关于数字PCR扩增过程中气泡产生机理及抑制方法研究

doi:10.19586/j.2095-2341.2025.0010

生物技术进展 ›› 2025, Vol. 15 ›› Issue (4): 693-701.DOI: 10.19586/j.2095-2341.2025.0010

• 研究论文 • 上一篇

关于数字PCR扩增过程中气泡产生机理及抑制方法研究

贾艳艳¹(), 端木路阳²()

^1.吉林医药学院附属医院资产管理与物资保障科，吉林吉林 132000
^2.长春理工大学物理学院，长春 130000

收稿日期:2025-02-11 接受日期:2025-03-28 出版日期:2025-07-25 发布日期:2025-09-08
通讯作者: 端木路阳
作者简介:贾艳艳 E-mail: 1019570283@qq.com；

Study on the Mechanism of Bubble Generation and Inhibition Method During Digital PCR Amplification Process

Yanyan JIA¹(), Luyang DUANMU²()

^1.Asset Management and Material Security Section，Affiliated Hospital of Jilin Medical College，Jilin Jilin 132000，China
^2.School of Physics，Changchun University of Science and Technology，Changchun 130000，China

Received:2025-02-11 Accepted:2025-03-28 Online:2025-07-25 Published:2025-09-08
Contact: Luyang DUANMU

摘要/Abstract

摘要：

数字PCR（digital PCR，dPCR）技术的出现显著推动了核酸检测的发展，然而微滴在微流控芯片中的移动、破碎和气泡产生问题又限制了该技术的发展，因此解决现有技术存在的问题具有深刻意义。构建了一种基于恒压调控微流控芯片中微滴PCR的装置及方法，对气泡的存在原理进行了分析，并对芯片中的气泡进行仿真验证。结果表明，通过气源装置对芯片中的压力进行调整，可实现无气泡产生的微滴数字PCR。利用该装置和芯片对人表皮生长因子受体（epidermal growth factor receptor, EGFR）外显子18基因进行定量检测，3 min内可将20 μL样品分布到约5万个油包水微滴中，微滴在芯片中排列紧密无气泡，并且实验检测信号与DNA浓度在10¹~10⁵拷贝·μL^-1范围内具有良好的线性关系（R²=0.999）。与目前去除气泡的研究相比，dPCR技术避免了微滴的破碎融合，并可对结果进行标准化，在核酸定量检测中具有广阔的应用前景。

关键词: 恒压调控, 微滴数字PCR, 荧光定量检测, 微流控芯片

Abstract:

The emergence of digital PCR （dPCR） technology has significantly promoted the development of nucleic acid detection. However， the problems of droplets moving， breaking and generating bubbles in microfluidic chips have limited the development of digital PCR technology and it is of great significance to solve the existing problems. The article constructed a device and method for droplet PCR in microfluidic chips based on constant pressure regulation analyed the existence principle of bubbles， and the bubbles in the chip were simulated and verified. The results showed that adjusting the pressure in the chip through the air source device could achieve bubble-free generation of droplets and PCR amplification. The quantitative detection of human epidermal growth factor receptor （EGFR） exon 18 gene was carried out by using the device and the chip. Within 3 minutes， 20 μL of the sample was distributed into about 50 000 water-in-oil droplets， which were tightly arranged without bubbles in the chip. The results showed that the experimental detection signal had a good linear relationship with DNA concentration in the range of 10¹~10⁵ copies·μL^-1 （R²=0.999）. Compared with current research on bubble removal， dPCR technology avoids the fragmentation and fusion of micro-droplets and can standard the results， which has broad application prospects in nucleic acid quantitative detection.

Key words: constant pressure regulation, droplet digital PCR, fluorescence quantitative detection, microfluidic chip

中图分类号:

Q-33

贾艳艳, 端木路阳. 关于数字PCR扩增过程中气泡产生机理及抑制方法研究[J]. 生物技术进展, 2025, 15(4): 693-701.

Yanyan JIA, Luyang DUANMU. Study on the Mechanism of Bubble Generation and Inhibition Method During Digital PCR Amplification Process[J]. Current Biotechnology, 2025, 15(4): 693-701.

图/表 13

图1 气泡示意图

Fig. 1 Schematic diagram of bubbles

图2 微流控芯片设计图

Fig. 2 Design drawing of microfluidic chip

图3 芯片制作过程A：由SU8雕刻微通道；B：浇筑COC；C：打孔；D：键合；E：制作好的芯片

Fig. 3 Chip fabrication process

图4 恒压调控装置示意图

Fig. 4 Schematic diagram of the constant-pressure control device

图5 二维微腔气泡模型注：Wc —微腔水平宽度；Wd —微腔高度；Ro —气泡半径。

Fig. 5 Two-dimensional microcavity bubble model

表1 气泡和溶液的物理属性

Table 1 Physical properties of bubbles and solutions

名称	值	单位	描述
R₀	30	μm	气泡初始半径
ρ	1 000	kg·m^-3	溶液的密度
ε₀	7.28×10^-2	N·M^-1	液体初始表面张力
μ	0.001	Pa×S	液体动力粘度
P_f0	1.013 3×10⁵	Pa	气泡内初始压力
P_f	$P f 0 × s t e p 2 (t [1 / s])$	Pa	液体内压力
P_N0	1.013 3×10⁵	Pa	液体内初始压力
k	1	1	气体多变指数
T₀	293	K	初始液体温度
K_S	1.52×10^-4	kg·(S²·K)^-1	液体扩散系数

表1 气泡和溶液的物理属性

Table 1 Physical properties of bubbles and solutions

名称	值	单位	描述
R₀	30	μm	气泡初始半径
ρ	1 000	kg·m^-3	溶液的密度
ε₀	7.28×10^-2	N·M^-1	液体初始表面张力
μ	0.001	Pa×S	液体动力粘度
P_f0	1.013 3×10⁵	Pa	气泡内初始压力
P_f	$P f 0 × s t e p 2 (t [1 / s])$	Pa	液体内压力
P_N0	1.013 3×10⁵	Pa	液体内初始压力
k	1	1	气体多变指数
T₀	293	K	初始液体温度
K_S	1.52×10^-4	kg·(S²·K)^-1	液体扩散系数

图6 液体温度随时间变化关系

Fig. 6 Liquid temperature changes with time

图7 气泡半径随温度变化关系

Fig. 7 Relationship between bubble radius and temperature changes

图8 微流控芯片组成及工作过程A：微流控芯片结构示意图；B：微滴生成的流聚焦结构，蓝色箭头表示油路流动方向，黄色箭头表示样本的流动方向；C：在收集室中单层分布的微滴

Fig. 8 Composition and working process of microfluidic chip

图9 不同压力下的ddPCR结果A：生成完好的核酸微滴；B：0 mbar压力；C：700 mbar压力；D：1 150 mbar压力；E：1 500 mbar压力；F：2 000 mbar压力

Fig. 9 ddPCR results of microdroplets under different pressures

图10 荧光信号采集系统

Fig. 10 Fluorescence signal acquisition system

图11 检测不同DNA浓度下EGFR外显子18基因的荧光图像

Fig. 11 Fuorescence images for simultaneous detection of EGFR exon18 gene with various DNA concentrations

图12 集成ddPCR基因芯片的观测值与期望值注：误差线代表95%置信区间。

Fig. 12 The observed values of the integrated ddPCR gene chip are consistent with the expected values

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关于数字PCR扩增过程中气泡产生机理及抑制方法研究

Study on the Mechanism of Bubble Generation and Inhibition Method During Digital PCR Amplification Process

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