纳米生物探针现场快速检测活性蓖麻毒素研究

As a consequence of its easy availability and very high cytotoxicity, ricin is the sole protein toxin listed in Schedule I of the Chemical Weapons Convention (CWC).For the purpose of military service and anti-bioterrorism, it is in urgent need to development methods for detecting active ricin in the real biological samples. Current methods for detecting active ricin based on mass spectrometry or immunoassays suffer from requiring costly chemicals and instruments, cumbersome operation and technical difficulty. The goal of this research is to develop a series of nano-biosensors, based on the exceptional optical properties of gold nanoparticles (AuNPs), for convenient, rapid and unambiguous on-site detection of active ricin at low levels in complex matrices. Firstly, a simple and stable single strand DNA substrate with high reactivity is obtained via optimization of the structure of a previously characterized stem-loop DNA. This optimized ssDNA substrate is further linked to a series of AuNPs with different diameters to build nano-biological probes for sensitive on-site detection of active ricin. Benefit from the high extinction coefficient and the strong size- and distance-dependent optical properties of AuNPs, a simple label-free colorimetric sensing method for active ricin based on the aggregate of ssDNA substrate capped AuNPs will been established. The method mechanism is that the active ricin specific depurination process results in the reduction of stability and thus aggregate of AuNPs, allowing the development of this visual assay. By using the large enhancement factor and good reproducibility of AuNPs as SERS substrates, and the aggregation of the ssDNA-modified AuNPs on Si slice after reacting with active ricin and being, a method for sensitive and selective detection of active ricin will be accomplished by monitoring the distinct decreasing raman peaks related to adenine..The realization of this research is of great significance to ensure the national security, safeguard the human life and property, and prevent terrorist attacks.

蓖麻毒素（ricin）不仅容易获得且具有极高的细胞毒性，是联合国禁止化学武器公约附表1清单化学品中唯一的蛋白类毒素。现有测定活性蓖麻毒素的方法存在仪器昂贵、步骤繁琐和技术要求高等局限性。本课题旨在建立适用于战争和突发事件等紧急状态下，活性蓖麻毒素灵敏、可靠的现场快速甄别新策略。拟基于活性蓖麻毒素脱嘌呤反应特性，通过优化毒素作用底物结构，获得结构简单、性质稳定的脱氧寡核苷酸序列，并将其与不同粒径纳米金（AuNPs）偶联构建纳米生物探针；基于活性蓖麻毒素在"纳米-生物"界面发生脱嘌呤反应后导致AuNPs耐盐性减弱，通过盐诱导AuNPs凝集变色实现对其可视化检测；基于AuNPs优良的表面增强拉曼散射效应，通过活性蓖麻毒素在纳米界面上发生脱嘌呤反应引起底物序列拉曼特征峰的显著变化，实现对其快速灵敏的定性、定量鉴定。本课题的实施对保障国家和人民生命财产安全、防范恐怖袭击和保证食品安全等具有重要意义。

活性蓖麻毒素（ricin）具有极高的细胞毒性，是联合国禁止化学武器公约附表1清单化学品中唯一的蛋白类毒素。现有的测定活性蓖麻毒素的方法具有仪器昂贵、步骤繁琐和技术性强等局限性，难以满足战争及突发事件等特殊环境下现场快速侦检的要求。本课题旨在发展基于金纳米颗粒构建的活性蓖麻毒素现场快速监测新方法，基于活性蓖麻毒素能够进行体外脱嘌呤反应的特性，通过优化和改进毒素作用底物结构，获得结构简单、性质稳定和使用方便的寡核苷酸序列（ssDNA），并将其与AuNPs偶联构建纳米生物探针，同时通过可控性修饰纳米材料的界面结构提高探针的性能；在验证并优化活性蓖麻毒素对AuNPs表面底物酶解反应的基础上，将反应后底物结构变化设计转化为获取直接、结果可靠的监测信号，据此建立表面增强拉曼散射法的活性蓖麻毒素快速定性与定量分析方法，为战争和突发事件等紧急状态下快速甄别残留活性毒剂提供灵敏、可靠的新途径，本课题对保障国家和人民生命财产安全、防范恐怖袭击和保证食品安全等具有十分重要的意义和广阔的应用前景。