Fluorescence and absorbance dual-mode immunoassay for detection of Ochratoxin A based on 2-aminoterephthalic acid and p-nitrophenol

Abstract

In this work, a dual-mode immunoassay to detect Ochratoxin A (OTA) was developed relied on p-nitrophenyl phosphate (PNPP) and 2-aminoterephthalic acid (PTA-NH2). This proposed immunoassay based on microplate immunoenzyme technique used alkaline phosphatase (ALP) as a label for forming immune complexes. The ALP was used to catalyze the dephosphorization of PNPP to generate yellow p-nitrophenol (PNP). Since the absorption wavelength of PNP overlapped with the fluorescence emission of PTA-NH2, the fluorescence of PTA-NH2 can be quenched by PNP. The color of PNP and the fluorescence of PTA-NH2 were used as double signals to detect OTA. In this dual-mode immunoassay, the limit of detection (LOD) and the linear detection range of fluorescence mode were 0.57 ng/mL (about 0.57 μg/kg) and 1.56–50.00 ng/mL (about 1.56–50.00 μg/kg), respectively. For absorbance mode, the LOD and linear range were 0.07 ng/mL (about 0.07 μg/kg) and 0.39–25.00 ng/mL (about 0.39–25.00 μg/kg), respectively. Both detection modes showed good selectivity for OTA. The recoveries rates of fluorescence mode and absorption mode in maize flours were 91.80–128.85% and 100.10–114.00%, respectively.