(168j) Multiplex ELISA in a Single Microfluidic Channel

Immunoassays are of vital interest to the scientific and biomedical community due to their ability to quantitate proteins in various biological samples. Among the different kinds of immunoassays developed till date, enzyme linked immunosorbant assay or ELISA is arguably one of the most practiced ones. The key advantage to using ELISA methods is the inherent signal amplification in this technique which allows one to reliably quantitate very small amounts of proteins present in biological samples. Today, ELISA methods are commonly practiced in commercially available micro-well plates that typically require sample volumes of the order of 100µL. While this may not be a large number in itself, it multiplies with the number of proteins that one need to quantitate in the sample of interest. For example, if one has to assay for 10 different proteins in a biological fluid, the total sample requirement goes up by an order of magnitude. Because the sample available for most immunoassays is often limited, there has been a significant effort towards miniaturizing ELISA methods without compromising on their sensitivity. Microfluidics is one such platform that has shown a lot of promise in this regard. While the implementation of ELISA methods in microfluidic channels has proved useful in decreasing the sample volume requirement, it is nevertheless desirable to further miniaturize this immunoassay in order to be able to extend its applicability to various research and biomedical applications.

In this work, we demonstrate a novel approach to implementing multiplex ELISA in a single microfluidic channel by exploiting the slow diffusion of the enzyme reaction product across the different assay segments. The functionality of the reported device is realized by creating short regions within a straight conduit that are selectively patterned with chosen antibodies/antigens by simply flowing these biomolecules through the fluidic network in a desired fashion. The different analytes are then captured in their respective assay regions by incubating a single aliquot of the sample in the analysis channel for a prescribed amount of time. Once the ELISA surfaces have been completely prepared and the enzyme substrate introduced into the analysis channel, ELISA reaction product (resorufin) proportional to the respective analyte concentration is generated at the center of each assay region provided that the ELISA reaction time (τ_R) is kept much shorter than the time required by the resorufin molecules to diffuse across the assay segments (τ_D). For operating conditions (τ_RD), the reported device has been shown to have a limit of detection which is similar to that of commercial micro-well plates but requiring less than a hundredth of the sample volume.