| Aptamers are oligonucleotides that screened by SELEX(Systematic Evolution of Ligands by Exponential Enrichment)technology and can specifically recognize targets.Aptamers have the properties of good stability,affinity and specificity,easy chemical modification and lack of immunogenicity that have been widely used in the fields of disease diagnose and therapy,bioimaging,analysis and detection and so on.Food safety hazards refer to biological,chemical or physical factors contained in food that have potential adverse effects to human health.Among them,the residues of antibiotics and contamination of mycotoxins in food are two important parts of food safety monitoring.This thesis took chloramphenicol and mycotoxins as the research objects,truncation optimization and virtual mutation were performed to improve the affinity of the aptamer and the binding mechanism of aptamer and target were investigated by isothermal titration calorimetry,circular dichroism,fluorescent labeling,enzyme digestion,molecular docking and molecular dynamics.At the same time,based on the binding mechanism of aptamers,sensitive,accurate and rapid detection methods was established by combining polymerase chain reaction,nuclease signal amplification technology and hybrid chain reaction,which provide new avenue and theoretical basis for the application of aptamer in the detection of food safety hazards.Firstly,a fluorescence polarization aptasensor was constructed for the detection of chloramphenicol by using the 80 mer aptamer obtained from SELEX technology.Aptamer and chloramphenicol were incubated in binding buffer and free aptamer were removed by the adsorption of graphene oxide.Polymerase chain reaction(PCR)amplification was conducted by using aptamer-chloramphenicol as template,fluorophore FAM labeled primer as original fluorescence polarization signal.Then,streptavidin was added to further amplify the molecular weight of the PCR product by using the streptavidin-biotin interaction.Fluorescence polarization signal was dual amplified by PCR and streptavidin.Under the optimal conditions,the fluorescence polarization value had a good linear relationship with the logarithmic value of chloramphenicol concentration,where the linear detection range was 0.001 nmol/L-200 nmol/L,the detection limit was 0.5 pmol/L and the recovery rate in honey samples was 95%-107%.Secondly,based on the binding domain of chloramphenicol and aptamer,a fluorescence aptasensor with the dual amplification of exonuclease I(Exo-I)and hybridization chain reaction(HCR)was constructed by using the 40 mer aptamer without primer region.The binding mechanism of aptamer and chloramphenicol was first explored by isothermal titration calorimetry,circular dichroism and molecular docking and the binding domain of aptamer was determined.The initiator of HCR was blocked by the binding domain of aptamer through base pairing.When aptamer bind with chloramphenicol,it undergoes conformational changes and dissociated with the initiator of HCR.Exo-I digested the aptamer-chloramphenicol complex to release chloramphenicol and the recycling of chloramphenicol will expose more initiator of HCR.After adding the two stem-loop chains,HCR was triggered and the fluorescence signal was amplified.Under optimized conditions,the detection linear range was 0.001 nmol/L-100nmol/L,detection limit was 0.3 pmol/L and recovery rates in milk samples was 91%-109%.Thirdly,chloramphenicol aptamer was systematically truncated by gradually removing bases at both ends of the sequence,a simple fluorescence polarization aptamer sensor was constructed based on the structure-switching binding mode of the truncated 30 bases aptamer LLR10.The binding mechanism of LLR10 and chloramphenicol was studied by using circular dichroism,UV-vis and molecular docking.It was found that the concentration of cations in buffer,especially Mg2+plays a vital role in the interaction of LLR10 and chloramphenicol,the minor groove region of LLR10 was binding domain.A label-free fluorescence polarization chloramphenicol aptasensor was developed based on the recognition mechanism of LLR10 by using LLR10 and SYBR Green I.Under optimal conditions,the detection linear range was 0.1nmol/L-10 nmol/L,detection limit was 0.06 nmol/L and recovery rates in milk and honey samples were 95%-98%and 94%-108%,respectively.This aptasensor was easy to operate,time saving,sensitive,accurate and separation free.Fourthly,aptamer sequence of T-2 toxin and aflatoxin B1(AFB1)were directly truncated by the guidance of molecular docking.A sensitive and fast fluorescence detection system was constructed to detect T-2 toxin based on truncated aptamer and SYBR Green I.According to the predicted binding domain of aptamer and target by molecular docking,aptamer T40(40bases)of T-2 toxin and B32(32 bases)of AFB1 were obtained by twice and once truncation,respectively.The binding mechanism of T40 to T-2 toxin and B32 to AFB1 were systematically studied using circular dichroism,fluorescent labeling,enzyme digestion and molecular docking.It was found that the binding domain of T40 to T-2 toxin located in the stem region of T40 and the binding domain of B32 to AFB1 located in the loop region of B32.Rapid and sensitive detection of T-2 toxin was achieved by using T40 and SYBR Green I,the linear detection range was 0.03 nmol/L-30 nmol/L,the detection limit was 0.01 nmol/L,the recovery rate in beer samples was 94%-107%.The established aptasensor had the advantages of high detection sensitivity,simple operation and time saving.Fifthly,the binding mechanism of aptamer B32 to AFB1,AFB2,AFM1 and AFG1 were systematically investigated by circular dichroism and molecular dynamics simulation.The affinity order of B32 to the four targets was AFB2,AFB1,AFM1 and AFG1,where B32 has the best affinity for AFB2 and the worst affinity for AFG1.The circular dichroism data showed that after B32 bound to the four targets,the loop structure of B32 changed and the base stacking effect of the stem was enhanced.It was inferred that B32 had the same binding mode with the four targets.Molecular dynamics studies had found that B32 has the same binding domain to the four targets.Van der Waals energy and electrostatic energy provided the main contribution to the binding of B32 to the four targets.Non-polar interaction was beneficial to the binding of B32 to the four targets.The affinity difference of B32 to the four targets was due to the slight differences in the molecular structure of the four targets,which makes the four targets have different steric hindrances in the binding domain,resulting in the four key base of C14,G20,G22 and T23 in the binding domain have different energy contributions to the binding of the four targets.Finally,based on the binding domain of B32 and AFB1,a mutant aptamer M1 with 2-fold increased affinity was obtained by virtual mutation.The upper C14,G20 bases and the bottom G12,T23 bases in B32"sandwich"binding domain maintained standard base complementary pairing in random mutations,the T13,T21 and G22 bases in the middle were randomly mutated and a total of 400 mutation sequences were obtained.According to the binding free energy of the mutant-AFB1 complex,8 sequences were screened.After affinity verification,a mutant aptamer M1 was finally obtained and the affinity of M1 was 2-fold higher than that of B32.After binding with AFB1,compared with B32,the circular dichroism data showed that the negative peak position of the of M1 shifted to the left by 5 nm.It is speculated that the binding mode of M1 to AFB1 or the conformational change during the binding process was different from that of B32 to AFB1.Molecular dynamics results showed that the increased affinity of M1to AFB1 was because that the G22 base of M1 had a stronger electrostatic interaction with AFB1,which made the G22 base contribute more binding free energy to the binding of M1 to AFB1.In summary,this thesis truncated and optimized the aptamer sequence of chloramphenicol,T-2 toxin and AFB1,explored the recognition mechanism of aptamer to chloramphenicol,T-2toxin and AFB1,and an aptamer sequence with increased affinity to AFB1 was obtained through virtual mutation screening.At the same time,combined with the recognition mechanism of the aptamer to the target and the fluorescence analysis technology,the sensitive and accurate detection of chloramphenicol and T-2 toxin were realized.This thesis provided a theoretical and practical basis for the application of aptamers to the detection of food hazards such as chloramphenicol,T-2 toxin and AFB1. |
PDF Full Text Request