基于表面等离子体共振的微流控芯片快速检测赤潮贝毒素

There is no in situ rapid detection method for red tide shellfish poisoning, and the present detection methods have the defects such as nonspecific adsorption and low sensitivity. In this project, the immunological detection and the surface plasmon resonance (SPR) of microfluidic chip was combined to realize the in situ rapid detection of red tide shellfish poisoning. This method could perform multi-channel parallel detection for crude extraction samples, with high sensitivity, high specificity. A small amount of sample is needed and without label. This method is rapid, easy to operate, and with low cost. This paper is about: (1) The shellfish poisoning was purified from crude extraction by the design and optimize of microfluidic purify chip and the select of different methods of antibody fixtion, desorption, and elution. (2) Paralytic shellfish poisoning(PSP) and Diarrhetic shellfish poisoning(DSP) were used as examples. The sensitivity and specificity of the detection was enhanced by the design of high sensitive SPR sensing system, multi-channel parallel detection system, establishment of reference channel, the adoption of immuno-capture method, and using gold nanoparticles. The objective of this study is to achieve the minimum limit of national environmental protection industry standard. The detection time of a group of samples is 10~20 min. (3) To study the influence of detection conditions on its sensitivity, the combined prediction model of multi shellfish poisoning was constructed by the coalition analysis of the SPR standard curve, stable value and trend line of resonance light signal. This paper can provide feasible method and technical support for portable detector of red tide shellfish poisoning.

针对目前赤潮贝毒素无法实现现场快速检测、存在非特异性吸附和灵敏度低的现状，本项目提出结合免疫检测技术和微流控芯片的表面等离子体共振（SPR）方法现场快速检测赤潮贝毒素，该方法可对粗提样品多通道并行检测，高灵敏度，高特异性，样品使用量少且无需标记，检测速度快，操作简便，费用低。具体研究：（1）通过设计与优化微流控纯化芯片，优化抗体固定和解吸附方法，实现从粗提液中纯化贝毒素；（2）以贝毒PSP和DSP为例，通过设计高精度SPR传感系统和多通道并行检测系统、设立参比通道、采用捕获式免疫方法、引入纳米金颗粒，提高检测的灵敏度和特异性，目标是达到国家环境保护行业标准规定的检测下限，一组样品检测时间为10-20分钟；（3）通过SPR 标准曲线族、共振光信号的稳定值和趋势线联合分析方法，建立多种贝毒联合预测模型，研究测量条件和实验参数对检测灵敏度的影响。为开发便携式的赤潮贝毒素检测仪提供方法和技术支持。

针对目前赤潮贝毒素无法实现现场快速检测和灵敏度低的现状，本项目提出结合免疫检测技术的表面等离子体共振（SPR）方法现场快速检测毒素，并对该方法进行改进。（1）自行组建了两套高精度的SPR检测系统，一套采用点扫描方式，一套采用SPR结合全息三维成像技术。（2）结合数字全息的SPR技术可同时获得样品的振幅和相位信息，提高了灵敏度，扩大了检测样品的折射率范围，降低了噪声。以Nacl溶液为例，实时监测其在蒸发过程中反射率和相位随时间的变化，分析了随样品折射率变化和时间变化的检测灵敏度。（3）研究阵列样品的扫描方法，研究自适应光学技术。采用棱镜耦合方式的SPR检测系统，当入射光角度改变的时候，入射光在样品平面的入射点通常会漂移，课题组提出了解决方案，即将棱镜沿特定方向平移，对该方法进行了理论分析和实验验证。（4）前期实验用瘦肉精（克伦特罗）、过敏源、农药（蝇毒磷）、玉米赤霉烯酮等摸索实验方法。（5）毒素样品采用微囊藻毒素，检测效果良好。（6）针对瘦肉精进行实际样品的现场快速检测。在市场上买新鲜的猪肉，进行样品处理，检测效果良好。发表标注课题号的论文8篇，其中3篇SCI，1篇EI，3篇核心期刊，1篇已经录用，待发表，该期刊为EI收录。申请发明专利3项，1项已授权。