A Novel Label-Free Fluorescent Turn-on Method for the Determination of Guanine Using Levodopa

A novel simple, direct, selective and rapid fluorometric method is described for the determination of guanine. The method is based on a "turn-on" response by the enhancement of the fluorescence of levodopa (L-3,4-dihydroxyphenylalanine (L-DOPA), under optimized conditions. The reaction is subjected to detailed spectroscopic and thermodynamic investigation. Upon addition of guanine, to levodopa, a significant enhancement of the fluorescence intensity is observed, accompanied by a slight bathochromic shift of ~ 10 nm in the emission maximum. This fluorescence response is consistent with an interaction-dependent change in the local environment of L-DOPA. Spectroscopic and association analyses support non-covalent association under the investigated conditions, although the available data do not establish a unique molecular binding geometry or identify the specific functional groups involved. Among the tested nucleobases, guanine produced the most pronounced fluorescence enhancement, demonstrating a preferential analytical response under the optimized conditions. A linear relationship between fluorescence intensity and guanine concentration is obtained over the concentration range of 0-280 µM guanine, with a detection limit of 0.088 µM (0.0133 µg/ml). The proposed method is validated following the standards of ISO/IEC 17025, AOAC, USP, U.S.EPA, and U.S.FDA by measuring data trueness, accuracy, bias, precision, within-day repeatability, between-days reproducibility, standard deviations, robustness, sensitivity, range, durability, stability, and limit of detection. The results reveal good data quality, reliability, and consistency. The method is applied for the determination of guanine in human serum samples using the standard addition (spiking) technique. An average accuracy of (93.3-102.2%) with good precision (mean RSD = 0.67%, n = 3) are obtained. The method provides a simple, label-free, and low-complexity approach for guanine determination in aqueous media, offering a practical alternative to more elaborate sensing platform.