Development of enzyme immobilized monolith micro-reactors integrated with microfluidic electrochemical cell for the evaluation of enzyme kinetics

This paper describes a simple and efficient method for producing an on-chip enzyme immobilized monolith micro-reactor that integrates a microfluidic electrochemical cell for rapid characterization of enzymatic kinetics. The monolith was generated using a sol-gel method, followed by PEI functionalization and enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. Using the proposed immobilization strategy, a glucose oxidase (GOD) immobilized monolith micro-reactor has been produced with the controllable porosity that gives better enzyme kinetics compared to previously reported devices. This can be attributed to a favourable enzyme-substrate affinity in which more than 98% of the immobilized enzyme remains in an active conformation. The kinetic studies conducted have identified that a similar value of the k (cat) is obtained for immobilized GOD (13.4 s(-1)) and GOD free in solution (14 s(-1)) whilst the immobilized Michaelis constant K (m(app)) (7.2 mM) is similar to 4 times lower than GOD in solution (25 mM). In addition, the immobilized GOD exhibits increased stability, retaining at least 95% of the initial activity when stored of 30 days at 4A degrees C, compared to only 60% for GOD in solution. Furthermore, the same enzyme immobilization strategy has been used for choline oxidase immobilization and similar kinetics to choline oxidase in solution were observed, once again indicating better maintenance of the enzyme conformation provided by the proposed method.