Dual-mode visual detection strategies of viable pathogens for point-of-care testing

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 for-mation - using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: re-action 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 degrees 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 con-taining 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/mu L 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.