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<p>Bacterial and viral co-infections significantly exacerbate morbidity and mortality. Rapid, sensitive, and parallel detection of these pathogens remains a critical challenge. Here, an orthogonal <b>C</b>RISPR/Cas system facilitated <b>d</b>ual-color fluorescence <b>f</b>iber-embedded <b>o</b>ptofluidic nano-<b>b</b>iochip (CD-FOB) was fabricated. Leveraging the time-resolved effect, the CD-FOB achieved ultrasensitive parallel detection of <i>Escherichia coli</i> O157:H7 (<i>E. coli</i> O157:H7) and SARS-CoV-2 based on a multiple signal enhancement strategy, including the collateral cleavage activity of CRISPR/Cas, evanescent wave fluorescence enhancement, DNA-mediated signal amplification, and air-displacement fluorescence enhancement. Without the need for amplification, the CD-FOB system has a detection limit of 643&#xa0;CFU/mL for <i>E. coli</i> O157:H7 and 3.48 copies/μL for SARS-CoV-2 within 50&#xa0;min&#xa0;analysis time. To enable rapid on-site detection, a lyophilized CRISPR/Cas assay was prepared using stabilized freeze-dried reagents for detecting <i>E. coli</i> O157:H7 and SARS-CoV-2 in actual samples, achieving recoveries&#xa0;ranging from 70.5% to 200.5%. The unique combination of technical simplicity, multiplexing capability, and operational robustness positions CD-FOB as a versatile solution for combating current and future pathogen threats.</p> Graphical Abstract <p></p>

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Orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip for parallel amplification-free on-site detection of bacterium and virus

  • 期刊论文

  • 发表日期：

    2025年6月12日

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  • Dan Song,
  • Wenjuan Xu,
  • Yuxin Zhuo,
  • Anna Zhu,
  • Feng Long

摘要

Bacterial and viral co-infections significantly exacerbate morbidity and mortality. Rapid, sensitive, and parallel detection of these pathogens remains a critical challenge. Here, an orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip (CD-FOB) was fabricated. Leveraging the time-resolved effect, the CD-FOB achieved ultrasensitive parallel detection of Escherichia coli O157:H7 (E. coli O157:H7) and SARS-CoV-2 based on a multiple signal enhancement strategy, including the collateral cleavage activity of CRISPR/Cas, evanescent wave fluorescence enhancement, DNA-mediated signal amplification, and air-displacement fluorescence enhancement. Without the need for amplification, the CD-FOB system has a detection limit of 643 CFU/mL for E. coli O157:H7 and 3.48 copies/μL for SARS-CoV-2 within 50 min analysis time. To enable rapid on-site detection, a lyophilized CRISPR/Cas assay was prepared using stabilized freeze-dried reagents for detecting E. coli O157:H7 and SARS-CoV-2 in actual samples, achieving recoveries ranging from 70.5% to 200.5%. The unique combination of technical simplicity, multiplexing capability, and operational robustness positions CD-FOB as a versatile solution for combating current and future pathogen threats.

Graphical Abstract