集成流体与温度控制的液滴型数字PCR微流控芯片设计与应用

doi:10.3969/j.issn.1004-132X.2025.10.025

摘要/Abstract

摘要：

数字聚合酶链式反应（dPCR）因高灵敏度、高准确度以及绝对定量的特点，成为分子生物学和临床诊断领域的重要工具，但仍面临集成度不高、样本使用量大及流体控制难等问题。设计了一种集成快速阀门系统和自动加热系统的液滴型dPCR微流控芯片，同时优化了该芯片的制备工艺。研究结果表明，该芯片能够高效、均匀地生成液滴，最大生成频率达5.132 kHz，液滴直径变异系数（CV）值仅为1.67%；形变阀可在0.1 s内快速实现流体通断；集成的片上循环加热系统可实现1.2 ℃/s的温度升降，温度误差在±0.6 ℃以内；改进PDMS配方还可有效防止流体蒸发；液滴识别算法准确率高达99.7%。

关键词: 微流控芯片, 微液滴, 数字聚合酶链式反应, 形变阀, 集成化

Abstract:

Digital PCR （dPCR） had emerged as a vital tool in molecular biology and clinical diagnostics due to the high sensitivity， accuracy， and absolute quantification capabilities. However， challenges had persisted regarding integration level， sample volume requirements and fluid control. An integrated droplet-based dPCR microfluidic chip incorporating a rapid valve system and automated heating system was presented， along with optimized fabrication processes herein. The results show that the chip may generate droplets efficiently and uniformly， with a maximum generation frequency of 5.132 kHz and a droplet diameter CV（the coefficient of variation） of only 1.67%. Rapid fluid control within 0.1s is enabled by the deformable valve， while temperature ramping rates of 1.2 ℃/s with temperature variations within ±0.6 ℃ are achieved by the integrated on-chip thermal cycling system. Fluid evaporation is effectively prevented by an improved PDMS formulation， and the accuracy of 99.7% is achieved by the droplet recognition algorithm.

Key words: microfluidic chip, microdroplet, digital polymerase chain reaction（PCR）, deformable valve, integration

中图分类号:

O652.9

何春华, 姚金辉, 聂磊, 廖广兰, 史铁林, 刘智勇. 集成流体与温度控制的液滴型数字PCR微流控芯片设计与应用[J]. 中国机械工程, 2025, 36(10): 2359-2368.

Chunhua HE, Jinhui YAO, Lei NIE, Guanglan LIAO, Tielin SHI, Zhiyong LIU. Design and Applications of Droplet-based Digital PCR Microfluidic Chip with Integrated Fluid and Temperature Control[J]. China Mechanical Engineering, 2025, 36(10): 2359-2368.

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链接本文: https://www.cmemo.org.cn/CN/10.3969/j.issn.1004-132X.2025.10.025

https://www.cmemo.org.cn/CN/Y2025/V36/I10/2359

图/表 14

图1 液滴dPCR芯片的结构

Fig.1 Structure of droplet digital PCR chip

图2 芯片制备

Fig.2 Chip preparation

图3 液滴dPCR微流控芯片测试系统

Fig.3 Droplet digital PCR microfluidic chip test system

表1 循环扩增反应程序

Tab.1 Procedure of cyclic amplification reaction

步骤	温度	持续时间	循环数
预变性	95 ℃	2 min	1
变性	95 ℃	15 s	30
退火	55 ℃	30 s	30
延伸	72 ℃	30 s	30

表2 分散相与连续相的液体成分及物性参数

Tab.2 Liquid composition and physical property parameters of dispersed phase and continuous phase

两相液体

物质

密度/

（kg·m^-3）

黏度/（mPa

·

表2 分散相与连续相的液体成分及物性参数

Tab.2 Liquid composition and physical property parameters of dispersed phase and continuous phase

两相液体

物质

密度/

（kg·m^-3）

黏度/（mPa

·

s）

界面张力/（mN·m^-1）

分散相

PBS缓冲液

1260

1.005

15.21

连续相

矿物油

935

表3 实验所用的生物试剂

Tab.3 Biological reagents used in the experiment

组分	体积
Genomic DNA	10 µL
Primer F	10 µL
Primer R	10 µL
2× Universal SYBR qPCR Mix	250 µL
去离子水	220 µL

图4 液滴在喷嘴处的受力分析

Fig.4 Force analysis of the droplet at the nozzle

图 5 不同喷嘴结构液滴生成的仿真图

Fig.5 Simulation diagram of droplet generation with different nozzle structures

图6 微流控芯片液滴生成性能分析

Fig.6 Performance analysis of droplet generation in microfluidic chip

图7 基于PDMS的微流控阀门仿真结果分析

Fig.7 Analysis of PDMS-based microfluidic valve simulation results

图8 流体控制阀门性能测试图

Fig.8 Fluid control valve performance test diagram

图9 防蒸发测试图及PCR发光测试

Fig.9 Anti-evaporation test diagram and PCR luminescence test

表4 近期dPCR研究直径CV对比

Tab.4 Comparison of diameter CV values in recent dPCR studies.

来源	CV值	年份
文献［23］	12.44%	2024
文献［24］	9.68%	2023
文献［25］	7.78%~10.52%	2016

图10 液滴自动计数检测

Fig.10 Automatic droplet counting and detection

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