Thermally Triggered Double Emulsion-Integrated Hydrogel Microparticles for Multiplexed Molecular Diagnostics

During the COVID-19 pandemic, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most reliable diagnostic tool. However, there is a need to develop multiplexed assays capable of analyzing multiple genes simultaneously to expand its application. To address this, a multiplexed RT-qPCR using a double emulsion (DE)-based carrier and a polymer microparticle reactor, termed primer-incorporated network tailored with Taqman probe (TaqPIN) is developed. The DE securely stores nucleic acid reagents like primers and probes within the polymer network until heating releases them for the reaction. The TaqPIN RT-qPCR demonstrates an amplification efficiency of 93.8% and can detect as few as 20 copies/µL. By loading the multiple microparticles into a single reaction, a multiplexed assay with only one optical channel is enabled. In practice, a nine-plex assay is designed to distinguish between variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Even subtle variations of a single nucleotide can be simultaneously detected. Testing on 75 nasopharyngeal swab samples yields 100% sensitivity and specificity for SARS-CoV-2 detection and 94% accuracy in variant discrimination.