Enzyme-linked immunosorbent assays (ELISA) are critically important tools in biological research, allowing the presence and concentrations of a wide variety of key biochemical intermediates to be determined. While the signal amplification that is the core advantage of ELISA methods is impressive, it is nevertheless the case that it is insufficient for some particularly demanding challenges in terms of sensitivity, assay time, or sample size. In this paper, we discuss three different approaches developed in our laboratory that can improve the sensitivity of ELISA methods by 2–3 orders of magnitude. Two of these approaches have been shown to reduce the minimum detectable concentration of the target analyte in the system through trapping of the analyte species and the enzyme reaction product around a semi-permeable membrane. The third approach, on the other hand, focuses on reducing the sample volume requirement in these assays by implementing multiplex ELISA methods in a single microfluidic channel using the same enzyme label. This multiplex technique relies on the slow diffusion of the enzyme reaction product across adjacent assay segments for accurate quantitation and has been demonstrated to have a limit of detection substantially better than that of commercial microtiter plates. We believe the combination of these approaches could significantly extend the applicability of the ELISA technique to more challenging assays than is currently possible.

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