Partitioning Indicator Displacement Assay

Indicator displacement assays work by detecting the replacement of an indicator from a binding site by an analyte. The binding sites are typically located on individual receptor molecules capable of carrying out molecular recognition. Here, we describe a new transduction mechanism based on analyte partitioning in a two-phase system, which we refer to as a partitioning indicator displacement assay (PIDA). In this assay, an indicator that is partitioned into the organic phase along with organic solutes is displaced by an analyte introduced into the aqueous phase. The indicator concentration in the receptor phase can be monitored optically, leading to the label-free detection of analytes. A sensor using cetyltrimethylammonium bromide (CTAB), octanol, and Blue 1 as the receptor phase was found to be selective for the small molecule Red 40 when compared to other similarly charged analytes. Fits to the sensorgrams reveal a quantitative trend consistent with a self-organized liquid crystalline layer whose thickness corresponds to organic solute concentrations. Coarse-grained molecular dynamics simulations provide insights into the mechanism behind the supramolecular aggregation that occurs at the interface, leading to the selectivity of the assay. The idea that a fluid organic phase could be selective in this way, depending on additives, provides a new way of thinking about biomolecular recognition in terms of small-molecule-dependent partitioning. The assay described here has potential for the detection of a range of small molecules and has implications for biological signaling in phase-separated systems.