Research On The Selection And Application Of Aptamers Targeted To Fusarium Mycotoxin

Aptamers are short oligonucleotides screened through an in vitro selection progress called SELEX(Systematic Evolution of Ligands by EXponential enrichment), which can speci?cally recognize and bind to target molecules as a result of their intrinsic properties to form elaborate three-dimensional structures. For development of over two decades, aptamers have been widely applied as new recognition elements not only in medical diagnosis and clinical therapy, but also in analysis chemistry and sepalation/purification field, because of their advantages of high specificity and affinity, low-cost and rapidly reproducible synthesis, simple and controllable modification. However, the lack of aptamers targeted to mycotoxin severely limits the potential application of such super-recognition elements in the area of mycotoxin detection. In this study, high-affinity DNA aptamers recognizing Fusarium mycotoxin(zearalenone, fumonisin B1, or T-2 toxin) were screened using improved SELEX technology. Then, a series of novel and sensitive analysis method for detecting mycotoxin in food were established by combining the specific aptamer with magnetic separation and enrichment technique, electrochemical impedance spectroscopy(EIS), gold colorimetric analysis techniques. The study enriches the SELEX methods and expands the application of aptamers, laying a solid foundation for developing effective Fusarium mycotoxin detection technology in the further study.Firstly, DNA aptamers recognizing zearalenone were screened against immoblized targets using improved magnetic-SELEX method, and the best aptamer Z8 was used as recognition element in the detection of zearalenone. The target molecules were immobilized on amine functionalized magnetic beads. Then after 14 rounds of rigorous screening, the enriched aptamer library were cloned and sequenced. The best aptamer Z8 specificialy binding to zearalenone was finally confirmed after the identification of affinity and specificity of the resulting aptamer candidates. Subsequently, capture probes were constructed by immobilizing aptamer Z8 on the magnetic nanoparticles, which bound selectively with zearalenone molecules in the samples. The zearalenone captured by aptamer Z8 were released in a reduced-volume buffer, of which the fluorescence of zearalenone was detected. Under the optimized experimental conditions, the correlation between the fluorescence intensity and logarithm concentration of zearalenone was observed to be linear with in the range of 3.14 nmol~31.4 μmol/L, and the detection limit of the method was 785 pmol/L. The developed method has been successfully applied to detect zearalenone in beer samples.Secondly, DNA aptamers recognizing fumonisin B1 were screened against immoblized targets using improved magnetic-SELEX method. The target fumonisin B1 molecules were immobilized on amine functionalized magnetic beads by glutaraldehyde. After 13 rounds of rigorous screening, the enriched aptamer library were cloned and sequenced. The affinity and specificity of the aptamer candidates were identified by using binding dissociation experiments and competitive dissociation experiments. Furtherly, the binding mechanism between free fumonisin B1 and aptamers were analysed by secondary structure analysis and circular dichroism spectrum. A best aptamer named F10 specificialy binding to fumonisin B1 was finally confirmed, which has laid a good foundation for the development of aptamer-based detection method for fumonisin B1.Thirdly, an aptasensor for the sensitive determination of fumonisin B1 via electrochemical impedance spectroscopy(EIS) and applying aptamer-based biorecognition has been developed. A thiolated aptamer for fumonisin B1(ap F10) applied as recognition element was anchored onto the surface of gold nanoparticles on a glassy carbon electrode. During detection of FB1, the changes in interfacial electron transfer resistance(Ret) using a redox couple of [Fe(CN)6]3-/4- as the probe were monitored. Under the optimized experimental conditions, a significant increase in resistance is found on interaction with fumonisin B1 in the 0.1 nmol/L to 100 μmol/L concentration range, and the detection limit is as low as 2 pmol/L. The assay has been applied to determine fumonisin B1 in spiked maize samples.Fourthly, DNA aptamers recognizing T-2 toxin were screened against non-fixed targets using improved GO(Graphene oxide)-SELEX method. Given the property of T-2 toxin, the SELEX screening program was designed based on the principle that GOs strongly adsorb single-stranded DNA but hardly adsorb target-combining DNA complexes. After 10 rounds of rigorous screening, the enriched aptamer library were cloned and sequenced. The affinity and specificity of the full-length sequence and the truncated sequences of aptamer candidates were examined, and a best-fit aptamer named Seq.16 was confirmed. This aptamer specifically targeted to T-2 toxin has laid a good foundation for the establishment of aptamer-based detection methods for T-2 toxin.Fifthly, a gold nanoparticles colorimetric assay for visually detecting T-2 toxin has been developed based on the inhibition of acetylcholinesterase-catalyzed hydrolysis by the aptamer-enriched T-2 toxin. Aptamer Seq. 16 for T-2 toxin was applied as recognition and concentration elements, combined with efficient magnetic separation/enrichment technology, the analyte T-2 toxin was purified from the complex system of sample matrix and pre-concentrated in the buffer system for colorimetric detection. The enriched T-2 toxin inhibits the activity of acetylcholinesterase, reducing the hydrolysate thiocholine thus preventing the aggregation of gold nanoparticles. Under the optimized experimental conditions, the correlation between the gold nanoparticles solution absorbance at A520 nm and logarithm concentration of T-2 toxin was observed to be linear in the range of 107 pmol/L ~2145 pmol/L, and the detection limit of the method was 107 pmol/L. The developed method has been successfully applied to detect T-2 toxin in beer samples.In conclusion, immobilized and non-immobilized SELEX method suitable for screening aptamers recognizing mycotoxin have been established, and specific aptamers targeted to zearalenone, fumonisin B1 or T-2 toxin have been respectively screened by the developed SELEX method. Taking advantages of high affinity and specificity to targets, and easy chemical synthesis and modification, the selected aptamers have been successfully applies as high-effect recognition elements in the detection of target Fusarium mycotoxin from food matrices, combined with efficient and convenient magnetic separation technology, sensitive fluorescence analysis, electrochemical impedance spectroscopy, and gold nanoparticles colorimetric assay. The study expands the application range of aptamers in the field of mycotoxin detection.