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<p>Digital formats of bioassays offer substantial advantages over conventional nucleic acid amplification tests and immunoassays, including absolute quantification of pathogen DNA or antigen, and improved limits of detection. In this work, we describe a simplified integrated microfluidic digital assay system with performance comparable to conventional digital benchtop instruments based on Recombinase Polymerase Amplification (RPA). For digital analysis, we fabricate a simple single layer polydimethylsiloxane (PDMS) microfluidic chip and develop a novel and simple sample compartmentalization strategy. An air-permeable polytetrafluoroethylene (PTFE) membrane embedded in the chip can minimize sample loss. Surfactant-modified PDMS is adopted to ensure successful RPA amplification with hydrophilic characteristic. The sample solution is partitioned into 20,032 microchamber arrays by developing the state with negative pressure inside the microfluidic chip before vacuum-based sample sucking. For the detection device of digital analysis, we have designed a fully automated and integrated digital RPA instrument that includes three modules: sample injection, amplification, and imaging analysis. We evaluate the quantitative capabilities of the system by analyzing African swine fever DNA samples and demonstrate excellent analytical accuracy and specificity. This integrated and fully automated diagnostic system represents a promising diagnostic platform for quantitative nucleic acid testing.</p>

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An automated, dead-volume reduced, isothermal digital nucleic acid quantification system based on recombinase polymerase amplification

  • 期刊论文

  • 发表日期：

    2025年1月25日

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  • Zhongping Zhang,
  • Rui Zhu,
  • Tianwei Li,
  • Nan Wang,
  • Lulu Zhang,
  • Michael G. Mauk,
  • Guijun Miao,
  • Xianbo Qiu

摘要

Digital formats of bioassays offer substantial advantages over conventional nucleic acid amplification tests and immunoassays, including absolute quantification of pathogen DNA or antigen, and improved limits of detection. In this work, we describe a simplified integrated microfluidic digital assay system with performance comparable to conventional digital benchtop instruments based on Recombinase Polymerase Amplification (RPA). For digital analysis, we fabricate a simple single layer polydimethylsiloxane (PDMS) microfluidic chip and develop a novel and simple sample compartmentalization strategy. An air-permeable polytetrafluoroethylene (PTFE) membrane embedded in the chip can minimize sample loss. Surfactant-modified PDMS is adopted to ensure successful RPA amplification with hydrophilic characteristic. The sample solution is partitioned into 20,032 microchamber arrays by developing the state with negative pressure inside the microfluidic chip before vacuum-based sample sucking. For the detection device of digital analysis, we have designed a fully automated and integrated digital RPA instrument that includes three modules: sample injection, amplification, and imaging analysis. We evaluate the quantitative capabilities of the system by analyzing African swine fever DNA samples and demonstrate excellent analytical accuracy and specificity. This integrated and fully automated diagnostic system represents a promising diagnostic platform for quantitative nucleic acid testing.