Study On Novel Electrochemical Sensors For DNA Methyltransferase And Dopamine

Deoxyribonucleic acid?DNA?methylation is one of the most pivotal epigenetic events that occurs in many biological processes.DNA methyltransferases?DNA MTase?catalyzed a methyl group from S-adenosylmethionine?SAM?transfer to cytosine or adenine at particular short palindromic sequences.It is reported that aberrant DNA methylation is recognized as a potential biomarker in early diagnosis of cancer,and also regarded as hallmark of other diseases.Dopamine?DA?is a pivotal catecholamine neurotransmitter molecule with a variety of functions in the central nervous system.It is a pivotal catecholamine neurotransmitter molecule in maintaining the functional activities of cardiovascular,nervous,and renal systems.Deficiency of DA may cause several diseases such as schizophrenia,Parkinson's disease and attention deficit hyperactivity disorder.Therefore,it is of great importance to detect DNA MTase activity and DA levels in organism.Electrochemical sensors have attracted great attention due to th eir advantages such as low cost,fast response,high sensitivity and miniaturization.In this paper,several novel electrochemical sensors have been constructed for the sensitive and rapid detection of DNA MTase activity and DA concentration.The specific work is as follows:1.A new electrochemical strategy for the simple and ultrasensitive evaluation of DNA methyltransferase?MTase?activity was developed based on electrocatalytic oxidation of ascorbic acid by graphene.In addition,the suitability of thi s sensing platform for MTase inhibitor screening was also demonstrated?Fig.1?.In this strategy,two strands of DNA?a long-chain single-stranded DNA?ss-DNA??S1?and a short-chainssDNA?S2??wereused.Then,theS1/S2hybridand6-Mercapto-1-hexanol?MCH?were immobilized on the gold?Au?electrode surface through the Au–S.After incubating with M.SssI CpG methyltransferase?M.SssI MTase?,which catalyzed the methylation of the specific CpG dinucleotides,led to HpaII can not catalyze cleavage of methylated part.Thus,a larger amount of ds-DNA remained on the electrode surface after the HpaII treatment,leading to the adsorption of a larger amount of graphene on the adsorption part of S1 and thus the larger oxidation current of AA catalyzed by graphene.The catalytic oxidation current of AA directly reflected the methylation event and M.SssI MTase activity.2.A new electrochemical method for rapid and sensitive evaluation of DNA methyltransferase?MTase?activity based on DNA strand displacement reacti on?SDR?recycling signal amplification strategy was developed.Briefly,DNA adenine methyltransferase?Dam MTase?can catalyze the methylation of adenine into N6-methyladenine adenine?m6A?in the specific 5?-GATC-3?of the hairpin DNA probe 1?H1?.Methylated H1 could be specifically recognized and cleaved by the restriction endonuclease DpnI,which released a unstable loop DNA and quickly converted to a single-stranded DNA?S1?.Then S1 can hybridize with hairpin DNA probe 2?H2?on the electrode and open the hairpin structure of H2,and hairpin DNA probe 3?H3?in the solution would hybridize with H2 to form a double-strand DNA?dsDNA?,freeing S1 to trigger another reaction cycle and generating a significant response signal.The results demonstrated that the developed method could be used in highly sensitive detection of Dam MTase with a low detection limit of 0.03 U mL^-1.Furthermore,the proposed method suggested that 5-Fluorouracil could inhibit the Dam MTase activity with the half maximal inhibitory concentration(IC₅₀)0.6?M,implying its promising application in screening of suitable inhibitors for Dam MTase.3.A dual-signaling electrochemical ratiometric biosensor based on DNA walker-assisted signal amplification strategies was developed for Dam MTase activity detection.Ferrocene?Fc?-labeled S1 hybridized with methylene blue?MB?-labeled S2to form anchor dsDNA 1.S3 which contained a swing-arm chain and blocker can hybridize with blocker S4 to form dsDNA 2.dsDNA 1 and dsDNA 2 were immobilized on the electrode through Au-S bond,lead to MB close to electrode and Fc far from electrode.DNA adenine methyltransferase?Dam MTase?can catalyze the methylation of adenine into N6-methyladenine adenine?m6A?in the specific5?-GATC-3?of the blocker part in dsDNA 2.Restriction endonuclease DpnI specifically recognized and cleaved the methylated dsDNA 2,and remaining a blunt3?-termini of S4 to initiate the exonuclease III?EXO III?digestion progress.Therefore the released swing arm hybridized with the anchor dsDNA 1 to form a blunt 3?-termini of S2,initiated the EXO III digestion progress,releasing MB-labeled DNA 1 and swing arm DNA 3,thus DNA walker was activated.The peak current change of MB and Fc associated with the Dam MTase activity,the linear range was 0.02-5 U mL^-1,and the detection limit was 0.012 U mL^-1.Also the biosensor had potential to apply in screening of suitable inhibitors for Dam MTase.4.A novel dual-signaling electrochemical ratiometric method and a dual isothermal amplification strategy assisted by EXO III was designed for simple and sensitive screening MTase activity.Briefly,Dam MTase catalyzed methylation on hairpin DNA?H1?,then DpnI could specifically recognize and cleave methylated H1into an unstable loop DNA and convert to a single DNA?S1?.S1 hybrided with single strand part of S2S3 and a blunt end at the 3?-termini of S2S3 was formed,which triggered the Exo III cleavage of S3 and released S1 and S2.S1 returned to reaction solution to realize isothermal amplification cycle 1,S2 in reaction buffer c ould further hybrid with single strand part of S4S5 to form a blunt end at the 3?-termini of S4S5,which triggered the Exo III digestion of Fc-labeled S4,releasing S2 returned to reaction solution to realize isothermal amplification cycle 2.MB-labeled S5 was remain on the AuNPs/GC electrode could thus tend to form a hairpin structure with the help of Mg²⁺.Therefore,the peak current of MB increased and peak current of Fc decreased because MB closed to the electrode and Fc was far away from it.The detection range of Dam MTase activity was 0.003 to 0.5 U mL^-1 with a detection limit of 0.0015 U mL^-1.Due to the specific recognition sequence present in the H1,this method also showed high selectivity for Dam MTase.In addition,inhibition studies with 5-fluorouracil demonstrated the capability of inhibition screening using this method.5.A simple,label-free electrochemical biosensor based on a dopamine?DA?DNA aptamer immobilized on a Au electrode through the Au–S bond was developed.Methylene blue?MB?was used as the intercalating probe,and DA was sensitively and selectively detected.The developed electrochemical DA aptasensor showed a good linear response to DA from 5-150 nM as well as having a low detection limit of 1.0nM?S/N=3?.The biosensor also exhibited satisfactory selectivity and could be successfully used to detect DA in human blood,suggesting that the aptasensor was a promising application for the analysis of DA in real clinical samples.6.The initial precursor Al-PCP contained Al sites with the organic ligands?1,4-naphthalene dicarboxylic acid??Al?OH??1,4-NDC?₂H₂O?formed ordered three-dimensional pore structure,after carbondizion washed with an HF solution to remove the residual Al component obtained high-level porous carbon materials?HPC?.The HPC showed uniform morphology,the surface area reached 969 m² g^-1.HPC materials were rich in pores,mainly composed of mesopores,and their pore diameters were mostly distributed in the 20-40 nm range.Theo micropores?<2 nm?and macropores?>50 nm?were also exsiting.Glassy carbon electrode modified with HPC?HPC/GC?exhibited good electrochemical properties toward ascorbic acid?AA?,dopamine?DA?and uric acid?UA?,and can be found three well-separated voltammetry peaks,the peak separations between AA and DA,DA and UA were 236mV,120 mV respectively.The HPC/GC electrode showed a good linear response to AA,DA and UA,the linear ranges were 50-3000?M,0.2-30?M and 1-50?M,respectively,and the detection limits?S/N=3?were 40.2?M,0.13?M and 0.58?M,respectively.The HPC provided potential applications in the simultaneous determination of UA,AA and DA.