Bilha Willner

Biomolecule-Based Nanomaterials and Nanostructures

Biomolecule-nanoparticle (or carbon nanotube) hybrid systems provide new materials that combine the unique optical, electronic, or catalytic properties of the nanoelements with the recognition or biocatalytic functions of biomolecules. This article summarizes recent applications of biomolecule-nanoparticle (or carbon nanotubes) hybrid systems for sensing, synthesis of nanostructures, and for the fabrication of nanoscale devices. The use of metallic nanoparticles for the electrical contacting of redox enzymes with electrodes, and as catalytic labels for the development of electrochemical biosensors is discussed. Similarly, biomolecule-quantum dot hybrid systems are implemented for optical biosensing, and for monitoring intracellular metabolic processes. Also, the self-assembly of biomolecule-metal nanoparticle hybrids into nanostructures and functional nanodevices is presented. The future perspectives of the field are addressed by discussing future challenges and highlighting different potential applications.

Biomaterials integrated with electronic elements: en route to bioelectronics.

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. Bioelectronic sensing devices, biosensors of tailored sensitivities and specificities, are being developed.

Nanoparticle–enzyme hybrid systems for nanobiotechnology

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.

Biomolecule–nanoparticle hybrids as functional units for nanobiotechnology

Biomolecule-metal or semiconductor nanoparticle (NP) hybrid systems combine the recognition and catalytic properties of biomolecules with the unique electronic and optical properties of NPs. This enables the application of the hybrid systems in developing new electronic and optical biosensors, to synthesize nanowires and nanocircuits, and to fabricate new devices. Metal NPs are employed as nano-connectors that activate redox enzymes, and they act as electrical or optical labels for biorecognition events. Similarly, semiconductor NPs act as optical probes for biorecognition processes. Double-stranded DNA or protein chains that are modified with metallic nanoclusters act as templates for the synthesis of metallic nanowires. The nanowires are used as building blocks to assemble nano-devices such as a transistor or a nanotransporter.

Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction

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.

Electrical contact of redox enzymes with electrodes: novel approaches for amperometric biosensors

Abstract 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.

Assembly of functionalized monolayers of redox proteins on electrode surfaces: novel bioelectronic and optobioelectronic systems

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.

Artificial photosynthetic model systems using light-induced electron transfer reactions in catalytic and 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.

PHOTOSWITCHABLE BIOMATERIALS AS GROUNDS FOR OPTOBIOELECTRONIC DEVICES

Abstract 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.

Layered molecular optoelectronic assemblies

Layered functionalized electrodes are used as optoelectronic assemblies for the electronic transduction of recorded photonic signals. Functionalization of a Au electrode with a photoisomerizable redox-activated monolayer enables the amperometric transduction of the photonic information recorded by the interface. This is exemplified with the organization of a phenoxynaphthacenequinone monolayer (1a). Organization of a photoactivated command layer on an electrode can be used to control interfacial electron transfer and might be applied for the electrical transduction of recorded optical signals. This is addressed with the assembly of a nitrospiropyran photoisomerizable monolayer (2a) on a Au electrode which acts as a command surface for controlling by light interfacial electron transfer. The monolayer undergoes photoisomerization between the neutral state (2a) and the positively charged protonated merocyanine state (2b). The charged interface controls the oxidation of dihydroxyphenylacetic acid, DHPAA (3), and of 3-hydroxytyramine, DOPA (4), and the system is used for the electrochemical transduction of optical signals recorded by the monolayer. Functionalization of electrodes with a β-cyclodextrin monolayer or with an eosin π-donor layer enables the light-stimulated association or dissociation of the photoisomerizable N,N′-bipyridinium azobenzene (5t) and of bis-pyridinium azobenzene (8t) to or from the modified surfaces. Association and dissociation of the surface-associated supramolecular complexes are transduced by electrochemical or piezoelectrical signal outputs. The organization of a supramolecular system where a molecular component is translocated by light-signals between two distinct positions enables one to design ‘molecular machines’. This is exemplified by the organization of a molecular assembly consisting of a ferrocene-functionalized β-cyclodextrin (11) threaded onto an azobenzene-alkyl chain wire and stoppered with an anthracene barrier which acts as a nanoscale molecular machine, a light-stimulated ‘molecular train’. The ferrocene-functionalized β-cyclodextrin is reversibly translocated between the trans-azobenzene and the alkyl chain by cyclic light-induced isomerization of the photoactive monolayer. The position of the β-cyclodextrin receptor is transduced by its chronoamperometric response.