Multiplexed high-throughput electrokinetically-controlled immunoassay for the detection of specific bacterial antibodies in human serum

In previous studies we have developed a simple electrokinetically-controlled lab-on-a-chip for heterogeneous immunoassay. In that method, all the sequential operations in an immunoassay, such as reagent loading and washing, were performed automatically by electrokinetically controlling the flow in an H-shaped microchannel. Here, we demonstrated further development of a high-throughput immunoassay microfluidic chip, and the application of the new immunoassay microfluidic chip in assaying human serum. The microfluidic immunoassay analyzed ten samples in parallel in 22 min. Bacterial antibodies in samples were captured by antigens pre-patterned on the bottom wall of a microchannel and then bound with TRITC-labeled detection antibodies to generate fluorescent signals. With optimized surface concentration of antigen, the assay detected Escherichia coli O157:H7 antibody and Helicobacter pylori antibody from buffer solutions in concentration ranges of 0.02-10 μg mL⁻¹ and 0.1-50 μg mL⁻¹, respectively. Human sera that were E. coli-positive or H. pylori-positive were accurately distinguished from respective negative controls. Moreover, the two antibodies, anti-E. coli and anti- H. pylori antibodies, could be simultaneously detected from human serum. This electrokinetically-controlled immunoassay shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.