Ultrafast Real-Time PCR in Photothermal Microparticles.

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.

Highly Selective Multiplex Quantitative Polymerase Chain Reaction with a Nanomaterial Composite Hydrogel for Precise Diagnosis of Viral Infection.

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.