Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography

A chemiluminometric biosensor system for point-of-care testing has been developed using an immuno-chromatographic assay combined with an enzyme (e.g., horseradish peroxidase) tracer that produces a light signal measurable on a simple detector. Cross-flow chromatography, a method previously investigated by our laboratory, was utilized in order to accomplish sequential antigen-antibody binding and signal generation. This enzyme-linked immunosorbent assay (ELISA) was effectively carried out on a plastic chip that was redesigned to simplify the fabrication process. To enhance the sensitivity, biotin-streptavidin capture technology was employed in preparing an immuno-strip that was then incorporated onto the chip in order to generate the ELISA-on-a-chip (EOC) biosensor. Samples containing cardiac troponin I (cTnI) were analyzed using the EOC. A chemiluminescent signal proportional to the analyte concentration was produced by adding a luminogenic substrate to the tracer enzyme complexed with the analyte on the chip. The luminescent signal was detected in a dark chamber mounted with a cooled charge-coupled device and the signal was converted to optical density for quantification. This EOC biosensor system was capable of detecting cTnI present in serum at concentrations as low as 0.027 ng mL⁻¹, 30 times lower than those measured using the conventional rapid test kit with colloidal gold as the tracer. In addition, the final data was acquired within 30 s after the addition of the enzyme substrate, which was faster than the detection time required when using a colorimetric substrate with the same tracer enzyme.