Reversible covalent enzyme immobilization methods for reuse of carriers

Reversible immobilization techniques which allow for multiple use of the carrier are relevant for applications, such as enzymatic microreactors, biosensors with specific setups and for expensive carriers such as superparamagnetic particles. The activity of immobilized enzymes reduces with time, so that the introduction of fresh immobilized enzyme becomes necessary. Thus, methods for reversible immobilization and multiple carrier reuse can help to reduce purchase costs and facilitate reactor construction. In this work, we present a method that makes use of the reduction and oxidation of cystamine, a cleavable linker with disulfide bond and amine functionality. For a proof of principle, alpha-chymotrypsin was immobilized on polyethylene glycol with terminal epoxy groups using cystamine as a crosslinker. The enzyme was highly active and could be used in repeated cycles. After the enzymatic reaction was demonstrated, alpha-chymotrypsin was cleaved off the particle by reducing agents. The resulting thiols on the particle surface were oxidized to disulfides by means of cysteamine, the reduction product of cystamine. This way, an almost complete oxidation of surface thiols with cysteamine was possible, restoring amine functionalization for further reactions. Reduction and oxidation were repeated several times without a decrease in the extent of amine coupling. Finally, immobilization of alpha-chymotrypsin could be repeated with results comparable to first run.