title: 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.
authors.affiliation: Faculty of Chemistry, Northeast Normal University, 5268 Renmin Road, Changchun, Jilin 130024, P. R. China. yangl330@nenu.edu.cn.
authors.forename: Zhengri
authors.initials: Z
authors.lastname: Yin
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zhao
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Tian
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.forename: Liping
authors.initials: L
authors.lastname: Guo
authors.forename: Li
authors.initials: L
authors.lastname: Yang
mesh_terms: D000809:Angiotensins / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006108:Graphite / Q000737:chemistry*; D010087:Oxides / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity
chemical_list: D000809:Angiotensins; D004800:Enzymes, Immobilized; D010087:Oxides; D012710:Serum Albumin, Bovine; D006108:Graphite