A novel indirect competitive fluorescence immunoassay based on the quenching of I- to silver clusters (AgNCs) was developed for the highly selective and ultrasensitive detection of sulfamethazine (SMZ). In this system, alkaline phosphatase (ALP) was labeled on the secondary antibody (Ab2). And after a competition step, magnesium ascorbyl phosphate could be catalyzed to produce ascorbic acid under the catalysis of ALP. Subsequently, I2 was introduced and further reduced to I- in the presence of ascorbic acid, triggering the fluorescence quenching of AgNCs dispersed in isopropanol (IPA) buffer. More importantly, trace I- could lead to an obvious reduction in fluorescence signal, indicating the sensitivity of this method would be greatly improved. Under the optimal condition, this improved method for the SMZ detection has a lower detection of limit (LOD, 0.05000 μg/L) with a wider range (0.1400-71.71 μg/L). After an evaluation, the fluorescence ELISA proposed in this work has satisfactory accuracy and reliability (recoveries, 84.18-118.6%; CV, 2.03-7.64%), illustrating good performance and great potential for the detection of trace SMZ in environmental and biological samples.
Keywords: Alkaline phosphatase (ALP); ELISA; Environmental and biological samples; Immunoassay; Silver clusters (AgNCs); Sulfamethazine (SMZ).
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