【作者】 程欲晓；

【导师】 金利通；

【作者基本信息】 华东师范大学 ， 分析化学， 2009， 博士

【摘要】 水和食品中细菌的检测，特别是致病性细菌的检测，对于控制传染病、保护环境卫生和人民群众身体健康都有着重要的意义。大肠杆菌是人及各种动物肠道中的常居菌，常随粪便从人及动物体内排出，广泛散播于自然界。大部分大肠杆菌没有致病性，但是部分能产生肠毒素，导致人体肠胃炎等疾病。特别是O157型的大肠杆菌会引起腹泻、出血性大肠炎和溶血尿毒症等疾病。当他入侵到肠道外的其他组织器官时，会引起尿道炎、膀胱炎、阑尾炎等，对于免疫力下降的病人还可引起败血症。在卫生质量的评价和控制中，通常采用大肠杆菌作为指示菌，利用对指示菌的检测和控制来了解水体或食品等的受污染状况，从而评价其质量以保证卫生安全。因此，建立快速检测大肠杆菌的新方法成为环境监测和食品卫生领域专家的一个巨大挑战。电化学／生物传感器是近二、三十年来发展较快的电分析化学测试和检测技术，具有快速、准确、灵敏等优点。其所测的信号通常是电位、电流、电阻等的变化，可以直接测量，便于仪器自动化、小型化和智能化。此外，将纳米材料应用于电化学／生物传感器的制备，可以增大传感器电流响应，降低检测限，提高灵敏度，为电化学／生物传感器提供了无穷的发展空间。针对细菌的生物学特性，电化学／生物传感已广泛被应用于食品及环境中各种病菌中的快速检测，具有极大的应用前景。本论文是导师金利通教授领导的世博会专项和华东师范大学优秀博士生基金项目的部分工作内容。论文基于纳米材料构筑了多种性能稳定、灵敏度高的电化学／生物传感器，并把传感器应用于大肠杆菌的快速检测，为水体和食品中大肠杆菌的快速检测提供了新方法，还以这些电化学／生物传感器为基础开发研制了电化学细菌快速分析仪。本论文内容共分为五部分：Ⅰ．绪论（第一章）本部分内容简要介绍大肠杆菌的环境卫生学意义、生物学特性以及致病性，对当前大肠杆菌检测的研究现状与进展做了较为详细的概括，同时对电化学／生物传感器的工作原理及其在大肠杆菌检测中的应用进行详细的介绍与综述。Ⅱ．纳米修饰电极快速电化学检测大肠杆菌的研究（第二、三章）大肠杆菌在含有诱导剂异丙基硫代-β-D-半乳糖苷（IPTG）的培养液中培养，能够产生β-D-半乳糖苷酶（β-gal）；在膜渗透剂的作用下，β-gal从细胞体内释放到溶液中并催化底物对氨基酚-β-D-半乳糖苷（PAPG）水解产生对氨基酚（PAP），根据PAP在修饰电极上的响应电流可以确定大肠杆菌的浓度。第二章制备铂纳米颗粒修饰电极（PtNP／GCE），并应用于β-gal活性的检测，实现了对大肠杆菌的快速检测。实验中考察了工作电位、底物浓度以及膜渗透剂等条件对检测结果的影响。PtNP／GCE提高了PAP的检测灵敏度，从而提高大肠杆菌的检测灵敏度，并且缩短了分析时间。与传统方法相比，能更好地满足食品安全、公共卫生和临床医学等领域中快速检测的要求。第三章制备了多壁碳纳米管（MWNTs）／Nafion复合膜修饰玻碳电极，并对大肠杆菌进行快速安培检测。由于Nafion的阳离子交换能力和MWNTs的电催化能力，MWNTs／Nafion修饰电极提高对氨基酚（PAP）的检测灵敏度；对大肠杆菌进行浓缩和预培养可进一步提高了其检测灵敏度。此外，对实际水样进行测定，结果与标准方法（平板技术法）具有较好的一致性。Ⅲ．酪氨酸酶（Tyr）生物传感器快速检测大肠杆菌的研究（第四、五章）生物传感器具有专一、易操作、设备简单以及测量快速准确等优点，因此得到广泛的应用。本部分制备了两种Tyr生物传感器，并应用于β-gal活性的检测，进而快速检测大肠杆菌。第四章我们把Tyr固定在MWNTs-壳聚糖（Chit）复合膜修饰的玻碳电极上，制备了Tyr／MWNTs-Chit／GCE生物传感器。该生物传感器对苯酚具有高的灵敏度、宽的线性响应范围和低的检测限。把该生物传感器应用于大肠杆菌的检测，其检测的原理是把β-gal作为大肠杆菌的标记物，催化phenol-β-D-galactopyranoside（PG）水解产生苯酚，通过生物传感器检测苯酚可得到大肠杆菌的浓度。实验结果表明，电流响应与大肠杆菌的浓度在10⁴～10⁷cfu／mL范围内呈线性关系。对大肠杆菌进行5．0h的预培养，可以检测出低至10cfu／mL的大肠杆菌。第五章基于Fe₃O₄磁性纳米颗粒（MNPs）-碳纳米管（CNTs）复合物，制备了一种新型的Tyr生物传感器，并进一步应用于流动注射法（FIA）检测大肠杆菌。CNTs表面修饰了阳离子聚合物聚二甲基二丙烯氯化铵（PDDA），因此带负电荷的Fe₃O₄MNPs在静电作用下可以均匀地吸附于CNTs表面上。将制备的Fe₃O₄ MNPs-CNTs纳米复合材料修饰于光玻碳电极上后，把Tyr通过戊二醛交联固定在此修饰电极上制得了Tyr／Fe₃O₄ MNPs-CNTs／GCE生物传感器。该生物传感器对苯酚的检测具有宽的线性范围(1．0×10^-8～3．9×10^-5M)、低的检测限(5．0×10^-9M)以及高的灵敏度（516mA／M）。把该Tyr生物传感器与FIA相结合应用于大肠杆菌的快速检测。实验结果表明，细菌浓度在20到1×10⁵cfu／mL范围内与电流响应呈良好的线性关系，其最低检测限达10cfu／mL，总的分析时间在4h内。这种生物传感器-FIA检测系统在快速临床诊断和水质分析方面具有良好的应用前景。Ⅳ．Au纳米标记物增强电化学免疫分析大肠杆菌的研究（第六章）本章在Au纳米颗粒表面修饰了辣根过氧化酶（HRP）标记的大肠杆菌抗体制备了一种新型的Au纳米标记物，并将该纳米标记物应用于增强电化学免疫分析大肠杆菌。经过酶联免疫反应后，Au纳米标记物、免疫磁性颗粒（IMB）和大肠杆菌形成了IMB／抗体-大肠杆菌-Au纳米标记物的三明治式免疫复合物。以3，3，5，5-四甲基联苯二胺（TMB）溶液作为底物，采用电化学与流动注射检测（FIA）相结合的技术测定HRP的活性。检测到的电流大小与免疫复合物上HRP的量成正比，从而与大肠杆菌的浓度成正比。Au纳米颗粒增加了HRP的负载量，增强电化学信号，大大提高了大肠杆菌的检测灵敏度。实验结果表明，大肠杆菌浓度在1．0×10²～5．0×10⁵cfu／mL范围内与电流大小成线性相关，最低检测限达50cfu／mL，总的分析时间比其他方法短，在1h内就能完成对大肠杆菌的快速检测。Ⅴ．电化学细菌快速分析仪的研制（第七章）本章基于细菌的生物学特性以及电化学工作原理开发研制了细菌快速分析仪，仪器对细菌的检测具有快速、灵敏、准确等特点，不但可检测水体中细菌总量，而且还可检测水体中的大肠杆菌的浓度。饮用水中细菌总数的检测范围为1．0×10²cfu／mL～1．0×10⁵cfu／mL，检测限为100cfu／mL；并且能在5个小时内检测水体中20cfu／mL～1．0×10⁶cfu／mL的大肠杆菌，检测限为20cfu／mL。该仪器体积小，携带方便，操作简便，性能稳定，且质量可靠。更多还原

【Abstract】 The timely detection of pathogens in foods and waters is a subject of greatimportance for the protection of environmental sanitation and our health.Escherichiacoli （E.coli） which are found in large numbers among the intestinal of humans andother warm-blooded animals spread abroad in natural environment.Most of E.colican not cause diseases,while part of them produces enterotoxin and are the majorcause of infection outbreaks with serious consequences.In particular,the serotypeO157 associate with several human diseases including diarrhoea,hemorrhagic colitisand hemolytic-uremic syndrome.The presence of E.coli is routinely used as anindicator to monitor potential enteric pathogen contamination of waters and foods.Therefore,it is a challenge for experts to develop new methods for rapid detection ofE.coli in the field of environmental monitoring and food safety.Electrochemical sensors and biosensors,an electrochemical analysis technology,develop very fast in the recent years.They have some advantages over otheranalytical systems in that they can operate in turbid media,offer comparableinstrumental sensitivity,and more amenable to miniaturization.Moreover,theapplication of nanomaterials in the preparation of electrochemical sensors hasprovided a new opportunity for their development.Combining with the biologicalcharacters of bacteria,the electrochemical sensors and biosensors have been widelyused to rapidly detect pathogens.In this dissertation,some sensitive and stable electrochemical sensors andbiosensors were fabricated based on novel nanomaterials,and were further applied torapidly detect E.coli in foods and water samples.Furthermore,an electrochemicalinstrument for the detection of bacteria was developed.Totally,this dissertation fallsinto five parts.Part 1:Preface （Chapter 1）In this part,we introduce briefly the biological characters and pathogenicity of E.coli in environment and attempt to review the current development of rapid detectionmethods of E.coli.Moreover,a critical regard to electrochemical sensors andbiosensors is given,and the application of electrochemical sensors to rapid detection of E.coli is also introduced.Part 2:Novel Nanomaterial Modified Electrode for Rapid detection of E.coli（Chapter 2 and 3）In the E.coli solution,β-D-galactosidase （β-gal） was released form E.coli by theeffect of permeabilizers,and catalyzed the hydrolysis ofp-Aminophenyl-β-D-galactopyranoside （PAPG） to produced p-aminophenol （PAP）.So,the concentration of E.coli could be calculated by the current responses of PAPon the modified electrodes.In Chapter 2,a platinum nanoparticles chemical modified electrode （PtNP/GCE）was fabricated by electro-deposition and used to detect the concentration of E.coli.The influence of work potential,substrate concentration and permeabilizers wereinvestigated.Compared with conventional methods,the electrochemical technologycould be suitable for rapid detection of E.coli in the fields of food industry,environmental monitoring and clinic biomedicine.In Chapter 3,a multi-wall carbon nanotubes （MWNTs）/Nafion modified glassycarbon electrode （GCE） was fabricated for the rapid amperometric detection of E.coli.Due to the cation-exchange capacity of Nafion and the electrocatalytic ability ofMWNTs,the detection sensitivity of PAP was improved and the detection time of E.coli was shortened.Under the optimized experiment conditions,10 cfu/mL E.colicould be detected after 4.5 h enrichment.The electrochemical sensor was further usedto detect E.coli in real sample,and the results were consistent to that obtained byplated count method.Part 3:The fabrication of tyrosinase （Tyr） biosensor and its application to rapiddetection of E.coli （Chapter 4 and 5）Biosensors have several advantages to other analytical methods in that they canoperate in turbid media,offer comparable instrumental sensitivity,and more amenableto miniaturization.In this part,two Tyr biosensors were fabricated based on novelnanomaterials and further applied to rapid detection ofE.coli.In Chapter 4,a Tyr biosensor was fabricated by immobilizing Tyr on the surface ofMWNTs-chitosan （Chit） composite modified glassy carbon electrode （GCE）.TheMWNTs-Chit composite film provided a biocompatible platform for the Tyr to retain the bioactivity and the MWNTs possessed excellent inherent conductivity to enhancethe electron transfer rate.The Tyr/MWNTs-Chit/GCE biosensor showed highsensitivity （412 mA/M）,broad linear response (1.0×10^-8～2.8×10^-5 M),low detectionlimit （5.0 nM） and good stability （remained 93% after 10 days） for determination ofphenol.Then,the biosensor was further applied to rapid detection of the E.coli.Thecurrent responses were proportional to the quantity of E,coli in the range of 10⁴-10⁷cfu/mL.After 5.0 h of incubation,E.coli could be detected as low as 10 cfu/mL.In Chapter 5,a novel tyrosinase （Tyr） biosensor was developed based on Fe₃O₄magnetic nanoparticles （MNPs）-coated carbon nanotubes （CNTs） nanocomposite andfurther applied to detect the concentration of coliforms with flow injection assay （FIA）system.Negatively charged MNPs were absorbed onto the surface of CNTs whichwere wrapped with cationic polyelectrolyte poly（dimethyldiallylammonium chloride）（PDDA）.The Fe₃O₄ MNPs-coated CNTs nanocomposite was modified on the surfaceof the glassy carbon electrode （GCE）,and Tyr was loaded on the modified electrodeby glutaraldehyde.The immobilization matrix provided a good microenvironment forretaining the bioactivity of Tyr,and CNTs incorporated into the nanocomposite led tothe improved electrochemical detection of phenol.The Tyr biosensor showed broadlinear response of 1.0×10^-8～3.9×10^-5 M,low detection limit of 5.0×10^-9 M and highsensitivity of 516 mA/M for the determination of phenol.Moreover,the biosensorintegrated with a FIA system was used to monitor E.coli.The detection principle wasbased on determination of phenol which was produced by enzymatic reaction in the E.coli solution.Under the optimal conditions,the current responses obtained in FIAsystem were proportional to the concentration of bacteria ranging from 20 to 1×10⁵cfu/mL with detection limit of 10 cfu/mL and the overall assay time of about 4 h.Thedeveloped biosensor with the FIA system was well suited for quick and automaticclinical diagnostics and water quality analysis.Part 4:Gold Nanolbels for Enhanced Electrochemical Immunoanalysis ofEscherichia coli （Chapter 6）In this part,a novel nanolabel based on gold nanoparticle modified with anti-E.coli peroxidase （HRP）-conjugated antibody is reported.It was used for enhancedelectrochemical immunoanalysis of E.coli.After the immuno-reaction,gold nanolabel,immunoparamagentic bead （IMB） and E.coli formed sandwich-typeimrnunocomplex.3,3,5,5-tetramethylbenzidine （TMB） was used as substrate for HRPin the presence of H₂O₂ and the enzyme activity of HRP was measured byelectrochemical detection coupled with flow injection assay （FIA）.The concentrationof E.coli could be confirmed by amperometric response,which was in proportion tothe quantity of HRP on the sandwich-type immunocomplex.Gold nanoparticles wereused as carriers of antibodies resulting in the increase of antibodies attached to E.coli.So,the amperometric signal was enhanced and the detection sensitivity of E.coli wasimproved greatly.It showed a good linear relationship between amperometricresponses and the logarithmic value of E.coli concentration ranging from 1.0×10² to5.0×10⁵ cfu/mL,with detection limit of 50 cfu/mL.The rapid detection could befinished within 1 h,shorter than other methods.Part 5:Development of electrochemical instrument for rapid detection ofbacteria （Chapter 7）In this part,we develop an electrochemical instrument for rapid detection ofbacteria by combining electrochemical techniques and biological characters ofbacteria.The instruments not only can detect total number of bacteria in the range of1.0×10² cfu/mL～1.0×10⁵ cfu/mL,but also can detect the concentration of E.coliwithin 5 h in the range of 20～1.0×10⁶ cfu/mL.The instrument was small,convenientand reliable.更多还原