基于点击化学介导金纳米簇聚集发光增强的超灵敏无酶免疫分析新方法

Immunosorbent assay is one of the most popular immunological screening technique which has been widely used in food safety, clinical diagnosis and environment monitoring. While traditional enzyme labeled immunosorbent assay (ELISA) suffers from low detection sensitivity due to its low intensity of colorimetric signal and poor robustness arising from the poor stability of natural enzymes. Herein, an ultrasensitive and robust enzyme-free immunoassay is proposed for the detection of three mycotoxins in food (aflatoxin B1, ochratoxin A and zeaxanone), where aggregation induced enhanced emission of thiol ligands protected gold nanoclusters (AuNCs) is used as a signal output, with a signal amplification based on an inorganic catalyst involved reaction. Firstly, thiol ligands AuNCs are modified with azide groups and alkyne groups separately. And then, a novel signal transition mechanism based on aggregation induced emission amplified with inorganic catalyst is established, by using Copper-catalysed azide–alkyne cycloadditions to trigger the aggregation of AuNCs modified with clickable groups. Then a polyethyleneimine (PEI) mediated in-situ reduction of Cu(II) ion on the surface of silica nanoparticles would be conducted to prepare Silica@PEI@CuNPs nanoparticle, which can be used as a heterogeneous catalyst and a carrier of competitive antigens for the novel proposed fluorescence immunoassay.

免疫吸附技术是目前实验室免疫筛查分析最常用的方法之一，在食品安全、临床诊断和环境监测等领域发挥着极其重要的作用。然而传统酶联免疫吸附法存在因比色信号强度弱导致的检测灵敏度偏低、以及因生物酶稳定性差导致的方法鲁棒性不足等问题。本研究拟基于巯基配体金纳米簇的聚集发光增强原理，结合无机催化剂介导的信号放大机制来构建高灵敏、高鲁棒性的无酶免疫检测方法，并实现食品中三种真菌毒素（黄曲霉毒素B1,赭曲霉毒素A和玉米赤霉烯酮）的高灵敏检测。首先，在巯基配体金纳米簇表面分别修饰叠氮基和端炔基两种点击化学基团；然后基于铜催化叠氮炔环加成反应介导金纳米簇聚集，构建基于无机催化剂介导聚集诱导发光原理的新型信号输出体系；最后在包覆支化聚乙烯亚胺的二氧化硅纳米颗粒表面原位还原Cu(II)，制备可介导金纳米簇聚集的无机非均相纳米催化剂，进一步以其为载体制备竞争抗原构建新型竞争免疫荧光分析技术。

酶联免疫吸附法（ELISA）作为目前实验室最常用的筛查分析方法，在基层真菌毒素筛查检测中发挥着极其重要的作用。然而，传统用于真菌毒素检测的竞争ELISA存在因比色信号强度弱导致的检测灵敏度偏低、因竞争抗原制备带来的二次污染以及因生物酶稳定性差导致的方法鲁棒性不足等问题。针对以上瓶颈问题，本项目致力于绿色高灵敏、鲁棒性好的真菌毒素快速检测方法的开发，主要从以下几方面进行了深入研究。（1）以双功能M13噬菌体为生物相容性的竞争抗原和葡萄糖氧化酶的载体，构建2种绿色、高灵敏ELISA方法，包括基于H2O2敏感的量子点的荧光ELISA和基于酶催化介导AuNPs聚集的比色光热双信号ELISA。所构建方法均较传统ELISA灵敏度提高10~30倍。（2）基于Cu(I)催化叠氮炔环加成反应（CuAAC）、金纳米颗粒纳米酶催化反应等无机催化反应介导信号输出，构建2种高鲁棒性的分析方法，分析灵敏度较常规ELISA略有提高。（3）针对上述新型ELISA方法过程较为复杂，检测时间较长的缺陷（60~90 min），本研究基于高亮度聚集诱导发光（AIE）微球探针构建高灵敏荧光免疫层析技术，其可在15min内实现玉米中OTA的高灵敏检测，且灵敏度较常规ELISA提高3倍。总之，本项目为食品中真菌毒素等小分子危害物的绿色高灵敏及快速检测提供了很好的理论基础以及应用参考。项目执行期间发表SCI论文9篇，申请专利2项，其中授权1项。