脲酶/蔗糖酶介导金纳米花金属化的新型等离子体共振ELISA超灵敏检测三种食源性致病菌的研究

Enzyme-Induced Metallization of Gold Nanoparticles is a new method to generate the localized surface plasmon resonance (LSPR), which commonly reduces Ag+ ions on the surface of gold nanoparticles (AuNPs) to form Au@Ag core–shell NPs by the addition of silver nitrate to a solution containing citrate-stabilized AuNPs and some reducing agents, such as hydrogen peroxide, ascorbic acid and p-aminophenol. But, their applications were limited in practice because of poor stability. Glucose has better stability, and Ag+ ions can be reduced by the aldehyde group of glucose in the presence of ammonia. In the preliminary experiments, we found that the change of ammonia or glucose concentration can regulate the reduction rate of Ag+ ions on the surface of the gold nano-flowers (AuNFs), and produce a colorful color. Therefore, the applicant proposes a new ideal to develop a sensitive pELISA based on enzyme-induced metallization of AuNFs, in which the urease or sucrase will be introduced into a conventional immunoassay for producing NH3 or glucose, and thereby regulating the NH3 or glucose concentration. In order to further improve the sensitivity of pELISA, the silica nanoparticles carrying poly(acrylic acid) brushes will be suggested as the "container" of urease or sucrase to increase the enzyme loading capacity on the surface of bacteria. The improved pELISA will exhibit the ultra-sensitivity for detection of E.coli O157:H7、LM and Salmonella with the detection limit of 10E+0 ~ 10E+1CFU / mL. Taken together, this proposal aims to establish an ultrasensitive immunoassay to shorten the detection time of foodborne pathogens, and provides a new idea for rapid screening of foodborne pathogens.

AuNPs金属化是调控胶体金LSPR的一种新方法。该方法通常采用双氧水、抗坏血酸及对氨基苯酚等还原剂将银离子在AuNPs表面还原形成银包金的核壳纳米结构，产生LSPR及颜色变化。但以上还原剂稳定性差，导致在实践中应用受到限制。银离子在氨存在下可被葡萄糖醛基还原形成单质Ag，而葡萄糖具有更好的稳定性。申请人前期实验发现，改变氨或葡萄糖浓度可调控银离子在金纳米花（AuNFs）表面的还原速度，产生丰富的颜色变化。依据此特性，申请人首次提出以脲酶或蔗糖酶替代常规ELISA中辣根过氧化酶，实现对氨或葡萄糖的精细调控，以此建立基于AuNFs 金属化的pELISA新方法；在此基础上进一步提出以硅球聚丙烯酸分子刷为pELISA载酶“集装箱”，通过增大菌体表面酶载量实现E.coli O157:H7、LM以及Salmonella的超灵敏检测（灵敏度达到10E+0~10E+1CFU/mL）。

食源性致病菌污染是我国食品安全的重大问题之一。目前检测食源性致病菌的“金标准”仍是传统的培养法。通常完成一次检测需要5～7d的时间，不能即时反馈检测结果。免疫学检测技术可用于食源性致病菌的早期筛查，但由于常规ELISA方法检测灵敏度的限制，一般仍需12～24 h的前增菌过程。因此，如何有效地提高现有检测方法的灵敏度，缩短食品中致病菌的检测时间是建立快速筛查方法面临的共性科学问题。.AuNPs的局域表面等离子共振峰会随粒子的大小、形状以及粒子间的距离而发生偏移并显示出显著的颜色差异；利用AuNPs颜色差异，结合免疫学中抗原/抗体反应建立的表面等离子共振ELISA（，pELISA），可大大提高ELISA方法的检测灵敏度。本项目利用酶法催化反应，建立了基于金纳米粒子聚集、蚀刻、生长以及金属化等四种类型的pELISA方法。其中利用脲酶催化尿素产生氨气，通过氨与AgNO3发生银镜反应，构建了基于金纳米棒金属化裸眼和定量检测猪霍乱沙门氏菌的新方法。该方法裸眼检测猪霍乱沙门氏最低检测限为1.2×10E+2 CFU/mL，仪器判读最低检测限为1.2×10E+1 CFU/mLCFU/mL，其灵敏度较常规ELISA提高2~3个数量级。尽管pELISA方法有着较高的检测灵敏度，该方法显色信号无法实现终止反应，需要严格限定检测时间，针对以上问题，本研究进一步建立了通过单链DNA诱导金纳米粒子各向异性生长的pELISA方法，实现了pELISA检测信号的稳定输出。该方法检测婴幼儿奶粉中克罗诺杆菌杆菌最低检测限达到1.6×10E+2 cfu/mL，灵敏度较常规ELISA提高了2个数量级。在此基础上，进一步以金纳米粒子光散射为输出信号，其灵敏度较等离子共振信号提高了一个数量级，检测克罗诺杆菌的最低检测限达到3.9×10E+1 cfu/mL。针对pELISA需要酶催化调控等离子信号，检测时间长的缺陷（60~90 min），本研究利用胶体金免疫层析技术平台，通过建立了金纳米粒子表面金、铜原位生长的信号放大策略，实现了免疫层析试纸条法超灵敏检测牛奶中大肠杆菌O157:H7的新方法。该方法检测牛奶中大肠杆菌O157:H7最低检测限达到1.25×10E+1cfu/mL 以及数个菌的检测，较常规ELISA灵敏度提高了2~3个数量级。