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Metallic nanoparticles (NPs) of unique electronic and catalytic properties, or supramolecular nucleic acid-based nanostructures, provide new materials for electroanalytical applications. Metallic NPs are used for the electrical contacting of redox enzymes with electrodes and as electrocatalysts for the development of amperometric biosensors. DNA is used as a template for electroanalytical applications, such as the electrical contacting of enzymes with electrodes. Finally, an electrochemical method to synthesize molecularly imprinted Au NPs composites on surfaces is described, and the imprinted composites enable the selective and ultrasensitive detection of explosives, or their use as electrochemically triggered sponges for the uptake and release of substrates.

The electrical contacting of redox enzymes with electrodes is the most fundamental requirement for the development of amperometric biosensors and biofuel cell elements. For the effective electrical communication of redox enzymes with electrodes the use of electron relay units that transport the electrons between the enzyme redox center and the conducting surface is essential. Also, the structural alignment of the redox enzyme units in respect to the electrode in a configuration where the enzyme redox center is in close proximity to the conductive surface is needed. The present report summarizes the reconstitution paradigm developed by our laboratory in the last decade as a versatile method to electrically contact redox enzymes with electrodes, and as a generic approach to develop amperometric biosensors and biofuel cell elements. The process is based on the reconstitution of the apo-enzyme on a relay-cofactor monolayer on thin film-functionalized electrode. Different relay units were used to electrically communicate flavin adenine dinucleotide (FAD)-containing enzymes (flavoenzymes) or pyrroloquinoline quinone (PQQ)-containing enzymes with electrodes. This included molecular redox-active relays, molecular redox-active ‘shuttles’, redox-active polymers (e.g., polyaniline), Au nanoparticles, and carbon nanotubes. The reconstitution of different apo-enzymes on these relay-cofactor-functionalized electrodes led to unprecedented efficient electrical contacting between the redox centers of the enzymes and the electrodes. Besides very sensitive amperometric biosensors that emerged from this method, the resulting amperometric biosensors revealed high selectivity and specificity. A related approach to establish electrical contact between redox enzymes dependent on diffusional cofactors and electrodes and to develop an integrated bioelectrocatalytically active enzyme electrode was developed. The method involved assembly of a relay-cofactor diad on the electrode, and the surface crosslinking of an affinity complex generated between the enzyme and the surface-confined cofactor units. This method was successfully applied to electrically contact nicotinamide adenine dinucleotide (phosphate) NAD(P)+-dependent enzymes and cytochrome c-dependent enzymes. For example, enzyme-modified electrodes for the bioelectrocatalyzed oxidation of alcohol, lactate and malate were fabricated by the electrical contacting of the respective NAD(P)+-dependent dehydrogenases. Similarly, the bioelectrocatalytic reduction of O2 was accomplished by an integrated cytochrome c/cytochrome oxidase-functionalized electrode. The electrically contacted enzyme electrodes were also used to develop noncompartmentalized biofuel cell elements. Biofuel cell elements consisting of electrically contacted reconstituted enzyme electrodes were constructed. Glucose or alcohol were used in these systems as fuel substrates and O2 as oxidizer.

Recent advances in nanobiotechnology involve the use of biomolecule-nanoparticle (NP) hybrid systems for bioelectronic applications. This is exemplified by the electrical contacting of redox enzymes by means of Au-NPs. The enzymes, glucose oxidase, GOx, and glucose dehydrogenase, GDH, are electrically contacted with the electrodes by the reconstitution of the corresponding apo-proteins on flavin adenine dinucleotide (FAD) or pyrroloquinoline quinone (PQQ)-functionalized Au-NPs (1.4 nm) associated with electrodes, respectively. Similarly, Au-NPs integrated into polyaniline in a micro-rod configuration associated with electrodes provides a high surface area matrix with superior charge transport properties for the effective electrical contacting of GOx with the electrode. A different application of biomolecule-Au-NP hybrids for bioelectronics involves the use of Au-NPs as carriers for a nucleic acid that is composed of hemin/G-quadruplex DNAzyme units and a detecting segment complementary to the analyte DNA. The functionalized Au-NPs are employed for the amplified DNA detection, and for the analysis of telomerase activity in cancer cells, using chemiluminescence as a readout signal. Biomolecule-semiconductor NP hybrid systems are used for the development of photoelectrochemical sensors and optoelectronic systems. A hybrid system consisting of acetylcholine esterase (AChE)/CdS-NPs is immobilized in a monolayer configuration on an electrode. The photocurrent generated by the system in the presence of thioacetylcholine as substrate provides a means to probe the AChE activity. The blocking of the photocurrent by 1,5-bis(4-allyldimethyl ammonium phenyl)pentane-3-one dibromide as nerve gas analog enables the photoelectrochemical analysis of AChE inhibitors. Also, the association CdS-NP/double-stranded DNA hybrid systems with a Au-electrode, and the intercalation of methylene blue into the double-stranded DNA, generates an organized nanostructure of switchable photoelectrochemical functions. Electrochemical reduction of the intercalator to the leuco form, -0.4 V vs. SCE, results in a cathodic photocurrent as a result of the transfer of photoexcited conduction-band electrons to O(2) and the transport of electrons to the valance-band holes by the reduced intercalator units. The oxidation of the intercalator, E 0 V (vs. SCE), yields in the presence of triethanolamine, TEOA, as sacrificial electron donor, an anodic photocurrent by the transport of conduction-band electrons, through intercalator units, to the electrodes, and filling the valance-band holes with electrons supplied by TEOA. The systems reveal potential-switchable directions of the photocurrents, and reveal logic gate functions.

Redox enzymes in bioelectronic devices usually lack direct electrical contact with electrodes, owing to the spatial separation of their redox centers from the conductive surfaces by the protein shells. The reconstitution of apo-enzymes on cofactor-functionalized nanostructures associated with electrodes provides a means to align the biocatalysts on the conductive surface and to electrically contact redox enzymes with electrodes. The reconstitution of apo-enzymes on cofactor-functionalized gold nanoparticles or carbon nanotubes has led to effective electrical communication between the redox proteins and the electrodes. Alternatively, the reconstitution of redox enzymes on molecular wires that enable electron tunneling or dynamic charge shuttling represent supramolecular biocatalytic nanostructures exhibiting electrical contact. The bioelectrocatalytic activities of the electrically wired reconstituted enzymes on electrodes have allowed the development of amperometric biosensors and biofuel cell elements.

Tailored sensoric, electronic, photoelectrochemical, and bioelectrocatalytic functions can be designed by organized molecular or biomolecular nanoparticle hybrid configurations on surfaces. Layered receptor-cross-linked Au nanoparticle assemblies on electrodes act as specific sensors of tunable sensitivities. Layered DNA-cross-linked CdS nanoparticles on electrode supports reveal organized assemblies of controlled electronic and photoelectrochemical properties. Au nanoparticle-FAD semisynthetic cofactor units are reconstituted into apo-glucose oxidase (GOx) and assembled onto electrodes. The resulting enzymes reveal effective electrical contacting with the electrodes, and exhibit bioelectrocatalytic functions toward the oxidation of glucose to gluconic acid. Magneto-switchable electrocatalysis and bioelectrocatalysis are accomplished by the surface modification of magnetic particles with redox-relay units. By the attraction of the modified magnetic particles to the electrode support, or their retraction from the electrode, by means of an external magnet, the electrochemical functions of the magnetic particle-tethered relays can be switched between "ON" and "OFF" states, respectively. The magneto-switchable redox functionalities of the modified particles activate electrocatalytic transformations, such as a biocatalytic chemoluminescence cascade that leads to magneto-switchable light emission or the activation of bioelectrocatalytic processes.

Tailoring of electrically contacted enzyme electrodes provides the grounds for bioelectronic and biosensor systems. Redox-enzymes organized onto electrodes as monolayer assemblies, and chemically functionalized by redox-relay groups, yield electrically contacted enzyme electrodes exhibiting bioelectrocatalytic features. The sensitivity of the enzyme electrode can be enhanced, or tuned, by the organization of multilayer enzyme electrodes and the application of rough metal supports. Enzyme electrodes of extremely efficient electrical communication with the electrode are generated by the reconstitution of apo-flavoenzymes onto relay-FAD monolayers associated with electrodes. The reconstitution process results in an aligned enzyme on the surface, and its effective electrical contact with the electrode yields selective enzyme electrodes of unprecedented high current responses. Integrated electrodes consisting of relay-NAD(P)+-cofactor and enzyme units are generated by the reconstitution of NAD(P)+-dependent enzymes onto a relay-NAD(P)+ monolayer assembly followed by lateral crosslinking of the enzyme network.

Photoisomerizable nitrospiropyran (SP)/nitromerocyanine (MR) monolayer assembled on Au-electrodes provides active interfaces for controlling, by light electron transfer, reactions at the electrode surface. The functionalized electrodes act as `photo-command' interfaces for the amperometric transduction and amplification of recorded optical signals. The nitrospiropyran monolayer, SP state, undergoes light-induced isomerization to the protonated nitromerocyanine monolayer, MRH + state. The positively charged MRH + -monolayer interface, by means of electrostatic interactions, allows control of electrochemical transformation at the electrode interface. Electrooxidation of 3-hydroxytyramine (dopamine), ( 3 ), is retarded at the MRH + -monolayer electrode as compared to its electrooxidation by the SP-monolayer electrode. In contrast, electrochemical oxidation of 3,4-dihydroxyphenylacetic acid, DHPAA, ( 4 ), is enhanced at the MRH + -monolayer electrode as compared to the SP-electrode state. By cyclic photoisomerization of the monolayer between the MRH + and SP states, the amperometric responses of the electrode are tuned to high and low values in the presence of the two substrates. Another photo-command surface includes a mixed monolayer of pyrroloquinoline quinone, PQQ, and nitrospiropyran units. In the PQQ-SP-monolayer configuration, effective electrocatalyzed oxidation of NAD(P)H proceeds in the presence of Ca 2+ ions. Photoisomerization of the monolayer to the PQQ-MRH + state blocks the electrocatalytic oxidation of NADPH. The system is used for the cyclic amplified amperometric transduction of optical signals recorded by the monolayer. The SP/MRH + -monolayer electrode is also employed to control bioelectrocatalyzed transformations. Electrostatic attraction of ferrocene-modified glucose oxidase, Fc-GOx, by the MRH + -monolayer electrode, facilitates the electrocatalyzed oxidation of glucose, whereas in the presence of the SP-monolayer electrode the bioelectrocatalytic process is inhibited. The enzyme Fc-GOx, enables the cyclic, amplified amperometric transduction of optical signals recorded by the photoactive monolayer. A mixed monolayer consisting of nitrospiropyran and pyridine units assembled on a Au-electrode provides a functionalized interface that controls the binding of cytochrome c (Cyt. c ) to the monolayer and the resulting electrical contact of Cyt. c with the electrode. With the pyridine-SP monolayer configuration, Cyt. c associates to the pyridine sites and reveals effective electrical communication with the electrode surface. In the pyridine-MRH + -monolayer state, Cyt. c is electrostatically repelled from the pyridine sites and its electrical contact with the electrode is blocked. The photostimulated association and dissociation of Cyt. c to and from the photoisomerizable monolayer is microgravimetrically analyzed by a quartz-crystal microbalance.

Optobioelectronic systems provide a means for the electronic transduction of recorded optical signals. The methodologies to assemble optobioelectronic devices are reviewed. One method involves the chemical modification of redox enzymes with photoisomerizable units and their integration with electrode surfaces. In one photoisomer state the protein structure is perturbed and its bioelectrocatalytic activities are blocked. In the second photoisomer state, the tertiary structure of the protein is retained and the enzyme exhibits bioelectrocatalytic functions. This method is exemplified by the chemical modification of glucose oxidase (GOx) with photoisomerizable nitrospiropyran units and with the reconstitution of apo-GOx with the photoisomerizable nitrospiropyran-FAD (flavin adenine dinucleotide phosphate) cofactor. The two systems enable the cyclic amplified amperometric transduction of optical signals recorded by the photoactive proteins. The second approach to assemble optobioelectronic systems involves the organization of photoisomerizable monolayers on electrode surfaces acting as ‘optical command surfaces’ for the control of the electrical contact between redox proteins (or redox enzymes) and the electrode. This method is exemplified by the control of the electrical contact of cytochrome c (Cyt. c) with a functionalized electrode consisting of a mixed monolayer of pyridine/nitrospiropyran assembled onto an Au-electrode. The ON-OFF photostimulated electrical contact of Cyt. c with the electrode is coupled to mediated electron transfer cascades, i.e. bioelectrocatalyzed reduction of oxygen by cytochrome oxidase (COx). The systems allow the amplified amperometric transduction of optical signals recorded by the photoisomerizable monolayer-electrode. The photochemically-triggered activation or deactivation of the electrical communication between Cyt. c and the electrode, originate from the association or repulsion of the protein from the photoisomerizable monolayer-electrode interface. This enables the microgravimetric transduction of the optical signals recorded by the monolayer using a quartz crystal microbalance (QCM). The photostimulated electrical contact of Cyt. c and the electrode, and the subsequent activation of an electron transfer cascade via the electrobiocatalyzed reduction of oxygen by COx, allow the amplified amperometric transduction of optical signals recorded by the monolayer-functionalized-electrode. Reversible immunosensors based on photoisomerizable antigen monolayers assembled onto electrodes represent another configuration of an optobioelectronic device. Assembly of an antigen monolayer on electrodes yields an active interface for the amperometric transduction of the association of the complementary antibody (Ab) to the monolayer. Binding of the Ab to the monolayer blocks the electrical contact of a redox enzyme, i.e. ferrocene-modified glucose oxidase (Fc-GOx), with the electrode, and inhibits the electrobiocatalyzed oxidation of glucose. A photoisomerizable antigen assembled as a monolayer on an electrode allows the tailoring of reversible immunosensing interfaces. In one photoisomer state, the monolayer acts as an active interface for amperometric detection of the antibody. The complementary photoisomer state lacks affinity for the Ab and allows the washing-off of the antibody and the regeneration of the active antigen monolayer by a second illumination cycle. This approach is exemplified by the application of a dinitrospiropyran monolayer assembled onto an Au-electrode as a reversible sensing interface for the dinitrophenyl antibody (DNP-Ab). The dinitrospiropyran monolayer, SP-state, acts as an active interface for the association of the DNP-Ab. Photoisomerization of the monolayer to the dinitromerocyanine configuration, MRH+-state, allows the washing-off of the DNP-Ab and regeneration of the active SP-monolayer electrode by a secondary photoinduced isomerization. The reversible photostimulated binding and dissociation of DNP-Ab to and from the electrode is transduced by the application of Fc-GOx as redox probe and is further supported by microgravimetric QCM analyses.

Electrical communication of the redox-active center of enzymes with an electrode surface is a fundamental element for the development of amperometric biosensor devices. Different methods to assemble enzyme-electrodes exhibiting electrical contact between the redox protein and electrode surface are discussed with specific examples for tailoring glucose sensing electrodes. By one approach, a multilayer array of glucose oxidase is assembled on a Au-electrode. The number of enzyme layers is controlled by the synthetic methodology to assemble the electrode. Electrical contact between the enzyme array and the electrode is established by chemical modification of the protein layer with N -(2-methyl-ferrocene)-caproic acid, acting as an electrode is established by chemical enzyme layers associated with the electrode allows one to control the sensitivity of the resulting enzyme electrode. A further means of enhancing the sensitivities of enzyme electrodes involves the application of rough Au-electrodes as base-support to assemble the enzyme network. The high surface area of these electrodes (roughness factor ≈ 20) allows the increase of the biocatalyst content in a single monolayer, and the resulting amperometric responses of the electrodes are ca. 8-fold enhanced compared to enzyme layers assembled on smooth electrodes of identical geometrical areas. A novel method to electrically wire flavoenzymes with electrode surfaces was developed by reconstitution of the apo-flavoenzyme with a ferrocene-tethered FAD diad. Reconstitution of apo-glucose oxidase with the ferrocene-FAD diad yields an active bioelectrocatalyst of direct electrical communication with the electrode, ‘electroenzyme’. The reconstitution methodology was further applied to tailor enzyme-electrodes of superior properties for electrical contact with the electrodes. A pyrroloquinoline quinone-FAD diad monolayer was assembled on a Au-electrode. Apo-glucose oxidase was reconstituted on the surface with the FAD-cofactor site to yield the aligned biocatalyst on the electrode. The pyrroloquinoline quinone. PQQ, redox unit acts as an electron relay that electrically contacts the FAD redox-site of the enzyme with the electrode. The surface reconstituted enzyme exhibits direct electrical communication with the electrode and acts as bioelectrocatalyst for the oxidation of glucose. The electrical communication of the reconstituted glucose oxidase on the PQQ-FAD monolayer is extremely efficient. The experimental current density at a glucose concentration of 80 mM is 300 ± 100 μ A · cm −2 . This value overlaps the theoretical current density of glucose oxidase electrode (290 ± 60 μ A · cm −2 ) taking into account the limiting turnover-rate of the enzyme, 900 ± 150 s −1 (at 35°C). The extremely efficient electrical contact of the reconstituted enzyme and the electrode yields an enzyme-electrode that is insensitive to oxygen and is not affected by glucose-sensing interferants such as ascorbic acid. The application of the different enzyme-electrode configurations as bioelectronic devices for the determination of glucose is addressed.

Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid. Photoswitchable redox proteins integrated with electrode surfaces act as active systems for the amplified amperometric transduction of recorded optical signals. Chemical modification of glucose oxidase by photoisomerizable nitrospiropyran units or reconstitution of apo-GOx with a photoisomerizable nitrospiropyran-FAD diad, yield photoisomerizable glucose oxidase with photoswitchable biocatalytic features. Assembly of the photoactive enzymes as monolayers on the Au-electrode results in functionalized surfaces for the cyclic ‘ON-OFF’ amplified amperometric transduction of recorded optical signals. A further method to photostimulate the electrical contact between a redox protein and an electrode involves the functionalization of the electrode with a photoisomerizable monolayer interface. A mixed monolayer consisting of pyridine and nitrospiropyran units was used to photoregulate the association and dissociation of cytochrome c to and from the monolayer assembly. The photostimulated electrical contact of cytochrome c with the monolayer electrode was employed to mediate the bioelectrocatalyzed reduction of oxygen in the presence of cytochrome oxidase, COx. The latter system provides an assembly for the cyclic amperometric transduction of recorded optical signals.

Electrical communication between the redox centres of enzymes and electrodes is an essential function for the development of enzyme electrodes for biosensors or biofuel cell elements.1–3 Redox proteins usually lack direct electrical communication between their redox sites and the electrode support. ...

Publisher Summary This chapter reviews different analytical schemes for the amplified electrochemical detection of DNA. It addresses the sensitivities of the different methods and describes the complexity of the different analytical schemes by detailing the amplification steps. An approach for the amplified bioelectrocatalytic detection of DNA, through mediated electrical contacting of a redox enzyme with the electrode, was demonstrated by the incorporation of redox mediator units into the replicated DNA hybrid complex. A different method to employ enzymes for the amplified detection of DNA involves the application of the biocatalyst label that stimulates the precipitation of an insoluble product on the electrode support. The formation of an insulating film represents an amplification path, as numerous insoluble molecules are generated by the enzyme label as a result of a single recognition event. The use of metal or semiconductor NPs for the amplified detection of DNA involves two steps. In the first step, the nucleic acid functionalized nanoparticles (NPs) are used as labels that hybridize with analyte DNA on a surface. In the second step, the NPs are dissolved by electrochemical or chemical means. The use of enzymes, NPs, liposomes, nanorods, or carbon nanotubes represents a few labels that altered the sensitivity regions for analyzing DNA.

Bioelectronics is a rapidly developing interdisciplinary research area aimed to integrate biomaterials with electronic elements in order to transduce electronically biological recognition events or biocatalytic transformations [1]. Mother Nature has evolutionary tailored biomaterials of highly specific and selective recognition properties, transport properties and catalytic properties. The unique recognition properties of antigens- antibodies, nucleic acid-DNA or hormone-receptors, the transport properties of ion- channels or the catalytic function of enzymes, represent some of these biomaterial functionalities. Electronic elements that could be applied as components of bioelectronic systems include electrodes, piezoelectric crystals, field-effect transistors and others. The simplest bioelectronic device is schematically depicted in Figure 1 where a redox-enzyme is immobilized on an electrode. Oxidation (or reduction) of the enzyme redox-center could activate the bioelectrocatalyzed oxidation (or reduction) of the substrate that corresponds to the biocatalyst. As a result, the generated current should relate to the substrate concentration in the medium, and the enzyme-electrode should function as a specific sensor for the quantitative analysis of the substrate. Although this simple scheme outlines the practical potential of such devices, the “real” operation of the system is far more complex. For example, the redox centers of enzymes usually lack electrical contact with the electrode surface, and upon the application of positive or negative potentials on the electrode, the oxidation or reduction of the active center is prohibited. The theoretical explanation for the lack of electrical contact between the redox-site and the electrode rests on the Marcus electron-transfer theory

The application of redox enzymes as bioactive matrices for biosensor design is of substantial basic and practical importance.1-4 Two basic configurations for the use of enzymes in biosensor devices are outlined in Fig. 4.1. In Fig. 4.1 A, the biocatalyst generates a redox-active product that undergoes a redox transformation at the electrode interface and yields a current or a potential response. For example, biocatalyzed oxidation of substrates such as glucose or amino acids by molecular oxygen in the presence of glucose oxidase (GOx)5-8 or L-amino acid oxidase (AAOx),9 respectively, generates H2O2 as an electroactive product. The amperometric response due to the reduction of H2O2 is proportional to the substrate concentration.10 Potentiometric detection of enzymatically produced H2O2 was also used.11-12 Alternatively, the depletion of oxygen monitored at an oxygen-sensitive electrode represents a potential transduction of the substrate concentration.13 This approach was used to develop the first generation of electrochemical biosensors and the methodology has been extensively reviewed.14 The second approach to designing amperometric biosensors is shown in Fig. 4.1B. Electrocatalyzed oxidation (or reduction) of the enzyme redox center stimulates the oxidation (or reduction) of the substrate, and the resulting current is proportional to the substrate concentration. The development of such enzyme/electrode systems suffers from the basic limitation that redox enzymes usually lack direct electrical contact with electrode surfaces.15