Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Highly Adsorptive Au-TiO2 Nanocomposites for the SERS Face Mask Allow the Machine-Learning-Based Quantitative Assay of SARS-CoV-2 in Artificial Breath Aerosols.

Human respiratory aerosols contain diverse potential biomarkers for early disease diagnosis. Here, we report the direct and label-free detection of SARS-CoV-2 in respiratory aerosols using a highly adsorptive Au-TiO2 nanocomposite SERS face mask and an ablation-assisted autoencoder. The Au-TiO2 SERS face mask continuously preconcentrates and efficiently captures the oronasal aerosols, which substantially enhances the SERS signal intensities by 47% compared to simple Au nanoislands. The ultrasensitive Au-TiO2 nanocomposites also demonstrate the successful detection of SARS-CoV-2 spike proteins in artificial respiratory aerosols at a 100 pM concentration level. The deep learning-based autoencoder, followed by the partial ablation of nondiscriminant SERS features of spike proteins, allows a quantitative assay of the 101-104 pfu/mL SARS-CoV-2 lysates (comparable to 19-29 PCR cyclic threshold from COVID-19 patients) in aerosols with an accuracy of over 98%. The Au-TiO2 SERS face mask provides a platform for breath biopsy for the detection of various biomarkers in respiratory aerosols.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.