Design and Applications of Droplet-based Digital PCR Microfluidic Chip with Integrated Fluid and Temperature Control

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

Abstract

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

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

CLC Number:

O652.9

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.

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.

Figures/Tables 14

Fig.1 Structure of droplet digital PCR chip

Fig.2 Chip preparation

Fig.3 Droplet digital PCR microfluidic chip test system

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

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

·

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

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

Fig.4 Force analysis of the droplet at the nozzle

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

Fig.6 Performance analysis of droplet generation in microfluidic chip

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

Fig.8 Fluid control valve performance test diagram

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

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

Fig.10 Automatic droplet counting and detection

References 25

[1]	KUANG J， YAN X， GENDERS A J， et al. An Overview of Technical Considerations When Using Quantitative Real-time PCR Analysis of Gene Expression in Human Exercise Research［J］. PLoS One， 2018， 13（5）：e0196438.
[2]	IP B B K， WONG A T C， LAW J H Y， et al. Application of Droplet Digital PCR in Minimal Residual Disease Monitoring of Rare Fusion Transcripts and Mutations in Haematological Malignancies［J］. Scientific Reports， 2024， 14（1）：6400.
[3]	LEI S， CHEN S， ZHONG Q. Digital PCR for Accurate Quantification of Pathogens：Principles， Applications， Challenges and Future Prospects［J］. International Journal of Biological Macromolecules， 2021， 184：750-759.
[4]	HO D， QUAKE S R， MCCABE E R B， et al. Enabling Technologies for Personalized and Precision Medicine［J］. Trends Biotechnol， 2020， 38（5）：497-518.
[5]	MEHTA N. RT-QPCR Made Simple：a Comprehensive Guide on the Methods， Advantages， Disadvantages， and Everything in Between［J］. Undergraduate Research in Natural and Clinical Science and Technology （URNCST） Journal， 2022， 6（10）：163-169.
[6]	黄琴，黄乐阳，靳翔宇，等. 注射式微流控芯片全集成核酸分析系统与精准医疗应用［J］. 中国激光， 2024， 51（9）：198-207.
	HUANG Qing， HUANG Leyang， JIN Xiangyu， et al. Fully Integrated Nucleic Acid Analysis System Based on Syringe Microfluidic Chip and Application of Precision Medicine ［J］. Chinese Journal of Lasers， 2024， 51（9）：198-207.
[7]	LI S， MA Z， CAO Z， et al. Advanced Wearable Microfluidic Sensors for Healthcare Monitoring［J］. Small， 2020， 16（9）：1903822.
[8]	祁恒，王贤松，陈涛，等. PMMA基连续流式PCR微流控芯片的CO₂激光直写加工与应用［J］. 中国激光， 2009， 36（5）：1239-1245.
	QI Heng， WANG Xiansong， CHEN Tao， et al. Fabrication and Application of PMMA Continuous-flow PCR Microfluidic Chip with CO₂ Laser Direct-writing Ablation Micromachining Technique［J］. Chinese Journal of Lasers， 2009， 36（5）：1239-1245.
[9]	WANG P， JING F， LI G， et al. Absolute Quantification of Lung Cancer Related Microrna by Droplet Digital PCR［J］. Biosensors and Bioelectronics， 2015， 74：836-842.
[10]	PINHEIRO L B， COLEMAN V A， HINDSON C M， et al. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification［J］. Analytical Chemistry， 2012， 84（2）：1003-1011.
[11]	刘聪，蒋克明，周武平，等. 基于液滴微流控技术的集成式数字PCR芯片［J］. 新技术新工艺， 2019（5）：1-6.
	LIU Cong， JIANG Keming， ZHOU Wuping， et al. Integrated Digital Polymerase Chain Reaction Chip Based on Droplet Microfluidic Technology［J］. New Technology & New Process， 2019（5）：1-6.
[12]	QUAN P L， SAUZADE M， BROUZES E. DPCR：a Technology Review［J］. Sensors （Basel）， 2018， 18（4）：1271.
[13]	LEI S， CHEN S， ZHONG Q. Digital PCR for Accurate Quantification of Pathogens：Principles， Applications， Challenges and Future Prospects［J］. International Journal of Biological Macromolecules， 2021， 184：750-759.
[14]	CUI X， WU L， WU Y， et al. Fast and Robust Sample Self-digitization for Digital PCR［J］. Analytica Chimica Acta， 2020， 1107：127-134.
[15]	SCHAERLI Y， WOOTTON R C， ROBINSON T， et al. Continuous-flow Polymerase Chain Reaction of Single-copy DNA in Microfluidic Microdroplets［J］. Analytical Chemistry， 2009， 81（1）：302-306.
[16]	ZHAN M， CHINGOZHA L， LU H. Enabling Systems Biology Approaches through Microfabricated Systems［J］. Analytical Chemistry， 2013， 85（19）：8882-8894.
[17]	PAN Y， MA T， MENG Q， et al. Droplet Digital PCR Enabled by Microfluidic Impact Printing for Absolute Gene Quantification［J］. Talanta， 2020， 211：120680.
[18]	李埰明. 基于数字PCR的核酸检测定量参考品制备与应用［D］.北京：中国科学院大学， 2021.
	LI Caiming. Preparation and Application of Quantitative Reference Materials for Nucleic Acid Detection Based on Digital PCR［D］. Beijing：University of Chinese Academy of Sciences， 2021.
[19]	VIVEKANANTHAN V， VIGNESH R， VASANTHASEELAN S， et al. Concrete Bridge Crack Detection by Image Processing Technique by Using the Improved Otsu Method［J］. Materials Today：Proceedings， 2023， 74：1002-1007.
[20]	ZHANG C， XING D， LI Y. Micropumps， Microvalves， and Micromixers within PCR Microfluidic Chips：Advances and Trends［J］. Biotechnology Advances， 2007， 25（5）：483-514.
[21]	TIAN Q， SONG Q， XU Y， et al. A Localized Temporary Negative Pressure Assisted Microfluidic Device for Detecting Keratin 19 in A549 Lung Carcinoma Cells with Digital PCR［J］. Analytical methods， 2015， 7（5）：2006-2011.
[22]	DHAL K G， DAS A， RAY S， et al. Histogram Equalization Variants as Optimization Problems：a Review［J］. Archives of Computational Methods in Engineering， 2021， 28：1471-1496.
[23]	GANGULY R， LEE C S. A Poisson-independent Approach to Precision Nucleic Acid Quantification in Microdroplets［J］. ACS Applied Bio Materials， 2024， 7（5）：3441-3451.
[24]	TANG Z， LV F， REYNOLDS D E， et al. Highly Parallel， Wash-free， and Ultrasensitive Centrifugal Droplet Digital Protein Detection in Sub-microliter Blood［J］. Lab on a Chip， 2023， 23（12）：2758-2765.
[25]	BSOUL A， PAN S， CRETU E， et al. Design， Microfabrication， and Characterization of a Moulded PDMS/SU-8 Inkjet Dispenser for a Lab-on-a-printer Platform Technology with Disposable Microfluidic Chip［J］. Lab on a Chip， 2016， 16（17）：3351-3361.