Study On The Fluorescence Detection Method Of Domoic Acid Specific Specific Polypeptide Based On Molecular Simulation

Domoic acid（DA）is a powerful neurotoxin that can affect the digestive tract,cardiovascular system and central nervous system in humans and animals.The study found that the maximum limit that humans can tolerate through eating is 20 mg/kg.Therefore,rapid and accurate detection of DA is very necessary.Currently,among the methods for the detection of DA,the liquid chromatography method needs to be equipped with professional large equipment and professional technical personnel,which is not suitable for rapid on-site detection,while the immuno-detection method has a long antibody acquisition cycle and too many uncontrollable factors.Molecular simulation and fluorescence detection methods were used to screen the polypeptide that can specifically recognize DA.Firstly,the crystal structure of 1YAE protein was analyzed and the binding result with DA was obtained to determine the final parent polypeptide fragment GLU 738-LYS 762,and the polypeptide group containing this fragment was intercepted.Then,the virtual amino acid mutation was carried out.The polypeptide with good docking results was selected for virtual amino acid mutation based on affinity and stability,and the new result sequence was obtained.Thirdly,all polypeptide were integrated into the self-built polypeptide library and three polypeptide chains（P54,P92 and P93）with the best docking results with DA were screened.Finally,fluorescence method was applied to further screen the polypeptide:Application of fluorescein（QDs and FITC）respectively to modify the three peptides with same concentration.And the fluorescence spectrophotometer was used to compare the combination ability of three polypeptides with fluorescein and the specific recognition ability to DA,according to the fluorescence detection results to identify P92 as recognition elements for the establishment of the fluorescence detection method.Establishment of fluorescence detection method for QDs-polypeptide:The QDs-P92fluorescence probe was prepared,and important experimental conditions affecting DA recognition,such as the coupling ratio of the probe,the concentration and incubation time of GO,and the incubation time of DA were optimized,and a FRET-based fluorescence detection method was established.The method had a good linear relationship in the concentration range of 9-100μg/L（R²=0.9993）,and the detection limit（S/N=3）of the method was 8.59×10^-1μg/L.The specificity,accuracy and stability of the method were good,and the recoveries were in the range of 88.10%to 113.02%.Establishment of FITC-polypeptide fluorescence detection method:The FITC-P92fluorescence probe was prepared,and important experimental conditions affecting DA recognition such as the coupling ratio of the probe,the concentration and incubation time of GO,and the incubation time of DA were optimized to establish a FRET-based fluorescence detection method.The method had a good linear relationship in the range of 10-100μg/L（R²=0.9945）,and the detection limit（S/N=3）of the method was6.52×10^-1μg/L.The specificity,accuracy and stability of the method were good.The recoveries were in the range of 85.45%to 107.84%.