Ultrasensitive Detection of Escherichia coli O157:H7 by Immunomagnetic Separation and Selective Filtration with Nitroblue Tetrazolium/5-Bromo-4-chloro-3-indolyl Phosphate Signal Amplification

Here, we report an enhanced colorimetric method using enzymatic amplification with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation for the ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective filtration. Biotinylated anti-E. coli O157:H7 antibody and streptavidin-alkaline phosphatase were conjugated to the surface of magnetic nanoparticles, and E. coli O157:H7-conjugates complexes remained on the membrane filter surface. The resultant light brown spots on the membrane filter were amplified with NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with an increasing E. coli O157:H7 concentration. E. coli O157:H7 was detected in pure samples with limits of detection of 10 and 6.998 colony-forming units (CFU)/mL through visual observation and measurement of optical density, respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli O157-H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This enhanced colorimetric method has potential for on-site detection of food contaminants and environmental pollutants.