Photoelectrochemical Biosensor For The Detection Of Methylated Nucleotide And Assay Of Application

DNA methylation and RNA methylation are the important epigenetics events,and DNA methylation is responsible for transcription,genomic imprinting and cellular differentiation.Aberrant DNA methylation is always contacted with various diseases.Although various methods have been applied to DNA methyltransferase?MTase?detection,most of them are generally complicated and expensive.Among RNA methylation modifications,N6-methyladenosine?m6A?has been identified as the most frequent internal mRNA modification in all higher eukaryotes and viruses.According to recent reports,the m6A modification is associated with mRNA splicing,export,stability,and immune tolerance.Up to now,a few methods for m6A detection have been reported,mainly including thin layer chromatography,gas chromatography,column liquid chromatography,liquid chromatography coupled with mass spectrometry,capillary electrophoresis.Though the above-mentioned approaches have great effective for detecting m6A,some disadvantages are still existence.Thus,it is necessary to develop a sensitive,simple,and economical method for m6A detection.Besides,2'-O-methyl group on the 3'terminal nucleotide in plant microRNAs,as also one kind of RNA methylations,is caused by HEN1 RNA methyltransferase?HENMT1?,which is thought to be crucial for ribosome biogenesis and function.HEN1 methyltransferase?HENMT1?catalyzes S-adenosyl-L-methionine?SAM?-dependent 2'-O-methylation at the3'-termini of small double-stranded RNAs,HENMT1-mediated 2'-O-methylation has been demonstrated to be a key mechanism to protect plant microRNAs and small interfering RNAs as well as animal piwi-interacting RNAs from degradation and 3'terminal uridylation.Thus,it is reported that HENMT1 is closely associated with microRNA metabolism,and it is also been confirmed that micro RNAs play fundamental roles in plant development.Therefore,it is necessary to develop a method for HENMT1 detection.According to the matching principle between energy level of different photoelectric materials.Depended on heterojunction,doping of quantum dots and ion substitution and integrated to the HRP and HRP mimic enzyme Pt Cu NPs,five different photochemical biosensors were fabricated for the detection of the m6A,M.SssI methyltransferase and HENMT1.The application of the immunoreaction between antibody and antigen and the specific reaction between biotin and avidin greatly improved the specificity and sensitivity.In this work,the level of m6A in serum,maize seedling leaves and chicken embryo hepatocytes have been detected successfully.Different pesticides were selected to investigate the inhibition effect of M.Sss I transmethylase and HENMT1.It provides a new way to research the regulatory mechanism of m6A and diagnose animal and plant diseases.?1?DNA methylation is one of the important epigenetics events,and it is responsi ble for transcription,genomic imprinting and cellular differentiation.Aberrant DNA methylation is always contacted with various diseases.Although various methods have been applied to DNA methyltransferase?MTase?detection,most of them are generally co mplicated and expensive.Herein,a simple and low-cost photoelectrochemical?PEC?biosensing method is proposed for detection of DNA methylation,assay of DNA MTase activity and screening of MTase inhibitor,which is based on M.SssI MTase-Hpa II endonuclease system.The graphite-like C₃N₄?g-C₃N₄?and CdS quantum dots?CdS QDs?are used as photoactive materials.After the double-stranded DNA?dsDNA?were treated with M.Sss I MTase in the presence of S-adenosylmethionine,the methylated dsDNA cannot be digested by Hpa II endonuclease and dithiol group at the terminal of dsDNA can be retained.As a result,CdS QDs could be coupled successfully with methylated ds DNA by reaction between Cd S QDs capped TGA and dithiol of dsDNA,achieving the amplification of PEC response.Thus,the photocurrent of PEC biosensor is consistent with the M.Sss I activity.The increased photocurrent was in proportion to M.SssI activity in the linear range from 1 to 80 U/mL with a detection limit of0.316 U/mL.Furthermore,atrazine and azamethipos were selected and investigated their effect on M.Sss I MTase activity,which might provide useful information on carcinogenic mechanism of pesticide.Therefore,we think the PEC biosensor has potential to screen of DNA MTase inhibitor,providing valuable information for anti-cancer drug research and also for cancer therapy.?2?The first part of the experiment verified that CdS QDs could enhance the photoelectric signal of g-C₃N₄.Therefore,in this section,g-C₃N₄/CdS QDs heterostructure was prepared.N6-methyladenosine?m6A?is an enigmatic and abundant internal modification in eukaryotic messenger RNA?mRNA?,which could affect various aspects of RNA metabolism and mRNA translation.Herein,a novel photoelectrochemical?PEC?immunosensor was constructed for m6A detection based on the inhibition of Cu²⁺to the photoactivity of g-C₃N₄/CdS quantum dots?g-C₃N₄/CdS QDs?heterojunction,where g-C₃N₄/CdS QDs heterojunction was used as photoactive material,anti-m6A antibody as recognition unit for RNA containing m6A,Phos-tag-biotin as link unit and avidin functionalized CuO as PEC signal inhibitor.When CuO was captured on electrode through biotin-avidin affinity reaction and treated with HCl,Cu²⁺could be released and Cu_xS would be formed based on the selective interaction between CdS QDs and Cu²⁺,which could cause an obviously decrease of the photocurrent.Under the optimal detection conditions,the PEC biosensor displayed a linear range of 0.01-10 nM and a low detection limit of 3.53 p M for methylated RNA determination.Furthermore,the developed method can also be used to detect the expression level of m6A methylated RNA in serum samples of breast cancer patient before and after operative treatment.The proposed assay strategy has a great potentia l for detecting the expression methylation level of RNA in real sample.?3?Considering that the CdS QDs in the previous two experiments are toxic,in this part,Ru@Si O₂ were used to enhance the photoelectric signal of g-C₃N₄.A novel,simple and sensitive photoelectrochemical?PEC?immunosensing platform was constructed for detecting N6-methyladenosine?m6A?based on the carboxylated g-C₃N₄ and avidin functionalized Ru@Si O₂?avidin-Ru@Si O₂?nanocomposite.Herein,the N⁶-methyladenosine-5?-triphosphate?m6ATP?was selected as the detection target molecule,m6A antibody was used as recognition unit for m6A,the carboxylated g-C₃N₄ was not only used as the photoactive substance,but the substrate for the m6A antibody immobilization,avidin-Ru@Si O₂ was synthesized and used as signal amplification unit to improve the photocurrent of g-C₃N₄,Phos-tag-biotin was employed as bridge of m6ATP and Ru@Si O₂ through the specific interaction between phosphate group of m6ATP and Phos-tag,biotin and avidin,respectively.Under the optimal condition,the fabricated PEC biosensor showed a linear range of 0.01-10nM and high detection sensitivity with low detection limit of 3.23 pM for m6ATP.Besides,the developed strategy was also successfully applied to evaluate the content of m6A in human serum samples.?4?2'-O-methyl group on the 3'terminal nucleotide in plant microRNAs,as one kind of the RNA methylations,is caused by HEN1 RNA methyltransferase?HENMT1?,which is thought to be crucial for ribosome biogenesis and function.Herein,a simple and label-free PEC biosensing method was proposed for assay of HENMT1 activity and inhibitor screening based on peroxidase mimic PtCu nanoframes?Pt Cu NFs?catalytic signal amplification.In this work,MoS₂@Graphene quantum dots/Phosphorus-doped rodlike carbon nitride?MoS₂@GQDs/P-RCN?heterojunction was used as photoactive materials.With the doping of GQDs and the formation of heterojunction,the photoactivity of MoS₂ is greatly improved.After the double-stranded RNA?ds RNA?with 2 nt 3'overhangs was treated with HENMT1 in the presence of S-adenosylmethionine,the 3'terminal nucleotide of the unmethylated ds RNA could be extended under the catalysis of the poly?U?polymerase in the existence of UTP.Poly?A?nucleotide chain modified with carboxyl group was captured on the electrode surface through hybridization reaction and acted as a bridge for the immobilization of reticular DNA-functionalized PtCu NFs?Pt Cu@DNA?.Under the catalysis effect of peroxidase mimics PtCu@DNA towards hydrogen peroxide,O^2-was in situ generated as electron donor and a strong photocurrent was obtained.The proposed PEC bioassay exhibited high selectivity and low detection limit of 3.36 ng/mL for HENMT1 activity assay.Furthermore,the inhibition research indicated that chlorpyrifos could inhibit the HENMT1 activity with the IC₅₀ value of 59 nM.?5?In the first three parts experiments,the photoelectric activity materials were g-C₃N₄.In order to increase the specific surface area of the carbon nitride and facilitate the electron transfer,the carbon nitride bar was prepared and used for the HEN1 RNA methyltransferase?HENMT1?detection.Herein,a novel dual-signal amplified photoelectrochemical?PEC?bioanalysis was successfully developed for the highly selective detection of N6-methyladenosine?m6A?methylated RNA.The PEC biosensor comprised Bi VO₄-110-Ti O₂ heterojunction and gold nanoparticle decorated MoS₂?MoS₂-AuNPs?as the photoactive materials,horseradish peroxidase conjugated biotin?HRP-Biotin?as the enzymatic unit,Ag⁺-mediated cytosine pairs?C-Ag⁺-C?as the signal amplification unit,and the anti-m6A antibody as the m6A methylated RNA recognition unit.Following immunoreaction between m6A and the anti-m6A antibody,the C-Ag⁺-C structure of the hairpin DNA unfolded,yielding the duplex strand DNA?dsDNA?and releasing Ag⁺ions.Superoxide ions?O₂^-?generated by the action of HRP on H₂O₂ then served as an electron donor,resulting in the deposition of Ag on the surface of the AuNPs,resulting in an increased photocurrent.Based on this change in the photocurrent,m6A could be accurately assayed using this dual-signal amplified PEC biosensor.The biosensor showed high selectivity and a very low detection limit of 1.665 pM for m6A,and was successfully applied to evaluate the content change of m6A in leaves of maize seedling and chicken fetal hepatocytes samples after treatment with chemical mutagen of ethylmethane sulfonate and hormone of insulin,respectively.