Vanya D. Uzunova

• This doctoral thesis describes the development of supramolecular sensing ensembles, based on host-dye reporter pairs, for the sensing of biological analytes and monitoring enzymatic transformations. The rational design and selection of host-dye pairs are governed by the size-fit relationship and the favourable photophysical changes, exhibited by the dye upon complexation. The binding constants of the reporter pairs are examined by isothermal titration calorimetry (ITC), which reveals the thermodynamic forces of binding (Ka, delta H, and T delta S). Fluorescence-based techniques are used to study the photophysical characteristics of the dyes upon host complexation. The reporter pairs with pronounced fluorescence differentiation upon binding are selected for further sensing applications of biological analytes. The reporter pairs are used for the sensing of different types of analytes such as various amino acids, including lysine and its methylated derivatives, as well as choline-type of neurotransmitters. The analytes are further implemented in the development of supramolecular tandem assays for monitoring enzymatic activity. The design of tandem assays employs the differential binding affinity of the analysed substrates and products to the selected macrocyclic host. On the basis of the choline studies, a novel type of tandem assay for acetylcholinesterase has been developed. Due to the similar binding of the substrate (acetylcholine) and the product (choline) to the macrocycle of choice, the enzyme is coupled together with choline oxidase, which further converts choline to betaine (a weaker competitor). This constitutes a new tandem assay type, which works in an "enzyme-coupled-substrate-selective" mode. The determination of enzyme-kinetic parameters, the quantification of absolute choline and acetylcholine concentrations, and the screening for inhibitors for Alzheimer's disease is easily achieved with the novel assay.