核酸纯化沉淀阻塞的微流控核酸目视检测研究

doi:10.11949/0438-1157.20250437

摘要/Abstract

摘要：

在当前对快速、安全、便捷的核酸检测技术需求日益增长的背景下，借鉴乙醇纯化核酸的过程及微滤器的过滤原理，设计出利用核酸沉淀阻塞通道的目视检测微流控芯片。在目标核酸特异性扩增后，使用乙醇进行沉淀，基于多孔介质内非刚性颗粒对流动的物理阻塞机理，实现目标核酸的目视检测。使用PMMA微球作为微流控芯片中的阻塞结构，30组重复实验全部在10 min内成功检出，保证了该方案的可行性。该方法兼容恒温和非恒温核酸扩增技术，芯片内环境全程负压且多次进行酒精处理，提供双重安全保障，提高了POCT应用的安全性。

关键词: 微流控, 核酸检测, POCT, 核酸纯化, 病毒

Abstract:

With the increasing demand for rapid, safe, and convenient nucleic acid detection technologies, this study designs a visual detection microfluidic chip based on the principle of nucleic acid precipitation and channel blockage. Inspired by the ethanol purification process of nucleic acids and the filtration principle of microfilters, this method utilizes the physical blockage effect of precipitated nucleic acids to achieve visual detection. After specific amplification of the target nucleic acids, ethanol is used to precipitate them, thereby enabling visual detection of the target nucleic acid. We designed the microfluidic chip using poly(methyl methacrylate) (PMMA) microspheres as the porous medium. Thirty repeated experiments were conducted on this microfluidic chip, all of which successfully detected the target within 10 minutes, demonstrating practicality and high accuracy. This qualitative reading method is applicable to all pathogens in principle and is compatible with both isothermal and non-isothermal amplification techniques. Additionally, the chip's internal environment is maintained under negative pressure throughout the process, and it is treated multiple times with alcohol, providing dual safety measures and enhancing the safety of its application in point-of-care testing (POCT).

Key words: microfluids, nucleic acid testing (NAT), point-of-care testing (POCT), nucleic acid purification, virus

中图分类号:

TQ 021.1

张文普, 陈汾琳, 李雯曦, 马晟利, 葛文玉, 李彪. 核酸纯化沉淀阻塞的微流控核酸目视检测研究[J]. 化工学报, 2025, 76(11): 5799-5805.

Wenpu ZHANG, Fenlin CHEN, Wenxi LI, Shengli MA, Wenyu GE, Biao LI. Research on visual detection of nucleic acids using microfluidic chips with precipitation-induced obstruction for nucleic acid purification[J]. CIESC Journal, 2025, 76(11): 5799-5805.

图/表 9

表1 反应液成分

Table 1 Component of the reaction mixture

名称	说明
细胞裂解液	盐溶液，含Igepal CA-630、多羟基醇、EDTA、氯化钠等组分
扩增酶	酶混合物
扩增反应液	核糖核苷酸、脱氧核糖核苷酸、扩增引物
阴性质控物	盐溶液
阳性质控物	含有靶基因位点的假病毒和人Hep-2细胞混合物

图1 阻塞式微流控芯片设计图Ⅰ—进口;Ⅱ—填充段装配口;Ⅲ—多孔介质填充段;Ⅳ—微球阻塞段;Ⅴ—出口

Fig.1 The design diagram of obstructive microfluidic chipⅠ—inlet; Ⅱ—filling section assembly port; Ⅲ—porous medium filling section; Ⅳ—obstruction section for microsphere; Ⅴ—outlet

图2 (a)软光刻系统；(b)微流控系统及图像记录系统

Fig.2 (a) Soft lithography system; (b) Microfluidic system and imaging recording system

图3 核酸纯化沉淀阻塞微流控芯片实验示意图

Fig.3 The schematic diagram of nucleic acid purification and precipitation obstruction microfluidic chip experiment

图4 (a)核酸检测流程及原理图；(b)微流控芯片实物图；(c)检测结果对比图

Fig.4 (a) The schematic diagram of nucleic acid testing process and principle; (b) Photograph of the microfluidic chip; (c) Comparison of testing results

图5 (a)微滤截留机制；(b)微球颗粒多孔介质中核酸沉淀阻塞示意图

Fig.5 (a) The schematic diagram of microfiltration retention mechanism; (b) The schematic diagram of nucleic acid precipitation and obstruction in porous medium with microspheres

图6 显微镜下的核酸沉淀（上方为明场拍摄，下方为二值化处理后图像）

Fig.6 Microphotograph of nucleic acid precipitation (The upper image is captured in bright-field mode, while the lower image is captured in dark-field mode)

图7 阴性样本、阳性样本的实验检测结果图：(a)实物图；(b),(c)显微镜照片

Fig.7 The experimental detection results of negative and position samples: (a) photograph; (b),(c) microphotograph

图8 阴性样本全部通过微通道的时间

Fig.8 Time for negative samples to pass through the microchannel

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