An integrated microfluidic platform for multi-target nucleic acid detection based on rotational magnetic field-induced uniform bead distribution

Efficient multi-pathogen detection remains crucial during concurrent respiratory disease outbreaks, yet existing multiplex platforms are limited by complex fluorescent encoding and high reagent costs. This study introduces a novel microfluidic chip system incorporating rotational magnetic control for simultaneous multi-pathogen nucleic acid detection. The system utilized a unique rotating magnetic field to drive the magnetic beads to actively and dynamically capture nucleic acid in the solution, enabling a "one-to-four division" of the beads and subsequently achieving a "one-to-four division" of the nucleic acid sample. Under optimized conditions, a rotating magnetic field generated by four symmetrically arranged permanent magnets was employed to achieve precise manipulation of magnetic beads, enabling fully automated nucleic acid extraction (NAE) within 10 minutes. The extraction efficiency was demonstrated to be comparable to that of conventional manual methods utilizing commercial extraction kits, ensuring both reliability and consistency. This developed system demonstrated the ability to detect SARS-CoV-2 RNA at levels as low as < 10 copies/test and exhibited excellent specificity and a broad dynamic detection range (10 ¹–10⁵ copies/μL) in multiplex detection of 12 respiratory pathogens. Validation with clinical samples confirmed its accuracy in identifying both single and co-infections, with results fully consistent with clinical diagnoses. This study represents an initial demonstration of a technical paradigm for multi-target detection via physical sample allocation, implementing a "single-sample input, multi-result output" strategy with external amplification support. It provides a promising approach for the rapid and accurate clinical diagnosis of multiple pathogens.