Electrochemical Biosensors

Electrochemical biosensors, particularly enzyme electrodes, have enjoyed considerable popularity, culminating in commercial devices, ranging from self-testing meters for blood glucose to high throughput analyzers for various metabolites [ 51-55] . All molecular-based biosensors rely on highly-specific recognition events to detect target analytes. The essential role of the sensor is to provide a suitable platform that facilitates formation of the probe-target complex in such a way that the binding event triggers a usable signal for electronic read-out. Because electrochemical reactions give an electronic signal directly, there is no need for expensive signal transduction equipment. A system displaying high sensitivity, good selectivity, and long-term stability would require the optimization of the proper immobilization of the biomol-ecule while ensuring no or little loss in its biological activity, sensitivity, and stability. However, the overall performance and the development of these biosensors will depend not only on the biocompatibility of the material and the immobilization method, but also on the physicochemical characteristics of the material, and on the existence of redox mediators and stabilizers.

Nanocomposites and nanoparticles are extremely suitable for designing new and improved electrochemical biosensing devices. To this end, a wide variety of compositions and configurations have been used for different electrochemical sensing systems such as enzyme biosensors, inmunosensors and DNA sensors. The essential functions provided by these nanocomposites and nanoparticles include the immobilization of the biomolecules, the catalysis of electrochemical reactions, the enhancement of the electron transfer between the electrode surfaces and proteins, and the labeling of biomolecules for detecting biorecognition events [56]. Generally, metal nanoparticles and carbon nanotubes (CNT) have excellent conductivity and catalytic properties, which make them suitable for acting as "electronic wires" for enhanced protein-electrode electron transfer and as catalyst to increase electrochemical reactions. Oxide nanocomposites and CNT pastes are often used to immobilize biomolecules due to their biocompatibility, while semiconductor nanoparticles are mainly used as labels or tracers for electrochemical analysis. It is the aim of this section to summarize the recent advances in nanocomposite-based electrochemical biosensors and the role that the different materials play in the sensing systems

15.2.2.1 Metal and Semiconductor Nanoparticle-based Biosensors

The unique electronic and catalytic properties of metal and semiconductor nanopar-ticles render them ideal labels for biorecognition and biosensing processes. Thus, nanoparticles-biomolecule hybrids have been used to enhance electron transfer on biosensors, for biofuel-cell elements (i.e., a "wiring" of the enzyme redox centers to the macroscopic electrode) and for the detection of a biorecognition event on DNA and antibody-antigen analyses [54, 56- 58] .

Metal and Semiconductor Nanoparticle-enzyme Hybrids Systems

The electrical contacting of redox enzymes with electrodes is a key process in the design of enzyme electrodes for bioelectronic applications such as biosensors or biofuel-cell elements. Although redox enzymes usually lack direct electrical communication with electrodes due to thick insulation protein shells surrounding the enzyme-active centers, electrical contacting of the proteins and electrodes have been reported by the application of diffusional electron mediators, the immobilization of the enzymes in redox-active polymers or the reconstitution of apo-enzymes on relay cofactor monolayers associated with electrodes [54, 59-62]. Biocatalytic electrodes for biosensor applications have been prepared by the co-deposition of redox proteins and metal nanoparticles on electrode supports. A well-known example in which the metal nonoparticles act as a nanoelectrode to electronically link the enzyme redox site to the macroscopic electrode is shown in Fig. 15.6 [63]. The gold nanoparticles (1.4 nm) were functionalized with N-(2-aminoethyl)-flavin adenine dinucleotide (FAD cofactor amine derivative 1), and then reconstituted with apo-glucose oxidase and assembled on a thiolate monolayer associated with the gold electrode. Alternatively, the functionalized gold nanoparticles could be first assembled onto the electrode through a thiolate monolayer and reconstituting apo-glucose oxidase subsequently. The resulting nanoparticle-enzyme electrode exhibited unprecedented efficient electrical communication with the electrode (electron h/ -'Glucose acid

Fig. 15.6 Assembly of Au-NP-reconstituted GOx electrode by (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalised with FAD on the dithiol-modifed Au electrode followed by the reconstitution of apo-GOx on the functional NPs. (Reprinted with permission from [63]; copyright Science Magazine)

Fig. 15.6 Assembly of Au-NP-reconstituted GOx electrode by (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalised with FAD on the dithiol-modifed Au electrode followed by the reconstitution of apo-GOx on the functional NPs. (Reprinted with permission from [63]; copyright Science Magazine)

┬┐ecc transfer turnover rate ~5000 s-1); i.e., seven times faster than that between glucose oxidase and its natural substrate oxygen. Moreover, the enzyme electrode could be used for glucose detection without interference as the effective electrical contacting made it insensitive to oxygen or other common interferants, such as ascorbic acid.

Silver nanoparticles have also been employed to enhance the electron transfer between the electrode and different proteins such as, Cytochrome C, hemoglobin and myoglobin [64, 65]. Recently, the application of semiconductor nanoparticles for the enhancement of electron transfer between redox proteins and electrodes has been reported. Hemoglobin and CdS nanoparticles were mixed and immobilized onto pyrolytic graphite electrodes exhibiting direct electrochemistry [66]. Moreover, the use of enzyme nanoparticles hybrid systems in which the product generated by the catalytic process activates the photoelectrochemical functions of the nanoparti-cles has been demonstrated using acetylcholine esterase (AChE)-CdS nanoparticles hybrid monolayer on an Au electrode [67].

Metal and Semiconductor Hybrid Nanoparticles as Sensors of Biorecognition Events

Several electrochemical protocols currently offer great promise for ultrasensitive nanoparticle-based transduction of biological interactions (antigen-antibody and base-pairing of DNA). In such protocols the treated (dissolved) metal or semiconductor nanoparticles or polystyrene active beads can act as the reporter units of a recognition event. Stripping voltammetry (i.e., electroanalytical technique for trace metals measurements) has been particularly useful for detecting metal nanoparti-cles tags. Its remarkable sensitivity is attributed to the "built-in" preconcentration (electrodeposition) step during which the target metals are accumulated (plated) onto the working electrode lowering the detection limit by 3-4 orders of magnitude compared to pulse voltammetric techniques employed earlier to monitor DNA hybridisation [58]. The specific capturing of biomolecule-nanoparticles on the respective sensing interfaces allows the secondary dissolution of the captured nano-particles and thus enables the amplified detection of the respective analyte by the release of many ions/molecules as a result of a single recognition event. Demonstrations of the use of gold nanoparticles tracers for the stripping-based electrochemical detection of antibody-antigen interactions and DNA hybridization were reported recently [68-70]. Such protocols relied on capturing the metal nanoparti-cles at the hybridized target or captured antibody followed by their dissolution and then stripping voltammetric measurements of the trace a metal (Fig. 15.7).

Since the sensitivity of the bioassay depends on the size of the metallic tag, a dramatic amplification of a single hybridization event using single reporter unit, such as metal or semiconductor nanoparticles, is expected with larger nanomaterial tracers. The catalytic enlargement of the gold tracer in connection to nanoparticle-promoted precipitation of gold or hydroquinone-induced silver, has promoted a second generation of amplified bioassays with subpicomolar detection limits [71, 72] , Solid-state measurements of the metal nanoparticles tag have also been carried out through a magnetic collection of DNA-linked particle assembly onto an electrode transducer [73]. Such bioassays involved the hybridization of a target nucleotide to probe coated magnetic beads, followed by binding of the streptavidin-coated gold nanoparticles to the captured target, catalytic silver precipitation on the gold particle tags, magnetic collection of the DNA-linked particle assembly and constant current chronoamperometric detection (Fig. 15.8).

Fig. 15.7 Schematic of a noncompetitive heterogeneous electrochemical immunoassay based on a gold nanoparticle label. (Reprinted with permission from [68]; copyright ACS publications)

Fig. 15.8 Solid state magnetically induced electronic measurements of the metal nanoparticles tag. The catalytic growth of the gold tracer led to a second-generation bioassay. The external magnetic field is positioned under the electrode to attract the DNA-particle assembly to the surface and allows solid state detection. TEM micrograph of the DNA-particle assembly produced following a 20 min hybridization with the 50 mg/mL-1 target sample and 10 min silver precipitation. (Reprinted with permission from [73] ; copyright ACS publications)

Fig. 15.8 Solid state magnetically induced electronic measurements of the metal nanoparticles tag. The catalytic growth of the gold tracer led to a second-generation bioassay. The external magnetic field is positioned under the electrode to attract the DNA-particle assembly to the surface and allows solid state detection. TEM micrograph of the DNA-particle assembly produced following a 20 min hybridization with the 50 mg/mL-1 target sample and 10 min silver precipitation. (Reprinted with permission from [73] ; copyright ACS publications)

A triple amplification bioassay has been developed by coupling a polymeric carrier sphere amplifying unit (loaded with numerous gold nanoparticles tags) with the electrochemical stripping detection and the catalytic enlargement of the multiple gold nanoparticle tags [ 74] . The gold-tagged beads were prepared by binding biotinylated Au nanoparticles to streptavidin-coated polystyrene spheres (Fig. 15.9a[ . These beads were functionalized with a single-stranded oligonucleotide, which was further hybridized with a complementary oligonucleotide that was linked to a magnetic particle. The numerous Au nanoparticles labels associated with one oligonucleotide pair were enlarged by the electroless deposition of gold and transported to the electrode array with the use of the magnetic particle. Then the Au assembly was dissolved upon treatment with HBr/Br2 and electrochemically analyzed by using the electrochemical deposition/stripping procedure.

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