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

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

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

4. 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

5. 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

6. 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.

7. Redox enzymes electrically contacted with the use of electron transfer mediators

8. 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

9. 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

10. 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

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

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

14. 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.

15. 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

16. 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

17. 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

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

19. 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

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

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

22. The method allows application of the native FAD cofactor

23. Molecular shuttle unit transporting electrons between the enzyme and electrode

26. SPR spectra controlled by the bioelectrocatalytic oxidation of glucose

27. Contact angle controlled by the bioelectrocatalytic oxidation of glucose

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

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

31. 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

32. 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

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

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

40. Recommended reading:
I. Willner, E. Katz, Integration of layered redox-proteins and conductive supports for bioelectronic applications. Angew. Chem. Int. Ed. 2000, 39, (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
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