Investigation And Application Of Immobilized Enzyme Reactor And Paper-based Sensor Based On Penicillinase

Penicillinase is an important enzyme in the class of?-lactamase which hydrolyzes and inactivates penicillin.Because of this characteristic,penicillinase have been widely applied in a variety of research fields of analytical chemistry.Focusing on the penicillinase in immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation and fabrication of paper-based sensor for rapid naked-eye detection of penicillin.The contents were listed below:1.A novelcapillaryelectrophoresis-integratedimmobilizedenzymereactor?CE-integrated IMER?is developed using single-step in situ penicillinase-mediated alginate hydrogelation and enzyme encapsulation.Alginate hydrogelation with“egg-box”structure is triggeredinside a capillary with releasing of Ca²⁺by changing the pH of the sol solution,which is accomplished in situ by penicillinase catalyzed hydrolysis reaction of penicillin G to produce penicilloic acid.Penicillinase and any other enzyme initially contained in the sol solution[e.g.,xanthine oxidase?XO?]are efficiently encapsulated as the hydrogel network grows,forming CE-integrated IMERs without any additional manipulation process.Approximately 61.8%of the original enzyme in the sol mixture can be encapsulated,indicating high loading capacity for the CE-integrated IMERs.The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility,and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays.Enzymatic activities and inhibition of immobilized penicillinase and XO are analyzed,and the results are compared with those using free enzymes.The proposed method is a simple,efficient,and universal approach for online CE assays with immobilized enzymes,which can be widely applied in bioanalysis.2.We developed a paper-based sensor for rapid naked-eye detection of penicillin.In our work,we ultilized a commercial filter paper as support,and used sodium alginate containing calcium carbonate as the medium,utilizing that penicillinase catalyzed penicillin G generating penicilloic acid,which can induce calcium carbonate in alginate into calcium ions forming gel,and the degree of gel formation is different,and the fluidity of the liquid is variant in the system,causing that the diameter of liquid dropped on paper is different.The larger the penicillin concentration,the smaller the diameter.We measured the linear equation.In addition,the sensor was successfully applied in penicillin detection of milk and pork sample,with the recovery ranged from 92%to 112%.The above results indicate the paper-based sensor is expected to applied in the on-site detection of the penicillin.