Rapid determination of total hardness in water using fluorescent molecular aptamer beacon

A double-labelled synthetic oligonucleotide is used as a fluorescent molecular aptamer beacon for the reagentless determination of total hardness in tap and bottled waters. Modified thrombin binding aptamer (5′-NH-C3-GGTTGGTGTGGTTGG-C3-SH-3′) carrying 6-carboxyfluorescein (FAM) and 7-amino-4-methyl-coumarin labels at 5′ and 3′, respectively, was used for the simultaneous combined measurement of Mg²⁺ and Ca²⁺ cations. Interference from the K⁺ cation is eliminated via selective tuning of the assay conditions, increasing the temperature beyond the melting point of the potassium-stabilised quadruplex facilitating its liberation from the quadruplex, whilst maintaining the integrity of the magnesium/calcium-stabilised structure. No interference from other cations found in tap or bottled water was observed. The detection limit of the aptamer beacon is 0.04 mmol L⁻¹, with a dynamic linear range of 0-0.5 μM and is very reproducible, with an R.S.D. = 8%, n = 3. The fluorescent molecular beacon is applied to the determination of total hardness in tap and bottled waters and its’ performance compared to that of the standard method of complexiometric titration and atomic absorption spectroscopy, with an excellent correlation observed. Further work is focused on the immobilization of the aptamer for the development of a re-usable fluorescent/electrochemical aptasensor, for the determination of water hardness.