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

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.