ABSTRACT
Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society.
ABSTRACT
Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes – colorimetric and aggregate formation – using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into antipyrine red or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/μL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings. © 2022 Elsevier B.V.
ABSTRACT
Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society