As the turnaround time of diagnosis becomes important, there is an increasing demand for rapid, point-of-care testing (POCT) based on polymerase chain reaction (PCR), the most reliable diagnostic tool. Although optical components in real-time PCR (qPCR) have quickly become compact and economical, conventional PCR instruments still require bulky thermal systems, making it difficult to meet emerging needs. Photonic PCR, which utilizes photothermal nanomaterials as heating elements, is a promising platform for POCT as it reduces power consumption and process time. Here, we develop a photonic qPCR platform using hydrogel microparticles. Microparticles consisting of hydrogel matrixes containing photothermal nanomaterials and primers are dubbed photothermal primer-immobilized networks (pPINs). Reduced graphene oxide is selected as the most suitable photothermal nanomaterial to generate heat in pPIN due to its superior light-to-heat conversion efficiency. The photothermal reaction volume of 100 nL (predefined by the pPIN dimensions) provides fast heating and cooling rates of 22.0 ± 3.0 and 23.5 ± 2.6 °C s^-1, respectively, enabling ultrafast qPCR within 5 min only with optical components. The microparticle-based photonic qPCR facilitates multiplex assays by loading multiple encoded pPIN microparticles in a single reaction. As a proof of concept, four-plex pPIN qPCR for bacterial discrimination are successfully demonstrated.

Kwon DH, et al. Korean Circ J. 2013 Dec;43(12):804-810. http://dx.doi.org/10.4070/kcj.2013.43.12.804

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.