Enzyme-linked immunosorbent assays (ELISA) are critically important tools in medical diagnostic applications due to their ability to reliably detect pathogenic biomarkers in complex bodily fluids. However, the sensitivity of the ELISA method has often proven insufficient for diagnosing several lethal diseases at an early stage in spite of the inherent signal amplification in this assay. While miniaturization of the ELISA technique onto the microfluidic platform recently has allowed significant reductions in its sample volume requirement and incubation period, there is still a need for developing approaches that will enable this assay to detect important biomarkers at lower concentrations than currently possible. Here we present a simple approach to accomplishing this goal for bead-based ELISAs using a microfluidic device. Our work shows that analyte molecules may be significantly pre-concentrated over a bead surface by immobilizing these particles at the interface of a shallow and a deep microchannel, in conjunction with a sample flow around them via electrokinetic means. More interestingly, the use of flow pulses rather than a continuous sample transport was observed to improve the effectiveness of this pre-concentration strategy by over an order of magnitude. This occurs as the reported strategy offers sufficient time for the analyte molecules to diffuse to the bead surface during the no-flow period between the pulses as well as rapidly replenishes the captured analytes in the solution phase in the remaining time duration. In our current work, an 80-fold decrease in the LOD for the bead based ELISA technique was accomplished using this pulsed flow pre-concentration method for the cancer marker CA 19-9. Interestingly, the higher ELISA sensitivity was realized in our experiments using only 5�L of the sample which is about 20-fold lower than that required in traditional microwell plate based ELISAs.

Enhancement in the sensitivity of microfluidic bead-based ELISA using electrokinetic flow pulses

248th National Meeting (2014)

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