Single-Molecule Detection of Sars-Cov-2 RNA Enabled By Plasmonic Sensing of Amplified Nucleic Acids

Single-molecule detection of pathogens such as SARS-CoV-2 is key to combat infectious diseases outbreak and pandemic. Current gold standard for single-nucleic-acid test relies on polymerase chain reaction (PCR). However, its time-consuming protocols and the needs for laboratory infrastructure largely preclude it from point-of-care (POC) testing. Isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have emerged as an alternative to PCR and can be performed at POC without the need for thermal cycling. Although simple, LAMP is susceptible to non-template amplification and its by-product-based readouts (e.g., pH change) can produce false-positive results. Previous efforts in advancing the LAMP assay, based on gold nanoparticles (AuNPs)-based sensing of LAMP products, provide simple readouts (e.g., naked-eye detection and later flow strip). However, the weak optical response of AuNPs leads to little sensitivity enhancement, while the non-specific sensing of ionic strength change or molecule-labeled primers limits the detection specificity. Herein, we report an ultrasensitive diagnostic method based on target-specific sensing of LAMP amplicons using gold and silver (Au-Ag) nanoshells as labels, yielding a “naked-eye” recognition of 10 RNA copies per microliter for SARS-CoV-2 detection. The ultrasensitive detection arises from two distinctive features. First, nanoshells made of Au and silver (Ag) are employed as labels. They have stronger plasmonic extinction in the visible range over AuNPs and provide 20-times sensitivity enhancement in plasmonic sensing. Second, we introduce restrict enzyme digestion and heat denaturation in the detection approach, in order to cut the long concatemers into short repeats that are amendable for subsequent hybridization with plasmonic sensors. In contrast, previous studies relying on direct sensing of LAMP amplicons have met with limited success because of the amplicons’ stem-loop and cauliflower-like structure. The plasmonic sensing of LAMP amplicons eliminates the contamination from non-template amplification, improving the detection specificity and sensitivity. Our study takes a significant step toward advancing plasmonic sensing to single-molecule detection.