Intramolecular Electron Transfer through Poly-Ferrocenyl Glucose Oxidase Conjugates to Carbon Electrodes: 2. Mechanistic Understanding of Long-Term Stability

- Glucose oxidase (GOX)-ferrocene-containing redox polymer conjugates (GOX-pFcAc) used to fabricate glucose-based biosensors.
- Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide grown from surface of GOX using atom transfer radical polymerization.
- GOX-pFcAc crosslinked along with human serum albumin and drop cast onto carbon paper strips provided mediated-electron transfer for glucose sensing.
- GOX-pFcAc-based biosensors were resistant to conjugate leaching and GOX deactivation during operation.
- During operation electron transfer efficiency through pFcAc chains decreased over time due to ferrocenium degradation and nucleophilic anion uptake.

Enzyme-based biosensors have garnered intense research interest due to the catalytic properties and physiologic applicability of enzymes. However, the use of these biological catalysts also introduced persistent lifetime limiting stability issues that hinder device longevity. We report on the identification and understanding of sources of long-term stability loss within a glucose oxidase (GOX)-based biosensor. Through polymer-based protein engineering (PBPE), we grew poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide (pFcAc) directly from initiator sites at the GOX surface via atom-transfer radical polymerization. The resulting GOX-pFcAc conjugates were crosslinked along with human serum albumin (HSA) within chitosan (Chit)-containing solution and drop cast onto carbon paper strips. Chit-GOX-pFcAc-HSA-carbon paper biosensors generated current by intramolecular electron transfer and electron self-exchange through the GOX-pFcAc conjugates as well as through oxygen mediated electron transfer under varied cell potentials and solution conditions. A systematic characterization method identified the leading sources of instability within the enzyme-polymer conjugate system. We found that a physically crosslinked network prevented GOX leaching from the functionalized electrode. Pre-wetting the electrode also increased current outputs. A reduction in ferrocene-mediated current generation resulted from instability of the ferrocenium ion produced during operation. This issue could be solved through the use of freely diffusing ferrocene, which indicated a path toward improvement for enzyme-polymer conjugate systems with carefully engineered redox characteristics.