带核酸提取的一体化对流CPCR微流控核酸诊断方法研究

In order to achieve rapid, simple, efficient and low-cost nucleic acid analysis in point-of-care testing (POCT), a new method for integrated microfluidic nucleic acid analysis based on convective PCR with nucleic acid extraction will be studied. A couple of critical issues including rapid nucleic acid extraction compatible with convective PCR, an integrated single convective PCR reactor for nucleic acid detection, and the physical model for the single reactor and microfluidic chip integration will be studied in details. Nucleic acid extraction based on solid phase membrane with simplified purification will be studied to develop a simple and rapid nucleic acid extraction method compatible with convection PCR. Based on the concept with a multiple functional chamber, an innovative single convective PCR reactor with nucleic acid extraction, convective PCR amplification and real-time detection will be studied. The physical model for the single reactor will be studied to establish the mathematical models respectively for the flow field, temperature field and concentration filed, which can be used to optimize the single reactor. Integration of different functional modules will be studied to develop the integrated microfluidic system for nucleic acid diagnosis based on convective PCR. Based on the remarkable amplification efficiency of convective PCR, integrated convective PCR analysis with nucleic acid extraction is able to short detection time, simplify detection procedure, and reduce system complexity. This study will not only speed up the research on nucleic acid analysis at the point-of-care settings, but also accelerate the multidisciplinary research between medicine and engineering.

为了实现快速、简单、高效、低成本的POCT（Point-of-Care Testing）核酸诊断，研究带核酸提取的一体化对流CPCR微流控核酸诊断方法，主要解决：适于对流CPCR核酸快速提取、一体化对流CPCR单反应器核酸检测、及单反应器物理建模分析与芯片集成等关键问题。研究基于固相载体的核酸提取方法，同时简化核酸纯化过程，建立适于对流CPCR，简单快速的核酸提取方法；基于一腔多能概念，研究集成核酸快速提取、对流CPCR扩增反应、及实时荧光检测的一体化对流CPCR单反应器；研究对流CPCR单反应器物理模型，建立其流场、温度场及浓度场模型，指导单反应器优化设计，同时，研究芯片集成方法，构建对流CPCR核酸诊断集成芯片系统。依靠对流CPCR显著的扩增速度优势，实现一体化对流CPCR核酸诊断，有利于缩短检测时间，简化检测过程，降低系统复杂度。本项目能够促进POCT核酸检测研究与医工学科交叉融合。

基于PCR的核酸诊断能够实现高灵敏度与高特异性的疾病检测，在生命医学领域发挥了重要的作用。受流程复杂性及操作繁琐性、检测时长及检测成本等关键因素的制约，普通PCR核酸诊断难以在各类POCT应用场景中得到合理的推广与应用。因此，研究基于对流PCR（CPCR）的一体化核酸检测方法，不仅具有重要的学术意义，而且有利于提升POCT核酸检测水平。.研究内容包括：结合理论仿真模拟与实验验证，研究了水平式CPCR扩增方法、以及毛细管反应器的物理建模。研究了基于微流控芯片的POCT血浆快速提取方法。研究了基于人工智能算法、及优化光电检测策略的CPCR扩增方法与系统。研究了基于磁珠法的核酸快速提取微流控芯片，及手持式CPCR装置，实现了POCT核酸快速检测。基于简化核酸提取方法及便捷式流体驱动，研究了一体化全集成CPCR核酸检测系统。.利用COMSOL，对水平式、垂直式CPCR反应器的流场、温度场进行了建模仿真，构建了一个基于智能手机的水平式CPCR系统。研究发现，CPCR反应器的加热器高度、及其与毛细反应管的高度比，对于扩增效率具有显著影响。研究了一种基于多功能微流控芯片的血浆快速提取方法，借助重力与多孔过滤膜的双重作用，实现红细胞滤除，同时，借助弹性反应腔，8分钟内，实现血浆分离与驱动。研究了基于纵向光路的荧光采集方法，提升了CPCR检测灵敏度，结合智能手机，构建了便携式多通道CPCR扩增系统，同时，研究了基于人工智能算法与常规判定方法的复合判读算法，显著提升了检测准确性（特异性由75%提升到97%）。研究了基于3D磁场驱动的磁珠法核酸提取微流控芯片，提升了核酸提取效率，同时，研究了手持式CPCR检测装置，构建了包括核酸提取与核酸扩增的POCT核酸检测系统，50分钟内完成核酸检测。将与PCR兼容的核酸提取膜集成到反应器中，构建“提取-扩增”一体化CPCR反应器，依靠离心式流体驱动，结合智能手机荧光检测，构建了集成核酸检测系统，45分钟内，实现了“样本进-结果出”一体化检测。本研究对于推动POCT核酸诊断具有一定的科学意义与应用价值。