Highly Sensitive Dual-Probe Fluorescence Assay Based on an Aptamer for the Detection of Sulfasalazine

Sulfadiazine (SD) is extensively utilized in agriculture, aquaculture, poultry, medical, and other industries. Its residues pose a threat to human health by entering the food chain and can also be released into the environment through animal feces and urine, leading to ecotoxicological pollution. Consequently, there is an urgent need to establish an efficient method for detecting SD residues in the environment. In this study, a novel two-probe fluorescence assay for determining SD in the environment, based on magnetic separation and real-time quantitative PCR-TaqMan probe technology, was successfully developed. In the experiment, SD served as the target substance, and an aptamer (Apt) with high affinity for SD was synthesized. Additionally, a non-fully complementary chain (Cdna) with favorable hybridization properties with the aptamer was designed and synthesized to create a magnetic probe of magnetic beads@Apt@Cdna. When SD was introduced, Apt specifically bound to SD with a hairpin structure and was released from the magnetic probe, allowing SD detection via the PCR-TaqMan method. Factors affecting the determination accuracy of this assay system, such as Apt concentration, SD standard solution pH, and incubation time, were optimized. Under the optimized conditions, the assay demonstrated high sensitivity for SD, with a detection limit of 2.34 × 10^-5 ng/mL. Finally, the method was applied to detect SD in water samples from the Jialu River Basin in Zhengzhou City, yielding spiked recoveries of 88.82-117.05%. The results indicated that the detection system is a highly sensitive and specific method for determining SD residues in environmental water samples, showcasing its potential application in SD detection.