Rapid detection of gram-positive and -negative bacteria in water samples using mannan-binding lectin-based visual biosensor

Waterborne bacterial infection is a health threat worldwide, making accurate and timely bacteria detection crucial to prevent waterborne disease outbreaks. Inspired by the intrinsic capability of mannan-binding lectin (MBL) in recognizing the pathogen-associated molecular patterns (PAMPs), a visual biosensor is developed here for the on-site detection of both Gram-positive and -negative bacteria. The biosensor was synthesized by immobilization of the MBL protein onto the blue carboxyl-functionalized polystyrene microparticles (PSM), which is then used in a two-step assay to detect bacterial cells in water samples. The first step involved a 20 min incubation following the MBL-PSM and calcium chloride solution addition to the samples. The second step was to add ethanol to the resultant blue mixture and observe the color change with the naked eye after 15 min. The biosensor had a binary (all-or-none) response, which in the presence of bacterial cells kept its blue color, while in their absence the color changed from blue to colorless. Testing the water samples spiked with four Gram-negative bacteria including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and two Gram-positive bacteria of Enterococcus faecalis and Staphylococcus aureus showed that the biosensor could detect all tested bacteria with a concentration as low as 101.5 CFU/ml. The performance of biosensor using the water samples from a water treatment plant also confirmed its capability to detect the pathogens in real-life water samples without the need for instrumentation.