"Super Sandwich" Assay Using Phenylboronic Acid for the Detection of E. coli Contamination: Methods for Application

This paper proposes a method for E. coli detection in a microplate format using low-molecular-weight compounds that specifically interact with the lipopolysaccharides (LPSs) of E. coli cell walls. These compounds can amplify analytical signals by binding to multiple repeating cell surface structures, while the selectivity for E. coli is ensured by preliminary cultivation on selective media, such as Endo or MacConkey agar. 3-Aminophenylboronic acid (APBA) was selected as the binding reagent for detecting E. coli LPSs. Conjugates of streptavidin (STP) and bovine serum albumin (BSA) with APBA and conjugates of biotin and soybean trypsin inhibitor (STI) and BSA were synthesized. The conditions for the sequential formation of "sandwich" type complexes (BSA-APBA conjugate/E. coli/STP-APBA/STI-biotin/STP-peroxidase) and their colorimetric detection using chromogenic peroxidase substrate were determined. The detection limit was 3 × 10² cells/mL, and the range of quantitative determination covered five orders of magnitude-from 10³ to 10⁸ cells/mL. The developed assay was successfully tested using inactivated cells of pathogenic E. coli strains, confirming its potential for application. The assay was demonstrated to have universality, with the ability to detect E. coli, other bacterial pathogens, and LPS alone. This method could be adopted for the quantitative determination of different specific bacterial species using different selective media.