Antibody‐free bioimprint aided sandwich ELISA technique for cell recognition and rapid screening for bacteria

Abstract

AbstractWe have developed and tested a novel ELISA‐like approach for bacterial detection based upon selective adhesion of targeted bacteria to microwells with prefabricated bacterial bioimprints. Bioimprints were produced from three bacterial species; Escherichia coli (Gram‐negative), Rhodococcus rhodochrous (Gram‐positive) and Sarcina aurantiaca (Gram‐negative), by using molding with curable silicone from dense layers of bacterial cells deposited on a glass substrate. We demonstrated that the surface functionalized whole cell bioimprints were able to selectively recognize and bind their own bacterial cell type. In order to detect target bacteria that are bound to the matching bioimprint, we also developed silica nanoparticles dual‐functionalized with (3‐glycidyloxypropyl)trimethoxysilane (GLYMO) coupled with 4‐hydroxyphenylboronic acid (4‐HPBA), SiO2NPs/GLYMO/4‐HPBA, which were further conjugated with horseradish peroxidase (HRP). Bacterial detection was demonstrated to work in the established ELISA‐like protocol using the colorimetric reaction of the conjugated HRP with 3,3′,5,5′‐tetramethylbenzidine (TMB). The bioimprints were used instead of capture antibodies and HRP‐coated dual functionalized silica nanoparticles instead of a secondary antibody with TMB as the enzyme‐converted reagent, producing a colored byproduct. This bacterial bioimprint‐based detection method does not rely on any antibodies, uses stable and inexpensive reagents, and could potentially find application for rapid diagnostics of bacterial pathogens in clinical samples at the point of care.