The Development of a New, Rapid, Amperometric Immunosensor for the Detection of Low Concentrations of Bacteria Part II: Optimization of the System for Escherichia coli

Abstract

The optimization of a flow-through immunoassay system was conducted to achieve rapid detection of low concentrations of E. coli bacteria. The system includes a new immunosensor that uses newly developed, disposable immunofiltration membranes to yield a system that is sensitive as well as simple. Comparison of the performance of this system with conventional immunoassays demonstrated a significantly shorter time for the assay. Optimization of the parameters of the system, including specific flow rates and concentrations, was conducted, resulting in an overall assay time of 17 min when a flow of 200 μL min^-1 was used. The first stage of immunoreaction of immobilized antibodies with the analyte was found to saturate in 2 min. The second stage of immunoreaction with the enzyme-labeled conjugate exhibited a more complex behavior but became saturated in 6 min. The amperometric detection using the substrate gave a steady output signal after 3 min. All stages are separated by 2 min of washing by a rinsing buffer solution and the whole analysis is preceded by a 2-minute pre-washing stage in order to eliminate impurities which can lead to non-specific binding of immunoagents and substantial background noise. The linear working range for the immunoassay was found to be 50-1000 cells mL^-1. The disposable immunofiltration membranes were found to retain 75% of their activity over a period of 7 days when refrigerated at 4°C. The short assay time, in combination with the ability to detect small concentrations of bacteria, permits the application of the developed system to near-real time detection of a wide range of analytes in many different applications, such as medical diagnostics, environmental applications and food quality control.