基于复合纳米材料的食源性致病微生物超灵敏等温核酸生物传感器及智能定量装备的研究

Food microorganism pollution is a significant problem in the field of food safety. In order to solve a series of scientific problems in microbiological detection skillfully, including capture, enrichment, extraction, amplification, analysis, and on-site testing, we will provide a series of strategies. Firstly, culture-free detection will be established in this program on the basis of dual-function aptamer recognition model and exercise-rich model to allow the signal transduction for high specificity detection of foodborne pathogens. Secondly, extraction-free detection will be established with the help of overspeed isothermal asymmetric amplification technology. This method will use aptamers as primers to avoid nucleic acid extraction, which will alter cumbersome steps of traditional asymmetric amplification and enable overspeed detection by controlling template length and reaction time. Thirdly, power-free detection will be developed employing lateral flow nucleic acid biosensor, which will introduce novel composite nanomaterials, platinum-palladium nanozyme to improve analytical sensitivity under polymeric adenine base planting model. Fourthly, instrument-free quantitative equipment will be developed based on smartphones and 3D printing technology, which will meet the technical requirements for device assembly, image analysis and data processing. Overall, this project proposed the theory of “Four Free Technology”, including culture-free, extraction-free, power-free, and instrument-free, which will be an ultra-sensitive, practical and efficient solution to the screening and identification of foodborne pathogenic microorganisms in a fast, accurate and high-throughput pattern.

微生物污染一直都是食品安全中的一个突出问题。为解决微生物从识别、富集、提取、扩增、分析、现场检测的一系列科学难题，本项目建立高特异性的食源性致病菌的双功能适配体“免培养”识别模型及运动富集模型，实现微生物的信号转导检测；提出通用扩增理论，建立“免提取”超速等温非对称扩增技术，利用适配体作为引物，避免核酸提取，改变传统非对称扩增的繁琐步骤，通过控制模板长度及反应时间实现超速检测；引入新型复合纳米材料，建立基于铂钯纳米酶纸基侧流层析传感器的“免动力”分析技术，提高分析灵敏度；搭载智能手机和3D打印技术，研发“免仪器依赖”型定量装备，满足了器件组合、图像分析和数据处理等技术要求。本项目提出了上述“免培养、免提取、免动力、免仪器依赖”的“四免检测”理论和技术手段，为食源性致病菌的快速、准确、高通量筛查和鉴定提供超灵敏、实用、高效的解决方案。

为解决食品安全中微生物从识别、富集、提取、扩增、分析、现场检测的一系列科学难题，本项目设计了一整套解决策略。首先为了解决微生物检测前增菌问题，在前期食源性致病菌的高特异性功能核酸分子识别库基础上，结合磁纳米粒子，搭建了“免培养”功能核酸识别模型及磁富集模型，通过识别富集无需培养将食源性致病菌归一转化为核酸；为解决信号扩增慢、通用性低的问题，搭建了“免提取”超速等温扩增技术体系，将适配体作为通用引物，无需核酸提取，实现信号快速扩增；为解决免培养检测灵敏度低的问题，在传统纸基侧流层析传感器中引入新型复合纳米材料铂钯纳米酶，建立新型“免动力”纸基传感分析技术；为解决现场数据处理难的问题，搭载智能手机和3D打印技术，研发“免仪器依赖”型定量装备。.本项目研究内容主要分为“食源性致病菌功能核酸识别及磁富集传感”、“超速等温扩增”、“核酸试纸生物传感器”及“高通量筛查检测定量装备”四大部分，分别建立磁珠分离富集技术、超速PCR扩增技术、等温扩增技术、铂钯纳米酶核酸试纸及便携式试纸读取仪器。在完成核定任务的基础上，着重进行了基于功能核酸的新型复合纳米材料等温组装及生物传感应用。为微生物高通量现场可视化检测提供技术支撑。.本项目中通过将核心任务分解，巧妙的解决了微生物从识别、捕获、富集、扩增、现场检 测的一系列科学难题，为食源性致病菌的快速、准确、高通量筛查和鉴定提供超灵敏、实用、高效的解决方案。