G-quadruplex specific thioflavin T-based label-free fluorescence aptasensor for rapid detection of tetracycline

A label-free fluorescence aptasensor was developed for the rapid detection of tetracycline (TET) based on G-quadruplex structure of TET aptamers and G-quadruplex specific dye Thioflavin T (ThT). The fluorescence of free ThT is essentially weak in aqueous solution, whereas it selectively identifies the G-quadruplex of aptamers to form the G-quadruplex/ThT conjugates, resulting in an enormous increase of the fluorescence intensity. However, the fluorescence intensity of G-quadruplex/ThT conjugates was drastically suppressed due to the release of free ThT from G-quadruplex/ThT conjugates after the addition of TET via specific binding with TET aptamers. The key factors affecting sensitivity and selectivity including the reaction medium, binding time of ThT to TET aptamers, incubation time between TET aptamers and TET, concentration of ThT and TET aptamers were investigated in detail. The optimal conditions were as follows: ultrapure water as reaction medium, binding time of 5 min, incubation time of 1 min, 9.0 μmol/L ThT and 0.03 μmol/L aptamers. A good linear relationship (correlation coefficient of 0.9973) was obtained between the fluorescence quenching efficiency (F₀ - F) / F₀ and the logarithm of TET concentration in the range of 0.01-1.0 μmol/L. The limit of detection was 0.001 μmol/L (S/N = 3). The proposed assay was applied for the detection of TET in the spiked honey and milk samples with recoveries ranging from 93.5% to 106.9%. The developed label-free fluorescence aptasensor showed advantages of high specificity, low cost and short time-consuming, illustrating potential application for on-site detection of TET in foodstuffs.