Electrochemiluminescence Sensor For 5-hydroxymethylcytosine Detection Based On Nano-luminescent Materials

DNA methylation,one of the most notable parts of epigenetics,has been attracted enormous attention owing to its relationship with a variety of diseases.In addition,some researchers have demonstrated that 5-hydroxymethylcytosine was formed by the oxidation of ten-eleven translocation?TET?proteins to 5-methylcytosine?5mC?in the presence of iron?II?and?-ketoglutarate??-KG?.Subsequently,the oxidation products of 5hmC could be glycosylated by T4?-glucosyltransferase??-GT?,where the glucose molecule specifically transformed onto the hydroxyl group of 5hmC for the following chemically modification.More importantly,the mutations exhibited lower levels of 5hmC compared with 5mC,which make it more difficult for 5hmC detection.Therefore,it is important for sensitively detecting5hmC.To date,various strategies have been constructed for the detection of 5hmC.Such as thin layer chromatography?TLC?,single-molecule real-time?SMRT?sequencing,UV detection,immunofluorescence microscopy,and liquid chromatography/tandem mass spectrometry?LC/MS-MS?.Though these methods can make 5hmC detection available,but they are still suffering from limitations.For example,TLC is environmentally hazardous,which can cause all kinds of damage on biological tissue,owing to its radioactive substrates.SMRT is laborious,manipulate complex,requiring additional fluorescent tags.To overcome the intrinsic drawbacks,exploring new techniques and strategies is beneficial for detection of5hmC.?1?In this work,a novel electrochemiluminescence?ECL?biosensor was fabricated for the simultaneous detection of 5mC,TET 1 protein and T4?-glucosyltransferase??-GT?activities.Furthermore,the inhibitor screening of TET 1 and?-GT were evaluated.The ECL biosensor was designed using gold nanoparticles?AuNPs?as platform,the gold nanoclusters?AuNCs?as the signal source based on AuNCs-H₂O₂ interaction,where the ECL emission could be quenched by excited state of AuNCs.Next,the methylation of double-stranded DNA which was adsorbed onto the assembled electrode was catalyzed by TET 1,glycosylated by?-GT and sequentially modified by horseradish-peroxidase.After the fabricated electrode was immersed in detection buffer,more H₂O₂ was catalyzed,leading to an enhanced ECL response.Under the optimized conditions,the proposed assay demonstrated high selectivity for 5mC-DNA detection with a detection limit of 3.46 p M,which is potentially well in drug discovery.?2?Previous researchers have demonstrated that 5mC can be stepwise oxidized to5-hydroxymethylcytosine?5hmC?by Ten-eleven translocation?TET?protein.However,there few reports about 5hmC detection.Meanwhile,the mutations exhibited lower levels of 5hmC compared with 5mC,which make it more difficult for 5hmC detection.Thus,an ultrasensitive electrochemical biosensor was constructed for 5hmC-DNA detection based on 5hmC chemical modification and HRP catalyzed signal amplification.In the first step,the probe DNA S1 was assembled on the surface of graphene-perylenetetracarboxylic acid?GR-PTCA?composites and then hybrid with 5hmC-DNA.When it was treated by M.HhaI,the acquired amino-derivatized dsDNA can couple to Si O₂-SPAAB-HRP by amido bond with superior catalysis activity towards the oxidation of o-phenylenediamine?OPD?in presence of H₂O₂,leading to a color change and a electrochemcialreduction peak was acquired at-0.514 V.The fabricated biosensor presented excellent specificity and even discriminated the similar base of5mC with a detection limit of 0.23 nM?at an S/N ratio of 3??3?An ultrasensitive sandwiched electrochemiluminescence?ECL?immunosensor was developed for 5-hm-dCTP detection based on Experiment 2 in genomic DNA by using Fe₃O₄@Si O₂ core–shell magnetic nanomaterial as a immobilization matrix for anti-5hmC antibody,PAMAM conjugated avidin and Ru?bpy?₂?phen-5-NH₂??PF₆?₂ as signal amplification unit.Importantly,Fe₃O₄@Si O₂ nanoparticles were verified to not only possess enormous surface for loading antibody by amido link,but also exhibit excellent bioactivity.With the dual signal amplification strategy,the ECL immunosensor showed wide detection range from 0.1 to 30 nM with low detection limit of 0.047 nM?S/N=3?.Based on the specific immunoreaction,the developed method also illustrated excellent detection selectivity.The fabricated immunosensor was also applied to detect the 5hmC in genomic DNA of cancer tissue,which indicated that the immunosensor possess potential applications in c linical detection.?4?In this work,we develop a novel ECL strategy for the quantitative detection of 5hmC to improve the sensitivity based on the bond between phosphate radical of 5hmC and Zr⁴⁺ion and the Ag–poly-amidoamine?PAMAM?–N-doped graphene?PAMAM-Ag-NG?.The multi-walled carbon nanotubes?MWCNTs?was used as a platform to immobilize primary antibodies?Ab1?and accelerate the electron transfer on the electrode interface.And the,Zr⁴⁺was used as a link unit to make the PAMAM-Ag-NG assembled on the electrode.PAMAM-Ag-NG with large surface area was employed as labels for combining signal molecule and amplified signals to improve the sensitivity of the designed immunosensor.Under optimal conditions,the designed immunosensor exhibited a linear concentration range from 0.01 nM to 50 nM,with a low detection limit of 2.47 pM?S/N=3?for 5hmC.Additionally,the designed immunosensor showed acceptable selectivity,reproducibility and stability.The satisfactory results indicated potential application promising in clinical monitoring of tumor markers.