發(fā)布時(shí)間：2018-02-27 15:29

  本文關(guān)鍵詞： 酶免疫傳感器 大腸桿菌O157:H7 納米金 吸附法 L-半胱氨酸 分子自組裝 電沉積　出處：《華中科技大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 腸出血性大腸埃希菌(enterohemorrhagic Escherichia coli,EHEC)是近年來危害最嚴(yán)重的致病腸道菌之一,0157:H7是EHEC最主要的血清型,可引起出血性結(jié)腸炎(Hemorrhagic colonitis HC)、溶血性尿毒綜合征(uremic syndrome hemolytic HUS)和血栓性血小板減少性紫癜(thrombotic thrombocytopenic purpura TTP)。HC是最常見的臨床癥狀,HUS和TTP的病死率比較高,有時(shí)可高達(dá)30%以上。由于E.coli O157:H7感染劑量極低,且病情發(fā)展快,死亡率高,近年來歐美、日本等國(guó)都有多次爆發(fā)流行及散發(fā)病例報(bào)道,對(duì)人類健康構(gòu)成嚴(yán)重威脅,因此建立特異、靈敏及快速的E.coli O157:H7檢測(cè)方法顯得尤為重要。 目前E.coli O157:H7的主要檢測(cè)方法有:生化法、免疫法、分子生物法三大類。我國(guó)目前對(duì)E.coli O157:H7檢測(cè)大多數(shù)是采用生化法,從細(xì)菌分離、培養(yǎng)、鑒定需要l周左右的時(shí)間,耗時(shí)耗材,而且檢出率較很低,顯然對(duì)E.coli O157:H7等病原體的暴發(fā)診斷不適宜。其它方法如:膠體金試紙法靈敏度低且不能定量,而DNA探針技術(shù)、PCR技術(shù)等方法存在操作復(fù)雜、儀器設(shè)備及人員要求高的不足。 酶免疫傳感器是近年發(fā)展起來快速檢測(cè)致病微生物的一項(xiàng)新技術(shù),它將酶電極的電化學(xué)放大作用與免疫電極的特異性相結(jié)合,不但具有免疫反應(yīng)的特異性和電化學(xué)分析的靈敏性,而且具有檢測(cè)設(shè)備相對(duì)簡(jiǎn)單,使用方便,構(gòu)建酶免疫電極方法靈活,體系容易集成化,微型化等優(yōu)點(diǎn)。 它在食品致病菌分析方面已取得良好效果。在酶免疫傳感器的構(gòu)建中,其關(guān)鍵技術(shù)是酶的固定,使固定化狀態(tài)的酶呈現(xiàn)最大的生物活性,具有較高的穩(wěn)定性可在較寬的pH范圍或不同pH的下操作,并且盡量避免或減少酶的泄漏。本研究采用了三種不同的酶固定方法:吸附法、分子自組裝膜技術(shù)、電沉積法建立快速檢測(cè)E.coli O157:H7的新型酶免疫傳感器,并對(duì)其性能特點(diǎn)分別進(jìn)行研究探討。 第一部分吸附固定的E.coli O157:H7酶免疫傳感器的研究 目的:將免疫法特異性和電化學(xué)法靈敏性相結(jié)合,建立一種快速檢測(cè)E.coli O157:H7的新型酶免疫傳感器。方法:采用吸附法及抗原抗體特異性反應(yīng)將E.coli O157:H7單克隆鼠抗固定在辣根過氧化物酶標(biāo)記的羊抗鼠IgG修飾的玻碳電極表面,制備用于檢測(cè)E.coli O157:H7的酶免疫傳感器。通過循環(huán)伏安法和恒電位法測(cè)定E.coli O157:H7被固定在電極表面引起的電信號(hào)改變進(jìn)行定量分析。結(jié)果:該酶免疫傳感器的檢出限為5×102cfu/mL,檢測(cè)線性范圍為103～105cfu/mL,相關(guān)系數(shù)為R=0.970,表觀米氏常數(shù)Kamp p值為12.08mmol/L,傳感器響應(yīng)電信號(hào)2周后為初值的80%。結(jié)論:該傳感器制備方法簡(jiǎn)單,檢測(cè)時(shí)間短,操作簡(jiǎn)單,可進(jìn)一步進(jìn)行深入研究。 第二部分納米金/L-半胱胺酸修飾的E.coli O157:H7酶免疫傳感器的研究 目的:建立基于納米金/L-半胱胺酸修飾的E.coli O157:H7酶免疫傳感器。方法:采用分子自組裝膜技術(shù)和抗原抗體特異性反應(yīng)將E.coli O157:H7單克隆鼠抗固定在辣根過氧化物酶標(biāo)記的羊抗鼠IgG/納米金/L-半胱胺酸修飾的金電極表面,制備用于檢測(cè)E.coli O157:H7的酶免疫傳感器。通過循環(huán)伏安法和恒電位法測(cè)定E.coli O157:H7被固定在電極表面引起的電信號(hào)改變進(jìn)行定量分析。結(jié)果:該酶免疫傳感器的檢出限為2×102cfu/mL,檢測(cè)線性范圍為103～105cfu/mL,相關(guān)系數(shù)為R=0.990,表觀米氏常數(shù)Kamp p值為9.69mmol/L,傳感器響應(yīng)電信號(hào)2周后保持不變。結(jié)論:與前面的吸附法相比較L-半胱胺酸和納米金的引入增強(qiáng)了電子在酶和電極之間的傳導(dǎo)能力,提高了酶和抗體的固定量及生物活性,使酶免疫電極的靈敏度和穩(wěn)定性增大。 第三部分基于納米金修飾的E.coli O157:H7酶免疫傳感器的研究 目的:建立基于納米金修飾的E.coli O157:H7酶免疫傳感器。方法:通過電沉積法及靜電吸附作用和抗原抗體特異性反應(yīng)將E.coli O157:H7單克隆鼠抗固定在辣根過氧化物酶標(biāo)記的羊抗鼠IgG/納米金修飾的玻碳電極表面,制備用于檢測(cè)E.coli O157:H7的酶免疫傳感器。通過循環(huán)伏安法和恒電位法測(cè)定E.coli O157:H7被固定在電極表面引起的電信號(hào)改變進(jìn)行定量分析。結(jié)果:在含0.1mol/LPBS(pH7.4),1mmol/L二甲氨基甲基二茂鐵,0.2mmol/L H2O2,30℃的底液中該酶免疫傳感器的檢出限為102cfu/mL,檢測(cè)線性范圍為103～105cfu/mL,相關(guān)系數(shù)為R=0.996,表觀米氏常數(shù)Kampp值為9.02mmol/L,傳感器響應(yīng)電信號(hào)2周后為初值的85%。結(jié)論:該傳感器制備方法與前兩種相比更簡(jiǎn)單,酶的固定量更多,靈敏度更高。 綜上所述,酶的固定方法對(duì)制備E.Coli O157:H7酶免疫生物傳感器非常重要,在吸附法中未修飾金納米的酶免疫傳感器檢測(cè)的電信號(hào)較弱、穩(wěn)定性與靈敏度較低,而在分子自組裝方法和電沉積方法中,納米金顆粒的表面效應(yīng)使羊抗鼠IgG-HRP被大量吸附并保持其生物活性,且納米金良好的導(dǎo)電性也提高了酶免疫生物傳感器的電流響應(yīng)性能。在三種方法中電沉積法由于電極表面沉積納米金顆粒數(shù)量多,在固定酶和抗體同時(shí)保持其生物活性和穩(wěn)定性,增強(qiáng)電子傳導(dǎo)信號(hào)優(yōu)于前兩種方法。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378

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