【作者】 张云；

【导师】 韩恩；

【作者基本信息】 江苏大学 ， 食品科学与工程， 2021， 硕士

【摘要】 金黄色葡萄球菌是常见的食源性病原菌之一,由于其容易引发食物中毒,对人类健康构成了很大的威胁,因此方便、快速地检测金黄色葡萄球菌对于保障食品安全至关重要。电化学免疫传感器作为一种快速、高灵敏的检测方法,已广泛应用于食源性致病菌的检测。本文制备了三种不同的金属纳米复合材料,并分别构建电化学免疫传感器用于高灵敏检测食品中的金黄色葡萄球菌。本论文主要研究内容如下:1.金钯铂三金属功能化多壁碳纳米管纳米复合材料模拟酶的合成及其在金黄色葡萄球菌检测中的应用设计了一种基于金钯铂（AuPdPt）三金属纳米粒子功能化多壁碳纳米管（MWCNTs）纳米复合材料模拟酶的电化学免疫传感器,并用于食品中金黄色葡萄球菌的免标记检测。首先,通过原位生长的方法在MWCNTs表面组装AuPdPt三金属纳米粒子制备MWCNTs-AuPdPt纳米复合材料。该纳米复合材料具有良好的导电性,并对过氧化氢表现出良好的催化性能。接着将抗-金黄色葡萄球菌抗体功能化到具有良好生物相容性和高比表面积的MWCNTs-AuPdPt修饰电极表面以构建免疫传感平台。当固定的抗体通过免疫反应有效捕获样品中的金黄色葡萄球菌后,利用电流-时间曲线实现对金黄色葡萄球菌的快速、灵敏检测。在优化的条件下,所构建的电化学免疫传感器对金黄色葡萄球菌的线性检测范围为1.1×10²～1.1×10⁷ CFU/m L,检测限为39 CFU/m L。该传感器在实际乳制品检测中金黄色葡萄球菌的回收率为91.2%～103.0%,相对标准偏差为4.9%～7.5%。2.基于八面体Cu₂O-Au纳米复合材料模拟酶的电化学免疫传感器及其对金黄色葡萄球菌的检测构建了一种基于金纳米粒子功能化八面体氧化亚铜（Cu₂O-Au）纳米复合材料模拟酶的电化学免疫传感器,实现了对金黄色葡萄球菌的免标记、快速检测。本研究首先制备了尺寸均匀的八面体Cu₂O纳米晶体,然后通过还原反应将金纳米粒子原位组装到Cu₂O纳米晶体表面,所形成的八面体Cu₂O-Au纳米复合物具有高比表面积,优异的电催化性能以及良好的抗体负载能力。接着利用所制备的Cu₂O-Au纳米复合材料修饰电极,并进一步通过抗体功能化,实现特异性结合样品中的金黄色葡萄球菌。由于在电极表面形成的免疫复合物阻碍了底物扩散和电子传递,从而引起电化学信号的变化,最终实现对金黄色葡萄球菌的检测。结果表明,在最优条件下,所制备的免疫传感器检测金黄色葡萄球菌的线性范围为2.4×10²～2.4×10⁷ CFU/m L,检测限为44 CFU/m L。此外,该电化学免疫传感器已成功应用于实际样品中金黄色葡萄球菌的检测。3.金铂纳米粒子功能化聚苯乙烯-丙烯酸球模拟酶纳米探针的合成及其在金黄色葡萄球菌检测中的应用建立了一种基于金铂纳米粒子功能化聚苯乙烯-丙烯酸球（PSA-Au@Pt）模拟酶纳米探针的夹心式电化学免疫传感器,并实现了对食品中金黄色葡萄球菌的高灵敏检测。首先通过原位生长的方法制备具有高比表面积、形貌均一的PSA-Au@Pt纳米球,接着通过抗体功能化构建模拟酶纳米识别探针。该纳米探针对过氧化氢的还原具有优异的催化性能,能够实现免疫传感器检测信号的放大。同时,实验利用自组装的方法合成了金纳米粒子功能化氧化石墨烯（GO-Au NPs）纳米复合物。以GO-Au NPs修饰电极构建免疫传感平台,并结合PSA-Au@Pt-Ab模拟酶纳米探针构建了夹心免疫传感器,利用该纳米探针催化过氧化氢产生电信号实现对金黄色葡萄球菌的高灵敏检测。在优化的实验条件下,所设计的电化学免疫传感器对金黄色葡萄球菌的线性检测范围为1.6×10²～1.6×10⁷ CFU/m L,检测限为25 CFU/m L。另外,所构建的电化学免疫传感器具有较好的重现性、稳定性和高特异性,并且成功应用于实际样品中金黄色葡萄球菌的检测。更多还原

【Abstract】 Staphylococcus aureus（S.aureus）is one of the common food-borne pathogens,which can easily cause food poisoning and pose a huge threat to human health.Therefore,convenient and rapid detection of S.aureus is essential for food safety.As a rapid and sensitive detection method,electrochemical immunosensor has been widely used in the detection of foodborne pathogens.In this paper,three different metal nanocomposites were prepared to construct electrochemical immunosensors to detect S.aureus in food with high sensitivity.The main research contents of this paper are as follows:1.Synthesis of gold-palladium-platinum trimetal functionalized multi-walled carbon nanotube nanocomposites mimic enzymes and its application in detection of S.aureusAn electrochemical immunosensor based on gold-palladium-platinum（AuPdPt）trimetallic nanoparticles functionalized multi-walled carbon nanotubes（MWCNTs）nanocomposites mimic enzymes was designed for the label-free detection of S.aureus in food.Firstly,the nanocomposite of MWCNTs-AuPdPt was prepared by an in situ growth method of loading AuPdPt trimetallic nanoparticles on the surface of MWCNTs.The nanocomposites featured good conductivity and superior catalytic performance for hydrogen peroxide.Then the anti-S.aureus antibody was functionalized on the surface of MWCNTs-AuPdPt modified electrode with good biocompatibility and high specific surface area to construct the immunosensing platform.After S.aureus was immobilized effectively captured by the immobilized antibody through immune reaction,the current-time curve was utilized to achieve rapid and sensitive detection of S.aureus.Under optimized conditions,the designed immunosensor could detect S.aureus in a linear range from 1.1×10²to 1.1×10⁷ CFU/m L with a low detection limit of 39 CFU/m L.The recoveries of S.aureus in dairy products were 91.2%～103.0%,and the relative standard deviations were 4.9%～7.5%.2.Electrochemical immunosensor based on octahedral Cu₂O-Au nanocomposites mimic enzyme for detection of S.aureusA label-free electrochemical immunosensor based on gold nanoparticles functionalized octahedral cuprous oxide（Cu₂O-Au）nanocomposites mimic enzymes was constructed for the detection of S.aureus.In this study,octahedral Cu₂O nanocrystals with uniform size were synthesized firstly,and then gold nanoparticles were assembled on the surface of Cu₂O nanocrystals by reduction reaction.The resulting octahedral Cu₂O-Au nanocomposites displayed large specific surface area,excellent electrocatalytic performance and good antibody loading capacity.Then the prepared Cu₂O-Au nanocomposites were employed to modify the electrode,and further functionalized by antibodies to achieve specific binding to S.aureus in samples.The immune complex formed on the surface of the electrode would hinder the substrate diffusion and electron transfer,resulting in changes of electrochemical signal,and finally realized the detection of S.aureus.Under the optimal conditions,the linear range of the prepared immunosensor was 2.4×10²～2.4×10⁷ CFU/m L,and the detection limit was 44 CFU/m L.Additionally,the electrochemical immunosensor had been successfully applied to the detection of S.aureus in actual samples.3.Synthesis of gold@platinum nanoparticles functionalized polystyrene acrylic spheres nanoprobe and its application in detection of S.aureusA sandwich electrochemical immunosensor based on gold@platinum nanoparticles functionalized polystyrene-acrylic sphere（PSA-Au@Pt）mimic enzyme nanoprobes was established for highly sensitive detection of S.aureus in food.Initially,the PSA-Au@Pt nanocomposites with high specific surface area were prepared by in-situ growth method.Then the nanospheres were functionalized with antibodies to construct mimic enzyme nanorecognition probes.The nanoprobes had excellent catalytic performance for the reduction of hydrogen peroxide,and can realize the amplification of the detection signal of the immunosensor.Meanwhile,gold nanoparticle functionalized graphene oxide（GO-Au NPs）was synthesized by self-assembly method.The GO-Au NPs modified electrode was utilized to construct the immunosensing platform,and the sandwich immunosensor was fabricated by combining with the PSA-Au@Pt-Ab mimic enzyme nanoprobes.The nanoprobes were used to catalyze hydrogen peroxide to generate electrical signals to realize highly sensitive detection of S.aureus.Under the optimized experimental conditions,the linear detection range of the designed electrochemical immunosensor for S.aureus was1.6×10²～1.6×10⁷ CFU/m L,and the detection limit was 25 CFU/m L.In addition,the constructed electrochemical immunosensor exhibited good repeatability,stability and specificity,and had been successfully used for the detection of S.aureus in actual samples.更多还原