Convenient and sensitive detection of viable Escherichia coli employing a sequential reaction between antibody-enzyme complexes.

We developed a rapid, convenient, and viable cell-selective sensing system for Escherichia coli (E. coli) based on a sequential enzymatic reaction on an intact cell surface. In food safety and infectious disease diagnostics, it is crucial to selectively detect viable cells that represent actual infection risk. However, conventional methods such as culture-based method and polymerase chain reaction are time-consuming and require specialized equipment, limiting their applicability for on-site testing. The proposed system is based on a cooperative reaction of antibody-enzyme complexes, in which chemiluminescent signals are generated only when two types of antibody-enzyme complexes localize in proximity on the bacterial surface. The system allows for homogeneous detection of E. coli simply by mixing reagents, eliminating washing and separation steps. The chemiluminescence intensity showed a concentration-dependent response and an approximately linear relationship with log (CFUmL-1) over the range of 1-104 CFU mL-1. Importantly, the signal was generated only in the presence of viable E. coli cells, indicating that the cooperative enzymatic reaction proceeds selectively on intact bacterial surface. This property allows clear discrimination between viable and dead cells, which has been difficult with conventional methods. Overall, the proposed approach provides a rapid, convenient, and viable-cell-selective platform for on-site microbial monitoring and risk assessment.