Photoelectrochemical Biosensors For DNA Hydroxymethylation Detection Using Bismuth-Based Perovskite

As an important epigenetic modification,5-hydroxymethylcytosine(5hm C)plays crucial roles in gene expression and regulation.Abnormal hydroxymethylation status may impact chromatin structure,result in tissue-related disease phenotypes.As an ideal biomarker,if5hm C can be sensitively detected,gene expression changes in relevant cell states could be recapitulated for effective early detection,diagnosis and prognosis.Thus it is imperative to analyze the level of 5hm C sensitively.With the virtues of inexpensive apparatus,simple operation and high selectivity,photoelectrochemical(PEC)technique has been aroused extensive interest.To achieve sensitive detection of 5hm C using PEC method,the photoactive material plays crucial role in the construction of PEC biosensor.Appropriate photoactive material can greatly improve the detection sensitivity.In the terms of selection for PEC sensors substrate materials,Bi₄NbO₈Cl and Bi₄TaO₈Cl have the properties of stable photoactivity and suitable bandgap,possessing obvious superiority.In order to enhance the photoelectric property,some methods can be used,including energy bandgap matching with narrow bandgap semiconductor and dye sensitization.Herein,the PEC biosensors were built based on the 3,4,9,10-perylenetetracarboxylicacid(PTCA)sensitized Bi₄TaO₈Cl,the Bi₄NbO₈Cl@Bi₂S₃and the amino-terminated perylene derivative(PTC-NH₂)sensitized Bi₄NbO₈Cl as substrate materials,which can detect 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate(5hmd CTP)and 5-hydroxymethyl-2'-deoxycytidine(5hmd C),realizing quantitative determination of 5hm C.Moreover,the PEC biosensors realized actual sample detection of 5hm C level.In order to confirm the practical application,the biosensors were used to detect samples from cultivated plants seedlings.And the biosensors can be applied to investigate the influence of heavy metal(Cd²⁺and Pb²⁺),perfluorooctanoic acid(PFOA),and exogenous phytohormone(3-indoleacetic acid,gibberellin,abscisic acid,and 6-benzylaminopurine)on 5hm C content in the genomic DNA of plants(rice or wheat)seedlings roots and leaves.This paper is divided into three parts:(1)A photoelectrochemical immunosensor was built for detecting 5-hydroxymethylcytosine,where the perovskite Bi₄TaO₈Cl sensitized by 3,4,9,10-perylenetetracarboxylicacid was implemented as photoactive material.Firstly,5hm C was recognized and captured in the light of the special recognition between antigen and antibody,where the phosphate group of 5hmd CTP was away from the electrode surface.After that,ZrO₂ and AMPA were captured based on the covalent binding between tetravalent Zr and phosphate group.Finally,Fc-COOH was modified onto the electrode surface by means of the covalent linkage between the carboxyl and amino.Employed as electron donor to harvest photogenerated hole,ferrocene can hinder photogenerated electron-hole recombination,accelerating electron transfer and amplifying photocurrent response.To certify the successful preparation of nanomaterials,some related tests were executed for perovskite Bi₄TaO₈Cl,PTCA and ZrO₂.Under the optimal condition,the biosensor manifested wide detection range from 0.02-100 n M with the low detection limit of 5.2 p M(S/N=3),validating the eminent detection selectivity,stability,and reproducibility.Moreover,the applicability of the biosensor was demonstrated by investigating the effect of heavy metal Cd²⁺and perfluorooctanoic acid on 5hm C content in the leaves and roots of wheat seedlings.The results indicated 5hm C level in leaves and roots of wheat seedlings decreases with the incubation of Cd²⁺.This work provides a new idea for the ecotoxicological effects of heavy metals and perfluorooctanoic acid.(2)A photoelectrochemical biosensor was established based on the in-situ generated heterojunction of Bi₄NbO₈Cl@Bi₂S₃,which was employed as the substrate material with excellent photoelectric property.Firstly,the heterojunction Bi₄NbO₈Cl@Bi₂S₃is used as substrate material to modify the surface of ITO electrode.Next,because Bi₄NbO₈Cl@Bi₂S₃ is dispersed in chitosan solution,the electrode surface is also modified with amino groups.TGA can be modified on the electrode by the covalence of amino and the carboxyl,resulting in covering sulfydryl on the electrode surface.Then,according to the research that–CH₂OH of5hm C can link with–SH under the catalysis of M.Hha I,the 5hm C can be further immobilized on the TGA/BNBS/ITO electrode specifically.Finally,the ZrO₂ can be combined on the electrode surface based on the combination between the tetravalence zirconium and the phosphate group of 5hm C.When the ZrO₂ is linked on the electrode,the photocurrent enhanced obviously to realize signal amplification.The experimental results demonstrated well specificity and sensitivity of this biosensor.Under optimal conditions,the linear relationship between the photocurrent and the logarithm value of 5hm C concentration was constructed with the range from 0.3-300 n M and the detection limit of 0.0779 n M.Significantly,this biosensor was applied to a preliminary study that the heavy metal Pb²⁺and the perfluorooctanoic acid influence the expression of 5hm C in the genomic DNA of wheat seedling roots and leaves.Compared to control group,the 5hm C level in the leaves increased in dealing with single Pb²⁺treatment,contrary to the level in the roots.When the wheat was treated by the single PFOA,the 5hm C content both declined in the leaves and roots.(3)Based on Bi₄NbO₈Cl and PTC-NH₂ as the base materials,a PEC biosensor was designed to detect 5-hydroxymethyl-2'-deoxycytidine.After Bi₄NbO₈Cl and PTC-NH₂heterojunction were modified one by one on the substrate electrode,thioglycolic acid and cysteamine hydrochloride were captured by forming amide bond,where the sulfhydryl group was employed as the specific recognition of 5hmd C.Under M.Hha I catalysis,the–CH₂OH of5hmd C reacted selectively with the–SH.Then,4-formylphenylboronic acid(FPBA)possessed two active groups of formyl and boronic acid group,reacting with glycosyl of5hmd C and amino of Fe-MIL-88-NH₂.Finally,Fe-MIL-88-NH₂ was modified on the electrode based on the crosslinker of FPBA.Under the catalysis effect of Fe-MIL-88-NH₂,benzo-4-chlorohexadienone(4-CD),an insoluble substance,developed on the electrode surface during the oxidation of 4-CN by H₂O₂,hindering the electron transfer rate of the electrode,resulting in distinct lessening of PEC response.Under optimal conditions,the photocurrent reveals a linear relationship with the logarithm value of 5hmd C concentration from 0.01-100 n M with a low detection limit of 0.00648 n M.More importantly,this method can be applied to investigate the influence of exogenous phytohormone on 5hmd C content in the genomic DNA of rice seedling roots and leaves.As for 7 days,four exogenous phytohormones presented obvious inhibition activity on 5hmd C expression in the leaves and roots of rice seedlings.It provides a new biosensor to the research for exogenous phytohormone pollution to rice.