Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Surface display of a redox enzyme and its site-specific wiring to gold electrodes.

The generation of a current through interaction between bacteria and electrodes has been explored by various methods. We demonstrate the attachment of living bacteria through a surface displayed redox enzyme, alcohol dehydrogenase II. The unnatural amino acid para-azido-L-phenylalanine was incorporated into a specific site of the displayed enzyme, facilitating electron transfer between the enzyme and an electrode. In order to attach the bacteria carrying the surface displayed enzyme to a surface, a linker containing an alkyne and a thiol moiety on opposite ends was synthesized and attached to the dehydrogenase site specifically through a copper(I)-catalyzed azide-alkyne cycloaddition reaction. Using this approach we were able to covalently link bacteria to gold-coated surfaces and to gold nanoparticles, while maintaining viability and catalytic activity. We show the performance of a biofuel cell using these modified bacteria at the anode, which resulted in site-specific dependent fuel cell performance for at least a week. This is the first example of site-specific attachment of a true living biohybrid to inorganic material.

Surface display of redox enzymes in microbial fuel cells.

A novel concept for a biofuel cell is presented. Enzyme based fuel cells suffer from enzyme instability when a long time of operation is required. Hence, a system that will continuously produce the biocatalyst needed for the system is necessary. A hybrid of an enzyme-based microbial fuel cell was developed. The redox enzyme glucose oxidase from Aspergillus niger was displayed on the surface of Saccharomyces cerevisiae using the Yeast Surface Display System in a high copy number and as an active enzyme. We have demonstrated its activity both biochemically and electrochemically and observed much higher activity over yeast cells not displaying glucose oxidase as well as over purified glucose oxidase from Aspergillus niger. Further, we were able to construct a biofuel cell, where the anode was comprised of the yeast cells displaying glucose oxidase in the presence of a mediator (methylene blue) and the cathode compartment was comprised of the oxygen reducing enzyme laccase from Trametes versicolor and a redox mediator. Our constructed biofuel cell displayed higher power outputs and current densities than those observed for unmodified yeast and a much longer time of operation in comparison with a similar cell where the anode is comprised of purified glucose oxidase.

Biofuel cell controlled by enzyme logic systems.

An enzyme-based biofuel cell with a pH-switchable oxygen electrode, controlled by enzyme logic operations processing in situ biochemical input signals, has been developed. Two Boolean logic gates (AND/OR) were assembled from enzyme systems to process biochemical signals and to convert them logically into pH-changes of the solution. The cathode used in the biofuel cell was modified with a polymer-brush functionalized with Os-complex redox species operating as relay units to mediate electron transport between the conductive support and soluble laccase biocatalyzing oxygen reduction. The electrochemical activity of the modified electrode was switchable by alteration of the solution pH value. The electrode was electrochemically mute at pH > 5.5, and it was activated for the bioelectrocatalytic oxygen reduction at pH < 4.5. The sharp transition between the inactive and active states was used to control the electrode activity by external enzymatic systems operating as logic switches in the system. The enzyme logic systems were decreasing the pH value upon appropriate combinations of the biochemical signals corresponding to the AND/OR Boolean logic. Then the pH-switchable electrode was activated for the oxygen reduction, and the entire biofuel cell was switched ON. The biofuel cell was also switched OFF by another biochemical signal which resets the pH value to the original neutral value. The present biofuel cell is the first prototype of a future implantable biofuel cell controlled by complex biochemical reactions to deliver power on-demand responding in a logical way to the physiological needs.

Yeast surface display of dehydrogenases in microbial fuel-cells.

Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3µWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9µWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

In†Situ fuel processing in a microbial fuel cell.

A microbial fuel cell (MFC) was designed in which fuel is generated in the cell by the enzyme glucoamylase, which is displayed on the surface of yeast. The enzyme digests starch specifically into monomeric glucose units and as a consequence enables further glucose oxidation by microorganisms present in the MFC anode. The oxidative enzyme glucose oxidase was coupled to the glucoamylase digestive enzyme. When both enzymes were displayed on the surface of yeast cells in a mixed culture, superior fuel-cell performance was observed in comparison with other combinations of yeast cells, unmodified yeast, or pure enzymes. The feasibility of the use of the green macroalgae Ulva lactuca in such a genetically modified MFC was also demonstrated. Herein, we report the performance of such fuel cells as a proof of concept for the enzymatic digestion of complex organic fuels in the anode of MFCs to render the fuel more available to microorganisms.

Immobilization strategies of Brucella particles on optical fibers for use in chemiluminescence immunosensors.

Immunosensors are powerful analytical tools in clinical and veterinary diagnostics. This has led us to design a chemiluminescent immunosensor aimed at identifying anti-Brucella antibodies using optical fibers as the transducer. In order to develop the optimal transducer, to achieve an optimal chemical modification thereby allowing an optimal covalent binding of the protein receptor, several cleaning strategies and silane coupling agents were investigated. Brucella killed organisms were used as a model receptor for quantifying anti-Brucella IgG antibodies in a suspension compared to conventional colorimetric and chemiluminescent ELISA. A silane-benzophenone derivative was selected as the best performing silane coupling agent: the optical fiber immunosensor (OFIS) has showed the lowest limit of detection at 0.207 microg/ml, compared to 0.828 microg/ml and 0.414 microg/ml achieved by colorimetric and chemiluminescent ELISAs, respectively. These results, together with the additional advantages of rapidity, lower reagent volumes and moderate operating conditions, have set the grounds for further study in order to adapt this platform for on-site diagnostics of brucellosis disease markers.