Co-assembly modulates a single dye to achieve complex functional outcomes: turn-on sensing, turn-off sensing, and analyte specificity through complexity.

Differential sensing with macrocyclic host molecules has enabled the detection of diverse chemical structures and complex mixtures, quickly advancing towards clinical applications. However, current systems are typically constrained by the binding properties of their individual sensor components, limiting practical implementation. In this work, we introduce a fundamentally different strategy: functional complexity is conferred onto a single reporter host through non-covalent co-assembly with a second host that brings both high potency and inherently high promiscuity-sulfo-pillar[n]arene. This co-assembly approach augments the performance of a DimerDye-class sensor by increasing its sensitivity (>100-fold in some cases) and by creating dramatic variations in its responses to different analytes. We present evidence that the co-assembled sensor forms ternary complexes with certain analytes, and that its new emergent properties include increased signal intensity and unique changes in fluorescence output. This means the performance typically achieved by sensor arrays built from several distinct host-type sensors can now be achieved using only a single DimerDye sensor that is simply mixed with unadorned sulfo-pillar[n]arenes under two different conditions. We report the mechanisms by which this emergent sensing occurs and demonstrate that a simple co-assembly enables differentiation of complex biological mixtures, reliably distinguishing between illicit drugs and whole-tissue samples of wild mushrooms.