Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties

The requirement for an enzyme label to carry out a chemical reaction directly at the signaling region of the enzyme substrate in order to produce a large change in its detectability places a significant constraint on the scope of enzyme-linked immunosorbent assays (ELISAs). In particular, this requirement limits the kinds of enzyme label-substrate couples employable in ELISAs and prevents their independent optimization with respect to the enzyme reaction and the detectability of the enzyme reaction substrate/product. The detection limit and multiplexing capabilities of the assay are consequently restricted in addition to rendering the technique applicable to a narrow range of assay conditions/samples. Attempting to address some of these limitations, the current article describes a microfluidic ELISA method that does not require the enzyme label to act around the signaling region of the substrate molecule. A highly detectable rhodamine based substrate was synthesized to demonstrate the reported assay which upon cleavage by the enzyme label, alkaline phosphatase, transformed from a monoanionic to a monocationic species, both of which had nearly identical fluorescence properties. These species were later separated based on their charge difference using capillary zone electrophoresis in an integrated device yielding a quantitative measure for the analyte (human TNF-α) in our sample. Impressively, the noted approach not only enabled the use of a new kind of enzyme substrate for ELISAs but also allowed the detection of human TNF-α at concentrations over 54-fold lower than that possible on commercial microwell plates primarily due to the better detectability of the rhodamine dye.