新型表面增强拉曼散射-ELISA乳品掺假蛋白检测技术研究

Dairy protein adulteration is harmful to national health and has attracted much social attention. ELISA technology is very suitable for the field detection of adulterated proteins for the good performance of specificity and flexibility, However, due to the low catalytic efficiency and high cost of biological enzymes, it is difficult to detect adulterated proteins in dairy products rapidly, which makes it become a difficulty to detect the authenticity of dairy products. It is urgent to establish a highly specific and sensitive method to detect adulterated dairy protein. Aiming at these detction problems, A new detection method is proposed in this project: High-resolution and high-sensitivity detection of target adulterated proteins in dairy products can be achieved by using immunomagnetic particles to enrich target proteins, high-efficiency catalysis of nano-enzymes and by combinating of two technologies of SERS and ELISA. Firstly, immunomagnetic particals with strong specific recognition ability were prepared to enrich and separat target proteins rapidly. Secondly, the functionalization methods of nano-enzymes with high catalytic performance and stability to replace biological enzymes were studied. the structure and properties of nano-enzymes were optimized, the characteristics of SERS signal of catalytic substrate were studied, the conversion mechanism of high resolution SERS signal without interference through substrate catalytic reaction was investigated, and the SERS substrates were optimized and SERS signals were enhanced. Finally, a new detection method of adulterated proteins in dairy products was established through the detection mode of "rapid enrichment of protein by immunomagnetic particles - high-efficiency catalytic reaction of nano-enzymes - SERS sensitive detection signal". This project will provide reliable detection proposal and key technique support to break through the bottleneck of detection technology of the adulterated protein detection in dairy products and ensure the authenticity of dairy products.

乳品蛋白掺假危害国民健康，备受社会关注。ELISA技术特异性强，灵活性高，非常适合掺假蛋白现场检测，但因生物酶催化效率低、成本高等缺陷影响，难以快速甄别乳品掺假蛋白，使其成为乳品真实性检测的难点。亟需建立一种高特异性高灵敏度的方法检测乳品掺假蛋白。项目提出一种新型方法：利用免疫磁珠富集靶蛋白，纳米酶高效催化和SERS-ELISA技术有机融合实现乳品中靶掺假蛋白的高分辨高灵敏度检测。首先，制备特异性强的免疫磁珠，实现靶蛋白的快速富集和分离。其次，探究高催化高稳定纳米酶替代生物酶的功能化实现，优化纳米酶结构和性能，研究催化底物SERS信号特征，探讨获得无干扰高分辨SERS信号的转化机制，优化SERS基底、增强SERS信号。最终，通过“免疫磁珠快速富集蛋白--纳米酶高效催化反应--SERS灵敏信号输出”的模式，建立乳品中掺假蛋白的新型检测方法。旨在解决乳品掺假蛋白检测技术瓶颈，保障乳品真实性。

乳及乳制品是日常饮食中重要的蛋白质来源，因此成为最易受掺假影响的食品之一。乳品掺假危害国民健康，影响乳品企业信誉，备受社会关注。项目主要围绕乳品掺假检测技术开展研究。以牛奶中蛋白质为目标检测物，项目分别构建了两种酶联免疫吸附测定法-表面增强拉曼光谱（ELISA-SERS）联用技术用来检测牛奶中的蛋白。采用间接竞争免疫法及显色/SERS双模信号输出方式构建免疫检测传感方案，检测牛乳中β-酪蛋白（β-CN）。为改善生物酶稳定性低的不足，项目筛选并制备出高催化活性、高稳定性的纳米酶结构（新型CeO2纳米酶），并将它代替生物酶应用在免疫分析中催化底物触发拉曼信号变化，从而检测牛乳中α-乳清蛋白（α-LA）。以上两种方法具有较好的特异性、灵敏度和适应性。为避免由于蛋白质结构变化对检测结果的影响，进一步提高检测的准确率，项目进一步探索了一种基于DNA探针、CRISPR/Cas12a驱动和SERS光谱检测的快速检测技术并将其应用在检测真实样品中的牛奶掺假成分。结合化学计量分析，利用SERS特征结合主成分分析方法实现了不同奶粉样品的快速准确高效鉴别，及奶粉中三聚氰胺的快速检测。为减少食品基质的干扰，项目也同时开展了高效前处理净化技术研究, 制备出两种具有高效分离功能的磁性纳米颗粒应用于食品样品前处理过程。项目成果有望为食品监督部门在乳品真实性检测中提供新的检测手段和思路。.项目总体执行情况良好，经费预算执行正常，重点研究工作进展顺利。该项目顺利完成了申请书中的全部研究内容。在该项目资助下，撰写论文7篇, 发表SCI l论文5篇（4篇已发表，1篇已接收, 在审论文2篇）。授权专利1项。培养在读研究生1名，协助指导研究生3名、博士研究生1名。