| Cytometric bead array (CBA) is a new analytical technique, which can achieve real-time and rapid detection of targeted components in a small amount of sample. With many advantages of high throughput screening, high specificity and sensitivity, low cost, easy operation and good repeatability, this CBA technique has been widely used for the screen and detection of mycotoxins in wheat, corn, peas and agricultural products, with the advantage of simultaneous detection of various mycotoxins. While, this CBA technique has not been applied for the detection of exogenous harmful pollutants of mycotoxins in traditional Chinese medicine. Ochratoxin A is the most compelling harmful mycotoxins and listed as a group B carcinogen by the International Agency WHO for research in Cancer. Many countries have established regulations to govern the OTA level to reduce the harm to people and animals. Traditional Chinese medicine malt has been used for the study to build a rapid, sensitive, specific, quantitative determination of ochratoxin A and to lay the foundation for the establishment of multi-mycotoxin residues detection platform in the CBA technique,the main contents are as follows.1. A cytometric bead array with fluorescence-encoded polystyrene microspheres as the carrier based on indirect competition mode has been developed for the sensitive and rapid detection of ochratoxin A (OTA) in malt.The malt samples were extracted with 60% methanol/PBS by vortex and ultrasonic, the extracts were diluted five times by 20% methanol/PBS, then the supernatant was collected to prepare sample solution for analysis. Afterwards, the microsphere surface was labeled with the bovine serum albumin-OTA (BSA-OTA) to compete the same monoclonal antibody (mAb) with OTA in samples. Then FITC-IgG was added to bind with the captured mAb on the microsphere. Finally, the mean fluorescence intensity from FITC on the surface of microsphere was detected by a flow cytometer for accurately qualitative and quantitative analysis of OTA. The results showed that the half inhibitory of concentration (IC5o)was 1.20 ng/mL with correlation coefficient (R2) of 0.989, and the limit of detection (LOD) for OTA was 0.12 ng/mL. The average recoveries ranged from 93.9% to 97.4% with relative standard deviations (RSDs) from 0.85% to 3.56%. Sixteen malt samples were analyzed and OTA were detected in 11 and 14 batches with the contents less than 3.83 μg/kg, which were lower than the legal maximum residue levels (5.0 μg/kg) proposed by the European Union. All the positive samples were confirmed by LC-MS/MS. In this study, the CBA technique based on indirect competition was developed for the first time for successful detection of OTA in malt samples. The method was easy, rapid, sensitive and reliable with high potential for the qualitative and quantitative of multiple mycotoxins in other complex matrices.2. A cytometric bead array (CBA) with fluorescence-encoded magnetic microspheres as the carrier based on indirect competition mode has been developed for the sensitive and rapid detection of ochratoxin A (OTA) in malt.Fluorescence-encoded magnetic microspheres has been used to make up for the shortcomings and drawback of fluorescence-encoded polystyrene microspheres in the process of washing, centrifugation, coupling, and immune reaction. And also, The results showed that the half inhibitory of concentration (IC50)was 0.463 ng/mL with correlation coefficient (R2) of 0.978, and the limit of detection (LOD) for OTA was 0.03 ng/mL. The average recoveries ranged from 89.6% to 96.46% with relative standard deviations (RSDs) from 2.05% to 3.73%. Sixteen malt samples were analyzed and OTA were detected in 11 and 14 batches with the average contents of (2.71±0.6、1.96±0.38)μg/kg, which were lower than the legal maximum residue levels (5.0μg/kg) proposed by the European Union. All the positive samples were confirmed by LC-MS/MS. In this study, the CBA technique with fluorescence-encoded magnetic microspheres as the carrier based on indirect competition has been established. with the advantage of simple, rapid, short time, low cost, robust and reliable results which provides a new carrier for rapid detection and analysis.The method was easy, rapid, sensitive, specific and reliable with high potential application and prospects in the favor of one-site rapid detection of mycotoxins in traditional Chinese medicine. |
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