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目的建立酶热生物传感器法用于快速检测酸奶中β-内酰胺酶。方法酸奶样品于4℃下5 000 r/min离心10 min,取中间层清液与一定量青霉素G混合,室温震荡反应3 h,使样品中β-内酰胺酶充分酶解加入的青霉素G底物,然后直接通过酶热生物传感器进行分析。酶热生物传感器中装备有专一性酶柱,能够特异性地与青霉素G进行反应,并对其含量进行测定。根据震荡反应前后酸奶样品中青霉素G含量的变化,可以推算出酸奶样品中β-内酰胺酶的活性。结果该方法样品检出限为2 U/ml,定量限为4 U/ml;线性范围4～20 U/ml,3个加标水平(6 U/ml、10 U/ml、18 U/ml)的平均回收率在85%～110%之间,相对标准偏差小于20%。结论本文所建立的酶热生物传感器法能够快速测定酸奶中β-内酰胺酶活性,与传统微生物法相比,操作简单、仪器可便携,显著节约了检测时间和成本,是传统方法的有力补充,更适合于大规模样品筛查和现场检测。
Objective To establish an enzyme thermosensor biosensor for the rapid detection of β-lactamase in yogurt. Methods The yoghurt samples were centrifuged at 5 000 r / min for 10 min at 4 ℃. The supernatant of the yogurt was mixed with a certain amount of penicillin G and reacted for 3 h at room temperature. The samples were thoroughly digested with β-lactamase to obtain penicillin G The material is then analyzed directly by the enzyme thermosensor. Enzyme thermal biosensor equipped with a specific enzyme column, can be specifically reacted with penicillin G, and its content was determined. According to changes in penicillin G content of yogurt samples before and after shaking reaction, the activity of beta-lactamase in yogurt samples can be calculated. Results The detection limit of the method was 2 U / ml and the limit of quantification was 4 U / ml. The linear range was 4 ~ 20 U / ml. ) Of the average recovery was 85% to 110%, the relative standard deviation of less than 20%. Conclusion The enzyme thermosensor established in this paper can rapidly determine the β-lactamase activity in yoghurt. Compared with the traditional microbial method, the enzyme thermosensor has the advantages of simple operation, portable instrument, significant saving on testing time and cost, and is a powerful supplement to traditional methods. More suitable for large-scale sample screening and on-site testing.