Single-step ELISA capillary sensor based on surface-bonded glucose oxidase, antibody, and physically-adsorbed PEG membrane containing peroxidase-labeled antibody

A single-step enzyme-linked immunosorbent assay (ELISA) capillary immunosensor that exploits the combination of surface-bonded glucose oxidase (GOD), antibody and physically-adsorbed poly(ethyleneglycol) (PEG) membrane containing peroxidase (POD)-labeled antibody in a single capillary, is presented. The present simple detection technique involves a proximity-based two-enzyme system where the product of the first enzyme reaction is used as a substrate into the second enzyme reaction. Since the degree of antigen–antibody reaction controls the second enzymatic reaction process, amount of final fluorescent product varies with antigen concentration that leads to the making ELISA procedure single step. Sample solution that contains antigen, glucose, ascorbic acid and a fluorescent substrate (amplex red) is simply introduced into the capillary immunosensor by capillary action. In the presence of an antigen in the nanoliter sample volume, the released POD-labeled antibody forms a sandwich immunocomplex on the surface bearing the antibody. This leads to a proximity of the POD and GOD. We confirmed that the rate of conversion of a non-fluorescent probe, amplex red, to a red fluorescent dye, resorufin, is dependent on the two-enzyme proximity in a certain experimental condition. Preliminary results yielded an approximate detection limit of around subnanogram per milliliter concentration for human IgG. The relative standard deviation of the fluorescence response was ca. 4.1% for the capillary pieces prepared by cutting a long immunosensor capillary. This approach could be an enabling technology applicable to microfluidic device integration for multiple analyte sensing. Realization of such an endeavor could be very promising for drug screening and clinical diagnostic applications.