Biosensors For 5hmC Detection Based On WS₂ And C₃N₄

5-Hydroxymethylcytosine?5hmC?is hydroxylated form of 5-methylcytosine?5mC?,which is produced by the oxidation of 5-methylcytosine during the catalysis of TET protein.Many studies have shown that 5hmC is relatively high in neuronal cells and embryonic stem cells,indicating that it plays an important role in mammalian embryonic development,nuclear reprogramming,gene expression regulation,and cancer progression.As an intermediate product of demethylation,it plays a vital role in the life activities of animals and plants.Therefore,5hmC is regarded as the sixth base of DNA.However,since 5hmC is similar in structure to 5mC and its content is low in plants and animals,the detection requires high sensitivity and specificity.Now,the commonly methods for detecting 5hmC include single-molecule real-time sequencing,capillary electrophoresis,high performance liquid chromatography-mass spectrometry,thin-layer chromatography,and fluorescence.Although these methods can achieve accurate detection,there are still some problems,such as the large precision instruments,complicated operation steps,and expensive costs.Therefore,it is a research hotspot to find simple,low-cost,fast and sensitivity detection.?1?Herein,a novel photoelectrochemical?PEC?biosensor was developed for 5hmC detection based on WS₂ nanosheets as the photoactive material and boronic acid functionalized carbon dots?B-CDs?for signal amplification unit.This biosensor can also be used for T4-?-glucosyltransferase??-GT?activity assessment.Firstly,WS₂ nanosheets and gold nanoparticles?AuNPs?were immobilized on an ITO electrode surface.Then probe DNA was immobilized on this electrode surface via Au-S bond.Afterwards,the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization.Subsequently,?-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues.After glycosylation,B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent.The PEC biosensor afforded high selectivity,excellent sensitivity and good reproducibility,with detection limits of0.00340 nM and 0.0280 unit/mL for 5hmC and?-GT,respectively.Results demonstrate that the photoelectrochemical strategy introduced here based on WS₂ nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection,?-GT activity assessment and?-GT inhibitor screening.?2?A novel photoelectrochemical biosensor was fabricated for 5-hydroxymethylcytosine?5hmC?detection based on the photocurrent inhibition effect of ZnO on MoS₂/C₃N₄heterojunction.Firstly,the ITO electrode was modified successively with MoS₂ and C₃N₄ as photoelectric materials to deliver a strong photocurrent response.Next,the 5-hydroxymethyl group?-CH₂OH?of 5hmC was oxidized by KRuO₄ to produce an aldehyde group?-CHO?,where 5hmC was converted into 5-formylcytosine?5fC?.Based on the covalent reaction with between–CHO of 5fC and–NH₂ groups of C₃N₄,5fC can be captured on electrode surface.Finally,the ZnO-PAMAM composite was covalently attached to the phosphate group of the immobilized 5fC,which could decrease the electron transfer amount of C₃N₄ to MoS₂,absorption of light and consumption of electron donors thereby resulting the decrease of photocurrent.Under optimal conditions,the photocurrent shows a linear relationship with the logarithm value of 5hmC concentration from 0.01-200 nM with a low detection limit of 2.60pM.Moreover,this method was selective and allowed to discriminate between 5hmC and5-methylcytosine?5mC?in DNA.Finally,the photoelectrochemical biosensor was successfully applied to investigate the effect of heavy metal ion and phytohormones on 5hmC expression in rice seedlings leaves.?3?A sensitive electrochemiluminescence?ECL?method was reported for 5hmC detection based on the ECL inhibition effect of ferrocenedicarboxylic acid polymer?PFc?on C₃N₄.Firstly,TiO₂/MoS₂/C₃N₄/GCE was prepared as matrix electrode,where C₃N₄ was employed as the ECL active material,the MoS₂ nanosheets were used as co-catalyst,and TiO₂was adopted as phosphate group capture reagent.In the presence of co-reaction reagent of K₂S₂O₈,the matrix electrode achieved a strong ECL signal.Then,the composite of5-hydroxymethyl-2?-deoxycytidine-5?-triphosphateandsulfydrylfunctionalized ferrocenedicarboxylic acid polymer?5hmC-PFc?was synthesized based on the covalent reaction between the–CH₂OH of 5hmC and the–SH of PFc under the catalysis effect of HhaI methyltransferase.Finally,through the specific interaction between TiO₂ on the matrix electrode surface and the phosphate group of 5hmC-PFc composite,5hmC-PFc can be further modified on TiO₂/MoS₂/C₃N₄/GCE surface.The ECL signal of this electrode decreased by the quenching effect of PFc.Under optimal conditions,5hmC was detected in the range of0.01-500 nM and the detection limit of 3.21 pM?S/N=3?.More importantly,the reaction between 5hmC and PFc was carried out in the solution,which can keep the high activity of enzyme.In addition,the biosensor was successfully applied to detect 5hmC in genomic DNA of chicken embryo fibroblast cell.?4?Herein,an electrochemiluminescence sensor was constructed for 5hmC detection based on thiol functional Fe₃O₄ magnetic beads and covalent chemical reaction of-CH₂OH in5hmC.First,Fe₃O₄ magnetic beads were prepared and modified with thiol.Then,5hmC was captured on the surface of the magnetic beads by the reaction between-CH₂OH of 5hmC and-SH of the thiol-functionalized Fe₃O₄ under the catalysis of DNA methyltransferase?M.HhaI?.After that,through a series of reactions,phos-tag-biotin,avidin,and bis?hexafluorophosphate??Ru?bpy?₂?phen-5-NH₂??PF₆?₂??Ru?were further successively immobilized on the surface of the magnetic beads.More importantly,these reactions were carried out in a solution to ensure the activity of the biomolecules,and further to ensure that the reaction proceeded sufficiently.Finally,an ECL signal was generated by the introduction of Ru.The concentration of 5hmC presented a good linear relationship with the ECL signal intensity in the range of 0.01-500 nM,and the detection limit was 2.86 pM.Moreover,we also used this method to study the 5hmC content change in chicken embryo fibroblast cell infected with and without avian leukosis virus subgroup J.