Electron-rich anionic calixarenes and resorcinarenes are known receptors for trimethylammonium-containing neurotransmitters, but the development of practical sensor applications has been impeded by the lack of suitable supramolecular sensing ensembles as well as the low selectivity and sensitivity of the macrocyclic cation-receptor hosts. The host–guest complexes between p-sulfonatocalix[n]arenes (n = 4–5) and the cationic aromatic fluorescent dye lucigenin (LCG) have been characterised by optical spectroscopic techniques, NMR, cyclic voltammetry, isothermal titration calorimetry, and X-ray crystallography. The dye is complexed with binding constants of the order of 107 M−1 and undergoes a strong static fluorescence quenching (factor 140) upon complexation as a consequence of exergonic electron transfer within the complex. LCG has been utilised in combination with p-sulfonatocalixarene to set-up a refined reporter pair for label-free continuous real-time enzyme assays according to the supramolecular tandem assay principle. This affords product-selective tandem assays for amino acid decarboxylases with a one order of magnitude higher sensitivity and a 3 orders of magnitude lower host/dye concentration range, a convenient substrate-selective tandem assay for direct monitoring of choline oxidase, and a conceptually novel substrate-selective enzyme-coupled tandem assay for acetylcholinesterase. The applicability of the method to the measurement of enzyme-kinetic parameters, the screening for inhibitors of acetylcholinesterase, and the highly selective determination of absolute, low micromolar concentrations of both choline and acetylcholine by simple fluorescence measurements has been demonstrated. A domino tandem assay can be employed to measure the two analytes in the same sample. The described applications bypass problems related to the unselective binding of the macrocycle by coupling the signalling event with highly specific enzymatic transformations.
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