Nanoplasmonic amplification in microfluidics enables accelerated colorimetric quantification of nucleic acid biomarkers from pathogens.

Deployment of nucleic acid amplification assays for diagnosing pathogens in point-of-care settings is a challenge due to lengthy preparatory steps. We present a molecular diagnostic platform that integrates a fabless plasmonic nano-surface into an autonomous microfluidic cartridge. The plasmonic 'hot' electron injection in confined space yields a ninefold kinetic acceleration of RNA/DNA amplification at single nucleotide resolution by one-step isothermal loop-mediated and rolling circle amplification reactions. Sequential flow actuation with nanoplasmonic accelerated microfluidic colorimetry and in conjugation with machine learning-assisted analysis (using our 'QolorEX' device) offers an automated diagnostic platform for multiplexed amplification. The versatility of QolorEX is demonstrated by detecting respiratory viruses: SARS-CoV-2 and its variants at the single nucleotide polymorphism level, H1N1 influenza A, and bacteria. For COVID-19 saliva samples, with an accuracy of 95% on par with quantitative polymerase chain reaction and a sample-to-answer time of 13 minutes, QolorEX is expected to advance the monitoring and rapid diagnosis of pathogens.

A Versatile Biomimic Nanotemplating Fluidic Assay for Multiplex Quantitative Monitoring of Viral Respiratory Infections and Immune Responses in Saliva and Blood.

The last pandemic exposed critical gaps in monitoring and mitigating the spread of viral respiratory infections at the point-of-need. A cost-effective multiplexed fluidic device (NFluidEX), as a home-test kit analogous to a glucometer, that uses saliva and blood for parallel quantitative detection of viral infection and body's immune response in an automated manner within 11 min is proposed. The technology integrates a versatile biomimetic receptor based on molecularly imprinted polymers in a core-shell structure with nano gold electrodes, a multiplexed fluidic-impedimetric readout, built-in saliva collection/preparation, and smartphone-enabled data acquisition and interpretation. NFluidEX is validated with Influenza A H1N1 and SARS-CoV-2 (original strain and variants of concern), and achieves low detection limit in saliva and blood for the viral proteins and the anti-receptor binding domain (RBD) Immunoglobulin G (IgG) and Immunoglobulin M (IgM), respectively. It is demonstrated that nanoprotrusions of gold electrodes are essential for the fine templating of antibodies and spike proteins during molecular imprinting, and differentiation of IgG and IgM in whole blood. In the clinical setting, NFluidEX achieves 100% sensitivity and 100% specificity by testing 44 COVID-positive and 25 COVID-negative saliva and blood samples on par with the real-time quantitative polymerase chain reaction (p < 0.001, 95% confidence) and the enzyme-linked immunosorbent assay.

Protocol for nuclear export signal characterization of cGAS in mammalian cells.

The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and triggers the type I interferon production. We used ISD45 or inactivated Vaccinia Virus (VACV) to stimulate cGAS and monitored cellular localization by immunofluorescence microscopy, Operetta high-content screening, and cytoplasmic/nuclear fractionation. LocNES server was used to predict cGAS nuclear export signal (NES) sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. For complete details on the use and execution of this protocol, please refer to Sun et al. Sun et al. (2021).