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摘要
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 K-m (0.24 +/- 0.02 mM) and the maximum velocity V-max (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.
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