适配体筛选微分析系统的研制

To address the problems of aptamer selection system such as complicated structure, tedious and long process and non-achievement of fully automatic detection, this project intends to design and fabricate an automatic micro-equipment and microfluidic chip-based analysis system for aptamer selection. It integrates several laboratory functions including surface plasma resonance imaging (SPRI) detection, PCR amplification and detection. By using SPRi analytical technology, target-binding aptamer candidates can be detected with high sensitivity via magnetic nanoparticle-based signal amplification strategy. Especially in the process for aptamer selection, association, dissociation and elution of target molecules and oligonucleic acids can be monitored in real time. By employing On-Chip PCR technology and a constructed automatic system containing PCR amplification, detection and self-circulation, the selected aptamer candidates automatically enter into the PCR amplification part and are amplified. Then these the amplified molecules go once more into the SPRi part for the subsequent binding and selection process, and thus forming the SELEX cycle. On the basis of our group''s research works focusing on aptamer-bsed detection and separation, SPR imaging method and biochip, we seek to design and fabricate the optical detection devices for realizing SPR imaging, temperature control unit for PCR amplification, electromagnetic separation unit and microfluidic control unit, etc. Meanwhile, combining with microarray biochip technology, high-throughput and rapid aptamer selection for multi-target molecules can be achieved, and a fully automatic high performance aptamer screening equipment (platform) implementing the SELEX on-a-chip can be developed.

针对适配体筛选系统中存在的结构庞杂、流程冗长和难以完全实现自动化等不足，本课题以研制适配体自动化筛选微型设备为目标，设计与加工集成有表面等离子体成像（SPRi）监测、PCR扩增与检测功能一体化的微流体芯片分析系统。采用SPRi检测技术，通过磁性纳米信号放大方法达到对适配体候选分子的高灵敏检测；采用On-Chip PCR技术，通过构建的PCR扩增、检测和自循环系统，使筛选出的适配体候选分子自动进入PCR区域进行扩增；扩增后的候选分子将再次进入SPRi区域进行结合与筛选，从而形成SELEX循环。利用本课题组在适配体检测与分离、SPR成像方法及生物芯片的研究基础，设计并加工实现SPRi成像的光路与检测器件、PCR扩增控温单元、电磁性分离单元及微流路液体控制单元等，同时结合微阵列生物芯片技术，实现多靶标分子的高通量快速筛选，研制出SELEX-on-a-chip适配体全自动高效筛选仪器（平台）。

本课题围绕适配体筛选微分析系统装置研制与适配体筛选分析应用等开展了系统研究工作。该装置研制主要包括微流控芯片的设计与制备、表面等离子体共振成像检测系统、PCR荧光芯片分析系统、微纳磁珠样品在线分离系统及适配体微分析系统集成等。研制中创新设计与制作了含有正筛通道与负筛通道的微流控芯片；构建了krechamn棱镜型表面等离子体共振成像仪器，该系统可实时动态监测正筛微流控通道中靶分子特异性结合的核酸分子的数量变化；PCR荧光芯片检测系统分别由微流控PCR芯片、温度实时监控系统、恒压泵PC在线控制系统、PCR热循环程控系统及荧光实时监测系统五部分组成，通过得到的扩增曲线快速获取最优扩增条件，提高了筛选中PCR扩增速度。研制的微纳磁珠样品在线分离系统由自动化电子升降台、接触式超声波混合系统和微流控微纳磁珠芯片组成，使PCR反应样品dsDNA的分离和ssDNA的再生操作总时间缩短到35min，分离效率达到约90%，可有效进行dsDNA纯化分离及次级文库再生。研制中通过微流体自动进样与连接装置，采用计算机操作系统控制将上述部分集成组装构成适配体筛选微分析系统。该系统已成功应用于乳铁蛋白适配体的自动化筛选中，筛选获得的适配体具有高特异性与高亲和力，并成功应用于生物传感分析中。该适配体筛选微分析系统具有动态化监测、智能化控制、自动化再生、集成化操作等创新点，大大提高了筛选操作的可靠性，节省了操作时间与繁杂程度。研制该系统有利于高效制备适配体探针，为生物传感器研制、生物成像分析、生物标志物发现、医学诊断及新药研发等领域提供积极与重要的支持；也为生物分子检测的智能化制造提供了发展思路。

内容获取失败，请点击重试