Bifunctional linker-based immunosensing for rapid and visible detection of bacteria in real matrices.

Detection of pathogens present in food and water is essential to help ensure food safety. Among the popular methods for pathogen detection are those based on culture and colony-counting and polymerase chain reaction (PCR). However, the time-consuming nature and/or the need for sophisticated instrumentation of those methods limit their on-site applications. We have developed a rapid and highly sensitive immunosensing method for visible detection of bacteria in real matrices based on the aggregation of AuNPs without requiring any readout device. We use biotinylated anti-bacteria antibodies as bifunctional linkers (BLs) to mediate the aggregation of streptavidin-functionalized gold nanoparticles (st-AuNPs) to produce visually recognizable color change, due to surface plasmon resonance (SPR), which occurs in about 30min of total assay time when the sample is mildly agitated or within three hours in quiescent conditions. The aggregation of st-AuNPs, which produces the indication signal, is achieved very differently than in visual detection methods reported previously and hence affords ultrahigh sensitivity. While BLs can both bind to the target and crosslink st-AuNPs, their latter function is essentially disabled when they bind to the target bacteria. By varying the amount of st-AuNPs used, we can tailor the assay effectiveness improving limit of detection (LOD) down to 10CFUmL-1 of E. coli and Salmonella. Test results obtained with tap water, lake water and milk samples show that assay performance is unaffected by matrix effects. Further, in a mixture of live and autoclaved E. coli cells our assay could detect only live cells. Therefore, our BL-based immunosensor is suitable for highly sensitive, rapid, and on-site detection of bacteria in real matrices.