基于压电免疫生物传感器的大肠杆菌O157：H7检测方法研究
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摘要
大肠杆菌O157:H7作为食源性致病菌存在流行暴发的可能性和感染的严重性,急需研制一种快速准确的检测方法。本课题根据石英晶体微天平良好的质量响应特性,在QCM石英金电极表面采用四种不同的抗体固定方法,将抗体固定在电极表面,并通过两种分析方法来检测大肠杆菌O157:H7。根据不同浓度的菌体溶液响应值存在规律性来测定菌体浓度。建立了基于QCM的压电免疫生物传感器的微生物检测系统,能够快速准确的检测大肠杆菌O157:H7。
本课题的主要研究内容和研究结果如下:
1.采用四种抗体固定方法:硅烷固定法,聚乙烯亚胺固定法,3-巯基丙酸固定法和蛋白A固定法等将抗体固定于金电极表面,并通过两种分析方法:气相分析法和流动注射法来跟踪了解石英金电极表面每一步反应的频率变化。结果表明,蛋白A固定法与流动注射法联合使用是最佳的QCM检测大肠杆菌O157:H7方法。
2.利用蛋白A固定法和流动注射法联合使用时,蛋白A浓度和抗体的浓度对结果有影响,实验证明蛋白A最佳浓度为0.5mg/mL,抗体最佳浓度为1.0mg/mL。
3.选择最佳的抗体固定方法和分析方法,通过频率变化量的差异对不同浓度的大肠杆菌O157:H7溶液进行定量检测,检测限为7.33×10~3CFU/mL,检测范围为7.33×10~3CFU/mL～7.33×10~8CFU/mL,在此范围内,其频率变化值和浓度的响应方程式为:△f=6.9LogC-10.5。
以上实验研究,为今后压电免疫生物传感器在食品致病菌的检测研究应用打下基础,为从事食品安全研究与检测提供相应的技术支撑。
Escherichia coli O157:H7 is one of highly harmful food-borne disease-caused bacteria, for the probability of epidemic outbreak and the danger of infection. It is in dire need of a rapid and accurate method for E.coli O157:H7 detection. This research investigated the mass response characteristic of QCM for detection of E.coli Ol 57: H7. Four different immobilization methods were used to immobilize the monoclonal antibody onto a 8MHz AT-cut quartz crystal's surface, and two analytical procedures namely dip-and-dry and flow injection methods were used to detect E.coli O157.H7. The binding of target bacteria onto the immobilized antibody decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The biosensor was characterized with high sensitivity, relatively rapid and reliable analysis.Main contents and results were as follows:Firstly, four types of antibody immobilization methods included silanized layer, MPA self-assembled monolayer (SAM), PEI polymer membrane and Protein A were applied in this study. And learned the frequency shift from chemical reaction on the electrode's surface by dip-and-dry and flow injection methods. The results showed that the Protein A-based antibody immobilization method and flow injection method offered the best ways to provide a sensitive fast and reproducible detection results.Secondly, the effects of concentration of protein A and antibody on the frequency shift were also discussed. The results showed that the best protein A concentration was 0.50mg/mL, and the best antibody concentration was 1.0mg/mL.Thirdly, the immunosensor could detect E.coli O157:H7 with a detection limit of 7.33×10~3CFU/mL, in a range of 7.33×10~3CFU/mL to 7.33×10~8CFU/mL. The system was quite specific to E.coli Ol57: H7 and applicable for repetitive use after a regeneration step employing NaOH, HC1 and Piranha reagent.
本课题的主要研究内容和研究结果如下:
1.采用四种抗体固定方法:硅烷固定法,聚乙烯亚胺固定法,3-巯基丙酸固定法和蛋白A固定法等将抗体固定于金电极表面,并通过两种分析方法:气相分析法和流动注射法来跟踪了解石英金电极表面每一步反应的频率变化。结果表明,蛋白A固定法与流动注射法联合使用是最佳的QCM检测大肠杆菌O157:H7方法。
2.利用蛋白A固定法和流动注射法联合使用时,蛋白A浓度和抗体的浓度对结果有影响,实验证明蛋白A最佳浓度为0.5mg/mL,抗体最佳浓度为1.0mg/mL。
3.选择最佳的抗体固定方法和分析方法,通过频率变化量的差异对不同浓度的大肠杆菌O157:H7溶液进行定量检测,检测限为7.33×10~3CFU/mL,检测范围为7.33×10~3CFU/mL～7.33×10~8CFU/mL,在此范围内,其频率变化值和浓度的响应方程式为:△f=6.9LogC-10.5。
以上实验研究,为今后压电免疫生物传感器在食品致病菌的检测研究应用打下基础,为从事食品安全研究与检测提供相应的技术支撑。
Escherichia coli O157:H7 is one of highly harmful food-borne disease-caused bacteria, for the probability of epidemic outbreak and the danger of infection. It is in dire need of a rapid and accurate method for E.coli O157:H7 detection. This research investigated the mass response characteristic of QCM for detection of E.coli Ol 57: H7. Four different immobilization methods were used to immobilize the monoclonal antibody onto a 8MHz AT-cut quartz crystal's surface, and two analytical procedures namely dip-and-dry and flow injection methods were used to detect E.coli O157.H7. The binding of target bacteria onto the immobilized antibody decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The biosensor was characterized with high sensitivity, relatively rapid and reliable analysis.Main contents and results were as follows:Firstly, four types of antibody immobilization methods included silanized layer, MPA self-assembled monolayer (SAM), PEI polymer membrane and Protein A were applied in this study. And learned the frequency shift from chemical reaction on the electrode's surface by dip-and-dry and flow injection methods. The results showed that the Protein A-based antibody immobilization method and flow injection method offered the best ways to provide a sensitive fast and reproducible detection results.Secondly, the effects of concentration of protein A and antibody on the frequency shift were also discussed. The results showed that the best protein A concentration was 0.50mg/mL, and the best antibody concentration was 1.0mg/mL.Thirdly, the immunosensor could detect E.coli O157:H7 with a detection limit of 7.33×10~3CFU/mL, in a range of 7.33×10~3CFU/mL to 7.33×10~8CFU/mL. The system was quite specific to E.coli Ol57: H7 and applicable for repetitive use after a regeneration step employing NaOH, HC1 and Piranha reagent.
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