A directional self-priming continuous-driven compartmentalized microfluidic chip for multiplexed pathogen detection

Rapid and efficient screening of foodborne pathogens is crucial for preventing bacterial spread and food poisoning. However, developing a multi-detection method that is easy to operate, offers good stability, and achieves high efficiency remains an enormous challenge. Existing multiplexed nucleic acid detection methods suffer from complex designs, leading to complicated operations, and non-robust sample introduction, causing primer/probe crosstalk and false-positive results. To address these issues, we developed a directional self-priming continuous-driven multiplexed detection microfluidic chip based on a snowflake shaped-inspired design with six branches, and each branch carried a unique functional input inlet for different specific targets, enabling simultaneous detection of multiple targets from one sample using monochrome fluorescence. Based on the PDMS negative pressure storage capacity, this chip explored the self-priming method that enables the continuous loading of primers/probes and samples without requiring an external power source, effectively addressing key challenges in multiplexed detection. This chip with specially designed outlet-free and symmetric inverted T-shaped microchannels allows for multi-step reagent loading and 100% compartmentalization without reagent loss, significantly reducing reagent consumption and sample loss compared to current methods. For further validation, we tested three types of foodborne pathogens (Salmonella enterica, Staphylococcus aureus, and Listeria monocytogenes) to demonstrate the chip's multiplexed detection capability, achieving a notably high sensitivity of detection as low as 10 copies per μL. Our snowflake-shaped microfluidic chip platform shows great potential for future applications in point-of-care (POC) settings, offering a robust and efficient solution for detecting foodborne pathogens.