The Construction Of Photoelectrochemical Sensor Based On Tungsten Sulfide Composite Material For The Detection Of DNA Formylation

As an important epigenetic modification,5-formylcytosine(5fC)is produced by the oxidation of 5-methylcytosine(5m C)in genomic DNA by TET(ten-eleven translocation)protein.As an important intermediate,it participates in various biological processes,such as cell differentiation,gene regulation and even some human diseases.As the counterpart of 5fC nucleic acid base modification,5-formyluracil(5fU)has gradually attracted attention.It is the main oxidative damage of thymine(T)in DNA,which is produced by exposure to UV light,ionizing radiation,reactive oxygen species attack or enzyme oxidation.It can change the structure of DNA,lead to gene mismatch,regulate protein-DNA interaction and DNA dysfunction.However,its biological functions have not been deeply explored due to the lack of detection and localization methods.Sensitive and selective detection of DNA formylation(5fC and 5fU)can promote the understanding of epigenetics.DNA formylation exist in many cells and tissues,among which 5fC is about 0.02%?0.002%of cytosine,while 5fU is lower than 5fC.Therefore,a highly sensitive and specific DNA formylation detection method is needed for single base resolution detection of the whole genome.In this work,the excellent photoelectric properties of WS₂ were utilized,the photo-activity of it was improved by stripping,doping and compounding,and its function modification was completed.The prepared tungsten sulfide composite material was used as the substrate of the photoelectric sensor,the covalent reaction of specific aldehyde functional groups on DNA formylation(5fC,5fU)were used to realize its specific capture.Finally,nano-materials,artificial mimic enzymes or hemin/G-quadruplex complexes were used as signal amplification units to realize the specific detection of DNA formylation.At the same time,the effects of environmental pollutants antibiotics and heavy metals on DNA formylation expression in roots and leaves of crops were explored,and the feasibility of using it as a new biomarker of crop ecotoxicological effects was explored.Combining the existing forms of 5fU and 5fC in genomic DNA,5-formyl cytosine deoxyribonucleotide(5-formyl-2'-deoxycytidine-5'-triphosphate,5fd CTP),5-formyl cytosine deoxyribonucleoside(5-formyl-2'-deoxycytidine,5fd C)and 5-formyluracil deoxyribonucleotide(5-formyluracil-2'-deoxyuridine-5'-triphosphate,5fd UTP)were mainly used as detection target molecules.(1)Based on Ag₂S@WS₂ as photosensitive material,Au@AHMT as the specific recognition unit of 5fd CTP,and benzene-4-chlorohexanedione(4-CD)generated by H₂O₂oxidation of 4-chloro-1 naphthol(4-CN)catalyzed by artificial mimic enzyme Fe VO₄ as signal quencher constructed a new photoelectrochemical(photoelectrochemical,PEC)biosensor.The developed method showed a wide detection range from 0.1 to 400 n M,and the detection limit was 0.062 n M(3?).It exhibits great detection selectivity,reproducibility and stability.In addition,the change of 5fd CTP content in rice genome DNA after heavy metal(Hg and Cd)treatment was explored,which verified the applicability of this method.When the concentration of heavy metals gradually increased from 0.1 mg/L to 10 mg/L,Hg²⁺promoted the expression of 5fC in genomic DNA of rice leaves and roots.For Cd²⁺,the low concentration obviously promoted the expression of 5fC,while the promotion trend of high concentration weakened.(2)In view of the fact that the hydrolyzed product of genomic DNA is 5-formyl-2'-deoxycytidine-5'-monophosphate,which is different from that of 5fd CTP,which limits the application of the first work to some extent.To solve this problem,a new PEC biosensor was developed in this experiment with 5fd C as the target molecule,amino-functionalized Bi IO₄-WS₂as the photosensitive material and the capture unit of 5fd C,and amino-functionalized Cu O as the signal amplification material.Due to the energy band-matched structure between Cu O and Bi IO₄-WS₂,the electron transfer on the electrode surface was facilitated,thus improving the photoelectric conversion efficiency and the detection sensitivity.There is a good linear relationship between 5fd C concentration and PEC signal intensity in the range of 0.005-200n M,and the detection limit was 0.38p M(3?).In addition,the effects of antibiotics sulfadiazine(SDZ)and heavy metals(Hg)on the change of 5fd C content in wheat tissue genomic DNA were also investigated,and the feasibility of this work was proved.With the increasing of SDZ concentration from 0.2×10^-3 to 50 mg/L,the 5fC content in wheat leaf genomic DNA increased.For Hg²⁺,low concentration promoted the expression of 5fC,while high concentration inhibited the expression.This is similar to the trend of compound pollutants(50 mg/L SDZ mixed with different concentrations of Hg²⁺)on 5fC expression.In addition,the detection results were compared with ELISA method,which proved the detection accuracy of this method.(3)As the nucleobase modification counterpart of 5fC,5fU has received increasing research attention.However,the structure of 5fU is highly similar to that of 5fC,and its content in cells and tissues is lower,which makes it a challenge to realize the sensitive and selective detection.According to literature reports,the electron attraction of the 4'-carbonyl electron in5fU makes its 5'-aldehyde group exhibit higher reactivity than 5fC.The study found that 5fU can form a stable benzimidazole structure with o-phenylenediamine structure at room temperature of p H 7,and shows strong specificity.In this experiment,WS₂/Bi/Bi OBr nanocomposites were used as photoactive materials,3,4-diaminobenzoic acid(DABA)was used as recognition unit,5fd UTP was used as target molecule,and a dual signal amplification strategy was used to construct a highly sensitive PEC biosensor to realize quantitative detection of 5fd UTP.The matching energy band between b-Ti O₂ and WS₂/Bi/Bi OBr was used as the first signal amplification.Spherical b-Ti O₂ provided more binding sites for ss DNA ligation,which triggered the formation of the hemin/G-quadruplex complex,accelerateing the electron transfer.It was regarded as the second signal amplification.In addition,the content of 5fd UTP in genomic DNA of crop tissues was investigated by standard addition recovery experiments,in which the recovery rate was 84.8?114%and RSD was 0.27?4.62%,which proved the precision and accuracy of the method were evaluated.