【作者】 王家琪；

【导师】 干宁； 曹国洲；

【作者基本信息】 宁波大学 ， 工程硕士（专业学位）， 2021， 硕士

【摘要】 当前,食源性致病菌正对人体安全健康构成持续性威胁。食品中常见的致病菌包括沙门氏菌（salmonella species）金黄色葡萄球菌（staphylococcus aureus,S.aureus）,铜绿假单胞菌（pseudomonas aeruginosa,P.aeruginosa）,肠杆菌（enterobacter species）等细菌,常分布在肉制品、蛋制品、乳制品、蔬菜等食物当中,常以食物中毒的方式威胁人体健康。目前常用的检测方法检测时间较长,常常会延误最佳诊疗时间,因此开发快速的现场快速检测传感器以进行检测、识别和监控食品中致病菌的即时指标非常重要。点击化学反应（click reaction）由于其具有的小分子单元反应,高选择性,高产率,快速,无副反应产物,以及较为温和的反应条件,成为致病菌筛查的有力工具。在本论文中,开发了基于点击化学反应的细菌传感器,通过偶联不同信号标签以及信号放大策略,实现了对细菌的快速灵敏鉴定。主要工作分述如下:1.基于点击化学制备MOF@Fc-BPA复合探针,并建立了高灵敏,快速的S.aureus的电化学芯片传感器:首先制备了卵黄抗体（Ig Y）,并将Ig Y固定在丝网印刷电极表面,可用于食品样本中S.aureus的选择性捕获;其次,通过点击化学在Ui O-66金属有机框架（MOF）材料表面连接二茂铁（Fc）和苯硼酸（BPA）,获得了MOF@Fc-BPA复合探针。此探针与电极上的Ig Y共同识别并捕获S.aureus,同时在电极上产生二茂铁的氧化还原信号,完成检测。该S.aureus传感器的检测范围为10¹～10⁹ CFU/m L,检出限为3 CFU/m L,检测时间为20分钟。2.建立了基于DNAzyme催化的点击反应的新型灵敏快速细菌检测策略:通过简便的自聚合和戊二醛偶联方法合成包裹有卵黄抗体的聚多巴胺纳米铜载体（Cu-PDA）信号探针,同时制备了能靶向捕获革兰氏阳性细菌的搅拌棒,提高了检测的特异性和分离的易操作性。金黄色葡萄球菌存在时可形成能有效触发点击反应的夹心复合物并与DNAzyme协同催化点击反应。最后,通过微流控芯片（microfluidic chip）来监测点击化学反应进程以监控细菌浓度。采用实际样品考察了该检测策略的实际适用性。结果显示出低的检测限（3 CFU/m L）和较短的检测时间（40分钟）,表明该检测策略效果令人满意。3.基于“细菌表面的DNA walker”,构建了多种致病菌的同时检测策略:首先制备了苯硼酸修饰的搅拌棒,可以用于多种致病菌的富集;其次通过点击化学制备了一系列抗体-编码探针和信号探针。该抗体-编码探针可特异性识别并结合搅拌棒上的目标细菌。随后将三明治复合物引入到信号探针/Zn²⁺体系中并触发“DNAwalker”。经过多步步行后,信号探针被切割并产生三种长度的DNA片段,分别对应金黄色葡萄球菌,鼠伤寒沙门氏菌和单核细胞增生李斯特菌。最后通过微流控芯片跟踪验证DNA walker的反应进程,并实现三种致病菌的同时快速检测。更多还原

【Abstract】 Previously,food-borne pathogens are continuously threatening to human health.Food-borne pathogens including Salmonella species,Staphylococcus aureus（S.aureus）,Pseudomonas aeruginosa（P.aeruginosa）,Enterobacter species,etc.those bacteria often distributed in meat products,egg products,dairy products,vegetables and other foods,and threaten human health in the form of food poisoning.At present,the commonly used detection methods take a long time to detect those pathogens and often delay the best diagnosis time.Therefore,it is important to develop fast and on-site detection sensors to detect,identify and monitor real-time indicators of pathogenic bacteria in food.Click reaction has become a powerful tool for screening pathogenic bacteria for its small molecular unit reaction,high selectivity,high yield,high speed,no side reaction products,and relatively mild reaction conditions.In this thesis,rapid and sensitive sensors for bacteria was developed by coupling different signal amplification strategies with click chemistry reaction and yolk antibody.This thesis is divided into three parts to research:1.A high-sensitive and fast S.aureus electrochemical-chip sensor was established based on the MOF@Fc-BPA composite probe prepared thorough click chemistry.Firstly,the yolk antibody（Ig Y）was prepared and was immobilized on the screen-printed electrode（SPE）.The obtained electrode can be used for the selective capture of S.aureus in food samples;Secondly,click chemistry was utilized to linking ferrocene（Fc）and phenylboronic acid（BPA）and Ui O-66 metal organic framework（MOF）and prepared the MOF@Fc-BPA probe.The probe and the immune electrode jointly identify and capture S.aureus,and generate the redox signal of ferrocene on the electrode to detect the S.aureus.The detection range of the S.aureus sensor was 10¹～10⁹ CFU/m L,and the detection time was 20 minutes.2.A new sensitive and rapid bacterial detection strategy based on DNAzyme-catalyzed click reaction was established:The probe was made by modifying Ig Y on copper functionalized polydopamine nanoparticles（Cu-PDA）thorough glutaraldehyde linking strategy.Before detection,4-mercaptophenylboronic acid functionalized stir bar was used for specifically capturing and enriching S.aureus in samples.Cu-PDA was then conjugated with the captured S.aureus on the bar to form sandwich structures.The Cu（II）was washed out to trigger Cu AAC reaction and form DNAzyme-Streptavidin complex.A microfluidic chip was used to monitoring the reaction progress which was proportional to the concentration of S.aureus.The sensor could be used to quantify as low as 3 CFU/m L of S.aureus in real sample within 40 minutes.The sensor was suitable for on-site screening foodborne pathogens.3.A“DNA walker on the bacteria”strategy was established for simultaneous detection of multiple pathogenic bacteria:Firstly,a phenylboronic acid modified stirring bar was prepared,which can be used for the enrichment of multiple pathogenic bacteria;Secondly,a series of antibodies-coding probes and signal probes was prepared thorough click chemistry.The target bacteria could be specifically recognized and bind by the antibody-encoded probe and the stir bar,then the sandwich complex was formed.Thirdly,the sandwich complex was introduced into signal probe/Zn²⁺solution and triggering the walking process.After multiple steps of walking process,the signal probe could be cleaved into three different lengths of DNA fragments,which was corresponding to the concentration of S.aureus,S.typhimurium,and L.monocytogenes.The generated DNA fragments was separated and quantitative thorough a microfluidic chip which could be used for the detection of three pathogenic bacteria.更多还原