Point-of-Care Solid-Phase PCR in a Vertical Microfluidic Chip Integrated with All-Dielectric Nanostructured Metasurface for Highly Sensitive, Multiplexed Pathogen Detection.

Abstract

Multiplexed solid-phase polymerase chain reaction (SP-PCR) has emerged as an indispensable modality for concurrent amplification of multiple genetic loci within a singular reaction vessel, facilitating efficient molecular diagnostics. Nevertheless, SP-PCR has seldom been integrated into point-of-care diagnostic devices due to several technical challenges, such as bubble formation during PCR, long reaction time, and low fluorescence signals generated from the PCR products on a solid surface. To circumvent these constraints, we engineered a microfluidic chip comprising SP-PCR and nanophotonic enhancement to enable highly sensitive, high-throughput, and cost-efficient molecular diagnostics. The chip's vertical orientation integrates preloaded reagent chambers for sequential lysis, washing, elution, and amplification, driven by a synchronized stepper motor and air vacuum, achieving robust nucleic acid purification and reverse transcription-PCR, and enabling bubble-free, gravity-assisted fluid dynamics during the PCR thermocycling. Thermal cycling is expedited through a dual-heater configuration alternating at subsecond intervals, obviating active cooling and shortening the reaction time. All-dielectric nanostructured metasurface was incorporated beneath the PCR chamber, allowing for the facile immobilization of DNA arrays to conduct SP-PCR. Taking advantage of guided-mode resonance supported by the metasurface and the SP-PCR approaches permits multiplexed detection and achieves a detection limit of 10 copies/reaction, highlighting the platform's potential for point-of-care diagnostics, personalized medicine, and high-throughput pathogen surveillance. Facile fabrication and automation emphasize scalability for mass production and deployment and collectively represent an advancement in point-of-care diagnostics.