功能化纸基微流控芯片及其对食源性致病菌快速选择性检测方法的研究

Microfluidic Paper-based devices, which is simple, disposable and low cost comparing to the traditional vitro analysis devices. In recent years, microfluidic paper-based devices integrated with colorimetric assays or immunoassay has become a burgeoning research field for a variety of applications including health diagnostics, environmental monitoring as well as food quality testing. However, for practical applications of paper-based analytical devices such as for monitoring selective interactions of biomolecules, especially in complex media, unmodified cellulose is inadequate at resisting nonspecific adsorption, leading to high background noise, loss of target analyte, and a reduction in assay reproducibility. Additionally, the physical adsorption of molecular-recognition elements typically used for immobilization on cellulose often results in ligand drift, signal distortion, and loss of ligand bioactivity, all of which can be significantly improved or eliminated via covalent attachment while also further enhancing the limit of detection.. Zwitterionic poly(carboxybetaine) (PCB) is an attractive material for implication with paper-based devices due to its dual capacity for enabling fouling resistance and functionalization. Superhydrophilic PCB has a demonstrated fouling level of less than 5 ng/cm2 in the presence of 100% human blood plasma and serum. Furthermore, unlike other nonfouling materials, PCB has abundant functional groups (COOH) which can be employed for the convenient immobilization of MREs via conventional 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide coupling chemistry. Zerionic PCB is an attractive material for implication with paper-based devices due to its dual capacity for enabling fouling resistance and functionalization. In this project, we report a robust and effective surface modification method for the formation of PCB brushes onto cellulose paper (CP) using surface-initiated atom transfer radical polymerization. This study will improve our understanding of the fundamental mechanisms of PCB modified paper based microfluidic devices, so as to pave the way for materials design and application of paper sensors.To demonstrate the potential of the PCB modified cellulose paper as a highly sensitive diagnostic sensor, we constructed a high-performance microfluidic paper-based analytical device for the first time, combining with the fluorescence immunity analysis technology, it can measure different food-borne pathogens in parallel, the sensing mechanism of the paper sensor was revealed by testing the intermediate and final product using in situ IR , XPS and so on, providing a new method for detection of food-borne pathogens.

快速、低成本、高通量的纸基微流控芯片技术在食品安全检测等领域已展现出巨大的应用前景，但其本身难以结合待测生物分子，并且生物样品体系中非特异性吸附使其选择性差和灵敏度低，严重限制了纸基微流控芯片在食源性致病菌快速实时检测领域的应用。本项目拟设计合成羧基甜菜碱类聚合物，利用该材料对芯片分析区域进行功能化修饰，不仅能明显提高分析区域的灵敏度和选择性，而且其功能化表面易于连接食源性致病菌抗体等生物分子，进一步融合荧光免疫分析技术，实现对食源性致病菌高通量、快速选择性的检测。在此基础上，结合原位红外光谱、XPS等表征技术对敏感过程的中间产物和最终产物进行跟踪测试，重点揭示羧基甜菜碱类聚合物、免疫荧光探针、食源性致病菌的相互作用关系及其动态变化规律，阐明识别机制，揭示该类聚合物提高纸基微流控芯片灵敏度和选择性的一般规律，最终为实现纸基微流控芯片在食源性致病菌快速实时检测领域的广泛应用奠定坚实理论基础。

致病菌是引起食源性疾病的主要原因，对人们的身体健康造成巨大危害。因此，为了实现对食源性致病菌的实时、灵敏、准确的检测，迫切需要发展操作简单、便携、低成本的快速检测与分离技术。在此基础上，建立标准、快速有效的检测方法具有重要的现实意义。本项目利用原子转移自由基聚合反应的方法将具有双功能化的羧基甜菜碱类聚合物嫁接到纸基微流控芯片的表面，成功构建了双功能化的羧基甜菜碱类聚合物功能化纸基微流控芯片，使其芯片表面既具有优异的抗非特异性蛋白吸附的功能，对1mg/mL纤维蛋白原和溶解酵素的吸附值都低于3ng/cm2，又能够成功实现功能化连接预期目标生物分子。解决了因非特异性吸附引起的灵敏度低、选择性差等生物传感器领域的一些难题，为功能化纸基微流控芯片的设计合成和性能优化提供了理论指导和技术支持。合成了三种不同颜色、光谱相互独立的量子点，将上述合成得到的量子点与抗体偶联得到三种不同颜色荧光探针，并将目标检测的荧光标记物连接到纸基微流控芯片表面，实现了对三种食源性致病菌的快速检测。研究了功能化聚合物的厚度、空间密度分布、量子点的荧光强度及稳定性、量子点与抗体的偶联效率与功能化纸基微流控芯片敏感性能的相互作用关系，初步探明了上述关键参数相互影响的动态变化规律。拓展了功能化纸基微流控芯片的应用，实现了其对水果复杂体系中维生素C、葡萄糖、蔗糖和果糖的检测，构建了基于功能化纸基微流控芯片评价水果品质的快速检测技术。为保障食品安全、检测食源性致病菌提供了新的思路和检测方法。