Capillary electrophoresis‐integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L‐lactate dehydrogenase via hydrophobic interactions

Abstract We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE‐integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L ‐lactate dehydrogenase ( L ‐LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV‐vis are used to characterize the fabricated CE‐IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter‐day and batch‐to‐batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five‐day usage. The measured K m values of pyruvate and NADH of the immobilized L ‐LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π‐π stacking is significant between the GO backbone and the aromatic residues of L ‐LDH and favorable to fabrication of CE‐integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.