A novel laminar flow-based hierarchical microfluidic (LF-HM) chip for multiplex detection without cross-talk

Microfluidic technology provides a promising platform for point-of-care multiplexed testing with minimal reagent consumption, aligning with the growing demand for cost-effective diagnostics. However, integrating multiple assays into a single microchannel remains challenging due to cross-talk between incompatible detection modes. In this study, a novel laminar flow-based hierarchical microfluidic (LF-HM) chip was developed to address cross-talk by leveraging stable laminar flow to create two vertically separated reaction layers within a single microchannel, isolating incompatible assays. The design was validated through finite element simulation, which confirmed critical functionalities, including fluid interception and minimal flow disturbance. Experimental characterization verified stable laminar flow, defined an optimal operating temperature range of 15-26 °C to minimize cross-stream diffusion, and demonstrated the complete elimination of cross-talk. The chip's utility was conclusively shown by integrating the rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) assays, a well-known pair problematic for cross-talk in multiplexed detection, which exhibited excellent analytical agreement with single-plex tests (Passing-Bablok slopes of 0.99 and 1.02 for RF and ACPA, respectively) and strong correlation with reference methods in clinical validation (R² = 0.85 for RF; R² = 0.95 for ACPA). The LF-HM chip offers a robust and generalizable solution for cross-talk-free multiplexing, with broad potential in point-of-care diagnostics where cross-talk has traditionally limited integrated assay development.