Microfluidic detection based on size-encoded microbeads and rolling circle amplification for multiplex respiratory virus nucleic acid detection

Acute respiratory tract diseases (ARDs) are predominantly caused by viral infections, with one of their hallmark characteristics being multiple viral co-infections. Such multiple viral infections not only complicate therapeutic interventions but also lead to an increase in mortality rates. Most traditional bioassays, however, are limited to identifying a single type of virus, leading to missed diagnoses in samples with multiple respiratory pathogens. Consequently, there is an urgent need for the development of a detection method that exhibits high sensitivity, allows for constant-temperature operation, is user-friendly, and is capable of simultaneously detecting multiple viral nucleic acid targets. To address this challenge, we developed a novel method for the simultaneous detection of multiple respiratory viral nucleic acids using size-encoded microbeads and rolling circle amplification. Additionally, we designed a microfluidic chip featuring distinct spacer arrays that separate the microbeads into different regions based on their size, thereby generating characteristic signals indicative of varying biomarker levels. Rolling circle amplification significantly enhances the detection sensitivity on the microbeads, achieving a detection limit as low as fmol L⁻¹. In representative clinical samples, both the sensitivity and specificity for distinguishing target viral RNA from other respiratory diseases reached 100%. Importantly, this strategy enables simultaneous and in situ sensitive analysis of four viral nucleic acids. The throughput of pathogen nucleic acid detection can be further enhanced by incorporating additional microbeads of varying sizes and implementing a corresponding microchip design. We anticipate that this strategy holds substantial promise for clinical diagnosis of multiple respiratory viral infections.