Surface Display of a Redox Enzyme and its Site-Specific Wiring to Gold Electrodes

Abstract

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.

Measuring Localized Redox Enzyme Electron Transfer in a Live Cell with Conducting Atomic Force Microscopy

“…Our earlier studies with the same mutants have shown much lower bioelectrocatalytic activity for mutant D314Az. 20 …”

“…A 5 nm chromium adhesion layer and a 20 nm gold layer were deposited using K575X sputter coater (Quorum Technologies, Kent, UK). Bacteria were attached to the surface as described in ref (20). …”

Methodologies for “Wiring” Redox Proteins/Enzymes to Electrode Surfaces

“…This allows amino acids with novel functionalities to be introduced at specific locations within proteins, and such residues can be subsequently targeted in bio‐orthogonal chemical ligations . This is a rapidly expanding field of research and there are now examples of such methodologies being adapted for covalent crosslinking of redox proteins and enzymes to electrode surfaces . A practical consideration that makes the use of UAA mutagenesis potentially unsuitable for “wiring” redox metalloenzymes onto electrodes is that highly complex biosynthetic pathways can make protein overexpression challenging; in such scenarios substantial quantities of synthetic UAA may be required to generate useable quantities of UAA containing protein.…”

Rational Tuning of the Electrocatalytic Nanobiointerface for a “Turn‐Off” Biofuel‐Cell‐Based Self‐Powered Biosensor for p53 Protein

“…[4,9,10] These BFC-based sensors open the way for future point-of-care testinga nd might provide ah elpfuls olution to low-cost and simplea nalytical systems without the need of any additional externale nergy.T od ate, the studies on BFC-baseds ensors mainly focus on their long-term operation, miniaturization, and extendt he analytes scope. However,afew reports are focused on regulating/ort uning electrocatalytic processes at the electrode surface of BFCs, [15,16] whichi st he most fundamental factorf or the development of BFC-based biosensors.…”