A capillary electrophoresis-based immobilized enzyme reactor using graphene oxide as a support via layer by layer electrostatic assembly

Abstract

A novel capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) using graphene oxide (GO) as a support was developed by using a simple and reliable immobilization procedure based on layer by layer electrostatic assembly. Using trypsin as a model enzyme, the performance of the fabricated CE-based IMERs was evaluated. Various conditions, including trypsin concentration, trypsin coating time, number of trypsin layers and buffer pH, were investigated and optimized. The Michaelis constant Km (0.24 ± 0.02 mM) and the maximum velocity Vmax (0.32 ± 0.04 mM s(-1)) were determined using the CE-based IMERs, and the values are consistent with those obtained using free trypsin, indicating that enzyme immobilized via the proposed approach does not cause a significant structural change of the enzyme or any reduction of enzyme activity. The presented CE-based IMERs exhibit excellent reproducibility with RSD less than 2.8% over 20 runs, and still remain 79.5% of the initial activity after five days with more than 100 runs. Using the proposed CE-based IMERs, the digestion of angiotensin was completed within 3 min, while quite a number of trypstic peptides were observed for BSA on-line digestion with an incubation time of 30 min. As identified by MS analysis, the online digestion products of BSA using the present CE-based IMER are comparable with those obtained using free trypsin digestion for 12 h incubation. It is indicated that the present immobilization strategy using GO as a support is reliable and practicable for accurate on-line analysis and characterization of peptides and proteins.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…[128][129][130][131][132][133][134][135]. Another suitable approach was the adsorption of either the enzyme [136][137][138][139][140][141] or enzyme previously immobilized to gold nanoparticles [142][143][144] to a capillary which had been coated by polybrene or poly(ethyleneimine). Alginate encapsulation of the enzymes prior to adsorption to the capillary has also been applied [145].…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…Despite this, immobilized enzyme reactors relying on electrostatic interactions have been reported to maintain up to 73% of the original enzyme activity after 50 assays [22]. A separate approach using graphene oxide to immobilize cationic trypsin enabled the incubation time for a trypsin digest to be reduced from 12 h for the free solution to 30 min with the immobilized enzyme [17]. …”

Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions

“…After preconditioning, 2 cm of the capillary was coated with poly (diallyldimethylammonium chloride) (PDDA). This plug was in contact with the capillary for one hour after which graphene oxide was introduced and let to react with the PDDA part of the capillary for 30 min followed by trypsin immobilization (Fig. ).…”

“…Schematic representation of trypsin immobilization using graphene oxide as a support layer onto the capillary wall. Adapted with permission from .…”