Construction Of Electrochemical Biosensor Based On Nanomaterial And Its Detection Of Three Biomarkers

With the development of society economy and the improvement of living standard,people have higher and higher demands on their own health,but various diseases are still one of the important factors that endanger human health.And the occurrence of most diseases is usually related to the abnormality of certain biomolecules content,structure and function in the body.Guanine（G）and adenine（A）are two kinds of purine bases which exist in the molecular structure of nucleic acid.They are involved in energy conversion and signal transmission between cells.Abnormal changes of their contents usually associate with the deficiency of the immunity system and may suggest the occurrence of some diseases including cancer,epilepsy and HIV infection.DNA methylation is an important epigenetic modification phenomenon,which plays an important role in embryonic development,cell division and regulation of gene expression.It has been proved to be a typical tumor marker.The p53 gene is one of the most relevant genes associated with cancer.Once mutation occurs,tumor suppressor function will disappear and cancer cells will accelerate malignant growth.In recent years,electrochemical biosensor has attracted more and more attention in biomolecular detection and disease diagnosis because of its advantages such as simple operation,high sensitivity and easy miniaturization.In this paper,three kinds of electrochemical biosensors were constructed based on nanomaterial for purine bases,DNA methylation and p53 gene detection.The main contents of this work carried out as follows:（1）An electrochemical biosensor based on overoxidized polypyrrole,multi-walled carbon nanotubes and molybdenum disulfide composite modified glassy carbon electrode（PPyox/MWCNTs-MoS₂/GCE）was developed for simultaneous detection of G and A.The composite exhibited excellent electro-catalytic properties towards the oxidation of G and A due to its large surface area and fast electron transfer ability.The oxidation peak positions of G and A were at 0.73 V and 1.03 V,respectively,which allowed simultaneous detection of the two substances in coexistence solution.The designed biosensor exhibited linear responses to G and A both in the ranges of 5³0μM and 30¹20μM,with detection limits of 1.6μM（G）and 1.7μM（A）（S/N=3）,respectively.In addition,the sensing strategy showed good properties and was successfully used for the detection of G and A in real salmon sperm DNA sample,indicating a potential application for the simultaneous detection of G and A.（2）A novel electrochemical biosensor based on gold nanoparticles modified glassy carbon electrode（AuNPs/GCE）was developed to detect DNA methylation.The probe DNA S1 was self-assembled on the surface of the modified electrode via Au-S bonding,and then hybridized with the complementary DNA S2 to form specific sites of methyltransferase（M.SssI MTase）and restriction endonuclease（HpaII）.HpaII could not cleave the target sequence after it was methylated by M.SssI MTase,while the sequence without methylation could be cleaved.Here,we used methylene blue（MB）as an electrochemical indicator.The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL（S/N=3）.Moreover,screening of M.SssI MTase inhibitors 5-azacytidine and 5-Aza-2′-deoxycytidine were successfully investigated using the fabricated electrochemical biosensor,indicating potential application in detection of DNA methylation and discovery of new anticancer drugs.（3）Two kinds of conductive substrates,glassy carbon electrode（GCE）and carbon paper（CP）combined with molybdenum disulfide and gold nanoparticles（MoS₂-AuNPs）composite,were used to detect p53 gene.The DNA S1 was first immobilized onto electrode surface by Au-S bonds,and could further capture the biotin labeled signal probe DNA S3 in the presence of target sequence DNA S2 to form sandwich mode.Then avidin-HRP could be introduced via the specific conjugation of biotin and avidin.HRP could catalyze the reaction of 3,3’,5,5’-tetramethylbenzidine（TMB）in the presence of hydrogen peroxide（H₂O₂）and differential pulse voltammetry（DPV）was used to detect the electrochemical signal of TMB to realize the indirect detection of p53gene.The designed two biosensors exhibited linear responses to p53 gene both in the range of 1.0×10^-121.0×10^-6 M,and the detection limits were 77×10^-15 M（GCE）and45×10^-15 M（CP）（S/N=3）,respectively.Meanwhile,the biosensors had good properties and showed good recoveries in actual serum samples.In addition,compared with the traditional GCE,the disposable CP electrode was cheaper,easier to operate and had better electrochemical performance.