Carbon-coated magnetic nanoparticles as a removable protection layer extending the operation lifetime of bilirubin oxidase-based bioelectrode.

One of the factors hindering the development of enzymatic biosensors and biofuel cells in real-life applications is the time-dependant degradation of the biocatalysts on electrode surfaces. In this work, we present a new practical approach for extending the operation lifetimes of bioelectrocatalytic assemblies based on bilirubin oxidase (BOD). As evident by both spectroscopic and electrochemical measurements, an adsorption of carbon-coated magnetic nanoparticles (ccMNPs) onto a BOD/carbon nanotubes-deposited surface yields a stable bioelectrocathode system for mediatorless oxygen reduction. As compared to electrodes, which were stored without a preliminary interaction with the ccMNPs, an 80% increase in the active enzymatic content and the electrocatalytic performance was evident for the modified assemblies over a course of one month. As the full removal of the protective particles before the measurement requires only a single step applying an external magnetic force, the method is shown to be simple, reproducible, and easy to implement. Combined with the high efficiency in preserving the enzymatic stability and bioelectrocatalytic currents, the findings suggest a promising methodology for enhancing the lifetimes of bioelectronic applications.

Power Generation by Selective Self-Assembly of Biocatalysts.

Through a careful chemical and bioelectronic design we have created a system that uses self-assembly of enzyme-nanoparticle hybrids to yield bioelectrocatalytic functionality and to enable the harnessing of electrical power from biomass. Here we show that mixed populations of hybrids acting as catalyst carriers for clean energy production can be efficiently stored, self-assembled on functionalized stationary surfaces, and magnetically re-collected to make the binding sites on the surfaces available again. The methodology is based on selective interactions occurring between chemically modified surfaces and ligand-functionalized hybrids. The design of a system with minimal cross-talk between the particles, outstanding selective binding of the hybrids at the electrode surfaces, and direct anodic and cathodic electron transfer pathways leads to mediator-less bioelectrocatalytic transformations which are implemented in the construction of a fast self-assembling, membrane-less fructose/O2 biofuel cell.

Enzymatic self-wiring in nanopores and its application in direct electron transfer biofuel cells.

A synthetic enzymatic activity in nanopores leading to the direct fabrication of modified electrodes applicable as biosensors and/or biofuel cell elements is reported. We demonstrate the heterogeneous enzymatic implanting of platinum nanoclusters, PtNCs, in glucose oxidase, GOx, immobilized on mesoporous carbon nanoparticles, MPCNP-modified surface. As the pores confine the growth of the clusters, the PtNC@GOx/MPCNP assembly becomes electrically wired to the matrix, demonstrating direct electron transfer, DET, bioelectrocatalytic properties that correlate with the applied duration of synthesis and cluster size. This inside-out nanocluster growth from the cofactor to the matrix is investigated and further compared to a reversed outside-in strategy which follows the electrochemical deposition of the Pt clusters inside the pores and their electrically induced expansion towards the FAD center of the enzyme. While the inside-out and outside-in methodologies provide, for the first time, synthetic bidirectional direct wiring routes of an enzyme to a surface, we highlight an asymmetry in the wiring efficiency associated with the different assemblies. The results indicate the existence of a shorter gap between the FAD cofactor and the PtNCs in the enzymatically implanted assembly, resulting in elevated bioelectrocatalytic currents, lower overpotential, and a higher turnover rate, 2580 e- s-1. The implanted assembly is then coupled to a bilirubin oxidase-adsorbed MPCNP cathode to yield an all-DET biofuel cell. Due to the superior electrical contact of the inside-out-synthesized anode, this cell demonstrates enhanced discharge potential and power outputs as compared to similar systems employing electrochemically synthesized outside-in-grown PtNC-GOx/MPCNPs or even GOx-modified MPCNPs diffusionally mediated by ferrocenemethanol.

Magnetically induced enzymatic cascades - advancing towards multi-fuel direct/mediated bioelectrocatalysis.

A generic method to magnetically assemble enzymatic cascades on electrode surfaces is introduced. The versatile method enables the simultaneous activation of both direct and mediated electron transfer bioelectrocatalysis to harness different substrates, which can serve as multiple fuels and oxidizers in biofuel cells generating clean energy.

Switchable aerobic/anaerobic multi-substrate biofuel cell operating on anodic and cathodic enzymatic cascade assemblies.

Enzymatic fuel cells may become more accessible for applications powering portable electronic devices by broadening the range of potentially usable fuels and oxidizers. In this work we demonstrate the operation of an integrated, yet versatile multi-substrate biofuel cell utilizing either glucose, fructose, sucrose or combinations of thereof as biofuels, and molecular oxygen originating from solution phase and/or an internal chemical source, as the oxidizer. In order to achieve this goal we designed an enzymatic cascade-functionalized anode consisting of invertase (INV), mutarotase (MUT), glucose oxidase (GOX), and fructose dehydrogenase (FDH), deposited on top of a mesoporous carbon nanoparticle matrix, in which electron relay molecules had been entrapped. The anode was then conjugated to a compatible enzymatic cathode that employs a cascade of catalase (CAT) and bilirubin oxidase (BOD), allowing the cell to operate in an aerobic environment and/or to utilize, under anaerobic conditions for instance, hydrogen peroxide as a source for the oxygen oxidizer. While operated in the presence of the sugar mixture and hydrogen peroxide, the power output of the dually cascaded biofuel cell reaches a peak power density of 0.25 mW cm-2 and demonstrates an open circuit potential of 0.65 V. To our knowledge this is the first reported biofuel cell that discharges with both anodic and cathodic enzymatic cascade architectures and the first biofuel cell that is repeatedly switched between aerobic and anaerobic conditions without any significant decrease in the discharge performance.

Controlled Vectorial Electron Transfer and Photoelectrochemical Applications of Layered Relay/Photosensitizer-Imprinted Au Nanoparticle Architectures on Electrodes.

Two configurations of molecularly imprinted bis-aniline-bridged Au nanoparticles (NPs) for the specific binding of the electron acceptor N,N'-dimethyl-4,4'-bipyridinium (MV(2+) ) and for the photosensitizer Zn(II)-protoporphyrin IX (Zn(II)-PP-IX) are assembled on electrodes, and the photoelectrochemical features of the two configurations are discussed. Configuration I includes the MV(2+) -imprinted Au NPs matrix as a base layer, on which the Zn(II)-PP-IX-imprinted Au NPs layer is deposited, while configuration II consists of a bilayer corresponding to the reversed imprinting order. Irradiation of the two electrodes in the presence of a benzoquinone/benzohydroquinone redox probe yields photocurrents of unique features: (i) Whereas configuration I yields an anodic photocurrent, the photocurrent generated by configuration II is cathodic. (ii) The photocurrents obtained upon irradiation of the imprinted electrodes are substantially higher as compared to the nonimprinted surfaces. The high photocurrents generated by the imprinted Au NPs-modified electrodes are attributed to the effective loading of the imprinted matrices with the MV(2+) and Zn(II)-PP-IX units and to the effective charge separation proceeding in the systems. The directional anodic/cathodic photocurrents are rationalized in terms of vectorial electron transfer processes dictated by the imprinting order and by the redox potentials of the photosensitizer/electron acceptor units associated with the imprinted sites in the two configurations.

Layered Metal Nanoparticle Structures on Electrodes for Sensing, Switchable Controlled Uptake/Release, and Photo-electrochemical Applications.

Layered metal nanoparticle (NP) assemblies provide highly porous and conductive composites of unique electrical and optical (plasmonic) properties. Two methods to construct layered metal NP matrices are described, and these include the layer-by-layer deposition of NPs, or the electropolymerization of monolayer-functionalized NPs, specifically thioaniline-modified metal NPs. The layered NP composites are used as sensing matrices through the use of electrochemistry or surface plasmon resonance (SPR) as transduction signals. The crosslinking of the metal NP composites with molecular receptors, or the imprinting of molecular recognition sites into the electropolymerized NP matrices lead to selective and chiroselective sensing interfaces. Furthermore, the electrosynthesis of redox-active, imprinted, bis-aniline bridged Au NP composites yields electrochemically triggered "sponges" for the switchable uptake and release of electron-acceptor substrates, and results in conductive surfaces of electrochemically controlled wettability. Also, photosensitizer-relay-crosslinked Au NP composites, or electrochemically polymerized layered semiconductor quantum dot/metal NP matrices on electrodes, are demonstrated as functional nanostructures for photoelectrochemical applications.

Block Copolymer Patterns as Templates for the Electrocatalyzed Deposition of Nanostructures on Electrodes and for the Generation of Surfaces of Controlled Wettability.

ITO electrodes modified with a nanopatterned film of polystyrene-block-poly(2-vinylpyridine), PS-b-P2VP, where the P2VP domains are quaternized with iodomethane, are used for selective deposition of redox-active materials. Electrochemical studies (cyclic voltammetry, Faradaic impedance measurements) indicate that the PS domains insulate the conductive surface toward redox labels in solution. In turn, the quaternized P2VP domains electrostatically attract negatively charged redox labels solubilized in the electrolyte solution, resulting in an effective electron transfer between the electrode and the redox label. This phenomenon is implemented for the selective deposition of the electroactive Prussian blue on the nanopatterned surface and for the electrochemical deposition of Au nanoparticles, modified with a monolayer of p-aminothiophenol/2-mercaptoethanesulfonic acid, on the quaternized P2VP domains. The patterned Prussian blue-modified surface enables controlling the wettability properties by the content of the electrochemically deposited Prussian blue. Controlled wettability is unattainable with the homopolymer-modified surface, attesting to the role of the nanopattern.

Addressing, amplifying and switching DNAzyme functions by electrochemically-triggered release of metal ions.

The design of artificial cells, which mimic the functions of native cells, is an ongoing scientific goal. The development of stimuli-responsive chemical systems that stimulate cascaded catalytic transformations, trigger chemical networks, and control vectorial branched transformations and dose-controlled processes, are the minimum requirements for mimicking cell functions. We have studied the electrochemical programmed release of ions from electrodes, which trigger selective DNAzyme-driven chemical reactions, cascaded reactions that self-assemble catalytic DNAzyme polymers, and the ON-OFF switching and dose-controlled operation of catalytic reactions. The addressable and potential-controlled release of Pb2+ or Ag+ ions into an electrolyte that includes a mixture of nucleic acids, results in the metal ion-guided selection of nucleic acids yielding the formation of specific DNAzymes, which stimulate orthogonal reactions or activate DNAzyme cascades.

Photosynthetic reaction center-functionalized electrodes for photo-bioelectrochemical cells.

During the last few years, intensive research efforts have been directed toward the application of several highly efficient light-harvesting photosynthetic proteins, including reaction centers (RCs), photosystem I (PSI), and photosystem II (PSII), as key components in the light-triggered generation of fuels or electrical power. This review highlights recent advances for the nano-engineering of photo-bioelectrochemical cells through the assembly of the photosynthetic proteins on electrode surfaces. Various strategies to immobilize the photosynthetic complexes on conductive surfaces and different methodologies to electrically wire them with the electrode supports are presented. The different photoelectrochemical systems exhibit a wide range of photocurrent intensities and power outputs that sharply depend on the nano-engineering strategy and the electroactive components. Such cells are promising candidates for a future production of biologically-driven solar power.

Enzyme-capped relay-functionalized mesoporous carbon nanoparticles: effective bioelectrocatalytic matrices for sensing and biofuel cell applications.

The porous high surface area and conducting properties of mesoporous carbon nanoparticles, CNPs (<500 nm diameter of NPs, pore dimensions ∼6.3 nm), are implemented to design electrically contacted enzyme electrodes for biosensing and biofuel cell applications. The relay units ferrocene methanol, Fc-MeOH, methylene blue, MB(+), and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS(2-), are loaded in the pores of the mesoporous CNPs, and the pores are capped with glucose oxidase, GOx, horseradish peroxidase, HRP, or bilirubin oxidase, BOD, respectively. The resulting relay/enzyme-functionalized CNPs are immobilized on glassy carbon electrodes, and the relays encapsulated in the pores are sufficiently free to electrically contact the different enzymes with the bulk electrode supports. The Fc-MeOH/GOx CNP-functionalized electrode is implemented for the bioelectrocatalyzed sensing of glucose, and the MB(+)/HRP-modified CNPs are applied for the electrochemical sensing of H2O2. The ABTS(2-)/BOD-modified CNPs provide an effective electrically contacted material for the bioelectrocatalyzed reduction of O2 (kcat = 94 electrons·s(-1)). Integration of the Fc-MeOH/GOx CNP electrode and of the electrically wired ABTS(2-)/BOD CNP electrode as anode and cathode, respectively, yields a biofuel cell revealing a power output of ∼95 µW·cm(-2).

Switchable mechanical DNA "arms" operating on nucleic acid scaffolds associated with electrodes or semiconductor quantum dots.

Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a ß-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of "molecular arms" proceeds, transduced by electrochemical or optical signals.

Photosystem I (PSI)/Photosystem II (PSII)-based photo-bioelectrochemical cells revealing directional generation of photocurrents.

Layered assemblies of photosystem I, PSI, and/or photosystem II, PSII, on ITO electrodes are constructed using a layer-by-layer deposition process, where poly N,N'-dibenzyl-4,4'-bipyridinium (poly-benzyl viologen, PBV(2+) ) is used as an inter-protein "glue". While the layered assembly of PSI generates an anodic photocurrent only in the presence of a sacrificial electron donor system, such as dichlorophenol indophenol (DCPIP)/ascorbate, the PSII-modified electrode leads, upon irradiation, to the formation of an anodic photocurrent (while evolving oxygen), in the absence of any sacrificial component. The photocurrent is generated by transferring the electrons from the PSII units to the PBV(2+) redox polymer. The charge-separated species allow, then, the injection of the electrons to the electrode, with the concomitant evolution of O2 . A layered assembly, consisting of a PSI layer attached to a layer of PSII by the redox polymer PBV(2+) , leads to an anodic photocurrent that is 2-fold higher, as compared to the anodic photocurrent generated by a PSII-modified electrode. This observation is attributed to an enhanced charge separation in the two-photosystem assembly. By the further nano-engineering of the two photosystems on the electrode using two different redox polymers, vectorial electron transfer to the electrode is demonstrated, resulting in a ca. 6-fold enhancement in the photocurrent. The reversed bi-layer assembly, consisting of a PSII layer linked to a layer of PSI by the PBV(2+) redox polymer, yields, upon irradiation, an inefficient cathodic current. This observation is attributed to a mixture of photoinduced electron transfer reactions of opposing effects on the photocurrent directions in the two-photosystem assembly.

Light-induced and redox-triggered uptake and release of substrates to and from mesoporous SiO2 nanoparticles.

The photonic- and redox-triggered cyclic uptake and release of organic substrates in functionalized mesoporous SiO2 nanoparticles (NPs) is demonstrated. The mesoporous SiO2 NPs are functionalized with nitrospiropyran photoisomerizable units. Rhodamine B is encapsulated in the channels of the SiO2 NPs and trapped by the hydrophobic nitrospiropyran capping units. Photoisomerization of the capping units to the protonated nitromerocyanine groups opens the channels and releases the encapsulated dye. Similarly, modification of the SiO2 channels by chloronaphthoquinone units traps eosin Y in the channels, by means of donor-acceptor interactions. The reduction of the quinone units to the chloronaphth hydroquinone donor groups opens the channels and releases the encapsulated substrate. The novelty of the study rests on the demonstration of the reversible and cyclic photostimulated or redox-activated uptake and release of substrates from the mesoporous SiO2 NPs.

Electrochemical switching of photoelectrochemical processes at CdS QDs and photosystem I-modified electrodes.

Photoactive inorganic CdS quantum dots (QDs) or the native photosystem I (PSI) is immobilized onto a pyrroloquinoline quinone (PQQ) monolayer linked to Au electrodes to yield hybrid relay/QDs (or photosystem) assemblies. By the electrochemical biasing of the electrode potential, the relay units are retained in their oxidized PQQ or reduced PQQH(2) states. The oxidized or reduced states of the relay units dictate the direction of the photocurrent (anodic or cathodic). By the cyclic biasing of the electrode potential between the values E ≥ -0.05 V and E ≤ -0.3 V vs Ag quasi-reference electrode (Ag QRE), retaining the relay units in the oxidized PQQ or reduced PQQH(2) states, the photocurrents are respectively switched between anodic and cathodic values. Different configurations of electrically switchable photoelectrochemical systems are described: (i) the PQQ/CdS QDs/(triethanolamine, TEOA) or PQQ/PSI/(ascorbic acid/dichlorophenolindophenol, DCPIP) systems, leading to anodic photocurrents; (ii) the PQQ/CdS QDs (or PSI)/(flavin adenine dinucleotide) systems, leading to cathodic photocurrents; (iii) the PQQ/CdS QDs (or PSI)/(O(2)) switchable systems, leading to cyclic anodic/cathodic switching of the photocurrents.

Amplified surface plasmon resonance and electrochemical detection of Pb2+ ions using the Pb2+-dependent DNAzyme and hemin/G-quadruplex as a label.

The hemin/G-quadruplex nanostructure and the Pb(2+)-dependent DNAzyme are implemented to develop sensitive surface plasmon resonance (SPR) and electrochemical sensing platforms for Pb(2+) ions. A complex consisting of the Pb(2+)-dependent DNAzyme sequence and a ribonuclease-containing nucleic acid sequence (corresponding to the substrate of the DNAzyme) linked to a G-rich domain, which is "caged" in the complex structure, is assembled on Au-coated glass surfaces or Au electrodes. In the presence of Pb(2+) ions, the Pb(2+)-dependent DNAzyme cleaves the substrate, leading to the separation of the complex and to the self-assembly of the hemin/G-quadruplex on the Au support. In one sensing platform, the Pb(2+) ions are analyzed by following the dielectric changes at the surface as a result of the formation of the hemin/G-quadruplex label using SPR. This sensing platform is further amplified by the immobilization of the sensing complex on Au NPs (13 nm) and using the electronic coupling between the NPs and the surface plasmon wave as an amplification mechanism. This method enables the sensing of Pb(2+) ions with a detection limit that corresponds to 5 fM. The second sensing platform implements the resulting hemin/G-quadruplex as an electrocatalytic label that catalyzes the electrochemical reduction of H(2)O(2). This method enables the detection of Pb(2+) with a detection limit of 1 pM. Both sensing platforms reveal selectivity toward the detection of Pb(2+) ions.

Integrated photosystem II-based photo-bioelectrochemical cells.

Photosynthesis is a sustainable process that converts light energy into chemical energy. Substantial research efforts are directed towards the application of the photosynthetic reaction centres, photosystems I and II, as active components for the light-induced generation of electrical power or fuel products. Nonetheless, no integrated photo-bioelectrochemical device that produces electrical power, upon irradiation of an aqueous solution that includes two inter-connected electrodes is known. Here we report the assembly of photobiofuel cells that generate electricity upon irradiation of biomaterial-functionalized electrodes in aqueous solutions. The cells are composed of electrically contacted photosystem II-functionalized photoanodes and an electrically wired bilirubin oxidase/carbon nanotubes-modified cathode. Illumination of the photoanodes yields the oxidation of water to O(2) and the transfer of electrons through the external circuit to the cathode, where O(2) is re-reduced to water.

Biomolecule/nanomaterial hybrid systems for nanobiotechnology.

The integration of biomolecules with metallic or semiconductor nanoparticles or carbon nanotubes yields new hybrid nanostructures of unique features that combine the properties of the biomolecules and of the nano-elements. These unique features of the hybrid biomolecule/nanoparticle systems provide the basis for the rapid development of the area of nanobiotechnology. Recent advances in the implementation of hybrid materials consisting of biomolecules and metallic nanoparticles or semiconductor quantum dots will be discussed. The following topics will be exemplified: (i) The electrical wiring of redox enzymes with electrodes by means of metallic nanoparticles or carbon nanotubes, and the application of the modified electrodes as amperometric biosensors or for the construction of biofuel cells. (ii) The biocatalytic growth of metallic nanoparticles as a means to construct optical or electrical sensors. (iii) The functionalization of semiconductor quantum dots with biomolecules and the application of the hybrid nanostructures for developing different optical sensors, including intracellular sensor systems. (iv) The use of biomolecule-metallic nanoparticle nanostructures as templates for growing metallic nanowires, and the construction of fuel-driven nano-transporters.

Photochemical switching of the phase-transition temperatures of p-NIPAM-Pt nanoparticles thermosensitive polymer composites associated with electrodes: functional electrodes for switchable electrocatalysis.

The thermosensitive poly(N-isopropylacrylamide) (p-NIPAM) is electropolymerized onto Au surfaces. The incorporation of the photoisomerizable N-carboxyethyl nitrospiropyran compound into p-NIPAM allows the reversible photochemical control of the gel-to-solid phase-transition temperatures of the polymer. Whereas the gel-to-solid phase-transition temperature of the nitrospiropyran-modified p-NIPAM is 33±2 °C, the phase-transition temperature of the nitromerocyanine-functionalized p-NIPAM matrix corresponds to 38±1 °C. Upon the incorporation of Pt nanoparticles (NPs) into the photochemically controlled p-NIPAM, a hybrid photoswitchable electrocatalytic matrix is formed. At a fixed temperature corresponding to 38 °C, the effective electrocatalytic reduction of H(2)O(2), or the oxidation of ascorbic acid, proceeded in the presence of the nitromerocyanine-functionalized p-NIPAM, yet these electrocatalytic transformations were inhibited in the presence of the nitrospiropyran-modified p-NIPAM.

Amplified surface plasmon resonance based DNA biosensors, aptasensors, and Hg2+ sensors using hemin/G-quadruplexes and Au nanoparticles.

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.