008.4. An excellent strategy to accomplish full suppression of the initial fluorescence utilizes an AFP variant that was split into two non-fluorescent fragments. et al. 2009).5)P4. 1.4.3. et al.comRecent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules 125 phosphate derivative (Sakaguchi. The conformational change of the PH domain induced by the ligand-binding event was transduced to the structural change of the chromophore of cpGFP. and the novel terminal of cpGFP (purple) was fused to the split Btk PH domain (blue). Fig. and then resulted in the ratiometric fluorescence change of cpGFP. 2.5)P4 based on the split Btk PH domain-cpGFP conjugate (Sakaguchi. the conjugate Btk-cpGFP realized a ratiometric fluorescence detection of Ins(1. still give fluorescent AFPs constructs. have been developed by inserting appropriate receptor modules. by utilizing a newly designed split PH domain of Bruton’s tyrosine kinase (Btk) and cpGFP (Sakaguchi.

T. are associated by binding to the analyte. in which split AFP halves reform into a fluorescent structure via noncovalent association. 2005. -D. Hu. 2007. 2. et al. which connect each of the two binding subunits. C. Wilson. I. a receptor composed of two subunits that are associated by binding to the analyte can be converted into a fluorescent biosensor by connecting each of the two subunits with each split AFP fragment (Figure 2). I. 2007) and protein interactions (Nyfeler. Valencia-Burton.3 FRET based biosensor Non-radiative transfer of energy from an excited donor fluorophore to an acceptor chromophore is known as fluorescence resonance energy transfer (FRET). Unlike the above-mentioned split AFP reconstitution. T. 2006). As a result. 2006). T. et al. mRNA(Ozawa. G. DNA methylation(Stains. et al. C. Demidov. C. 2011). V. 2006. 2000). Schematic illustration shows split AFP based fluorescent biosensor. 2. B. et al. has been developed by Ozawa and co-workers (Ozawa. M. another reconstitution strategy. M. A fluorescent protein such as GFP is split into two halves [GFP(N) and GFP(C)]. et al. 2003.com126 Biosensors – Emerging Materials and Applications been developed for fluorescent detection of specific DNA sequences (Stains. In this strategy. et al. Fig. 2000) Based on this strategy.intechopen. More comprehensive information on this reconstitution strategy is available in other excellent reviews (Ozawa. C. Each split intein-EGFP fusion is attached to a protein of interest. In order to . 2005. V. et al. et al. several types of biosensors have www. The split inteins are brought into close proximity to trigger protein splicing when an analyte induces the association between proteins of interest. split inteins were fused to split EGFPs. et al. et al. 2004).background fluorescence in the separated state and a fluorescence emission was significantly recovered by the reassembly of the two fragments when they were placed in close proximity by strongly interacting antiparallel leucine zippers. I.(Ghosh. inteinmediated reconstitution. Actually. the two EGFP fragments are linked with a covalent bond and emit fluorescence. et al. Awais.

W.g. First. N. M. the two fluorophores are attached at two ends of a peptide sequence in the receptor protein or the concatenation of interacting domains. this strategy would be the most straightforward way to integrate the signal transduction function into the receptor of interest. et al. 2007). and the two fluorophores must be close in proximity (< 10 nm) and in a favorable orientation (Sapsford. et al. the following two issues in the sensor design should be considered. . P. 2008). W. 2001. various FRET biosensor for imaging intracellular events such as enzyme activities [e. 2002. kinase (Sato. AFPbased FRET biosensors can be divided into two classes. K. the donor and the acceptor fluorophores should be placed at a rational distance which can drastically change www. In the case of a receptor that displays a large structural change upon binding to the substrate. 1999. M. Based on the mechanism by which FRET efficiency changes. H. protease (Mahajan. Nagai. To obtain the expected energy transfer efficiency for biological applications. 2007) Therefore. respectively (Piston.comRecent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules 127 the efficiency of FRET before and after the sensing event. K.intechopen. 2000).induce FRET. Y. Ai. a FRET based biosensor can sense the analyte in a ratiometric manner by comparing the donor and acceptor emission intensities that are result from the analyte induced distance and/or conformational changes. an intramolecular and an intermolecular FRET systems (Figure 3). The feasibility of this strategy strongly depends on the magnitude of the structural change of the receptor. D. et al. et al. 2006). et al. The efficiency of FRET is sensitive to the distance and the orientation between the donor and acceptor groups. that is.. 2002. In the case of intramolecular FRET biosensors. Luo. Rehm. et al. et al. Q. E.(Ohashi. In the AFP-based FRET strategy. T. suitable FRET pairs in which the donor emission spectrum overlaps the acceptor absorption spectrum should be chosen. et al. Based on this strategy. et al. the excitation spectrum of the acceptor must overlap with the emission spectrum of the donor. Second. CFP and YFP mutants have been favorably utilized as a FRET donor and an acceptor.

g. www.com128 Biosensors – Emerging Materials and Applications obligatory conformational change in the receptor protein severely limits the choice of proteins available for the construction of FRET biosensors by this strategy. et al. V. 2000).Ca2+(Miyawaki. 1997. cGMP (Sato. Recently. 2002). More comprehensive . M. Zaccolo. cAMP (Nikolaev. is frequently necessary to realize the satisfactory response of the FRET change. respectively. the Fig. et al. Most importantly. et al. to which AFPs of FRET donor and acceptor are attached. (a) Intramolecular FRET-based biosensor: The protein domains with a large structural change upon the analyte binding event. they cause difficulty in analysis of the FRET efficiency changes. 2005)] have been developed. Although this strategy shows a potential to effect a dynamic FRET change by the analyte-induced association and/or dissociation of protein subunits. This biosensor can detect the rise of intracellular cAMP concentration by the decrease in the FRET efficiency induced by dissociation of the catalytic subunit from the regulatory subunit. M.V. A. Johnsson and co-workers have demonstrated a new type of FRET biosensor based on their SNAP-tag technique. M. et al. et al. the stoichiometry of the FRET donor and acceptor may vary between either cells or intracellular compartments. et al. R. 2008)] and dynamics of intracellular second messengers [e. 3. Zaccoro and co-workers constructed FRET biosensor for cAMP by applying this strategy to the regulatory and catalytic subunit of protein kinase A (PKA) (Zaccolo. 1997). M et al. M. Romoser. for which conformational changes upon analyte binding were not required (Brun. H. In these cases. A.phosphatase (Newman. such as tuning the position of AFPs relative to the sensing domain by changing the linker between each of protein units.intechopen. et al. et al. It should be noted that careful optimization. IP3 (Sato. AFP-fused FRET based biosensors. 2009). (b) Intermolecular FRET-based biosensor: The change of FRET efficiency is induced by the dissociation or association of the subunit upon the analyte-binding event. A. Intermolecular FRET biosensors have been developed by employing two protein domains separated from each other. 2000. 2004).

2001. 3. A. unlike AFPs based biosensor. et al. such as artificial receptors. O. Second. 2008. Zheng. such as electroporation (Marrero. Protein-based biosensor covalently modified with fluorescent artificial molecules Another useful strategy to construct fluorescent biosensors is a structure-based design of a protein covalently modified with a fluorescent dye. VanDngelenburg. et al. M. et al 2004). Carlson. 2010). could be introduced to the receptor protein. this type of protein-based biosensor generally require the invasive technique for translocating across the plasma membrane. microinjection (Abarzua.. 1998. et al. J. et al. B. et al. a superior characteristic of dye. the incorporation of small dye to proteins is also possible in the middle of loop regions or at close proximity to the binding pocket. 2007. the fluorescence changes in intensity and wavelength and the microenvironmental sensitivity such as pH. et al. Third. Fenton. et al.1 Introduction of a thiol reactive fluorophore on a unique cysteine residue of . K. Here.B. 1995. can be incorporated. et al. P. 2009). First. Sakaguchi. that is. In addition. On the other hands. and tagging cell-permeable peptide sequences (Wadia. Not only simple dyes but also functional molecules. S. 2003). E. et al. M. 1995). et al 2005. the central issue for the construction of these types of biosensors is the way to introduce a dye into the receptor protein site-selectively. Advantages for the use of fluorescent dyes are as follows. J. R. X.S. polarity or molecular recognition.and C termini of receptor proteins.information on dual FRET-based biosensors is available in other excellent reviews (Souslova. H. the relatively smaller size of the synthetic fluorophore is likely to less perturb the property of the original receptor protein. lipofection (Zelphati. 3. a variety of fluorescent biosensors that use fluorescent molecules is described according to a classification of the incorporation methodologies of fluorescent dye. Sugimoto. the attachment of dye to the protein framework is more flexible than the use of AFPs. While the attaching positions of AFP are generally limited to the N.

1994. et al. A. a representative protein scaffold. et al.comRecent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules 129 the nonspecific labeling of cysteine reactive fluorophores.intechopen. which can permit dynamic conformational changes induced upon ligand binding. but they are capable of recognizing the various . an environmentally sensitive fluorophore is positioned in the binding pocket so that the ligand-induced changes in the fluorescence are produced by the direct fluorophore-ligand interactions.engineered receptor protein The most important process to success this methodology is that all of the original cysteine residue of the receptor protein must be initially substituted with other amino acids to avoid www. Most of bPBPs consist of two domains connected by a hinge region. were converted to fluorophore-modified biosensors by Hellinga et al. (Dwyer. 2004) or others (Gilardi. M. In the first approach. Following the process. On the other hand. et al. a unique cysteine residue was introduced at specific position. there are number of proteins that do not undergo such a dynamic conformational change upon ligand binding. Hirshberg. This approach often has a disadvantage that unfavorable steric interactions between the introduced fluorophore and the ligand lower the binding affinity. 1998. G. As a pioneering work. M. Brune. This allosteric sensing mechanism shows an advantage that the ligand binding is essentially unaffected by introducing a fluorophore. M. et al. The position to introduce a fluorophore is most conveniently determined by the three-dimensional structure of the receptor protein. bPBPs (bacteria periplasmic binding protein). The second approach introduces environmentally sensitive fluorophore at the region that is distant from the ligand-binding site but exhibits dynamic domain movement in response to the ligand binding. with a ligand binding site located at the interface between the two domains. 1998). two distinct approaches are used to establish an efficient signal transduction mechanism that would sense the ligand-binding event. Therefore.

. 2004. et al.com130 Biosensors – Emerging Materials and Applications . 2003). 2002) and the general receptor for phosphoinositides 1 (GRP1) (Sakaguchi. 2010) (Figure 4).4. Sugimoto.4. A sitespecific mutagenesis with an expanded genetic code that employed an amber suppression method (Wang. R. The process might cause the instability of the receptor protein mutant. J.2 Site-specific unnatural amino acid mutagenesis with an expanded genetic code As mentioned above. et al.intechopen. et al.5trisphosphate [Ins(1.3. Xie. But several successful examples demonstrated that such a methodology is applicable for obtaining biosensors (Chan.4. et al. R.substances of biological importance. T. H. the post-labeling of unique cysteine residues required preliminary preparation that all of the original cysteine residue of the receptor protein must be substituted with other amino acids. respectively. A mutagenesis technique for direct incorporation of synthetic fluorophores as unnatural amino acids into desired positions in proteins can avoid such a problem. H. Morii and coworkers constructed novel biosensors for inositol 1. T. 2005. L.3. K. In these biosensors a synthetic fluorophore was attached at the proximity of the ligand-binding site based on the three dimensional structures of proteins so that the changes in orientation of the fluorophore induced by the substrate binding lead to a sufficient fluorescence response. et al. Chan. Nalbant. M. 2004. 2010.5)P4] by utilizing the pleckstrin homology (PH) domain of phospholipase C (PLC) . et al. et al. The useful methodology to convert such non-allosteric proteins to fluorescent biosensors is to introduce an environmentally sensitive fluorophore within the proximity of the ligand-binding site.5)P3] and 1. P. though this strategy might have some drawbacks as mentioned above. 2004. et al. P. et al. 3. 2008). P. 2006) or a four-base codon method (Hohsaka. et al.4. This structure-based design of synthetic fluorophore-modified biosensors is a powerful method to produce biosensors with high selectivity and appropriate affinity to target inositol derivatives in living cells (Sakaguchi. 2002) in cell-free translation systems has provided a variety of fluorescently modified www.5-tetrakisphosphate [Ins(1. Nishida.

though the examples of the construction of fluorescent biosensor based on these methods are still limited. 2002. A schematic illustration shows a fluorescent biosensor for Ins(1.3. R.3. The chemically engineered antibody. of which the proximal antigen-recognition site was modified by fluorescent molecule. J. 3.4. R. the original cysteine residues (cyan) of GRP PH domain were replaced with other amino acids. In that case. et al. can detect antigen binding by fluorescence decrease.Fig. In addition. 2010). This is a powerful tool for site-specific introduction of unnatural amino acids into protein. As an excellent example. an approach to site-specifically incorporate a signal transducer proximal to the binding pocket of intact receptor protein by using selective chemical modifications is valid. The local environmental change of the fluorophore induced by the ligand-binding event was transduced to the fluorescence enhancement. a unique cysteine residue (magenta) was introduced to the resultant mutant followed by labeling with thiol reactive fluorescein (green) as an environment sensitive fluorophore to give Ins(1. Kajihara D. et al. proteins (Anderson. Taki.4. two different position of which were replaced with unnatural amino acids bearing a FRET pair of BODIPY derivatives by using two sets of four-base codons.3 Covalent introduction of fluorescent molecules by chemical modification Modification of a protein by using genetic method often causes the lower activity or instability of the mutated protein as mentioned in the previous section. 1988). Second. Firstly. M. et al.5)P4 sensor. S. et al. Schultz and co-workers constructed an antibody-based fluorescent biosensor by using an affinity-labeling method (Pollack. et al. Hamachi and coworkers constructed a lectin-based fluorescent biosensor using an improved photo affinity . 4. the method is not appropriate when the three dimensional structure of a receptor protein is not known.5)P4 based on the GRP1 PH domain (Sakaguchi. Hohsaka and co-workers prepared a series of semisynthetic calmodulins. 2002. D. Some of the doubly modified calmodulin sensed calmodulin-binding peptide by substantial FRET signal changes. 2006). As the primary example.

2001. Y. an extensively www. which is composed of a ligand module. E. T. 4. et al. Nakata. et al. 2004) can be introduced to Con A. 2005. Signaling aptamers Protein based biosensors are generally constructed by using native or slightly modified . et al. Nakata. H. P-PALM was demonstrated by using concanavalin A (Con A).labeling method. et al. 2009). 2008) but also fluorescent artificial receptor (Nakata. artificial receptor) proximal to the active site of a target protein without genetically modifying the protein framework. 2003). I. Recently. et al. Nagase. not only environmental sensitive fluorophore (Koshi. 2009). E.intechopen. Because the initial PPALM strategy based on thiol chemistry shows limited bioorthogonality. Hamachi and co-workers also developed ligand-directed tosyl (LDT) chemistry-based approach as a more general and simple strategy of target selective chemical modification (Tsukiji. 2008) as bioorthogonal chemoselective modifications. et al. Hamachi group adopted the ketone/aldehyde-based hydrazone/oxime exchange reaction (Takaoka. Intact Con A can be converted to a various type of fluorescent biosensors that successfully sense the saccharide derivatives in different manners. Y. In a proof-of-principle experiment. Depending on the nature of the subsequent modification by a thiol reactive artificial molecule. S. E. Nagase. H. et al. 2006) and the organometallic Suzuki reaction (Wakabayashi. T. a photo reactive module and a cleavable disulfide module.comRecent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules 131 studied lectin (saccharide-binding protein). To overcome this drawback. E.g. termed as P-PALM (post-photoaffinity labeling modification) (Hamachi. et al. this method is not applicable to many proteins. et al. A detailed description of their strategies is described in other review articles (Nakata. Introduction of a thiol group as a chemoselective modification site in proximity of the ligand-binding pocket of Con A is conducted by a designed photoaffinity labeling molecule. Wang. fluorophore. This methodology can introduce artificial molecules (e. 2005. et al. 2000. 2007.

et al.1 Modular strategies for tailoring aptamer sensors Sophisticated design strategies have successfully provided fluorescent biosensors based on biomolecules such as DNA. D. Gold. A. Here we focus on unique modular strategies to construct aptamer sensors. aptamers that bind to the substrate of interest with tailor made functions. such as the specificity and the affinity toward the substrate. Therefore. but these strategies usually require the redundant optimization of sensor functions. et al.intechopen. Introduction of the signal transduction module such as a fluorophore at an appropriate site of the aptamer enables a read out of the ligand-binding event as a local environmental change of the fluorophore. J. such as the specificity and the affinity. Previous work indicated that most of the structurally characterized aptamers underwent induced-fit type of conformational change upon ligand binding [Westhof. 1994. E. D. Osborne. Wilson. 1997]. 4. et al. Thus. Some excellent reviews of aptamer sensors have already covered the selection and evolution techniques and sophisticated applications of the aptamer sensors [Liu. 1990. 1995. Unlike receptor proteins. et al. et al. W. Ellington. the function of the constructed biosensor. can potentially be generated through in vitro selection. 1997. introduction of the fluorophore often impairs the original receptor function and does not always ensure the fluorophorelabelled receptor to act as an expected sensor. For example.com132 Biosensors – Emerging Materials and Applications . L. RNA or proteins. S. E. S. 2008]. also known as SELEX (systematic evolution of ligands by exponential enrichment) (Ellington. It is quite difficult to empirically apply the www. depends on that of the native receptor.proteins as the scaffold. Mok. 1999). the design of aptamer-based fluorescent sensors represents an attractive and promising alternative to the protein-based sensors. A. et al 2009. D. et al. DNA or RNA based receptors (aptamers) which have appropriate affinity and specificity for various targets ranging from small molecules to proteins can be generated by using in vitro selection. That is. which would avoid the cumbersome trial-and-error process to construct a sensor with an optimized function.

N. In the second step. On the other hand. M. Later. The target binding aptamers were fused with the reporter dye binding aptamers. showing the generality of the approach. and the reporter dye binding was significantly enhanced upon target binding.obtained findings from the previously constructed biosensor to the other one. Furutani. 2005. W. K. et al. several groups reported various allosteric aptamer sensors based on the methodology (Kolpashchikov. T. which can drastically increase the fluorescent intensity of reporter dye. Xu. 2010). et al. Fluorescent sensors for adenosine triphosphate (ATP). et al. the Rev peptide was modified with a fluorophore as the transducer of binding event without greatly disturbing the affinity and specificity of the RNP receptor. because the communication between the substrate binding and the signal-transduction is not so simple and is unique to the individual biosensor. D. 2010. C. 2002). The constructed fluorescent RNP sensor showed the fluorescent intensity . et al. M. a randomized nucleotide sequence was introduced into the RNA subunit of RNP to construct RNP library. In vitro selection method was applied to the RNP library to afford a series of RNP receptors for a given target (Morii. 2004). In the first step to construct the fluorescent RNP sensor. et al. Morii and co-workers have recently developed a conceptually new strategy for preparation of fluorescent biosensors with diverse functions based on a framework of ribonucleopeptide (RNP). Stojanovic and co-workers have proposed a modular design of signaling aptamers based on the allosteric regulation of binding events (Stojanovic. a modular strategy that permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer has recently emerged as a new paradigm for a versatile design of fluorescent biosensors. flavin mononucleotide (FMN) and theophylline have been demonstrated based on this design. The application of the selection and evolution technique is not limited to obtain functional macromolecules solely composed of RNA or DNA. 2010). such as the structurally well characterized complex of the Rev Responsive Element (RRE)-HIV Rev peptide (Rev peptide) and RRE RNA (Figure 5) (Tainaka.

phosphotyrosine (Hasegawa. RNP sensors with desired affinity. www. et al. In similar to RNA aptamers. By taking the advantage of such the noncovalent nature of the RNP complex. 5. such as various fluorophore modified Rev peptide libraries with a variety of excitation and emission wavelengths. the RNP receptors. M. GTP (Hagihara. et al. Actually. are considered as a RNP receptor library. showing the generality of the approach. because a variety of RNA structures and reveal different affinity to the target molecule were included. The combined peptide subunit is also easily converted to functionalized Rev peptide libraries. 2006). the group showed that ATP-binding RNP sensor was rationally converted to GTP-binding RNP sensor to have realized the detail of the recognition mechanism (Nakano. 2011). T. et al. Recently. T. histamine (Fukuda. A covalently linking of RNA and peptide subunits without sacrificing the sensing function would overcome such disadvantages. which obtained by in vitro selection. a variety of fluorescent biosensors for targeting ATP (Hagihara.comRecent Progress in the Construction Methodology of Fluorescent Biosensors Based on Biomolecules 133 Fig.intechopen. for instance. 2006). 2005). 2009). et al. M. the RNP complex would dissociate to each component under reducing condition such as the nanomolar range. Though the noncovalent configuration conveniently provides fluorescent RNP sensors in the selection stage. M. 2008) have been produced by the group. selectivity and optical sensing properties could be selected in a high-throughput manner by combining a series of RNA subunits derived from each of the library. Combination between the RNA subunit library and several dye-labeled Rev peptide . M. et al. S. 2006]. et al. Screening methodology of a tailor-made RNP fluorescent sensor [Hagihara. it have a possibility to becomes a disadvantage for the practical measurements after optimization of the sensor function. et al. and phosphotyrosinecontaining peptide fragment (Hasegawa.changing upon binding to the target molecule as the result of the conformational change of RNA subunit by inducing target binding.

Further effort in the fields for establishing a general and simple strategy to construct usable biosensors will realize tailor-made fluorescent biosensors. 5. in the cell has been progressively clarified owing to significant contribution of these new biosensors. P. Such the drawbacks will be overcome by the improved selection and evolution technique to construct the aptamers that resist to the cellular degradation activity. Gubler. Aptamer-based biosensors have potential to realize the tailor-made biosensor with finely tuneable affinity and selectivity based on in vitro selection technique. M. J. 6. that is. & Neri.subunits generates combinatorial fluorescent RNP receptor libraries. A. www.. 3490–3494. such as optical property. and to visualize intracellular molecules. there is no doubt that these sensors represent the most practical and reliable tools for the real-time measurements of various biologically important molecules in living cells. which have both the advantages and drawbacks as mentioned above. protein-based biosensor and aptamer-based biosensor. are selected. Perspective Here we overviewed construction methodologies of fluorescent biosensor based on biomolecules. In the case of the protein based biosensor. Reference Abarzua. However. strongly indicated the lack of general approach to conjugate a recognition module with a signal transducer unit.com134 Biosensors – Emerging Materials and Applications .. affinity and selectivity.intechopen. from which RNP sensors with desired function... L. this type of sensor is practically passive with challenges in cell application owing to the inherent liability of RNA molecules in the intracellular condition. for example. However. the function of second messengers. Microinjection of Monoclonal Antibody PAb421 into Human SW480 Colorectal Carcinoma Cells Restores the Transcription Activation Function to Mutant p53. The systematic developments of these technologies have expanded the applicability of fluorescent biosensors. Cancer Res. (1995). 55(16). LoSardo. Actually. E. the wide varieties of the construction strategies.

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Campbell. 3(12). (2003). Y. M. Ting. Cancer Res. Cell Biol.Emerging Materials and Applications Edited by Prof. A. Shanghai.. 906–918. Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.comBiosensors .intechopen. Zheng. Yan An Road (West)..intechopen. 2011 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka. China Phone: +86-21-62489820 Fax: +86-21-62489821 A biosensor is a detecting device that combines a transducer with a biologically sensitive and selective . R. 2011 Published in print edition July..65.. Office Block. 63(20). 6909–6913. E. Creating new fluorescent probes for cell biology. www. Y. Lundberg. Hotel Equatorial Shanghai No. M. July.Zhang. Rev. X. J. 630 pages Publisher InTech Published online 18. Karlsson. 200040. Mol. & Tsien. Pier Andrea Serra ISBN 978-953-307-328-6 Hard cover. Nat. Johansson.. A. Lipid-Mediated Protein Delivery of Suicide Nucleoside Kinases.. (2002). R..com InTech China Unit 405.

The book consists of 27 chapters written by 106 authors and divided in three sections: Biosensors Technology and Materials. Available from: http://www. How to reference In order to correctly reference this scholarly work. Pier Andrea Serra (Ed. InTech. Recent progress in the construction methodology of fluorescent biosensors based on biomolecules. This book covers a wide range of aspects and issues related to biosensor technology. Biosensors for Health and Biosensors for Environment and Biosecurity. Prof.Emerging Materials and Applications.intechopen.). FongFong Liew. Shun Nakano and Takashi Morii (2011). chemical processes. Biosensors . ISBN: 978-953-307-328-6. Biosensors can measure compounds present in the environment.com/books/biosensors-emerging-materials-and-applications/recentprogress-in-theconstruction-methodology-of-fluorescent-b . feel free to copy and paste the following: Eiji Nakata.component. bringing together researchers from 19 different countries. food and human body at low cost if compared with traditional analytical techniques.