Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage.

The need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW) received in the proton-exchanged (SO ₃ H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA ⁺ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA ⁺ : SO ₃ ^- ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 and 500 cm ^-1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA ⁺ modified Aquivion ionomers were able to reach maximum power densities (P _max ) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA ⁺ modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW ^-1 ), where the greater density of SO ₃ ^- moieties in the Aquivion materials produces an environment more favorable to mass transport and higher TBA ⁺ concentrations.