ABSTRACT
Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.
Subject(s)
Body Temperature , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acids , Wearable Electronic Devices , COVID-19/diagnosis , DNA , Escherichia coli O157 , Humans , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/isolation & purification , Recombinases/chemistry , Recombinases/genetics , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
In this study, we introduce a paper microdevice fully integrating DNA extraction, loop-mediated isothermal amplification (LAMP), and Safranin O-based colorimetric detection of two major infectious pathogens, namely SARS-CoV-2 and Enterococcus faecium. The paper microdevice is composed of two parts: sample and reaction chambers. A sealing film acted as a bottom layer to allow foldable motion for transferring DNA from sample chamber to reaction chamber in a seamless manner. An FTA card was employed in the sample chamber for DNA extraction and purification from bacteria-spiked milk. After LAMP reaction at 65 °C for 30 min, a novel aggregation-based DNA detection was obtained by Safranin O polymerization in the reaction chamber. Specifically, Safranin O underwent polymerization by addition of oxidant to form Safranin O oligomers. The electrostatic interaction between the positively charged Safranin O oligomers and the negatively charged DNA comprising LAMP amplicons resulted in the aggregation with a dark red color. Meanwhile, in the absence of LAMP amplicons, Safranin O oligomers were well dispersed and displayed their original red color. By using Safranin O-based detection, SARS-CoV-2 and E. faecium were successfully identified by naked eye within 60 min, and the limits of detection were 10-4 ng/µL and 102 CFU/mL, respectively. These results indicate that a fully integrated paper microdevice plays an important role in sample-in-answer-out format in the genetic analyses of infectious disease and serves as a rapid tool for controlling the spread of diseases.
Subject(s)
Biosensing Techniques , COVID-19 , Communicable Diseases , Escherichia coli O157 , COVID-19/diagnosis , Escherichia coli O157/genetics , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Phenazines , Point-of-Care Testing , SARS-CoV-2/geneticsABSTRACT
A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core-shell nanostructures and confining free reduction and self-nucleation of Cu2+ for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL-1 for HIV-1 capsid p24 antigen and 6 CFU mL-1 for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.