ABSTRACT
Metal-reducing bacteria in the genus Geobacter use a complex protein apparatus to guide the self-assembly of a divergent type IVa pilin peptide and synthesize conductive pilus appendages that show promise for the sustainable manufacturing of protein nanowires. The preferential helical conformation of the Geobacter pilin, its high hydrophobicity, and precise distribution of charged and aromatic amino acids are critical for biological self-assembly and conductivity. We applied this knowledge to synthesize via recombinant methods truncated pilin peptides for the bottom-up fabrication of protein nanowires and identified rate-limiting steps of pilin nucleation and fiber elongation that control assembly efficiency and nanowire length, respectively. The synthetic fibers retained the biochemical and electronic properties of the native pili even under chemical fixation, a critical consideration for integration of the nanowires into electronic devices. The implications of these results for the design and mass production of customized protein nanowires for diverse applications are discussed.IMPORTANCE The discovery in 2005 of conductive protein appendages (pili) in the metal-reducing bacterium Geobacter sulfurreducens challenged our understanding of biological electron transfer and pioneered studies in electromicrobiology that revealed the electronic basis of many microbial metabolisms and interactions. The protein nature of the pili afforded opportunities for engineering novel conductive peptides for the synthesis of nanowires via cost-effective and scalable manufacturing approaches. However, methods did not exist for efficient production, purification, and in vitro assembly of pilins into nanowires. Here we describe platforms for high-yield recombinant synthesis of Geobacter pilin derivatives and their assembly as protein nanowires with biochemical and electronic properties rivaling those of the native pili. The bottom-up fabrication of protein nanowires exclusively from pilin building blocks confirms unequivocally the charge transport capacity of the peptide assembly and establishes the intellectual foundation needed to manufacture pilin-based nanowires in bioelectronics and other applications.
Subject(s)
Fimbriae Proteins/metabolism , Geobacter/metabolism , Peptides/metabolism , Amino Acids, Aromatic/metabolism , Electric Conductivity , Electron Transport/physiology , Fimbriae, Bacterial/metabolism , NanowiresABSTRACT
The metal-reducing bacterium Geobacter sulfurreducens produces protein nanowires (pili) for fast discharge of respiratory electrons to extracellular electron acceptors such as iron oxides and uranium. Charge transport along the pili requires aromatic residues, which cluster once the peptide subunits (pilins) assemble keeping inter-aromic distances and geometries optimal for multistep hopping. The presence of intramolecular aromatic contacts and the predominantly α-helical conformation of the pilins has been proposed to contribute to charge transport and rectification. To test this, we self-assembled recombinant, thiolated pilins as a monolayer on gold electrodes and demonstrated their conductivity by conductive probe atomic force microscopy. The studies unmasked a crossover from exponential to weak distance dependence of conductivity and shifts in the mechanical properties of the film that are consistent with a transition from interchain tunneling in the upper, aromatic-free regions of the helices to intramolecular hopping via aromatic residues at the amino terminus. Furthermore, the mechanistic stratification effectively "doped" the pilins at the amino terminus, favoring electron flow in the direction opposite to the helix dipole. However, the effect of aromatic dopants on rectification is voltage-dependent and observed only at the low (100 mV) voltages that operate in biological systems. The results thus provide evidence for a peptide environment optimized for electron transfer at biological voltages and in the direction needed for the respiration of external electron acceptors. The implications of these results for the development of hybrid devices that harness the natural abilities of the pilins to bind and reduce metals are discussed.
Subject(s)
Electric Conductivity , Fimbriae Proteins/chemistry , Electrodes , Geobacter , Gold/chemistry , Mechanical Phenomena , Protein Conformation, alpha-Helical , Recombinant Proteins/chemistryABSTRACT
Electrochemical activation of glassy carbon, carbon paper and functionalized carbon nanotubes via high-applied-potential cyclic voltammetry leads to the formation of adsorbed, redox active functional groups and increased active surface area. Electrochemically activated carbon electrodes display enhanced activity toward nicotinamide adenine dinucleotide (NADH) oxidation, and more importantly, dramatically improved adsorption of bioelectrochemically active azine dyes. Adsorption of methylene green on an electroactivated carbon electrode yields a catalyst layer that is 1.8-fold more active toward NADH oxidation than an electrode prepared using electropolymerized methylene green. Stability studies using cyclic voltammetry indicate 70% activity retention after 4000 cycles. This work further facilitates the electrocatalysis of NADH oxidation for bioconversion, biosensor and bioenergy processes.
Subject(s)
Carbon/chemistry , Electrochemical Techniques/methods , NAD/chemistry , Adsorption , Electrodes , Kinetics , Microscopy, Electron, ScanningABSTRACT
BACKGROUND: With the increase in production and use of engineered nanoparticles (NP; ≤ 100 nm), safety concerns have risen about the potential health effects of occupational or environmental NP exposure. Results of animal toxicology studies suggest that inhalation of NP may cause pulmonary injury with subsequent acute or chronic inflammation. People with chronic respiratory diseases like asthma or allergic rhinitis may be even more susceptible to toxic effects of inhaled NP. Few studies, however, have investigated adverse effects of inhaled NP that may enhance the development of allergic airway disease. METHODS: We investigated the potential of polyethylene glycol coated amorphous silica NP (SNP; 90 nm diameter) to promote allergic airway disease when co-exposed during sensitization with an allergen. BALB/c mice were sensitized by intranasal instillation with 0.02% ovalbumin (OVA; allergen) or saline (control), and co-exposed to 0, 10, 100, or 400 µg of SNP. OVA-sensitized mice were then challenged intranasally with 0.5% OVA 14 and 15 days after sensitization, and all animals were sacrificed a day after the last OVA challenge. Blood and bronchoalveolar lavage fluid (BALF) were collected, and pulmonary tissue was processed for histopathology and biochemical and molecular analyses. RESULTS: Co-exposure to SNP during OVA sensitization caused a dose-dependent enhancement of allergic airway disease upon challenge with OVA alone. This adjuvant-like effect was manifested by significantly greater OVA-specific serum IgE, airway eosinophil infiltration, mucous cell metaplasia, and Th2 and Th17 cytokine gene and protein expression, as compared to mice that were sensitized to OVA without SNP. In saline controls, SNP exposure did cause a moderate increase in airway neutrophils at the highest doses. CONCLUSIONS: These results suggest that airway exposure to engineered SNP could enhance allergen sensitization and foster greater manifestation of allergic airway disease upon secondary allergen exposures. Whereas SNP caused innate immune responses at high doses in non-allergic mice, the adjuvant effects of SNP were found at lower doses in allergic mice and were Th2/Th17 related. In conclusion, these findings in mice suggest that individuals exposed to SNP might be more prone to manifest allergic airway disease, due to adjuvant-like properties of SNP.
Subject(s)
Lung/drug effects , Nanoparticles/toxicity , Ovalbumin , Respiratory Hypersensitivity/chemically induced , Silicon Dioxide/toxicity , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/immunology , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Immunoglobulin E/blood , Inflammation Mediators/metabolism , Inhalation Exposure/adverse effects , Lung/immunology , Lung/metabolism , Lymph Nodes/drug effects , Lymph Nodes/immunology , Mice, Inbred BALB C , Neutrophil Infiltration/drug effects , Particle Size , Pulmonary Eosinophilia/blood , Pulmonary Eosinophilia/chemically induced , Pulmonary Eosinophilia/immunology , Respiratory Hypersensitivity/blood , Respiratory Hypersensitivity/genetics , Respiratory Hypersensitivity/immunology , Risk Assessment , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Time FactorsABSTRACT
A diverse range of molecular interactions can occur between engineered nanomaterials (ENM) and biomembranes, some of which could lead to toxic outcomes following human exposure to ENM. In this study, we adapted electrophysiology methods to investigate the ability of 20nm polystyrene nanoparticles (PNP) to induce pores in model bilayer lipid membranes (BLM) that mimic biomembranes. PNP charge was varied using PNP decorated with either positive (amidine) groups or negative (carboxyl) groups, and BLM charge was varied using dioleoyl phospholipids having cationic (ethylphosphocholine), zwitterionic (phosphocholine), or anionic (phosphatidic acid) headgroups. Both positive and negative PNP induced BLM pores for all lipid compositions studied, as evidenced by current spikes and integral conductance. Stable PNP-induced pores exhibited ion selectivity, with the highest selectivity for K(+) (PK/PCl~8.3) observed when both the PNP and lipids were negatively charged, and the highest selectivity for Cl(-) (PK/PCl~0.2) observed when both the PNP and lipids were positively charged. This trend is consistent with the finding that selectivity for an ion in channel proteins is imparted by oppositely charged functional groups within the channel's filter region. The PK/PCl value was unaffected by the voltage-ramp method, the pore conductance, or the side of the BLM to which the PNP were applied. These results demonstrate for the first time that PNP can induce ion-selective pores in BLM, and that the degree of ion selectivity is influenced synergistically by the charges of both the lipid headgroups and functional groups on the PNP.
Subject(s)
Ionophores/chemistry , Lipid Bilayers/chemistry , Nanoparticles/chemistry , Polystyrenes/chemistry , Chlorides/chemistry , Membrane Potentials , Phosphatidic Acids/chemistry , Phosphorylcholine/chemistry , Potassium/chemistry , Static ElectricityABSTRACT
Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.
Subject(s)
Biosensing Techniques/instrumentation , Electrochemistry/instrumentation , Microelectrodes , Biosensing Techniques/methods , Electric Conductivity , Electrochemistry/methods , Equipment Design , Equipment Failure Analysis , Surface PropertiesABSTRACT
We report the novel fabrication of a highly sensitive, selective, fast responding, and affordable amperometric glucose biosensor using exfoliated graphite nanoplatelets (xGnPs) decorated with Pt and Pd nanoparticles. Nafion was used to solubilize metal-decorated graphite nanoplatelets, and a simple cast method with high content organic solvent (85 wt %) was used to prepare the biosensors. The addition of precious metal nanoparticles such as platinum (Pt) and palladium (Pd) to xGnP increased the electroactive area of the electrode and substantially decreased the overpotential in the detection of hydrogen peroxide. The Pt-xGnP glucose biosensor had a sensitivity of 61.5+/-0.6 microA/(mM x cm(2)) and gave a linear response up to 20 mM. The response time and detection limit (S/N=3) were determined to be 2 s and 1 microM, respectively. Therefore, this novel glucose biosensor based on the Pt nanoparticle coated xGnP is among the best reported to date in both sensing performance and production cost. In addition, the effects of metal nanoparticle loading and the particle size on the biosensor performance were systematically investigated.
Subject(s)
Biosensing Techniques/instrumentation , Glucose Oxidase/chemistry , Glucose/analysis , Graphite/chemistry , Nanostructures/chemistry , Palladium/chemistry , Platinum/chemistry , Crystallization/methods , Electrochemistry/instrumentation , Electrochemistry/methods , Enzymes, Immobilized/chemistry , Equipment Design , Equipment Failure Analysis , Glucose/chemistry , Macromolecular Substances/chemistry , Materials Testing , Microelectrodes , Molecular Conformation , Nanostructures/ultrastructure , Nanotechnology/instrumentation , Particle Size , Reproducibility of Results , Sensitivity and Specificity , Surface PropertiesABSTRACT
Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2 degrees ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, beta-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than beta-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2 degrees ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2 degrees ADH). This mutation increased the Km(app) for NADP+ 200-fold while decreasing the Km(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2 degrees ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7 x 10(4) times, suggesting that the Y218F 2 degrees ADH-modified bioelectronic interface is stable.
Subject(s)
Alcohol Oxidoreductases/genetics , Biosensing Techniques , Phenylalanine/genetics , Thermoanaerobacter/enzymology , Thermoanaerobacter/genetics , Tyrosine/genetics , Alcohol Oxidoreductases/physiology , Electrochemistry , Mutagenesis, Site-Directed , NAD/genetics , NAD/metabolismABSTRACT
Neuropathy target esterase (NTE) is a membrane protein found in human neurons and other cells, including lymphocytes. Binding of certain organophosphorus (OP) compounds to NTE is believed to cause OP-induced delayed neuropathy (OPIDN), a type of paralysis for which there is no effective treatment. Mutations in NTE have also been linked with serious neurological diseases, such as motor neuron disease. This paper describes development of the first nanostructured biosensor interface containing a catalytically active fragment of NTE known as NEST. The biosensor was fabricated using the layer-by-layer assembly approach, by immobilizing a layer of NEST on top of multilayers consisting of a polyelectrolyte (poly-L-lysine) and an enzyme (tyrosinase). The biosensor has a response time on the order of seconds and gives a concentration-dependent decrease in sensor output in response to a known NEST (and NTE) inhibitor. Potential applications of the biosensor include screening OP compounds for NTE inhibition and investigating the enzymology of wild-type and mutant forms of NTE. Although the development of a NEST biosensor was the primary purpose of this study, we found that the approach developed for NEST could also be extended to measure the activity of other esterases involved in neural processes, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). On the basis of measured sensitivities, phenyl valerate was the preferred substrate for NEST and BChE, whereas phenyl acetate was better for AChE.
Subject(s)
Biosensing Techniques/methods , Carboxylic Ester Hydrolases/chemistry , Enzymes, Immobilized/chemistry , Nanostructures , Acetylcholinesterase/analysis , Acetylcholinesterase/chemistry , Butyrylcholinesterase/analysis , Butyrylcholinesterase/chemistry , Carboxylic Ester Hydrolases/antagonists & inhibitors , Catalytic Domain , Conductometry , Electrodes , Gold/chemistry , Humans , Peptide Fragments/chemistry , Phenylacetates/chemistry , Valerates/chemistryABSTRACT
Microarrays containing multiple, nanostructured layers of biological materials would enable high-throughput screening of drug candidates, investigation of protein-mediated cell adhesion, and fabrication of novel biosensors. In this paper, we have examined in detail an approach that allows high-quality microarrays of layered, bionanocomposite films to be deposited on virtually any substrate. The approach uses LBL self-assembly to pre-establish a multilayered structure on an elastomeric stamp, and then uses microCP to transfer the 3-D structure intact to the target surface. For examples, different 3-D patterns containing dendrimers, polyelectrolyte multilayers and two proteins, sADH and sDH, have been fabricated. For the first time, the approach was also extended to create overlaid bionanocomposite patterns and multiple proteins containing patterns. The approach overcomes a problem encountered when using microCP to establish a pattern on the target surface and then building sequential layers on the pattern via LBL self-assembly. Amphiphilic molecules such as proteins and dendrimers tend to adsorb both to the patterned features as well as the underlying substrate, resulting in low-quality patterns. By circumventing this problem, this research significantly extends the range of surfaces and layering constituents that can be used to fabricate 3-D, patterned, bionanocomposite structures. [image in text]
Subject(s)
Dimethylpolysiloxanes/chemistry , Drug Evaluation, Preclinical/methods , Microarray Analysis/methods , Nanocomposites/chemistry , Nylons/chemistry , Polymers/chemistry , Dendrimers , Microscopy, Fluorescence , Polyamines/chemistryABSTRACT
Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. However, these interfaces contain labile components, including enzymes and cofactors, which have limited lifetimes and must be replaced periodically to allow long-term operation. Current methods to fabricate bioelectronic interfaces do not allow facile replacement of these components, thus limiting the useful lifetime of the interfaces. In this paper we describe a versatile new fabrication approach that binds the enzymes and cofactors using reversible ionic interactions. This approach allows the interface to be removed via a simple pH change and then replaced to fully regenerate the biocatalytic activity. The positively charged polyelectrolyte poly(ethylenimine) was used to ionically bond a dehydrogenase enzyme and its cofactor to a gold electrode that was functionalized with 3-mercaptopropionic acid and the electron mediator toluidine blue O. By reducing the pH, the surface-bound 3-mercaptopropionic acid was protonated, disrupting the ionic bonds and releasing the enzyme-modified polyelectrolyte. After neutralization, fresh enzyme and cofactor were bound, regenerating the bioelectronic interface. Cyclic voltammetry, chronoamperometry, constant potential amperometry, electrochemical impedance spectroscopy, and Fourier transform infrared spectroscopy analyses were used to characterize the bioelectronic interfaces. For the two enzymes tested (secondary alcohol dehydrogenase and sorbitol dehydrogenase) and their respective cofactors (beta-nicotinamide adenine dinucleotide phosphate and beta-nicotinamide adenine dinucleotide), the reconstituted interface exhibited a surface coverage, an electron-transfer coefficient, and a turnover rate similar to those of the original interface.
Subject(s)
Alcohol Dehydrogenase/chemistry , Bacterial Proteins/chemistry , L-Iditol 2-Dehydrogenase/chemistry , Thermoanaerobacter/enzymology , Alcohol Dehydrogenase/genetics , Bacterial Proteins/genetics , Biosensing Techniques , Coenzymes/chemistry , Electric Impedance , Electronics , Enzyme Stability , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , Hydrogen-Ion Concentration , L-Iditol 2-Dehydrogenase/genetics , NAD/chemistry , NADP/chemistry , Polyethyleneimine/chemistry , Thermoanaerobacter/geneticsABSTRACT
This paper presents the formation of a novel biomimetic interface consisting of an electrolessly deposited gold film overlaid with a tethered bilayer lipid membrane (tBLM). Self-assembly of colloidal gold particles was used to create an electrolessly deposited gold film on a glass slide. The properties of the film were characterized using field-effect scanning electron microscopy, energy dispersive spectroscopy, and atomic force microscopy. Bilayer lipid membranes were then tethered to the gold film by first depositing an inner molecular leaflet using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (DPGP), and cystamine in ethanol onto a freshly prepared electrolessly deposited gold surface. The outer leaflet was then formed by the fusion of liposomes made from DPGP or 1,2-dioleoyl-sn-glycero-3-phosphocholine on the inner leaflet. To provide functionality, two membrane biomolecules were also incorporated into the tBLMs: the ionophore valinomycin and a segment of neuropathy target esterase containing the esterase domain. Electrochemical impedance spectroscopy, UV/visible spectroscopy, and fluorescence recovery after pattern photobleaching were used to characterize the resulting biomimetic interfaces and confirm the biomolecule activity of the membrane. Microcontact printing was used to form arrays of electrolessly deposited gold patterns on glass slides. Subsequent deposition of lipids yielded arrays of tBLMs. This approach can be extended to form functional biomimetic interfaces on a wide range of inexpensive materials, including plastics. Potential applications include high-throughput screening of drugs and chemicals that interact with cell membranes and for probing, and possibly controlling, interactions between living cells and synthetic membranes. In addition, the gold electrode provides the possibility of electrochemical applications, including biocatalysis, bio-fuel cells, and biosensors.
Subject(s)
Electronics/methods , Gold , Lipid Bilayers/chemistry , Phospholipids/chemistry , Colloids , Electrochemistry/methods , Microscopy, Electron, Scanning , Models, Molecular , Molecular Conformation , SolutionsABSTRACT
This paper presents novel methods to produce arrays of lipid bilayers and liposomes on patterned polyelectrolyte multilayers. We created the arrays by exposing patterns of poly(dimethyldiallylammonium chloride) (PDAC), polyethylene glycol (m-dPEG) acid, and poly(allylamine hydrochloride) (PAH) on polyelectrolyte multilayers (PEMs) to liposomes of various compositions. The resulting interfaces were characterized by total internal reflection fluorescence microscopy (TIRFM), fluorescence recovery after pattern photobleaching (FRAPP), quartz crystal microbalance (QCM), and fluorescence microscopy. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA) were found to preferentially adsorb on PDAC and PAH surfaces. On the other hand, liposome adsorption on sulfonated poly(styrene) (SPS) surfaces was minimal, due to electrostatic repulsion between the negatively charged liposomes and the SPS-coated surface. Surfaces coated with m-dPEG acid were also found to resist liposome adsorption. We exploited these results to create arrays of lipid bilayers by exposing PDAC, PAH and m-dPEG patterned substrates to DOPA/DOPC vesicles of various compositions. The patterned substrates were created by stamping PDAC (or PAH) on SPS-topped multilayers, and m-dPEG acid on PDAC-topped multilayers, respectively. This technique can be used to produce functional biomimetic interfaces for potential applications in biosensors and biocatalysis, for creating arrays that could be used for high-throughput screening of compounds that interact with cell membranes, and for probing, and possibly controlling, interactions between living cells and synthetic membranes.
Subject(s)
Electrolytes/chemistry , Lipid Bilayers/chemistry , Liposomes/chemistry , Microarray Analysis/instrumentation , Polymers/chemistry , Adsorption , Microarray Analysis/methods , Microscopy, Fluorescence , Models, Structural , Molecular Structure , QuartzABSTRACT
Bioelectronic interfaces that allow dehydrogenase enzymes to communicate with electrodes have potential applications such as biosensors and biocatalytic reactors. A major challenge in creation of such bioelectronic interfaces is to orient the enzyme, its cofactor, and an electron mediator properly with respect to the electrode in order to achieve efficient, multistep electron transfer. This paper describes a versatile, new method that uses cysteine, an inexpensive, branched amino acid having sulfhydryl, amino, and carboxyl functional groups, to achieve such orientation. This approach provides greater flexibility in assembling complex bioelectronic interfaces than previously reported approaches that bind the enzyme, cofactor, and mediator in a linear chain. Cysteine was attached to a gold electrode through the sulfhydryl groups, to the electron mediator toluidine blue O (TBO) through the carboxyl group, and to the cofactor (e.g., NAD(P)+) through the amino group. Cyclic voltammetry, impedance spectroscopy, chronoamperometry and quartz crystal microbalance gravimetry were used to demonstrate the sequential assembly steps and the electrical activity of the resulting bioelectronic interface.
Subject(s)
Bioelectric Energy Sources , Biosensing Techniques/instrumentation , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Boronic Acids , Cysteine , Electrochemistry , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Thermoanaerobacter/enzymology , Thermoanaerobacter/geneticsABSTRACT
A novel approach is presented that allows high-quality, 3D patterned bionanocomposite layered films to be constructed on substrates whose surface properties are incompatible with existing self-assembly methods.