2019年爆发的新冠疫情给我国人民的生产生活带来了巨大的冲击，严重威胁着我国人民的生命健康安全。面对疫情，高效快速的检测手段必不可少。核酸检测技术是进行RNA病毒临床诊断的重要手段。样本提纯这一步骤不仅是核酸检测过程中耗时最长的一步，而且其提纯的效率会对检测结果产生较大的影响。传统的核酸提纯和检测试剂盒尽管具有较高的提纯效率和检出准确性，但它们往往操作复杂，耗时较长，且气密性较差，存在交叉污染的潜在风险。相比之下，微流控技术能够很好地克服这些问题，其具备样本消耗量少、易于集成控制、适合于高通量检测等优点，因而成为近年来核酸提取和扩增临床应用的研究热点之一。本文的研究目标为开发一种集成核酸提取全部流程的自动化微流控芯片，以及配套的离心设备和检测手段。本文的研究具体所做的工作为：开发了一种基于表面修饰技术的核酸提纯方法，利用修饰在芯片流道表面的PAMAM提取核酸。这种方式不需要磁珠和硅珠，因而无需外源性的控制设备。PAMAM分子末端有着大量的可用官能团，因而这种方法不需要通过微阵列的方式增加流道表面积，从而进一步降低了芯片成本。为了便于控制和集成，我们设计了一套与提取方法配套的离心设备和芯片，将核酸提纯的各个步骤集成于一个平台之上。这种离心设备通过两个伺服电机提供驱动力，从而可以控制芯片与离心轴之间的偏转角，进而改变不同位置受到的离心力。芯片的各个腔室之间设计了升高的流道，当离心力够强液体才能通过。不同腔室和目标腔室之间流道的高度不同，进而实现差异化的控制。利用这种方式，我们实现了对芯片流体的时序控制，从而可以不借用磁场等设备实现核酸提纯和扩增所需要液体混合和分离等操作。最后，我们以新冠N基因质粒和HPV病毒为样本，进行了芯片上的核酸提取及扩增实验。实验结果表明，我们的方法具有良好的核酸提纯能力，并且不会引入任何可能会影响扩增的有机试剂，配套的离心设备也极大地简化了实验人员的操作。总体而言，我们的设备在提高了核酸提纯效率的同时降低了芯片的制造成本，除了适合应用于较大规模的临床检测以外，还可以用于制备实验室需要的核酸样本，因而具有一定的实用价值。
The outbreak of the New Coronavirus epidemic in 2019 has brought a huge impact on the production of the whole society, and is still seriously threatening the life and health safety of our people. In the face of the epidemic, efficient and rapid detection means are getting increasingly important. Nucleic acid detection technology is an important tool for conducting clinical diagnosis of RNA virus. The sample purification step is the most time-consuming step in the process of nucleic acid testing, while its efficiency often lays a great impact on the test results. Although traditional nucleic acid purification and detection kits have high purification efficiency and detection accuracy, they are often complex, time-consuming, and have poor gas tightness, with potential risk of cross-contamination. In contrast, microfluidic technology can overcome these problems, and it has the advantages of low sample consumption, easy integration and control, and suitable for high-throughput detection, thus becoming one of the research hotspots for clinical applications of nucleic acid extraction and amplification in recent years.The goal of this paper is to develop an automated microfluidic chip that integrates the entire process of nucleic acid extraction, along with supporting centrifugation equipment and assays. The specific work of this paper was developing a nucleic acid purification method based on surface modification technology, which used PAMAM modified on the surface of the chip flow channel to extract nucleic acids. This approach does not require magnetic or silica beads, thus eliminating the need for external control devices, and the large number of available functional groups at the end of the PAMAM molecule eliminates the need to increase the surface area of the flow channel by means of microarrays, thus further reducing the cost of the chip. To facilitate control and integration, we designed a centrifugation device and chip to accompany the extraction method, integrating the various steps of nucleic acid purification on a single platform. The centrifugation equipment is driven by two servo motors that control the deflection angle between the chip and the centrifugal shaft, thus changing the centrifugal force at different positions. The flow paths between the chambers of the chip are elevated, so that the liquid can pass through only when the centrifugal force is strong enough. The height of the flow channels of, thus enabling differentiated control. In this way, we have been able to achieve temporal control of the chip fluid so that operations such as mixing and separation of liquids required for nucleic acid purification and amplification can be performed without the use of magnetic fields or other equipment.Finally, we performed nucleic acid extraction and amplification experiments on the integrated chips using the new crown N gene plasmid and HPV virus as samples. The experimental results showed that our method had good nucleic acid purification capability and did not introduce any organic reagents that may affect amplification, and the accompanying centrifugation equipment greatly simplified the experimental personnel‘s operation. Overall, it has been shown that our equipment can improve the efficiency of nucleic acid purification while reducing the manufacturing cost of the microfluidic chips, and is of practical value as it can be used for preparing nucleic acid samples for laboratory needs in addition to larger scale clinical assays.
