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<p><i>Mycoplasma pneumoniae</i> (MP) is a major pathogen of community-acquired pneumonia, especially in children, but its early diagnosis remains challenging due to nonspecific symptoms and limitations of conventional methods. In this study, we present a portable, amplification-free microfluidic platform for ultrasensitive detection of both MP DNA and RNA. This system integrates a phosphate-responsive zirconium-based metal-organic framework (Zr-MOF) with CRISPR‒Cas12a to achieve cascade signal amplification. Specifically, the MOF is co-loaded with target-specific probes and a high density of activators. Upon stimulation with phosphate, the MOF structure disassembles, releasing a burst of activators that trigger the trans-cleavage activity of Cas12a, leading to an amplified fluorescence readout. Enabled by efficient mass transport in the microfluidic device, the assay reduces detection time and sample consumption. Without the need for thermal cycling or reverse transcription, the platform achieves limits of detection as low as 0.35&#xa0;fg/µL for DNA and 0.43&#xa0;fg/µL for RNA. The detection results for 50 clinical samples show excellent agreement with qPCR. Furthermore, the assay is compatible with a smartphone-based portable reader, making it ideal for point-of-care testing. This microfluidic MOF-CRISPR platform offers a promising strategy for rapid, low-cost, and amplification-free nucleic acid diagnostics in resource-limited settings.</p>

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Portable Microfluidic Detection of Mycoplasma Pneumoniae via MOF-CRISPR–Cas12a Cascade Signal Amplification

  • 期刊论文

  • 发表日期：

    2026年5月5日

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  • Xiaoqing Cao,
  • Yurou Kang,
  • Yong Wang,
  • Wen Bi,
  • Jingjing Li,
  • Xincheng Yang,
  • Yanfeng Gao,
  • Yujun Song,
  • Bangshun He

摘要

Mycoplasma pneumoniae (MP) is a major pathogen of community-acquired pneumonia, especially in children, but its early diagnosis remains challenging due to nonspecific symptoms and limitations of conventional methods. In this study, we present a portable, amplification-free microfluidic platform for ultrasensitive detection of both MP DNA and RNA. This system integrates a phosphate-responsive zirconium-based metal-organic framework (Zr-MOF) with CRISPR‒Cas12a to achieve cascade signal amplification. Specifically, the MOF is co-loaded with target-specific probes and a high density of activators. Upon stimulation with phosphate, the MOF structure disassembles, releasing a burst of activators that trigger the trans-cleavage activity of Cas12a, leading to an amplified fluorescence readout. Enabled by efficient mass transport in the microfluidic device, the assay reduces detection time and sample consumption. Without the need for thermal cycling or reverse transcription, the platform achieves limits of detection as low as 0.35 fg/µL for DNA and 0.43 fg/µL for RNA. The detection results for 50 clinical samples show excellent agreement with qPCR. Furthermore, the assay is compatible with a smartphone-based portable reader, making it ideal for point-of-care testing. This microfluidic MOF-CRISPR platform offers a promising strategy for rapid, low-cost, and amplification-free nucleic acid diagnostics in resource-limited settings.