单层滤纸三维微流控纸芯片的构建及用于复杂试样的在线净化和农药残留快速检测研究

In current project, a new protocol for the fabrication of 3D microfluidic paper-based analytical devices (3D uPADs) is established on single layer filterpaper substrate, to meet the sample purification demands in rapid detection. The new type of 3D uPAD contains only one single layer of filter paper substrate. It is fabricated by region-strength double selective surface modification of paper substrate, without involving any additional facilities like adhesive glues or chip holders for further chip stabilization. Sample pre-filtering function is integrated onto the fabricated 3D uPADs to achieve solid-liquid separation for agricultural product analysis, through flow manipulation by the 3D microchannel construction on the device. Samples are pipetted to sampling points on one side of the device and guided to permeate through the chip substrate by the hydrophilic paper fibers of the 3D micro-channels, during which solid impurities are filtered out before samples arrive at the detection points on the other side of the device. Filtering/separating efficiency of this process is characterized by image scanning of the detection areas and comparing the obtained images with those from normal sampling method (in which fluids do not penetrate through chip substrate during flow). In order to establish the solvent handling ability for further applications, channel walls of the new developed 3D uPADs could be Teflon-modified to acquire lipophobicity. High analytical sensitivity is expected in the detection of acetylcholinesterase inhibitors in tea samples through a unique strategy established in present project based on the special 3D uPADs, which combines online sample pre-purification, solvent isolation and enzyme inhibition measurement. Rapid detection ability and performance of the new founded 3D uPADs platform are evaluated. The new 3D uPADs are believed to have great perspective on rapid detection, as well as in future establishment of miniaturized and portable analytical equipment.

针对目前微流控纸芯片上比色法快速检测中集成化样品前处理难、对复杂样品检测性能差的问题，本项目拟采用区域和深度双重选择性的表面改性方法，在单层滤纸上构建三维微流控通道，研制由单层滤纸构成的新型三维微流控纸芯片；拟在纸芯片的正面进行进样和试样预净化、在背面进行显色反应和比色法检测，实现三维纸芯片上的集成化样品在线净化预处理，提高芯片在实际样品检测中的抗干扰能力；拟对研制的三维纸芯片通道壁进行疏脂功能化修饰，并利用纸芯片特性和有机溶剂易挥发的特点，建立“样品有机溶剂前处理-芯片上在线样品预净化和溶剂分离-酶抑制率测定”一体化的胆碱酯酶抑制剂类农药残留快速检测方法，实现实际样品中农药残留水平的高灵敏度、低检测限快速检测。最后，以实际茶叶样品中的农药残留快速检测为模型，研究芯片对实际样品的在线预净化和快速检测性能，为快速检测应用及未来仪器小型化开发奠定基础。

基于乙酰胆碱酯酶活性抑制的农药残留快速检测方法是目前应用最广的有机磷和氨基甲酸酯类农药残留速测方法。为了避免有机溶剂对酶试剂的影响，该方法在实际应用时一直采用水性溶剂进行样品前处理，因而对大部分潜在农残组分的提取率低下，严重制约了检测结果的可靠性；同时，快检产品用于现场速测时，受制于操作条件，通常难以对样品溶液进行常规净化，待测液中的固体杂质易对检测结果产生干扰。.本研究分别研究了“十八烷基三氯硅烷（OTS）整体修饰—空气等离子体区域选择性处理”和“灰度控制喷蜡打印”两种滤纸亲、疏水性改性方法，建立了单层滤纸上3D微流控纸芯片的制备方法，考察了芯片通道对固体杂质的在线分离能力，建立了基于3D通道构型的集成化样品净化方法，实现了对液体中固体颗粒的在线分离，提高了快检方法对样品溶液中固体杂质的抗干扰能力。.本项目利用芯片基底材料具有超大比表面积的优势，以及有机溶剂易挥发的特性，建立了基于有机溶剂样品前处理的有机磷和氨基甲酸酯类农药残留快速检测方法，显著提高了实际样品中农药残留快速检测的灵敏度和可靠性。0.80 mg/kg的加标水平下，应用本研究所建立的方法，生菜样品中的辛硫磷的回收率可达到107.5%（vs.传统快检方法18.6%）。本研究所建立的方法还可实现地对水、饮料等样品中超低浓度的农药污染的现场快速筛查。.基于以上研究，我们在传统的农药残留速测卡的基础上，通过集成独立的进样功能区，研发了可对有机溶剂提取的样品溶液直接检测的新型农药残留速测卡，实现了便携式快速检测产品与有机溶剂样品前处理方法的兼容，可显著提高对实际样品检测结果的可靠性，同时具有成本低、操作简单的优势，具有明显的应用和推广价值。下一步研究中，我们希望能进一步优化和完善该速测卡的进样区功能，在探明不同农产品基质对酶抑制法快速检测结果干扰的具体原因（干扰来源）及干扰水平的前提下，在进样区集成具有针对性的基质净化材料，实现对典型农产品中农药残留的快速、灵敏、可靠检测。

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