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A "sample-in-multiplex-digital-answer-out" chip for fast detection of pathogens.

Juxin Yin1, Zheyu Zou2, Zhenming Hu3

  • 1Research Centre for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, Zhejiang Province 310058, P. R. China. muying@zju.edu.cn and Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, National Ministry of Education), The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China and Institute of Translational Medicine, Zhejiang University, Hangzhou 310029, China.

Lab on a Chip
|February 1, 2020
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Summary

This study introduces an integrated microfluidic chip for rapid, multiplex, and absolute quantification of foodborne pathogens. The device enables point-of-care detection of multiple bacteria with high sensitivity and accuracy.

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Area of Science:

  • Biotechnology
  • Microfluidics
  • Molecular Diagnostics

Background:

  • Point-of-care (POC) testing requires rapid diagnostic results, but current quantification methods rely on standard curves and external references, lacking direct absolute quantification.
  • Existing methods for pathogen detection are often time-consuming and require complex laboratory equipment, limiting their application in resource-constrained settings.

Purpose of the Study:

  • To develop an integrated microfluidic chip for simultaneous, digital, and absolute quantification of multiple foodborne pathogens.
  • To create a "sample-in-multiplex-digital-answer-out" system for streamlined pathogen detection.
  • To demonstrate the chip's efficacy in detecting pathogenic bacteria in food matrices at the point-of-care.

Main Methods:

  • Fabrication of a multi-layer polydimethylsiloxane (PDMS) microfluidic chip using soft lithography, featuring a six-layer structure and screw microvalve control.
  • Integration of magnetic bead-based nucleic acid extraction (within 15 min) and multiplex digital recombinase polymerase amplification (dRPA) with fluorescence detection.
  • Utilized 12,800 chambers (2.7 nL each) for dRPA, with pre-embedded reagents controlled by screw valves and passively introduced via vacuum-based self-priming.

Main Results:

  • Successfully demonstrated simultaneous detection of three pathogenic bacteria species within 45 minutes in contaminated milk.
  • Achieved direct, absolute quantification without the need for standard curves, yielding digital results.
  • Established a detection limit of 10 bacterial cells per pathogen type.

Conclusions:

  • The integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip offers a rapid, sensitive, and accurate solution for foodborne pathogen detection.
  • The developed system enhances the applicability of point-of-care diagnostics for food safety.
  • Further development holds promise for feasible, rapid, multiplex, and accurate detection of foodborne bacteria.