Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase.

Rapid, low-cost and efficient assays for penicillinase activity and inhibition are of vital importance for therapeutics and diagnostics of bacterial resistance to antibiotics. Herein we report a novel approach for on-line enzyme assays for penicillinase utilizing capillary electrophoresis-integrated immobilized enzyme reactors (CE-IMERs). The CE-IMERs are fabricated based on penicillinase-mediated alginate hydrogelation, allowing single-step in-situ encapsulation of enzymes without any additional manipulation process. We show that the fabricated CE-IMERs have high enzyme loading capacity with approximately 61.8% of the original penicillinase in the sol mixture being encapsulated in the "egg-box" hydrogel matrix. Excellent intraday and interday stability and batch-to-batch reproducibility are proved, indicating the reliability of our method for accurate on-line enzyme assays for penicillinase. Enzymatic activities and inhibition of immobilized penicillinase are analyzed, the results of which are in good agreement with those using free enzymes. The proposed method is successfully used for determination of penicillin in pork samples, indicating the potential applications for analysis of complicated real samples.