| Ochratoxin A (OTA), as a kind of chlorophenolic mycotoxin, exist widely in plant origin food and is harmful to human. The current national standard of China stipulate that the maximum level of OTA at 5 ng-mL-1 for cereals, beans and its products. Under the consideration of the universality and perniciousness of OTA, the development of reliable and rapid detection methods of OTA in food samples is of great practical significance for food safety. Surface plasmon resonance (SPR) biosensor has some properties for label-free, real-time, sensitive and rapid and has emerged as a powerful technique in biological and chemical analyses. Unlike antibodies, aptamer can be chemically derivatized easily to extend their lifetimes and bioavailability. With respect to antibodies, aptamers have the following advantages:low cost, little or no batch-to-batch variation and easy modification. In our work, we have developed a new biosensor based on the (SPR) properties of gold surfaces and the peculiarity of aptamers. Detection of OTA in wine and peanut oil was further performed in the SPR biosensor using simple liquid-liquid extraction for sample pretreatments. The main results were as follows:1. Based on the investigation and research of literature, our project chooses the sequences of OTA-aptamer:5’-GATCGGGTGTGGGTG GCGTAAAGGGAGC ATCGGAC A-3’.2. To confirm binding of OTA to aptamer marked by biotin, ITC experiments were performed. The concentration of OTA and biotin-aptamer was 8 μM and 99 μM respectively and the results were optimized. All data could be fitted using the "one set of sites" model within the Origin software and giving an apparent equilibrium constant KA=8.83×106±1.20×106 M-1 with the Aptamer/OTA stoichiometry of 1.34±0.01. Our data showed an strong binding event between the OTA and biotin-aptamer and were consistent with the result reported in literature. From the ITC experiment results, it could be easily observed that OTA did not exhibit binding to the aptamer in the absence of magnesium ions.3. In this assay, the streptavidin protein as a cross-linker was immobilized onto the surface of a CM5 sensor chip using a standard amine coupling protocol. Streptavidin immobilized on the sensor chip surface can be used to capture biotinylated aptamer with high efficiency. Experiment Parameters, such as association time, dissociation time, regeneration project, were optimized. Moreover, the specificity of the SA cross-linker based SPR aptasensor for detection of OTA was checked by respectively introducing Kanamycin A, Aflatoxin B1, Ochratoxin B and N-acetyl-L-phenylalanine on the OTA-aptamer layered chip. Our findings demonstrate that the biosensor is specific for OTA detection. Each sample with different concentration was injected in triplicate and a calibration plot of the relative responses was plotted against OTA concentration. The biosensor exhibited a detection range from 0.094 to 100 ng·mL-1 (linear range from 0.094 to 10 ng·mL-1) of OTA with a lower detection limit of 0.005 ng·mL-1.4. Here, we assessed the feasibility of the analytical method in complex matrix samples by detecting OTA in red wine and peanut oil with the standard addition method. In our research, two simple but efficient liquid-liquid extraction methods were developed for the separation of OTA from complex components existing in wine and peanut oil samples. A commercial ELISA method was employed as a comparison to evaluate the validity of the developed SPR method with the same sample preparation process. The detection result of ELISA was higher than the real situation. The most probable cause was serious matrix interference that was negligible for SPR method. Free OTA were spiked in wine and peanut oil solutions with the final concentration of 3,40,75 ng·mL-1 and mean absolute recoveries were analyzed to be 86.92～116.54%. |
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