Photoelectrochemical Detection Of RNA Methylation Based On Black Titanium Dioxide And Bismuth Vanadate Heterojunction Materials

RNA methylation modification plays a crucial role in the normal function of RNA,affecting the molecular structure and stability of RNA and playing a potential role in embryonic development.Studies have shown that RNA methylation plays an important role in environmental stress,ribosome biogenesis,antibiotic resistance and aspects.It can be used as a tumor marker and has certain clinical application value for the prevention of cancer.Therefore,quantitative and qualitative detection of RNA methylation is very important.Currently,RNA methylation detection methods include two-dimensional thin-layer chromatography,high performance liquid chromatography,quantitative mass spectrometry,methylated RNA immunoprecipitation sequencing,electrochemical sensors and photoelectrochemical?PEC?sensors.The research group chose two major RNA methylation modifications,namely N⁶-methyladenine?m⁶A?and N¹-methyl adenine?m¹A?.Because two methylated molecules exist in the form of nucleotides in the RNA sequence,this paper uses m⁶A and m¹A nucleotides as the detection targets,respectively.?1?A novel photoelectrochemical immunosensor was constructed for N⁶-methyladenine?m⁶A?detection based on the photoactive materials of black titanium dioxide?B-TiO₂?and bismuth trioxide?Bi₂O₃?and the signal amplification unit of[Ru?bpy?₃]²⁺-doped metal organic framework?MOF?.The Bi₂O₃/B-TiO₂/ITO electrode was first fabricated,then decorated with gold nanoparticles?AuNPs?which provided sites for anchoring m⁶A antibodies.After the capture of m⁶A via immunoreaction with the antibody,the Zr-based metal organic framework?UiO-66?-[Ru?bpy?₃]²⁺compound was further attached specifically to the phosphate group of m⁶A.With visible light irradiation,a large and stable photocurrent response was produced in the presence of ascorbic acid?AA?.Under optimized experimental conditions,the linear range of the PEC biosensor was 0.05–30 nmol?L^-1,with a low detection limit of 16.7 pmol?L^-1?S/N=3?.This method showed high specificity,selectivity,stabilization and repeatability.Moreover,it has been successfully used in studies that evaluate the effect of Pb²⁺on m⁶A in total RNA in rice seedling leaves.The results showed that the expression level of m⁶A in rice seedling leaves decreased with the increase of Pb²⁺concentration.This work provides useful information for studying the ecotoxicological effects of Pb²⁺on rice development.?2?Black titanium dioxide(TiO_2-x)and molybdenum sulfide?MoS₂?heterojunction(TiO_2-x-MoS₂)was employed as photoactive material,m⁶A antibody was used as target molecule recognition reagent,biotin-functionalized phos-tag?Phos-tag-biotin?was adopted as bridging reagent to link m⁶A and avidin-functionalized alkaline phosphatase?avidin-ALP?.With the catalytic effect of ALP,its substrate of L-ascorbic acid 2-phosphate trisodium salt in detection buffer can be hydrolyzed to produce ascorbic acid?AA?,which will offer electron to capture the hole of photoactive material,improve the photoelectrochemical response and increase the detection sensitivity.Under the optimal conditions,the detection range for m⁶A is ranged from 0.005–35 nmol?L^-1,and the detection limit is 1.67 pmol?L^-1?S/N=3?.The biosensor has good selectivity,reproducibility and stability.Moreover,In addition,the effects of antibiotics on the content of m⁶A in total RNA of root,stem and leaf tissues of rice seedlings were studied by means of construction The results showed that amoxicillin,chloramphenicol and tobramycin reduced the content of m⁶A in roots,stems and leaves of rice seedlings.With the increase of antibiotic concentration,m⁶A content decreased.?3?Bismuth vanadate?BiVO₄?/graphitic carbon nitride?g-C₃N₄?heterojunction?BiVO₄/g-C₃N₄?was employed as photoactive material,polyamidoamine?PAMAM?and4-carboxyphenylboronic acid?CPBA?were used as m¹A antibody immobilization matrix,and Ti-based metal-organic framework encapsulated heterostructure of TiO₂@NH₂-MIL-125?Ti?was adopted as signal amplification.Based on the specific interaction between TiO₂ and phosphate group,TiO₂@NH₂-MIL-125?Ti?can be specifically captured on the electrode surface after immunoreaction between m¹A and its antibody,which can improve the photoelectric response of the sensor and further improve the detection sensitivity.Under optimal experimental conditions,the biosensor presented wide linear range from 0.05 to 35nmol?L^-1 with the low detection limit of 16.7 pmol?L^-1?S/N=3?.This detection strategy showed good detection reproducibility,stability and selectivity.This method was used to study the effect of different antibiotics on the m¹A content of total RNA in leaf,stem and root tissues of rice seedlings.The results showed that chloramphenicol,tobramycin and tetracycline reduced the content of m¹A,while roxithromycin and norfloxacin increased the content of m¹A in rice roots.