| 5-Formylcytosine（5f C）is the formylated form of 5-methylcytosine（5m C）,which is produced by the oxidation of 5m C through TET protein.5-Formylcytosine（5f C）is an important epigenetic modification that has been found in a variety of cell genomic DNA and is believed to be involved in DNA demethylation.As an important intermediate oxidation product in the process of DNA demethylation,studies have shown that 5f C not only participates in gene regulation and cell differentiation,but is also related to changes in DNA double helix structure and protein identification,and plays an important role in the growth and development of plants.Therefore,the analysis and detection of 5-formylcytosine is helpful to understand the biological process related to epigenetic modification and provide a basis for clinical diagnosis and treatment.At the same time,5f C is widely present in the genomic DNA of various plant tissues,in addition to direct DNA glycosylase cleavage and binding to the BER pathway,DNA demethylation in plants may also be carried out through other pathways similar to mammals.In addition,environmental stresses such as drought and salinization can change the content of 5f C in plants,indicating the functional role of 5f C in responding to environmental stress.In order to fully understand the biological functions of5f C,sensitive and selective techniques are needed.The content of 5f C is less than 1/10 of the content of 5hm C,and the content of 5hm C in mammalian cell genomic DNA can be as low as0.0004%of cytosine.At the same time,the structure of 5f C and 5hm C is similar.Therefore,the detection of 5f C is faced with severe challenges,high sensitivity and high specificity detection methods have become a research hotspot.This work mainly utilized the excellent optoelectronic properties of two-dimensional nanomaterials such as WS2,C3N4 and Mo S2 to modify and compound them.While improving its photoelectric performance,it also completes its functional modification.The prepared two-dimensional nanomaterial or its composite is used as the substrate of the photoelectrochemical sensor,and the specific aldehyde group on 5-formylcytosine is used to specifically capture it.Then,using black titanium dioxide,manganese dioxide and other nanomaterials as signal molecules or biocatalytic precipitation,a photoelectrochemical sensor was constructed to complete the detection of 5f C.In addition,we also applied the prepared sensor to the detection of 5f C content in plant genomes,by studying the effects of environmental contaminants antibiotics and heavy metals on 5-formylcytosine in maize roots,stems and leaves,5-formylcytosine was applied as a new biomarker for crop ecotoxicological effects.（1）A novel PEC biosensor was constructed using P-g-C3N4-WS2 nanocomposite as the photoactive material.First,Au NPs/P-g-C3N4-WS2/ITO electrodes were prepared as substrate electrodes.Secondly,the probe DNA and the complementary DNA containing 5f C bases were modified to the electrode surface through the formation of Au-S bonds and base complementary pairing principle.Finally,through the interaction of the aldehyde groups on the 5f C with the amino groups on the Mn O2,the amino-functionalized Mn O2 were further modified on the electrode surface.Using Mn O2 nanomaterials to oxidize ascorbic acid,the electron donor in the system disappears,which can quench the photoactivity of the P-g-C3N4-WS2 nanocomposite,and can realize the sensitive and specific detection of 5f C.The detection range of the sensor is 0.01-200 n M,and the detection limit is 3.8 p M.In addition,the sensor has good detection specificity,stability and reproducibility.（2）Based on Mo S2 nanosheets and polydopamine-coated tungsten disulfide complex（PDA@WS2）,this study developed a simple photoelectrochemical method to detect 5f C-DNA strands,in which Mo S2and WS2 were used as photosensitive materials,and PDA is used as a photosensitive promoter,solid-state electron donor and 5f C recognition reagent.The nanomaterials prepared were characterized by scanning electron microscope,transmission electron microscope,X-ray powder diffraction,and UV-Vis diffuse reflection.The mechanism of PDA promoting the photosensitivity of WS2 nanosheets was studied in detail.Under the best experimental conditions,the linear range of the biosensor is from 0.005 to 200 n M,and the detection limit is as low as 1.6 p M（S/N=3）.The developed method also shows good detection selectivity and can even distinguish 5f C and other cytosine derivatives.The applicability of this method is compared with other detection methods,which proves the sensitivity of this method.（3）A novel nanocomposite CN-WS2 with excellent photoelectric activity was prepared by thermal polymerization.Through the new strategy of simultaneously loading C and N elements on pure WS2,the formation of CN-WS2 brings about high mobility,ultra-high-speed carrier separation and transport,and reduces the recombination rate of electrons and holes.Then,a novel photoelectrochemical biosensor was established using CN-WS2 composite material as photosensitive material,aldehyde-based reaction probe ARP was used as a capture reagent for 5-formylcytosine,and Zn Fe2O4 as artificial mimic enzyme was used as signal amplification.Under the catalysis of Zn Fe2O4,4-chloro-1-naphthol is rapidly oxidized by H2O2 to produce insoluble substance benzo-4-chlorohexadienone,which is deposited on the surface of the electrode,resulting in a decrease in PEC response.Under the best experimental conditions,the biosensor showed a detection limit of 3 p M.This method can also be used to identify cytosine and other cytosine derivatives.More importantly,the applicability of the method was verified by evaluating the effects of antibiotics（amoxicillin,chloramphenicol,tobramycin）on the content of 5-formylcytosine in the roots,stems and leaves of maize seedlings.（4）A novel photoelectrochemical（PEC）biosensor was constructed for 5f C detection,in which the Mo S2/WS2 heterojunction was used as the photosensitive material,and the amino-functionalized Fe3O4 and SMCC were used as the linking agent.4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole was used as a 5f C recognition reagent,and black Ti O2（B-Ti O2）was used as a signal amplification unit.Under the best experimental conditions,this PEC biosensor shows a wide linear range of 0.01-200 n M and the lowest detection limit is 2.7 p M（S/N=3）.Due to the specific covalent reaction between-NH2 and-CHO,the biosensor has high detection sensitivity and can even distinguish between 5f C and other cytosine derivatives.Finally,the photoelectric biosensor was used to successfully study the effect of heavy metal ions(Cd2+)on the expression of 5f C in the roots,stems and leaves of maize seedlings. |
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