Electrically Contacted Redox Enzymes: Biosensor and Bioelectronic Applications Part 1: Mediated electron transfer

We will discuss: Electrical contacting of redox enzymes using electron transfer mediators Supramolecular bioelectrocatalytic systems based on molecular architecture and their applications for biosensors

Electrical contacting of enzymes is needed to transduce biocatalytic processes electronically Electrode

Direct, non-mediated electron-transfer between proteins/enzymes and electrodes Direct electron-transfer between an electrode and an enzyme redox-center located in close vicinity to the surface Application of the direct electrical ‘wiring’ of HRP for the detection of H2O2 produced by an oxidase in response to a primary substrate

Alignment of proteins at an electrode surface provides a short electron transfer distance Cytochrome c Only small redox proteins with non-symmetrical location of active centers could be contacted directly at electrodes Azurin Stellacyanin

MP-11 cyclic voltammetry and electrocatalytic H2O2 reduction. Super-small protein fragments could be directly electrically contacted at electrodes Microperoxidase-11 Microperoxidase-11 monolayer covalently bound to Au electrode MP-11 cyclic voltammetry and electrocatalytic H2O2 reduction.

Redox enzymes electrically contacted with the use of electron transfer mediators

Dissolved enzymes activated by diffusional mediators Ferrocene is a typical oxidative electron relay Cyclic voltammograms for the bioelectrocatalyzed oxidation of glucose by GOx mediated by soluble ferrocene relay

Monolayer- or multilayer-enzyme electrodes activated by diffusional mediators A bienzymatic network consisting of choline oxidase and acetylcholine esterase for the amperometric detection of acetylcholine using a diffusional electron relay

The electrical contacting of dissolved enzymes at mediator-functionalized electrodes Assembly of a C60-monolayer, and its use in mediating the bioelectrocatalytic oxidation of glucose

Bioelectrocatalytic reduction of nitrate by nitrate reductase mediated by a monolayer immobilized MP-11

The electrical contacting of mediator-modified enzymes: many electron relays are bound randomly, at non-optimized positions

Dissolved redox-enzymes functionalized with tethered electron-transfer mediators Electrical ‘wiring’ of glucose oxidase with ferrocene units tethered to lysine residues of the protein backbone Effect of the spacer on the efficiency of the electron transfer: (b) n = 2; (c) = 3; (d) n = 8 W. Schuhmann, A. Heller, et. al.

Monolayer- and multilayer-enzyme assemblies functionalized with tethered electron-transfer mediators The assembly of an electrically-contacted glutathione reductase monolayer on an electrode The effect of the spacer length on the rate of the bioelectrocatalytic reaction

The preparation of a non-ordered polymeric layer of glucose oxidase electrically ‘wired’ by ferrocene groups covalently tethered to the enzyme net The bioelectrocatalytic oxidation of glucose by the non-ordered electrically contacted enzyme net

The stepwise assembly and electrical contacting of a crosslinked organized multilayer array of glucose oxidase (GOx) on an Au-electrode Bioelectrocatalytic oxidation of glucose by the enzyme electrode in: (a) 1, (b) 4 and (c) 8 layer configurations

Polymer- and inorganic matrix-bound enzymes contacted by co-immobilized mediators

Enzymes were entrapped into redox-polymer and sol-gel matrices Redox-functionalized monomers applied for electropolymerization and the entrapment of redox-enzymes Encapsulation of enzymes into a sol-gel matrix containing redox-relay groups and conductive particles

Electrical “wiring” of redox enzymes by the reconstitution method The method requires a unique synthetic analog of the FAD cofactor

Electrical “wiring” and alignment of flavoenzymes by reconstitution in a monolayer on an electrode surface

The method allows application of the native FAD cofactor

Molecular shuttle unit transporting electrons between the enzyme and electrode

SPR spectra controlled by the bioelectrocatalytic oxidation of glucose

Contact angle controlled by the bioelectrocatalytic oxidation of glucose

Electrical “wiring” and alignment of NAD+ dependent enzymes by the affinity complex formation on an electrode surface

The assembly of an integrated nitrate sensor electrode by the crosslinking of a MP-11-NR affinity complex on a Au- electrode

The assembly of a nitrate sensing electrode by the crosslinking of an affinity complex formed between nitrate reductase and a Fe(III)-protoporphyrin reconstituted de novo four helix-bundle protein Cyclic voltammograms of the electrode at [NO3-]: (a) 0, (b) 12, (c) 24, (d) 46, (e) 68 mM

We discussed: Electrical contacting of redox enzymes: Direct electrochemical processes of small proteins/enzymes   Electron-relay mediated bioelectrocatalytic reactions Supra-molecular bioelectrocatalytic systems self-assembled on electrode surfaces Interfacial properties (refractivity, wettability, etc.) of modified electrodes controlled by bioelectrocatalytic reactions

Different chemical means to immobilize enzyme on electrodes in monolayer configurations:

We discussed: Monolayer immobilization of enzymes / proteins on various electrode surfaces

Recommended reading: I. Willner, E. Katz, Integration of layered redox-proteins and conductive supports for bioelectronic applications. Angew. Chem. Int. Ed. 2000, 39, 1180-1218. (available in pdf) E. Katz, A.N. Shipway, I. Willner, Mediated electron-transfer between redox-enzymes and electrode supports. In: Encyclopedia of Electrochemistry, Vol. 9: Bioelectrochemistry, G.S. Wilson, (Ed.), A.J. Bard, M. Stratmann (Editors-in-Chief), Wiley-VCH GmbH, Weinheim, Germany, 2002, Chapter 17, pp. 559-626. E. Katz, A.N. Shipway, I. Willner,The electrochemical and photochemical activation of redox-enzymes. In: Electron Transfer in Chemistry, Vol. 4, V. Balzani, P. Piotrowiak, M.A.J. Rodgers (Eds.), Wiley-VCH, Weinheim, Germany, 2001, pp. 127-201.