Miniaturized Flow Stacked Immunoassay for Detecting Escherichia coli in a Single Step

Commercially available systems that provide cost-effective, fast, simple, and portable solutions for health and environmental applications are few despite advancements in bioassays and biosensor research. We have developed a new system based on stacked membranes, each layer with a specific function. Samples were added onto the bottom-most layer, and as each layer becomes wet, the analyte pushes through to the next membrane layers. During migration, the analyte attaches with the corresponding antibody, itself conjugated with horseradish peroxidase (HRP) to produce a measurable signal. To prevent false positive results, blocking layer membranes are added to stop unbound antibodies from reaching the top membrane. Thus, only analyte/antibody-HRP complex will generate a signal. In order to prove this concept, Escherichia coli was used as the target analyte. After optimization, our immunoassay sensitivity was adjusted to 100 cells mL^-1. Different environmental water sources were also tested to demonstrate the sensitivity and specificity of our proposed stacked bioassay. Simplicity, low price, sensitivity, and modularity (capability to change to any target analyte) make this idea very promising for future commercialization.