Study On Screening Aflatoxin Specific Peptide Based On Molecular Simulation And Research On Sensor

Aflatoxin B1 is a carcinogenic substance that can cause liver cancer and even death.At present,the commonly used detection methods for aflatoxin B1 include chromatography and immunoassay methods.The chromatographic method is complicated and the detection cost is high.The preparation of specific antibodies in immunoassay is a key step.However,the antibody preparation process is complicated and random.Therefore,the search for substances with specific antibody functions has become a new research hotspot.In this study,we analyzed the binding site of aflatoxin B1 antibody obtained in the PDB library by computer-aided simulation technology,and intercepted the parental polypeptide fragment.The design and construction of amino acid site-directed mutation and random mutation of the parent polypeptide fragment were carried out by using Discovery Studio software.Specific polypeptide library;further using molecular docking technology,preliminary screening of six polypeptide sequences according to binding energy,named:S1-CRPEPKSIFRF,S2-CRRPEPKSIFRF,S3-CHISAFLSIFRF,S4-CNVLPFDSIFRF,S5-WNVLPFDSIFRFC and S6-KYLAYTSIFRFC;The binding energy is sorted from low to high S6<S5<S4<S3<S2<SI.The specific binding of six polypeptides was verified by electrochemical sensor method.First,since the specific polypeptide itself has a sulfhydryl group,it is directly modified to the surface of the gold electrode by a gold-sulfur bond.Cyclic voltammetry indicates that the six polypeptides have different degrees of specific binding effect on AFB1;further electrochemical impedance spectroscopy is used.The specific binding ability of the six polypeptides was be compared with each other.The results showed that the strong binding relationship of the six polypeptides to AFB1 specific binding ability:S4>S6>S5>S3>S2>S1;both methods indicated that the six polypeptides could be certain within the range specifically binds aflatoxin,of which S4 works best.By comparing with computer simulation results,two conclusions are obtained:one is that the closer of energy of specific polypeptide that binds to the afb1,the stronger the ability to bind AFB1;the second is that the lower the energy of polypeptide and AFB1,the better the binding ability of the polypeptide.An electrochemical sensor method based on specific polypeptide S4 was optimized.Firstly,the gold nanoparticles were modified to the surface of the glassy carbon electrode by electrodeposition in situ reduction composite method;then the specific polypeptide S4 was self-assembled to the surface of the modified electrode by gold-sulfur bond,and finally AFB1 was detected by differential pulse method.The time for depositing gold was 90 s,the concentration of the polypeptide solution was 0.5 mg/mL,the assembly time of the polypeptide solution was 1 h,the blocking time of mercapto hexanol was 60 min,and the binding time of aflatoxin was 2 h.Under the condition,an electrochemical sensor detection method based on the specific polypeptide S4 was established.The method has a good linear relationship(R2=0.9980)in the range of 10-2-10 μg/kg.and the minimum detection limit is 4×10-4 μg/kg,which has good stability,selectivity and reproducibility in the sample.In the addition and recovery experiments(polished glutinous rice,corn,rice),the recovery rate of the method was in the range of 89.34%-126.78%.Experiments have shown that the electrochemical sensor method based on the specific polypeptide S4 can be applied to the detection of actual samples.