Preparation And Analytical Applications Of The Electrochemical Biosensor Based On The Amplification Of Graphene Quantum Dot And Enzyme

Adenosine triphosphate(ATP)is the direct source of energy for all the life activities of cells and is known as molecular currency of intracellular energy transfer in all living cells.The storage and transfer of chemical energy,the synthesis processes of protein,fat,sugar,and nucleotide are required ATP to participate in.It also can promote the repair and regeneration of various cells in the body,enhance cell metabolic activity and has strong pertinence in the treatment of various diseases.It is well known that cancer cells are in a unique metabolic program that allows the rapid proliferation of cells,and the cancer cells often have more ATP.Therefore,according to the concentration of ATP in the cell can determine whether the function of the organism is normal,it also acts as an important basis for early diagnosis of disease.MicroRNA(miRNA)is a class of noncoding RNA molecules with a length of about 20-24 nucleotides,playing important roles in diverse biological processes.The main role of miRNA are three:first,'the target gene completely complementary combination;second,incomplete complementary bind with target gene,and translation repression.The expression of miRNAs plays a significant role in cell differentiation,developmental biology and disease occurrence development process.It will also enable the miRNA may become a new biological marker for diagnosis of diseases.Deoxyribonucleic acid(DNA)methylation is one of the most prominent form of epigenetic regulation and plays crucial roles in the regulation of gene transcription,gene expression,genomic imprinting,genomic stability,cellular differentiation,and cell development.The alterations in MTase activity usually occur far before other signs of malignancy and may be of potential use in early diagnosis of tumors.Aberrant DNA methylation is recognized as a potential biomarker in early diagnosis of cancer,and also regarded as hallmark of other diseases.In this paper,we use the electrochemical biosensor for the detection of ATP,miRNA-155and methyltransferase with the help of the graphene quantum dot(GQD)and the nicking endonuclease.The experimental part of this paper mainly includes the following three aspects:(1)In this paper,an electrochemical biosensor was developed for the detection of adenosine triphosphate(ATP)based on target-induced conformation switching and nicking endonuclease(NEase)-assisted signal amplification.The electrochemical biosensor was constructed by the base pairing and target recognition.After capture DNA hybridized with the gold electrode,a significant current of methylene blue(MB)was obtained by differential pulse voltammetry.In the presence of ATP,the hairpin DNA formed a G-quadruplex structure due to the specific recognition between hairpin DNA and ATP.Then the exposed part of target-aptamer complex hybridized with the 3'-terminus of capture DNA to form a specific nicking site for Nb.BbvCl,which led to the NEase-assisted target-aptamer complex recycling.The released target-aptamer complex hybridized with the left capture DNA.Nb.BbvCI-assisted target-aptamer complex recycling caused the continuous cleavage of capture DNA with MB at its 5'-terminus,resulting in releasing a certain amount of DNA fragment labeled with MB.Then the current value decreased significantly.The reduced current showed a linear range from 10 nM to 1 ?M with a limit of detection as low as 3.4 nM.Furthermore,the proposed strategy can be used for other similar substances detection.(2)A specific and sensitive method was developed for quantitative detection of miRNA by integrating horseradish peroxidase(HRP)-assisted catalytic reaction with a simple electrochemical RNA biosensor.The electrochemical biosensor was constructed by a double-stranded DNA structure and enzyme-assisted catalytic reaction.The structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes(capture DNA),assembled on the gold electrode surface,with target DNA and aminated indicator probe(NH2-DNA).After the construction of the double-stranded DNA structure,the activated carboxyl groups of GQD assembled on NH2-DNA.GQD was used as a new platform for HRP immobilization through noncovalent assembly.HRP modified biosensor can effectively catalyze the hydrogen peroxide(H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine(TMB),accompanied by a change from colorless to blue in solution color and an increased electrochemical current signal.Due to GQD and enzyme catalysis,the proposed biosensor could sensitively detect miRNA-155 from 1 fM to 100 pM with a detection limit of 0.14 fM.High performance of the biosensor is attributed to the large surface-to-volume ratio,excellent compatibility of GQD.For these advantages,the proposed method holds great potential for analysis of other interesting tumour markers.(3)A label-free electrochemical biosensor for methyltransferase(M.SssI MTase)activity assays was developed based on the amplification of endonuclease Hpall and GQD.The electrochemical biosensor was constructed by methylation sensitive restriction endonuclease Hpall and GQD as a new platform for immobilization.In the presence of M.SssI MTase,CpG site in the double-stranded DNA(ds-DNA)was methylated which consequently impeded endonuclease HpaII digestion process.More ds-DNA was protected,and then more GQD and HRP were modified on the electrode,resulting a higher reduction current.This method could be used to determine M.SssI MTase activity in the range of 1-40 U mL-1 with a low detection of 0.3 U mL-1.For the high performance of the biosensor,the method may be a potential stategy for clinical diagnosis in the future.