Suraniti E., Studer V., Sojic N., Mano N.
Anal. Chem. 2011, 83, 2824–2828

Immobilization and electrical wiring of enzymes is of particular importance for the elaboration of efficient biosensors and can be cumbersome. Here, we report a fast and easy protocol for enzyme immobilization and as a proof of concept we applied it to the immobilization of Bilirubin Oxidase, a labile enzyme. In the first step, bilirubin oxidase is mixed with a redox hydrogel “wiring” the enzyme reaction centers to electrodes. Then, this adduct is covered by an outer layer of PEGDA made by photoinitiated polymerization of poly(ethylene-glycol) diacrylate (PEGDA) and a photoclivable precursor, DAROCUR®. This 2 steps protocol is 18 times faster than the current state-of-the-art protocol and leads to currents 25 % higher. In addition, the outer layer of PEGDA acts as a protective layer increasing the lifetime of the electrode by 100 % when operating continuously for 2000 s and by 60 % when kept in dry state for 24 hours. This new protocol is particularly appropriate for labile enzymes that quickly denaturate. In addition, by tuning the ratio PEGDA/DAROCUR®, it is possible to make the enzyme electrode even more active or more stable.