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Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer–DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, “DNA machines” that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.

Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer–DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, “DNA machines” that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.

Biomolecule-nanoparticle (NP) [or quantum-dot (QD)] hybrid systems combine the recognition and biocatalytic properties of biomolecules with the unique electronic, optical, and catalytic features of NPs and yield composite materials with new functionalities. The biomolecule-NP hybrid systems allow the development of new biosensors, the synthesis of metallic nanowires, and the fabrication of nanostructured patterns of metallic or magnetic NPs on surfaces. These advances in nanobiotechnology are exemplified by the development of amperometric glucose sensors by the electrical contacting of redox enzymes by means of AuNPs, and the design of an optical glucose sensor by the biocatalytic growth of AuNPs. The biocatalytic growth of metallic NPs is used to fabricate Au and Ag nanowires on surfaces. The fluorescence properties of semiconductor QDs are used to develop competitive maltose biosensors and to probe the biocatalytic functions of proteases. Similarly, semiconductor NPs, associated with electrodes, are used to photoactivate bioelectrocatalytic cascades while generating photocurrents.

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4 nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4 nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800 s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4 nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl4- or p-aminophenolphosphate/Ag+, allowed the synthesis of Au nanowires or Ag nanowires, respectively.

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

Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomaterials with electronic transducers such as electrodes, field-effect transistors or piezoelectric crystals. Surface engineering of biomaterials such as enzymes, antigen-antibodies or DNA on the electronic supports controls the electrical properties of the biomaterial/transducer interface and enables the electronic transduction of biorecognition events, or biocatalyzed transformation, on the transducers. The development of biosensor systems of tailored sensitivities and specificities represents a major advance in bioelectronics.

Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.

Amplified electronic transduction of DNA sensing is accomplished by the application of a biocatalytic probe that precipitates an insoluble product on the transducer as a result of the DNA sensing. Alternative amplification of DNA sensing is achieved by the use of functionalized liposomes that bind to the electrode upon the sensing process. Single base mutations in DNA are detected by the polymerase-induced coupling of a biotinylated base complementary to the mismatch site, followed by the biocatalyzed precipitation of an insoluble product on the transducer. DNA biosensors of unprecedented sensitivity and specificity are designed. Faradaic impedance spectroscopy and microgravimetric quartz-crystal-microbalance measurements are used for the electronic transduction of the DNA sensing.

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

Light energy can be coupled to biocatalytic assemblies by two basic features. First, solar light could provide the energy input for biocatalysed syntheses, similar to the performance of the photosynthetic cycle in nature. Thereby, light-induced biocatalysed processes mimicking photosynthesis could provide a novel biotechnological approach. Second, interaction of light energy with enzymes could eventually trigger their activities “on” or “off”. As a result, enzymes could be photoregulated by external light signals and the application of biocatalytic materials as biochemical switches in sensor devices or information storage systems seems feasible. This article will describe the progress of our laboratory in harnessing light energy in the performance of biocatalytic assemblies.

Artificial photosynthetic devices provide a means for the use of solar light in generating fuel materials and valuable chemicals and for the removal of environmental pollutants. Control of photosensitized electron transfer reactions and development of catalysts for utilizations of the intermediate electron transfer products are essential aspects in designing artificial photosynthetic systems. Homogeneous and heterogeneous catalysts as well as biocatalysts (enzymes and cofactors) can be coupled to photochemically induced electron transfer reactions and effect photosynthetic transformations such as hydrogen evolution, CO2-fixation, hydrogenation, and hydroformylation processes. The progress in tailoring artificial photosynthetic devices in the context of thermodynamic and kinetic limitations of such systems is described. Integrated systems, where catalytic performance and control of electron transfer reactions which occur in organized assemblies are specifically emphasized.

Photosensitized regeneration of NAD(P)H cofactors is accomplished by biocatalyzed and artificially catalyzed transformations in photochemical assemblies. Photogenerated N,N'-dimethyl-4,4′-bipyridinium radical cation, MV+., acts as electron carrier for the reduction of NADPH in the presence of the enzyme ferredoxin reductase and for the reduction of NADH in the presence of lipoamide dehydrogenase. For the photogeneration of MV+. and subsequent NADPH formation, three different photosensitizers are applied: Ru(bpz)23+, Ru(bpy)23+, and Zn—TMPyP4+. The highest quantum yield for NADPH formation is observed with Ru(bpz)32+ and is ϕ = 1.7 × 10−1. For NADH regeneration only Zn-TMPyP4+ can be applied. Ru(bpy)32+ and Ru(bpz)2+3 interact with NADH in their excited or oxidized forms and therefore cannot be used as light-active compounds in the system. The NADPH regeneration cycle has been coupled to the biocatalyzed synthesis of glutamic acid. Although Ru(bpz)32+ is 42.5-fold more efficient than Ru(bpy)32+ in the regeneration of NADPH, the synthesis of glutamic acid is improved only by a factor of 2 in the presence of Ru(bpz)32+, implying that the coupled process is rate limiting. Oxidative regeneration of the NAD+ cofactor is accomplished in a photosystem that includes Ru(bpy)32+ as photosensitizer. The photoprocess is coupled to dehydrogenation of ethanol, propanol, lactic acid, and alanine with concomitant H2 evolution. A photosystem that includes Ru(bpy)32+ as photosensitizer, ascorbate as electron donor, and chloro-tris-(3-diphenylphosphinobenzene sulfonate)Rh(I), RhCl(dpm)33-, is catalytically active in the photoinduced regeneration of NAD(P)H cofactors. Mechanistic investigations show that photogenerated Ru(bpy)3+ mediates the generation of a hydrido-rhodium complex that acts as a charge relay for the production of NAD(P)H.

The photoisomerizable electron acceptors, trans-4,4′-bis(N-methylpyridinium)azobenzene, (1t), and trans-3,3′-bis(N-methylpyridinium)azobenzene, (2t), exhibit photoswitchable binding affinities to eosin. While the trans isomers, (1t) or (2t) exhibit high association affinities (Ka = 8.3 × 103 M−1 and 3.8 × 104 M−1, respectively), the cis isomers (1c) or (2c) reveal lower binding affinities (Ka = 3.4 × 103 M−1 and 1.4 × 104 M−1, respectively). The formation of the supramolecular donor–acceptor complexes between the eosin and the electron acceptors is associated with an absorbance change of the chromophore. This enables the cyclic spectroscopic transduction of the optically-induced formation and dissociation of the complexes upon photoisomerization between the respective trans and cis states. An eosin monolayer was assembled onto the Au-electrodes associated with a quartz piezoelectric crystal. The eosin-functionalized crystal was employed for the microgravimetric, quartz-crystal-microbalance analyses of the association of (1t), (2t), (1c), or (2c) to the crystal interface. The microgravimetric analyses allowed the characterization of the kinetics of association of the electron acceptors to the monolayer and the association constants between the isomers (1t), (2t), (1c), and (2c) with the eosin monolayer. By cyclic photoisomerization of the electron-acceptor between the trans and cis states, reversible piezoelectric transduction of the formation of the complexes with (1t) or (2t) at the monolayer interface, and their dissociation upon photoisomerization to (1c) or (2c) was accomplished.