基于“法拉第笼式”免疫分析的海洋致病菌高灵敏多通道快速检测技术研究

There is a need for sensitive, accurate, simple, rapid and multiplex detection and monitoring of marine pathogenic bacteria as existing methods cannot fully satisfy the requirement. Electrochemical and electrochemiluminescent biosensors based on conventional "On-Electrode" "Sandwich-Type" immunoassay mode appear promising for this purpose; however, the sensitivity must be improved. The major factors restricting the improvement of the sensitivity are: (1) number of electrochemical/electrochemiluminescent labels in biosensor is limited, (2) only a part of the labels coated on the signal probes participate in the electrode reaction, and (3) voltammetry used with normal scanning speed is difficult to effectively enhance the intensity of electrochemical/electrochemiluminescent signals. Therefore to overcome these limitations and realize the benefits of the biosensor-based approach, this proposal aims at combining functionalized bionanomaterials and fast scan voltammetry to establish a new-concept "In-Electrode" "Faradaycage-Type" immunoassay mode. In this approach, magnetic graphene material coated with the first antibody of marine pathogenic bacteria is used as capture probes, graphene materials simultaneously coated by the second antibody of marine pathogenic bacteria and electrochemical/electrochemiluminescent labels is used as signal probes, and fast scan voltammetry is used as excitation source. Thus, the three key bottlenecks above-mentioned obstructing the improvement of the sensitivity can be eliminated, and an ultrasensitive, accurate, simple, rapid and multiplex detection method for marine pathogenic bacteria can be obtained. Through optimizing integrated circuit and building instrument platform, rapid on-site detection and streamlining detection of marine pathogenic bacteria can be both achieved. Based on monitoring data, monitoring mechanism and early-warning mechanism of marine pathogenic bacteria can be preliminarily studied. Thus, this proposal has high scientific significance, public interest and application prospect.

海洋致病菌的灵敏、准确、简单、快速、多通道检测与监测是重大需求，现有方法难以全面满足。基于传统的“电极上”“三明治式”免疫分析模式的海洋致病菌光电化学生物传感器，可准确、简单、快速、多通道检测，但其灵敏度提高存在三个瓶颈制约因素：光电标记物标记数量有限、标记的光电标记物不能全部参与电极反应、常规扫描速度伏安法难以有效增强光电信号。本项目拟结合功能化生物纳米材料和快速扫描伏安法，以海洋致病菌第一抗体包被的磁性石墨烯材料为捕获探针，以光电标记物和海洋致病菌第二抗体同时包被的石墨烯材料为信号探针，以快速扫描伏安法为激发源，创立全新概念的“电极内”“法拉第笼式”免疫分析新模式，消除瓶颈制约因素，实现海洋致病菌的超高灵敏、准确、简单、快速、多通道检测。通过优化一体化电路、构建仪器平台，实现现场快速检测、大量样品流程化检测。基于监测数据，初步探讨监测及预警机制。具有重要科学意义、社会效益和应用前景。

围绕海洋致病菌快速检测与监测的重大需求，本项目开展了四个方面的研究：功能化生物纳米材料的制备、“电极内”“法拉第笼式”免疫分析新模式的创立、海洋致病菌快速高灵敏多通道传感方法与平台的构建、海洋致病菌快速高灵敏多通道监测及预警机制的开发。取得以下三点重要突破：（1）首次提出“法拉第笼式”免疫分析概念，创立“电极内”“法拉第笼式”免疫分析新模式，这是除竞争式、三明治式之外的第三种免疫分析模式；（2）采用“法拉第笼式”免疫分析新模式，开发了一系列生物传感器，可以应用于检测致病菌、蛋白质、多肽、DNA/RNA、重金属离子等；（3）检测灵敏度提高了几十到几百倍，可检测1 CFU/mL致病菌、ag/mL蛋白质/多肽、fmol/L DNA/RNA、pmol/L-fmol/L重金属离子。研究成果已在《Analytical Chemistry》、《Chemical Communications》、《Biosensors & Bioelectronics》等期刊发表SCI论文21篇、申请专利11项并已获授权8项，为广泛的现场检测和诊断应用开辟了新领域，兼具重要科学意义与应用前景。