Modular Microfluidic Sensor Integrating Nucleic Acid Extraction, CRISPR/Cas13a, and Electrochemiluminescence for Multichannel RNA Detection

Rapid and accurate screening of pathogens is crucial for disease detection. Here, a modular microfluidic sensor has been constructed for RNA detection, with integrated nucleic acid extraction, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a reaction, and electrochemiluminescence (ECL) detection. The sensor consists of nucleic acid processing and detection modules. The nucleic acid processing module is used for nucleic acid extraction, RNA distribution, and the CRISPR/Cas reaction. Specifically, immiscible filtration assisted by surface tension is employed for nucleic acid extraction, significantly reducing the extraction time. Magnetic force is utilized for RNA distribution and transportation, minimizing the need for microstructures, such as microvalves and micropumps. Multichannel CRISPR/Cas13a reactions enable biological recognition, signal amplification, and multiplex detection. The fiber material-based detection module controls fluid flow and performs dry chemistry-based ECL detection. A novel multichannel closed bipolar electrode-based ECL (MCBPE-ECL) system is employed, with simplified experimental operations and enhanced sensitivity. Together, the multichannel CRISPR/Cas13a reactions and MCBPE-ECL enable the sensor's multiplexed detection. Under optimized conditions, the sensor can complete RNA extraction and detection in 30 min, with a detection limit of 0.372 fM for <i>Escherichia coli</i> 16S rRNA. Furthermore, in human blood samples, the detection limit for <i>E. coli</i> is 63.8 cfu/mL. Notably, the sensor can simultaneously determine the growth curves of single colonies of <i>E. coli and Staphylococcus aureus</i> strains in the same culture medium, demonstrating its multiplexed detection capability.