大肠杆菌O157：H7酶免疫传感器的研究

发布时间：2018-02-27 15:29

本文关键词： 酶免疫传感器大肠杆菌O157:H7 纳米金吸附法 L-半胱氨酸分子自组装电沉积　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 肠出血性大肠埃希菌(enterohemorrhagic Escherichia coli,EHEC)是近年来危害最严重的致病肠道菌之一,0157:H7是EHEC最主要的血清型,可引起出血性结肠炎(Hemorrhagic colonitis HC)、溶血性尿毒综合征(uremic syndrome hemolytic HUS)和血栓性血小板减少性紫癜(thrombotic thrombocytopenic purpura TTP)。HC是最常见的临床症状,HUS和TTP的病死率比较高,有时可高达30%以上。由于E.coli O157:H7感染剂量极低,且病情发展快,死亡率高,近年来欧美、日本等国都有多次爆发流行及散发病例报道,对人类健康构成严重威胁,因此建立特异、灵敏及快速的E.coli O157:H7检测方法显得尤为重要。目前E.coli O157:H7的主要检测方法有:生化法、免疫法、分子生物法三大类。我国目前对E.coli O157:H7检测大多数是采用生化法,从细菌分离、培养、鉴定需要l周左右的时间,耗时耗材,而且检出率较很低,显然对E.coli O157:H7等病原体的暴发诊断不适宜。其它方法如:胶体金试纸法灵敏度低且不能定量,而DNA探针技术、PCR技术等方法存在操作复杂、仪器设备及人员要求高的不足。酶免疫传感器是近年发展起来快速检测致病微生物的一项新技术,它将酶电极的电化学放大作用与免疫电极的特异性相结合,不但具有免疫反应的特异性和电化学分析的灵敏性,而且具有检测设备相对简单,使用方便,构建酶免疫电极方法灵活,体系容易集成化,微型化等优点。它在食品致病菌分析方面已取得良好效果。在酶免疫传感器的构建中,其关键技术是酶的固定,使固定化状态的酶呈现最大的生物活性,具有较高的稳定性可在较宽的pH范围或不同pH的下操作,并且尽量避免或减少酶的泄漏。本研究采用了三种不同的酶固定方法:吸附法、分子自组装膜技术、电沉积法建立快速检测E.coli O157:H7的新型酶免疫传感器,并对其性能特点分别进行研究探讨。第一部分吸附固定的E.coli O157:H7酶免疫传感器的研究目的:将免疫法特异性和电化学法灵敏性相结合,建立一种快速检测E.coli O157:H7的新型酶免疫传感器。方法:采用吸附法及抗原抗体特异性反应将E.coli O157:H7单克隆鼠抗固定在辣根过氧化物酶标记的羊抗鼠IgG修饰的玻碳电极表面,制备用于检测E.coli O157:H7的酶免疫传感器。通过循环伏安法和恒电位法测定E.coli O157:H7被固定在电极表面引起的电信号改变进行定量分析。结果:该酶免疫传感器的检出限为5×102cfu/mL,检测线性范围为103～105cfu/mL,相关系数为R=0.970,表观米氏常数Kamp p值为12.08mmol/L,传感器响应电信号2周后为初值的80%。结论:该传感器制备方法简单,检测时间短,操作简单,可进一步进行深入研究。第二部分纳米金/L-半胱胺酸修饰的E.coli O157:H7酶免疫传感器的研究目的:建立基于纳米金/L-半胱胺酸修饰的E.coli O157:H7酶免疫传感器。方法:采用分子自组装膜技术和抗原抗体特异性反应将E.coli O157:H7单克隆鼠抗固定在辣根过氧化物酶标记的羊抗鼠IgG/纳米金/L-半胱胺酸修饰的金电极表面,制备用于检测E.coli O157:H7的酶免疫传感器。通过循环伏安法和恒电位法测定E.coli O157:H7被固定在电极表面引起的电信号改变进行定量分析。结果:该酶免疫传感器的检出限为2×102cfu/mL,检测线性范围为103～105cfu/mL,相关系数为R=0.990,表观米氏常数Kamp p值为9.69mmol/L,传感器响应电信号2周后保持不变。结论:与前面的吸附法相比较L-半胱胺酸和纳米金的引入增强了电子在酶和电极之间的传导能力,提高了酶和抗体的固定量及生物活性,使酶免疫电极的灵敏度和稳定性增大。第三部分基于纳米金修饰的E.coli O157:H7酶免疫传感器的研究目的:建立基于纳米金修饰的E.coli O157:H7酶免疫传感器。方法:通过电沉积法及静电吸附作用和抗原抗体特异性反应将E.coli O157:H7单克隆鼠抗固定在辣根过氧化物酶标记的羊抗鼠IgG/纳米金修饰的玻碳电极表面,制备用于检测E.coli O157:H7的酶免疫传感器。通过循环伏安法和恒电位法测定E.coli O157:H7被固定在电极表面引起的电信号改变进行定量分析。结果:在含0.1mol/LPBS(pH7.4),1mmol/L二甲氨基甲基二茂铁,0.2mmol/L H2O2,30℃的底液中该酶免疫传感器的检出限为102cfu/mL,检测线性范围为103～105cfu/mL,相关系数为R=0.996,表观米氏常数Kampp值为9.02mmol/L,传感器响应电信号2周后为初值的85%。结论:该传感器制备方法与前两种相比更简单,酶的固定量更多,灵敏度更高。综上所述,酶的固定方法对制备E.Coli O157:H7酶免疫生物传感器非常重要,在吸附法中未修饰金纳米的酶免疫传感器检测的电信号较弱、稳定性与灵敏度较低,而在分子自组装方法和电沉积方法中,纳米金颗粒的表面效应使羊抗鼠IgG-HRP被大量吸附并保持其生物活性,且纳米金良好的导电性也提高了酶免疫生物传感器的电流响应性能。在三种方法中电沉积法由于电极表面沉积纳米金颗粒数量多,在固定酶和抗体同时保持其生物活性和稳定性,增强电子传导信号优于前两种方法。
[Abstract]:Enterohemorrhagic Escherichia coli (enterohemorrhagic Escherichia, coli, EHEC) is one of the most serious hazards of pathogenic intestinal bacteria in recent years, 0157:H7 EHEC is the most predominant serotypes, can cause hemorrhagic colitis (Hemorrhagic colonitis HC), hemolytic uremic syndrome (uremic syndrome hemolytic HUS) and thrombotic thrombocytopenic purpura (thrombotic thrombocytopenic purpura TTP.HC) is the most common clinical symptoms of HUS and TTP, the mortality rate is relatively high, sometimes as high as 30%. E.coli O157:H7 due to the low infectious dose, and rapid progression and high mortality, in recent years, Europe and the United States, Japan and other countries have repeatedly reported outbreaks and sporadic cases, pose a serious threat for human health, therefore establish a specific, sensitive and rapid E.coli O157:H7 detection method is particularly important.
At present, the main method for detection of E.coli O157:H7: biochemical method, immunoassay, molecular biological method three categories. China's current E.coli O157:H7 detection is most by biochemical method, separation, cultivation from bacteria, identification of needs l weeks, and time-consuming consumables, the detection rate is very low, obviously on E.coli O157:H7 the diagnosis of pathogens outbreak is not appropriate. Other methods such as: colloidal gold test method of low sensitivity and not quantitative, and DNA probe technology, PCR technology and other methods such as complex operation, lack of equipment and personnel requirements.
Enzyme immunosensor is a new technique developed in recent years, the rapid detection of pathogenic microorganisms, it will amplify the specific electrochemical enzyme electrode effect and immune electrode combination, sensitivity analysis not only has the specificity of immunoreactions and electrochemical testing equipment, and has a relatively simple, easy to use, flexible construction of enzyme electrode method system, easy integration and miniaturization.
It has achieved good results in the analysis of pathogens in food. In the construction of enzyme immunosensor, the key technique is to fix enzyme, immobilized enzyme showed the largest state of biological activity, has high stability in operation under a wide range of pH or pH, and try to avoid or reduce enzyme leakage. This study used three different fixation methods: enzyme adsorption, molecular self-assembly technique, electrodeposition method to establish the rapid detection of E.coli O157:H7 enzyme immunosensor, and studied on its performance characteristics.
Study on the first part adsorption and immobilization of E.coli O157:H7 enzyme immunosensor
Objective: immune specificity and sensitivity of electrochemical method combined with the establishment of a new type of enzyme immunosensor for rapid detection of E.coli O157:H7. Methods: the method of adsorption and antigen antibody specific reaction to the surface of a glassy carbon electrode E.coli O157:H7 monoclonal mouse fixed on horseradish peroxidase labeled Goat anti mouse IgG modification, preparation for enzyme immunosensor for detection of E.coli O157:H7. E.coli O157:H7 was immobilized on the electrode surface caused by signal changes were quantitatively analyzed by cyclic voltammetry and potentiostatic method. Results: the enzyme immunosensor for the detection limit of 5 * 102cfu/mL, the linear range of detection is 103 ~ 105cfu/mL, the correlation coefficient is R=0.970, the apparent Michaelis constant the Kamp p value is 12.08mmol/L, the sensor response signal after 2 weeks was 80%. conclusion: the initial sensor preparation method is simple, short detection time, simple operation, can be a Step in - depth study.
Study on the second part of the E.coli O157:H7 enzyme immunization sensor modified by nanoscale /L- cysteamine
Objective: to establish a E.coli O157:H7 enzyme immunosensor gold nanoparticles /L- cysteine modified. Methods: using molecular self-assembly technique and antigen antibody reaction to the surface of the gold electrode E.coli O157:H7 monoclonal mouse fixed on horseradish peroxidase labeled Goat anti mouse IgG/ gold nanoparticles /L- cysteine modification, preparation preparation for enzyme immunosensor for detection of E.coli O157:H7. E.coli O157:H7 was immobilized on the electrode surface caused by signal changes were quantitatively analyzed by cyclic voltammetry and potentiostatic method. Results: the detection of the enzyme immunosensor for the detection limit is 2 * 102cfu/mL, the linear range is 103 ~ 105cfu/mL, the correlation coefficient was R=0.990, apparent Michaelis constant Kamp p value is 9.69mmol/L, the sensor response signals remained unchanged after 2 weeks. Conclusion: the introduction of adsorption method and compared with the L- in front of the cysteine and gold nanoparticles enhanced power The conduction ability between the enzyme and the electrode increases the fixed amount and biological activity of the enzyme and antibody, and increases the sensitivity and stability of the enzyme immunoelectrode.
The third part of E.coli O157:H7 enzyme immunosensor based on nano gold modification
Objective: to establish a E.coli O157:H7 enzyme immunosensor based on gold nanoparticles modified. Methods: by electrodeposition and electrostatic adsorption and antigen antibody reaction to the surface of a glassy carbon electrode E.coli O157:H7 monoclonal mouse fixed on horseradish peroxidase labeled Goat anti mouse IgG/ gold nanoparticles, preparation for enzyme immunosensor for detection of E.coli O157:H7. E.coli O157:H7 was immobilized on the electrode surface caused by signal changes were quantitatively analyzed by cyclic voltammetry and potentiostatic method. Results: in 0.1mol/LPBS (pH7.4), 1mmol/L two methylaminomethyl two ferrocene, detected the enzyme immunosensor 0.2mmol/L bottom H2O2,30 C in the detection limit is 102cfu/mL. The linear range is 103 ~ 105cfu/mL, the correlation coefficient is R=0.996, the apparent Michaelis constant Kampp value is 9.02mmol/L, the sensor response signal after 2 weeks for the initial 85%. on the The preparation method of the sensilla is simpler than the first two, with more immobilization and higher sensitivity.
In summary, fixation enzyme E.Coli O157:H7 enzyme immunosensor is very important for the preparation of gold nanoparticles modified enzyme immunosensor in adsorption method in the detection of weak signal, stability and low sensitivity, and the molecular self-assembly method and electrodeposition method, surface effect of nanometer gold particles made of Goat anti mouse IgG-HRP was adsorbed and retained its bioactivity, good conductivity and gold nanoparticles also improves the response performance of current enzyme immunosensor. In the three method in electro deposition of gold nanoparticles deposited on the electrode surface due to the quantity, in the immobilization of enzymes and antibodies while maintaining its biological activity and stability, enhanced electronic conduction the signal is better than the former two methods.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

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