Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria

Zhenqing Li; Yifei Wang; Zehang Gao; Shinichi Sekine; Qingxiang You; Songlin Zhuang; Dawei Zhang; Shilun Feng; Yoshinori Yamaguchi

doi:10.1016/j.aca.2023.340995

Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria

Anal Chim Acta. 2023 Apr 22:1251:340995. doi: 10.1016/j.aca.2023.340995. Epub 2023 Feb 24.

Authors

Zhenqing Li¹, Yifei Wang¹, Zehang Gao², Shinichi Sekine³, Qingxiang You¹, Songlin Zhuang¹, Dawei Zhang⁴, Shilun Feng⁵, Yoshinori Yamaguchi⁶

Affiliations

¹ Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, Shanghai Environmental Biosafety Instruments and Equipment Engineering Technology Research Center, University of Shanghai for Science and Technology, Shanghai, 200093, China.
² State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; Department of Clinical Laboratory, Third Affiliated Hospital of Guangzhou Medical University, Guangdong, 510150, China.
³ Department of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Osaka, Japan.
⁴ Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, Shanghai Environmental Biosafety Instruments and Equipment Engineering Technology Research Center, University of Shanghai for Science and Technology, Shanghai, 200093, China. Electronic address: dwzhang@usst.edu.cn.
⁵ State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China. Electronic address: shilun.feng@mail.sim.ac.cn.
⁶ Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, Shanghai Environmental Biosafety Instruments and Equipment Engineering Technology Research Center, University of Shanghai for Science and Technology, Shanghai, 200093, China; Graduate School of Engineering, Osaka University, Osaka, 565-0871, Japan. Electronic address: yoshi.yamaguchi@ap.eng.osaka-u.ac.jp.

PMID: 36925286
DOI: 10.1016/j.aca.2023.340995

Abstract

Background: Rapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation.

Results: Experiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 μm² (40 × 40 μm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11'16″ and observed the fluorescence from droplets.

Significance and novelty: Such a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.

Keywords: Continuous flow PCR; Droplet; Microfluidic chip; Nucleic acid; Periodontal pathogen; Polymerase chain reaction.

MeSH terms

Bacteria / genetics
Microfluidic Analytical Techniques*
Microfluidics*
Polymerase Chain Reaction
Water

Substances

Water