A scanner-based microplate platform for high-throughput oxytetracycline detection using diazotization-coupling chemistry with greenness evaluation

Routine oxytetracycline (OTC) monitoring of aquaculture effluents, water sources, and pharmaceutical products is essential for environmental management and regulatory compliance. However, existing analytical methods remain largely instrumental and low-throughput. Herein, a microvolume, high-throughput, scanner-based colorimetric microplate platform is developed for OTC determination using a sensitive and selective diazotization-coupling chemistry with RGB digital image analysis. Sulfanilamide is diazotized with nitrite to generate reactive diazonium species, which subsequently couple with OTC to form a strongly colored yellow-orange azo dye. Only microliter-scale volumes of reagents and samples are required in each well, and the reaction is completed within a few minutes, enabling simultaneous analysis of up to 96 samples. The microplate is imaged with a flatbed scanner, and color responses are quantified using ImageJ software. A linear calibration range of 1 − 40 μM was obtained with a detection limit of 0.33 μM. The platform was successfully applied to OTC determination in aquaculture effluents, tap water, drinking water, and pharmaceutical samples. It exhibited high selectivity against other antibiotics with no observable cross-reactivity. The green analytical procedure index (GAPI) confirmed the method’s environmental friendliness. Overall, the scanner-based microplate platform offers a simple, low-cost, portable, and high-throughput alternative to conventional OTC assays.