Pushbutton-activated microfluidic cell-free biosensor for multiplexed pathogen detection

In this paper, we have developed a novel cell-free biosensor based on a multiplexed pushbutton-activated microfluidic device (mPAMD) that enables simultaneous detection of multiple 16S rRNAs of pathogens in a single device. The multi-step target-responsive cell-free protein synthesis process was seamlessly integrated into a single microfluidic device with an intuitive finger-pumping mechanism, allowing simultaneous mixing, aliquoting, and detection of 16S rRNAs through the production of reporter proteins. The mPAMD incorporates multiplexed detection zones with pathogen-specific probes to enable the identification of multiple 16S rRNAs that allow a simple and intuitive diagnostic platform for cell-free biosensors. Microchannels were designed and optimized to achieve efficient sample mixing and even distribution of common reagents, ensuring uniform reaction conditions across all reaction channels. The developed system achieved a detection limit for 16S rRNA ranging from 1.69 to 7.39 pM, corresponding to approximately 10⁴ to 10⁵ CFU mL^-1 of pathogens_. These results address the growing demand for an accessible multiplexed diagnostic system while ensuring high sensitivity and specificity.