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 interday 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.Abbreviations: GO, graphene oxide; IMER, immobilized enzyme microreactor; L-LDH, L-lactate dehydrogenase; NAD + , β-Nicotinamide adenine dinucleotide hydrate; NADH, β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate; PDDA, poly (diallyldimethylammonium chloride); UV-vis, ultraviolet-visible * These authors contributed equally in this work.Color online: See the article online to view Figs. 1-3 and 5 in color.

Recent (2018–2020) development in capillary electrophoresis

“…To enhance enzyme loading, nanomaterials can be used as the support, providing high relative surface areas for higher loading capacity of the enzyme molecules. One good example is to use the hydrophobic interaction and hydrogen bonding between GO and the negatively charged enzymes to facilitate CE-IMER [56]. In this method, poly (diallyldimethylammonium chloride) (PDDA) was firstly adsorbed onto the capillary wall to form a film and attract GO via electrostatic interaction.…”

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

“…Ninety percent of the initial enzymatic activity remained after 5 days of usage which is far better than for the free enzyme. The IMER was successfully applied to determine pyruvate in beer samples [82].…”

Enzymatic Bioreactors: An Electrochemical Perspective

“…A disadvantage of wall-coated microreactors is that lower enzyme loadings occur in comparison with other microreactors. In order to increase enzyme loadings, different strategies can be used, including the deposition of nanostructured materials such as gold nanoparticles [107], nanosprings [131], graphene oxide [132] and dopamine [133,134] on the wall as well as using multiple layers immobilised with enzymes attached to the surface of the wall [106,135,136]. For example, Valikhani et al constructed a wall-coated microreactor with the use of silica nanosprings, comprised of helical silicon dioxide (SiO 2 ) structures grown via a chemical deposition process.…”