Porous charged polymer nanosheets formed via microplastic removal from frozen ice for virus filtration and detection.

We developed a method for producing porous charged polymer nanosheets using frozen ice containing microplastics. Upon assessing SARS-CoV-2 filtration using nanosheets with 100 nm-sized pores, a high rejection rate of 96% was achieved. The charged surfaces of nanosheets further enabled the electrophoretic capture of the virus using a portable battery with additional real-time sensing capability.

Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9.

Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.

Clustered Regularly Interspaced Short Palindromic Repeats-Mediated Surface-Enhanced Raman Scattering Assay for Multidrug-Resistant Bacteria.

Antimicrobial resistance and multidrug resistance are slower-moving pandemics than the fast-spreading coronavirus disease 2019; however, they have potential to cause a much greater threat to global health. Here, we report a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated surface-enhanced Raman scattering (SERS) assay for multidrug-resistant (MDR) bacteria. This assay was developed via a synergistic combination of the specific gene-recognition ability of the CRISPR system, superb sensitivity of SERS, and simple separation property of magnetic nanoparticles. This assay detects three multidrug-resistant (MDR) bacteria, species Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae, without purification or gene amplification steps. Furthermore, MDR A. baumannii-infected mice were successfully diagnosed using the assay. Finally, we demonstrate the on-site capture and detection of MDR bacteria through a combination of the three-dimensional nanopillar array swab and CRISPR-mediated SERS assay. This method may prove effective for the accurate diagnosis of MDR bacterial pathogens, thus preventing severe infection by ensuring appropriate antibiotic treatment.