Multiplex Determination of Pathogens via a Dual-Mode Microfluidic Platform Integrated with a Universal Sensing Strategy

Abstract Multiplex determination of pathogens is crucial for precise medical treatment of infectious diseases. To address this challenge, here we propose a CRISPR/Cas-assisted microfluidic multiplex sensing platform (CAMMSP) based on a nucleic acid amplification-free, universal, and modular sensing strategy. This strategy leverages the synergistic action of Cas12a and nanoporous materials to construct a target-responsive indicator release/enrichment system and achieve cascade signal amplification without nucleic acid amplification. By virtue of the programmability of the CRISPR system, this strategy is readily extended to different targets by replacing crRNA only. With the aid of a spatially divisional microfluidic platform, four common reproductive tract pathogens were synchronously detected as a proof-of-concept. The signal property of the methylene blue indicator was utilized to achieve electrochemical and optical signal cross-validation, guaranteeing the detection reliability at the readout level and offering flexible options for different scenarios. The developed CAMMSP exhibits broad linear ranges across 6 orders of magnitude and sub-femtomolar detection limits, with outstanding specificity in complex samples. Clinical sample analysis further validates its potential for practical applications, establishing a promising laboratory proof-of-concept and reliable technical foundation for developing multiplex point-of-care testing devices.