基于膜上富集与信号放大探针的病毒现场快速检测方法

复杂样品介质中痕量病毒的灵敏、特异检测是生物及环境分析领域的一个重要研究方向，对于传染性疾病的防控和病毒环境污染研究等方面具有重要的意义。本项目拟开发膜上靶向富集与信号放大探针相结合的方法，在微尺度条件下解决生物样本中痕量病毒的原位快速分析难题。项目选择寨卡病毒为研究对象，研制对寨卡病毒非结构蛋白Zika NS1具有高富集能力和高选择性的新型检测试纸，合成两种信号放大探针。在膜上构建生物素－链霉亲和素支架，实现寨卡病毒在空间维度上的靶向富集与分离。探针以纳米金为载体，在其表面修饰核酸适配体和大量信号分子。特异富集得到的寨卡病毒由探针识别，实现双信号输出途径：1）探针上的酶使显色底物变为蓝色；2）与纸喷雾质谱联用输出质谱信号。项目采用的膜上富集与信号放大探针相结合的策略有减少基质干扰、选择性强、富集能力强和信号放大等优势，为痕量病毒的现场分析提供了一种新型的技术手段。

Highly sensitive and specific detections of trace viruses in complicated matrix is an important research direction in the field of biomolecular recognition research, which is of important significance for the prevention and control of virus infection, and environmental virus research. This work is aimed to establish a rapid and on-spot analysis of trace viruses in biological samples under micro-scale conditions through enrichment of targeted viruses on the membrane with combination of signal amplification probes. In this present work, the Zika virus (ZIKV) was selected as the research object, and a new on-membrane test paper with high selectivity and enrichment effect of ZIKV was developed. Moreover, two types of signal probes with specific recognition and amplification functions for ZIKV were synthesized to perform the detection. A scaffold composed of biotin-streptavidin linking was constructed on the home-made membrane for catching more antibodies or nucleic acid aptamers that can specifically bind the ZIKV non-structural protein Zika NS1 in the spatial dimension on the membrane, enriching Zika NS1 proteins and separation from biological matrix. The probe was composed of a carrier (gold nanoparticles, AuNPs), Zika NS1 aptamers and signal molecules. The Zika NS1 proteins, which could be specifically enriched on the test paper, then recognized by the probes. There are two kinds of signal output ways in this work: 1) colorimetric analysis by a reaction with a mixed solution of the chromogenic substrate (3,3',5,5'-tetramethylbenzidine, TMB) and hydrogen peroxide; 2) coupling to paper spray mass spectrometry. The strategy of combining on-membrane enrichment of viruses and signal probing is endowed with the advantages of reducing matrix interference, high selectivity and enrichment ability of target viruses and signal amplification. The strategy can provide an effective new technical means for fast and accurate analysis of trace viruses.