One-step microfluidic technology for generating multifunctional quantum dot (QD) biomarker barcode particles for multiplex pathogen detection

High throughput, multiplex analysis, and operational convenience are essential for biomedical diagnostics. Quantum dot (QD) barcode particles hold significant promise for biomarker detection due to their powerful encoding capabilities. However, conventional QD encapsulation methods still face challenges such as complexity, low efficiency, and poor stability. Although microfluidic technology offers a promising solution for QD encapsulation, there remains a lack of integrated microfluidic platforms capable of the “one-step” preparation of barcode particles. Herein, based on the flow-focusing principle, we developed an automated multifunctional microfluidic platform that integrates fluid control, real-time imaging, in-situ photocuring, and automated product collection. By employing a multi-channel capillary microfluidic chip, we established a “one-step” microfluidic technology for the automated, high-throughput production of QD barcode particles. Owing to its high level of integration and automation, this platform enables efficient and highly stable production, with a fabrication rate exceeding 500 particles per minute. The resulting particles exhibit excellent monodispersity, with a coefficient of variation (CV) in diameter of less than 1.35% and a span of less than 0.04. Using this platform, we fabricated novel multifunctional QD barcode particles featuring a QD-encoded core and a shell loaded with magnetic nanoparticles (MNPs). The QDs provide unique optical barcodes for identification, while the MNPs confer excellent magnetic responsiveness and offer sites for biomolecule immobilization. Compared to existing QD particles, the core-shell structure not only provides a functional outer layer, but also effectively mitigates the risk of QD leakage and reduces the quenching effect of MNPs on QD fluorescence. By modifying the particle surface with molecular beacon (MB) probes, we developed a visual, multiplex nucleic acid detection technology based on polymerase chain reaction (PCR). As a proof of concept, we created specific barcoded particles for African Swine Fever Virus (ASFV) detection. This assay enables visual detection of PCR products for the B646L , CD2V , and MGF genes of ASFV within 30 min, with a limit of detection superior to 200 copies/mL. This technology provides a novel and efficient approach for multiplex nucleic acid detection.