ABSTRACT
Infectious diseases have always been a seriously endanger for human life and health. A rapid, accurate and ultra-sensitive virus nucleic acid detection is still a challenge to deal with infectious diseases. Here, a RNA extraction-free reduced graphene oxide-based reverse transcription-loop-mediated isothermal amplification (EF-G-RT-LAMP) fluorescence assay was developed to achieve high-throughput, rapid and ultra-sensitive SARS-CoV-2 RNA detection. The whole detection process only took â¼36 min. The EF-G-RT-LAMP assay achieves a detection limit of 0.6 copies µL-1 with a wide dynamic range of aM-pM. A large number (up to 384) of samples can be detected simultaneously. Simulated detection of the COVID-19 pseudovirus and clinical samples in nasopharyngeal swabs demonstrated a high-throughput, rapid and ultra-sensitive practical detection capability of the EF-G-RT-LAMP assay. The results proved that the assay would be used as a rapid, easy-to-implement approach for epidemiologic diagnosis and could be extended to other nucleic acid detections.
Subject(s)
COVID-19 , Graphite , Limit of Detection , Nucleic Acid Amplification Techniques , RNA, Viral , SARS-CoV-2 , Graphite/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/genetics , Nucleic Acid Amplification Techniques/methods , Humans , COVID-19/diagnosis , COVID-19/virology , RNA, Viral/analysis , RNA, Viral/genetics , Molecular Diagnostic Techniques/methods , FluorescenceABSTRACT
Coffee rings formed by evaporation of analyte-containing droplets are widely observed in micropatterning, bio-arrays, and trace detection. The coffee-ring effect caused by contact line pinning significantly affects the detection uniformity and sensitivity. Here, we propose a simple and operable method to effectively suppress coffee rings through controllable nanoparticles aggregation by superhydrophobicity-enabled dynamic evaporation. The gold nanoparticles (AuNPs) deposition footprint formed after dynamic evaporation on an integrated superhydrophobic surface was reduced by â¼3 orders of magnitude compared to that of non-interventional evaporation. Detailed experiments, numerical simulations, and theoretical studies have revealed that substrate wettability, temperature and droplet motion behaviors play significant roles in suppressing coffee-ring effect. More critically, based on the force mechanism of AuNPs at the interface/contact line, universal mathematical models and regime maps were established to classify the different deposition modes for AuNPs under different evaporation conditions by introducing dimensionless parameter G, revealing the enrichment mechanism of AuNPs in droplets under superhydrophobicity-enabled dynamic evaporation. The accuracy of the theoretical model and enrichment mechanism was demonstrated through the single-molecule detection of rhodamine 6G with excellent sensitivity (10-17 M, enhancement factor â¼1013) and perfect uniformity (relative standard deviation â¼5.57 %), which provides a valuable guide for research and applications of nanoparticle aggregation.