Machine vision-integrated portable colorimetric biosensing platform with triple-layered (Pt@ZIF-8)³@PDA nanozymes for...

Simple and in-field detection of multiple pathogenic bacteria is urgently needed. In this work, a biosensor using a nanozyme-based colorimetric immunoassay was developed for sensitive detection of multiple pathogenic bacteria. Firstly, a nanocomposite (Pt@ZIF-8)³@PDA-Ab was synthesized based on nanozyme coated with multiple layers of metal-organic frameworks to recognize and probe target bacteria. Additionally, leveraging the catalytic properties of Pt nanoparticles with different substrates (TMB, OPD, AEC, and ABTS), a multicolor sensor array, a portable imaging device, and an in-house coded machine vision algorithm were developed for colorimetric detecting multiple pathogenic bacteria. For single target pathogen detection, this approach exhibits a favorable linear relationship within the range of 10-10⁶ CFU/mL, with a detection limit (LOD) as low as 2 CFU/mL for Salmonella Typhimurium. Furthermore, the (Pt@ZIF-8)³@PDA-Ab-based biosensor exhibited excellent selectivity and ambient storage stability for as long as 60 days. The colorimetric biosensing platform demonstrates excellent detection capabilities of multiple pathogens bacteria, showing a favorable linear relationship within the range of 10-10⁶ CFU/mL, with LOD as low as 10, 7, 5, and 3 CFU/mL for Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli, and Vibrio parahaemolyticus, respectively. The proposed sensor exhibits excellent anti-interference capability (RSD<5.8%). When applied in detecting carrot, lake water and artificial saliva samples, the method demonstrated satisfactory accuracy (RSD<5.8%) for four types of bacteria. This work highlights the synergistic integration of a structurally engineered ambient-stable nanozyme, a multicolor sensor array, and a machine vision-enabled portable device, offering a promising and generalizable strategy for point-of-need pathogen detection in food safety, environmental monitoring, and clinical diagnostics.