Improving Affinity Of Aptamer Based On Molecular Simulation Technology And Developing Electrochemical Aptasensors For Sensitive Detection Of Carcinoembryonic Antigen

As a wide-spectrum tumor marker,carcinoembryonic antigen(CEA)is very important for cancer diagnosis.So,many efforts have been made to develop CEA biosensors with high sensitivity and accuracy.However,most of the reported biosensors still have some limitations.Firstly,the long-term stability and the synthesis reproducibility of antibody,which is one of the main components in the widely used immunoassays,are not very satisfactory.Secondly,although aptamer have more advantages than antibody,their affinity needs to be improved.Thirdly,the enzyme covalent labeling strategy in normal biosensor fabrications is expensive,complex and time-consuming.Fourthly,the CEA biosensors developed at present are difficult to integrate the advantages of sensitivity,accuracy,simplicity,stability,rapidity and low-cost.Objectives:This study aims to replace antibody with aptamer to fabricate CEA biosensors;using molecular simulation technology to re-screen the CEA aptamer sequence obtained by exponential enrichment ligand system evolution(SELEX)to improve its affinity;making use of the natural properties of horse radish peroxidase(HRP),the unique properties of DNA aptamers and nanomaterials,to fabricate antibody-free aptasensors with integrated advantages of sensitivity,accuracy,simplicity,stability,rapidity and low-cost.Methods:1.Fabrication of Concanavalin A(ConA)-based and label-free sandwich electrochemical aptasensor for sensitive detection of CEA,based on the specific affinity of ConA to sugar residuesThe affinities of ConA,one kind of lectin,to glycoproteins,CEA and HRP,were verified by gel electrophoresis experiments.The aptasensor fabrication process was characterized by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS)techniques.The differential pulse voltammetry(DPV)was used to optimize the fabrication conditions and evaluate the performances of the aptasensor in terms of sensitivity,linearity,specificity,reproducibility,stability,and applicability in human serum samples.2.In-silico post-SELEX screening based on molecular simulation for acquisition of high affinity aptamers against CEAA DNA mutant library was firstly established through nucleotide base substitution or addition on a parent aptamer(P)against CEA,which was selected by SELEX.Mfold web server was utilized to predict the secondary structures of the DNA sequences.The RNA Composer server and MOE software were used for prediction and optimization of three-dimensional(3D)DNA structures.Afterwards,the docking model and binding ability of the 3D DNA structures with CEA were simulated and predicted by using ZDOCK program.High ZDOCK score DNA mutants were then tested by using biolayer interferometry technique(BLI),to determine their affinity to CEA.Finally,the obtained high affinity DNA aptamers were characterized and verified in our electrochemical aptasensor application.3.Fabrication of a fast,sensitive,and multimode-detection aptasensor based on HGNPs and HRP for dual signal amplifications,and based on the quick separation ability of magnetic beads,and structure-switching property of DNA aptamersHGNPs were synthesized from the silver templates via chemical replacement reaction.The properties of HGNPs were characterized by UV-vis spectroscopy,high-resolution transmission electron microscopy(HRTEM)and energy dispersive system(EDS).The aptasensor fabrication process was characterized by UV-vis spectroscopy,field emission scanning electron microscopy(FESEM)and energy dispersive X-ray detector(EDX).The fabrication conditions were optimized by DPV technology.Then,the semi-qualitative CEA detection ability of the aptasensor at colorimetry detection mode was evaluated.Finally,the sensitivity,linear range,specificity,reproducibility,stability,and applicability in human serum samples of the aptasensor for CEA detection at both UV-vis spectrometry and DPV detection modes were investigated.Results:1.A lectin-based and label-free sandwich electrochemical aptasensor for sensitive detection of CEAThe results of gel electrophoresis indicate that ConA could bind with HRP and with CEA.CV and EIS results suggest the successful binding of each corresponding material onto the electrode surface in our aptasensor fabrication.Under optimum conditions,the electrochemical detection signal increased monotonically and linearly with the CEA concentration ranging from 5 to 40 ng/mL CEA.The limit of detection(LOD)was calculated to be 3.4 ng/mL,lower than the threshold level in human serum for cancer patients(～10 ng/mL).Finally,a lectin-based and label-free sandwich electrochemical aptasensor for sensitive detection of CEA was developed,which also showed very good specificity,reproducibility,stability,and applicability in human serum samples.2.In-silico post-SELEX screening based on molecular simulation for acquisition of high affinity aptamers against CEATwo mutation sequences(P-ATG and GAC-P)exhibited significantly higher ZDOCK scores than P in the in-silico molecular simulation.The dissociation constant of P-ATG and GAC-P to CEA was determined to be 4.62 and 3.93 nM by the BLI assay,obviously superior to that of P(6.95 nM).The LOD of them in the lectin-based and label-free aptasensor application was 1.5 and 1.2 ng/mL,respectively,markedly better than that based on P(3.4 ng/mL).3.A multimode-detection aptasensor based on HGNPs and HRP for fast and sensitive detection of CEAThe UV-vis spectra show that the surface plasmon resonance position of HGNPs could be tuned to cover the entire visible to close to NIR region.The HRTEM images and EDS results revealed that the HGNPs were spherical Au/Ag composite nanoparticles with hollow/porous core.The UV-vis spectra,FESEM and EDX results indicate the successful fabrication of the aptasensor.Under optimum conditions,different CEA levels could be easily differentiated by colorimetry.Under UV-vis spectroscopy mode,the absorbance peak intensity was linearly proportional to CEA concentration from 10 to 250 ng/mL,with a LOD of 8.6 ng/mL.In contrast,DPV peak current was linearly correlated with CEA concentration in the range of 2-250 ng/mL.The LOD was 0.78 ng/mL.Moreover,sample detection could be accomplished within 45 min.Finally,a fast,sensitive,and accurate CEA aptasensor compatible with multiple detection modes was developed based on the dual signal amplification strategy of HGNPs and HRP,which also had the advantages of good specificity,reproducibility,stability,and applicability in human serum samples.Conclusion:A novel,ConA-based,and label-free sandwich electrochemical aptasensor for CEA detection has been developed based on the specific binding between ConA and glycoproteins.Attributed by the obviations of usage of antibody and labeling steps,the developed aptasensor has the advantages of stability,simplicity,and low-cost.After that,in order to improve the sensitivity of the aptasensor,an in-silico post-SELEX screening approach based on molecular simulation has been developed to improve the affinity of aptamer.Two DNA mutants exhibiting significantly higher binding ability to CEA than the parent aptamer are obtained.They have much better CEA detection performances in fabrication of aptasensors.Finally,a multimode-detection aptasensor based on the synergistic effect of HGNPs and HRP has been developed for fast and sensitive detection of CEA by making use of the quick separation property of magnetic beads and the structure-switching property of aptamer.The aptasensor also has the advantages of high accuracy,good stability and simple operation for CEA detection.In a word,we have used aptamers instead of antibody in fabrication of CEA biosensors,and successfully improved the affinity of CEA aptamer,and also realized the non-covalent labeling of enzyme.Finally,aptasensors with the advantages of sensitivity,accuracy,simplicity,stability,rapidity,economy and multi-mode detection have been developed,successfully solving some problems existed in CEA biosensor development.