食品中真菌毒素的多元、高通量纸基免疫层析微流控芯片检测方法研究

Microfluidic chip is one of the hot fields of "Micro Total Analysis system (μ-TAS)", and has been developed to be one of the most advanced technologies in the world. But the high fabrication cost of traditional microfluidic chip limited its scale application. The paper-based microfluidic chip developed in recently years, using filter paper as the microfluidic substrate is more suitable to be applied in low resource settings and has been used in the fields of chemistry, biology and medicine. However, the application of paper-based microfluidic chip in the field of food safety is barely reported, and the double sandwich assay format mainly adopted by conventional paper-based microfluidic devices is difficult being used in the detection of small molecular compounds which often cause problems of food safety. According to the present situation, we will combine the immunochromatographic strip with paper-based microfluidic chip to develop a paper-based immunochromatographic microfluidic chip. The novel chip using competitive assay format will not only have the advantages of high-throughout of conventional microfluidic chip but also possess the properties of paper test strips being suitable for small molecular compounds detection, and can realize quick and high-throughout detection of multiplex small molecular compounds in food. The project taking mycotoxins as target analytes mainly includes the following contents: antigen and antibody preparations of three mycotoxins (aflatoxin B1, ochratoxin A and zearalenone); nanogold and nanogold-antigen complex preparations; paper-based immunochromatographic microfluidic chip development and its application in food matrix.

微流控芯片是当前微全分析系统发展的热点领域，目前已发展为世界上最先进的技术之一。但传统微流控芯片的制作成本较高，限制了其大规模应用。近几年发展起来的纸基微流控芯片以滤纸为基材，更有利于在资源匮乏条件下使用，已被应用于化学、生物、医学等领域，然而，在食品安全领域的应用却鲜见报道，而且其主要采用的双抗体夹心分析模式也很难应用于引起食品安全问题的小分子化合物的检测。针对这一现状，本项目将免疫层析试纸与纸基微流控芯片相结合，研制纸基免疫层析微流控芯片。该芯片采用竞争分析模式，兼具有传统微流控器件通量高的优点，又结合了免疫层析试纸适用于小分子化合物检测的特点，能够实现食品中多种分析物的快速、高通量检测。本项目以真菌毒素为目标分析物，研究内容主要包括三种真菌毒素（黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮）抗原、抗体的研制；纳米金及其标记物的研制；纸基免疫层析微流控芯片的研制及在食品中的应用。

赭曲霉毒素 A（OTA）是曲霉菌属和青霉菌属的某些产毒菌株的次级代谢产物，对农作物的污染在全球范围内都比较严重，是一种强烈的肾毒素和肝毒素，还具有免疫抑制性，并有致畸、致癌和致突变的作用。OTA的产毒株广泛存在于自然界中，及时进行检测和分析是预防和控制其危害的有效手段。为此，本研究中首先制备了抗赭曲霉毒素的单克隆抗体，对抗体的参数进行了详细鉴定；之后，采用筛选出的抗赭曲霉毒素单克隆抗体，对酶联免疫吸附分析方法中可能影响到方法灵敏度的重要参数进行了优化、确定，建立了高灵敏度的赭曲霉毒素快速检测方法。最后，通过十六烷基三甲基溴化铵溶液和三甲氧基硅烷的庚烷溶液的处理得到亲、疏水基底，制备了纸基微流控芯片，并对芯片的制备工艺进行了优化，对亲疏水通路形成机理进行了探究。本研究中的芯片制备方法不需要任何昂贵的仪器、试剂和金属模具，制备方法新颖，简单，便捷，成本低，制作周期短。