Pathogens are a persistent threat to human health, causing infectious disease and millions of deaths annually. The accurate and timely detection of pathogens is crucial in disease prevention, treatment, and monitoring. Although reliable detection methods are well established, many of them are still limited in use to clinical laboratories due to the need for costly and specialized instrumentation. In this study, we demonstrate a handheld and low-cost pathogen sensor consisting of a paper-based analytical device (μPAD) that can perform immunoassays and quantify analyte concentration by integrating with an automated color detection system that analyzes the color intensity of the μPAD. The core of the proposed sensor is the portable color detection system that can read the red-green-blue color of the paper emitted light from fluorescent nanomaterials, including graphene quantum dots (GQDs) and gold nanoclusters (AuNCs), which are conjugated with antibodies to indicate the immunoassay results, converting the presence of a pathogen to a colorful fluorescence signal. By adopting GQDs and AuNCs with high quantum yield and relatively high fluorescence intensity as the sensing signal, the paper-based detection system decreases the detection limit to as low as subnanogram/mL. Furthermore, GQDs and AuNCs can emit distinguishable fluorescence under the same light source (UV light) and possess limited background interference from the cellulose, enabling two or more analytes to be simultaneously detected with one UV light. Furthermore, a reaction time of just 10 min is needed, enabling diagnoses to be made in a timely manner and with high sensitivity. As a result, the proposed handheld pathogen sensor can rapidly detect the presence of pathogens with enhanced sensitivity and multiplexity, along with low instrumentation requirements, making it suitable for use in resource-limited settings where medical infrastructure is lacking.
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