纳米孔微流通道多元SERS传感技术用于病原菌基因诊断研究

This project is supposed to construct nanopore based PDMS microfluidic channels for multiplexed gene diagnosis of microbial pathogens in food,which combines the merits of resistive effect of nanopore with biosensing signal amplification techniques and sensitive Surface Enhanced Raman Scattering (SERS) analysis. Firstly, the microfluidic channels with nanopore will be constructed using PDMS matrix by rapid prototyping method.Then confocal raman microscope is coupled with the PDMS microfluidic system to establish a platform for biosensing. The recognition events of biomolecules captures the target DNA strand before the nanopore,then some signal amplification technologies, such as nicking enzyme assisted isothermal strand-displacement polymerization and enzymatic recycling cleavage for amplification, are utilized to quantitatively cleavage and release Raman signal labeling molecules. So the recognition events of pathogen gene sequence could be transformed into the detection of raman labeling molecules released. Surface enhanced nano-substrates would be used to monitoring the SERS signal chances from the raman tags that translocated through the nanopore to the detection areas. In this way, novel highly sensitive gene diagnosis methods would be constructed for multiplexed detection of microbial pathogens in food. Proteins that can specifically recognize and bind mismatched or methylated bases combined with Raman-labeled nanoparticles are used to develop novel gene detection method for point mutation detection. The proteins modified magnatic bead could specifically concentrate ds-DNA with mismatch base or methlyated base onto its surface,and then nanoparticles with raman lablels are bonded for signal amplification through "SA-Biotin" interaction. The controlled release of the raman tags from the nanoparticles would be an effective way of amplified sensing. Novel "on-chip" gene detection techniques are also proposed on bases of allele-specific invasive-cleavage.The nucleotide with flap 5'-raman labels are immobilized on the magnetic beads, the 3'-end of the invader probe is designed to overlap one base into the downstream duplex formed by the 5'-raman labeled probe on beads and the target strand. Then the 5'-raman labeled end of the strands could continually be removed from the beads by a specific exonuclease to produce detectable SERS signals. Nanopore microfluidic systems with three channels or radial multiple channels are designed to develop highthroughput gene diagnosis techniques for the detection of microbial pathogens in food. Different kind of pathogens and mutation sites could be detected quickly with high sensentivity. Some key methods and techniques would be constructed with independent intellectual property. Then, a new platform would be developed for food safety monitoring and fast pathogen detection in food.

本项目拟构建基于纳米孔的PDMS微流通道系统，集成纳米孔阻滞效应、生物传感信号放大技术和表面增强拉曼光谱（SERS）分析，发展高通量的食源性病原菌快速基因诊断新技术。利用生物分子识别事件将目标DNA阻滞在纳米孔前，结合等温扩增和酶循环切割等生物信号放大技术，定量剪切释放拉曼信号标记分子，采用纳米增强基底监测信号分子穿越纳米孔产生的SERS变化，发展高灵敏的食源性病原菌基因检测新技术，在构建单通道基础上设计三通道和辐射状多通道系统，发展高通量多元分析技术；结合错配或甲基化碱基识别蛋白和拉曼标记纳米粒子信号放大技术发展基因突变多元分析新方法；基于等位基因内切酶侵入剪切技术发展快速简便的"on-chip"高通量基因突变检测新技术；实现对不同致病菌种和突变位点的快速诊断和高灵敏检测。以期形成具有自主知识产权的关键方法和技术，建立食品安全监测和食源性病原菌快速检测的新技术平台。

针对全球食源性病原菌事件频发的问题，本项目致力于发展基于DNA酶的简便、灵敏、高通量的诊断技术，构建病原菌快速检测新技术平台。以细菌特异性RNA-剪切-DNA酶探针为分子识别元件，在DNA酶修饰的磁珠上偶联脲酶，将细菌检测转换成pH值的增加，从而通过酸碱指示剂或pH试纸实现快速检测。该细菌比色分析技术从检测单个菌所需要时间上来看，优于传统的PCR(10小时)和ELISA（16小时），体现出简便、成本低，和普适性的优点，具有广泛的应用前景。提出了一种DLISA技术: 即基于DNA酶的ELISA（传统的蛋白酶联免疫分析），利用标记ZnS纳米晶体的离子交换溶出提供DNA酶催化单元所需的辅助因子，结合催化分子信标（CAMB）体系的循环放大，实现三重荧光信号扩增，比传统ELISA和其他单步信号扩增免疫分析技术相比灵敏度更高，同时在微孔板中构建了传感阵列，实现了多目标的高通量免疫分析。基于表面增强拉曼光谱（SERS）的生物分析新方法研究方面做了如下探索：将基于半自动点样的纸上SERS基底制备、磁性纳米颗粒的富集分离，以及酶催化底物产生高SERS活性产物的反应相结合，避免了内源荧光干扰，建立了一种简便的纸上表面增强共振拉曼散射光谱（SERRS）-酶联免疫分析新方法；制备了纳米银沉积聚苯乙烯微球，用于表面增强拉曼光谱编码和无标记快速检测，探索了该微球SERS基底在PDMS微流通道的应用；将比色法与SERS用于快速检测革兰氏阴性大肠杆菌DH5α；基于磁场诱导纳米金聚集的SERS传感器用于细菌DNA的检测。在多色荧光生物传感及成像新技术方面进行了以下研究：利用聚集态苝酰亚胺（PDI）作为高效猝灭剂，发展了多色荧光传感器用于多目标DNA/核酸内切酶分析平台；采用DNAzyme作为识别和信号放大器，DNA稳定的银簇作为信号报告部件，报道了无标记的催化分子信标信号放大检测技术；基于荧光探针和纳米金的免疫传感技术用于组蛋白去乙酰化酶检测；基于萘衍生物的双光子荧光探针的活细胞H2S高灵敏生物成像；基于聚T-DNA-银纳米簇的荧光生物传感器用于尿酸的高灵敏检测；基于碳量子点的荧光猝灭型传感器检测有机磷杀虫剂；研究了常见红色荧光蛋白在蛋白标记示踪和高分辨成像中的应用。该项目建立的一系列新技术为病原菌的快速检测提供了新策略，具有潜在应用价值。