Researchers at Columbia Engineering and Rover Diagnostics, a Columbia Technology Ventures company, used plasmonic nanoparticles with compact optics to develop a real-time, multiplexed, reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test. Weighing just two pounds, the ultrafast, portable PCR testing system is practical for use in decentralized and point-of-care (POC) settings. “Our aim was to create a platform that can be used in locations where rapid turnaround results are critical, at pharmacies, transportation hubs, public events, and companies screening employees coming back to work,” said professor Sam Sia, co-founder of Rover Diagnostics. The RT-PCR platform uses the photothermal process of plasmonic thermocycling to irradiate nanoparticles with infrared (IR) light and quickly generate heat from inside the reaction vessel. In tests, the platform rapidly detected SARS-CoV-2 RNA from human saliva and nasal specimens with 100% sensitivity and specificity, as well as two distinct SARS-CoV-2 variants. PCR testing, used to diagnose certain infectious diseases and genetic changes that could indicate disease, takes place primarily at large, centralized laboratories. The instruments for ramping up and ramping down the temperature in a controlled manner are too expensive, bulky, and technical to use in POC settings. Instead of relying on the Peltier effect and heat blocks to set the temperature of the reaction vessel, the researchers directly heated the solution via plasmonic nanoparticles. In the reaction vessels, they used gold nanorods with localized surface plasmon resonance in the near-infrared range, about 850 nm. This wavelength range allowed them to use fluorescent probes for real-time fluorescence detection without needing to remove the nanorods. A team from Columbia Engineering and Rover Diagnostics showed infrared heating of plasmonic nanoparticles facilitated multiplexed reverse transcriptase quantitative PCR for rapid detection of SARS-CoV-2. Courtesy of Abigail Ayers/Columbia Engineering and Nicoletta Barolini. To drive thermocycling, the researchers created an optical setup consisting of three IR LEDs, operating at 850 nm and concentrically positioned around a thin-walled PCR tube. A concentration of the gold nanorods that was high enough to achieve the photothermal effect without interfering with fluorescence measurements rapidly generated heat through the solution. During each cycle, cooling was achieved with a small 12-V fan. For real-time fluorescence monitoring, the researchers excited and detected up to three fluorescent probes with a common excitation source and detection source. The fluorescence setup consisted of a 488-nm laser diode as the excitation source and an optical-fiber-coupled spectrometer that could detect multiple wavelengths. The team performed RT-qPCR in a reaction vessel containing all the PCR reagents and obtained test results in 23 minutes. The Rover team is moving forward with a commercial product that can detect COVID-19, its variants, and other infectious diseases. The research was published in Nature Nanotechnology (www.doi.org/10.1038/s41565-022-01175-4).