Stopped-flow microarray immunoassay for detection of viable E. coli by use of chemiluminescence flow-through microarrays

Abstract

Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4 × 10(5) cells mL(-1). By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.