Antibody-Free Photoelectrochemical Biosensors Based On MoSe₂ For Detection Of N⁶-Methyladenine And Related Proteins

N⁶-methyladenosine（m⁶A）is the first discovered RNA methylation product with reversible chemical modification.Studies have shown that the expression level of m⁶A in RNA is closely related to gene expression regulation,animal and plant growth and development,and human diseases.The core molecule regulating the methylation of RNA m⁶A is Methyltransferase-like 3/Methyltransferase-like 14（METTL3/METTL14 protein）.METTL3/METTL14 protein has attracted more and more attention as a new drug target.As a m⁶A demethylase,fat mass and obesity-related protein（FTO protein）plays a key role in the physiological and biochemical processes of animals and plants,and can be used as a new diagnostic biomarker and promising therapeutic target.Escherichia coli toxin MazF protein is an RNA ribonuclease,which is sensitive to m⁶A modification in ACA sequence and participates in the growth regulation of stress response.Due to the inert reaction of m⁶A methyl,most of the existing m⁶A detection methods rely on the immunoprecipitation reaction of m⁶A antibody,but the antibody can not distinguish m⁶A and N⁶,2‘-O-dimethyladenosine（m⁶A_m）,and the antibody is expensive,difficult to save,large batch difference,high false positive.Therefore,in this paper,a m⁶A selective chemical labeling method（m⁶A-SEAL）using FTO protein,a detection method using METTL3/METTL14 protein and MazF protein were selected to achieve antibody-free and high-specific detection of m⁶A and related proteins.The research content of this paper is divided into the following four parts:（1）A novel photoelectrochemical biosensor was constructed by using demethylase FTO protein and dithiothreitol（DTT）to catalyze m⁶A and initiate m⁶A thiol-functionalized antibody-free detection strategy.Specifically,as a photosensitive material,Bi₂Mo O₆ greatly improved the photoelectric properties of MoSe₂.Si O₂@COOH was used as a linker to immobilize ss DNA that recognizes the m⁶A RNA sequence on the electrode surface.When m⁶A RNA was further captured on the electrode surface,the m⁶A base in the RNA sequence was oxidized to N⁶-hydroxymethyladenine（hm⁶A）in the presence of FTO protein.Then,hm⁶A was labeled by DTT-mediated thiol addition reaction to form stable N⁶-dithiolsitolmethyladenosine（dm⁶A）.At the same time,the free thiol group on the electrode surface specifically recognized Cd S,and the nanomaterial amplification strategy was used to further improve the PEC response.Through the change of photocurrent,the PEC biosensor can be used to quantitatively detect the concentration of m⁶A RNA and FTO protein.Under the optimal experimental conditions,the linear ranges for the detection of m⁶A RNA and FTO protein were 0.001-50 n M and 0.0005-500μg/L,respectively,and the detection limits were0.37 p M and 0.034 ng/L（S/N=3）,respectively.The potential application value of the biosensor was evaluated by studying the effect of entocapone on the activity of FTO protein.（2）Using a FTO protein-assisted m⁶A selective chemical labeling method（m⁶A-SEAL）and RNA hydrolysis technology,the specific quantitative detection of m⁶A at the nucleotide level was achieved,and the quantitative detection of FTO protein was also achieved.In the process of enzymatic m⁶A structural changes,the chemically inert m⁶A in the RNA sequence is first oxidized to an unstable N⁶-hydroxymethyladenine（hm⁶A）under the catalysis of FTO protein,and then a stable N⁶-dithiolmethyladenine（dm⁶A）is formed under the thiol addition reaction mediated by dithiothreitol（DTT）.After the above reaction,RNase A was used to catalyze the hydrolysis of dm⁶A RNA sequence to obtain N⁶-dithiolsitolmethyladenosine monophosphate（dm⁶AMP）for nucleotide level detection.In terms of detection methods,an antibody-free enzyme-assisted PEC biosensor was constructed by using non-metal doped Se-MoSe₂ nanoflowers as photoactive substances,gold nanoparticles（Au NPs）as recognition reagents,dm⁶AMP as target molecules,and Zn Se（which can form type II heterojunction with Se-MoSe₂）as signal amplification unit.Under the optimal experimental conditions,the biosensor can detect m⁶A RNA in a linear range of 0.01-50 n M with a detection limit of 6.3p M（S/N=3）.The biosensor can also be used to detect FTO protein with a linear range of 0.005-500μg/L and a detection limit of 0.78 ng/L（S/N=3）.In addition,the biosensor can also be used to reveal the effect of antibiotics on m⁶A content in total RNA of rice tissues and the effect of heavy metal ions on the oxidative activity of FTO protein,and to evaluate the potential application of the biosensor.（3）A novel photoelectrochemical biosensor for simultaneous detection of methylated RNA,METTL3/METTL14 protein and MazF protein was constructed by using the catalytic activity of m⁶A methylase METTL3/METTL14 protein on the methylation of adenine（A）in RNA sequence and the specific cleavage activity of methyl-selective ribonuclease MazF protein on RNA chain containing ACA sequence.The p-n heterojunction MoSe₂-Bi OI nanocomposites were used as photoactive materials for PEC biosensors.Using the methylation catalytic activity of METTL3/METTL14 protein and the specific cleavage activity of MazF protein,highly specific detection of methylated RNA,METTL3/METTL14 protein and MazF protein was achieved.Combined with the catalytic hairpin self-assembly strategy（CHA）,polyaspartic acid（PASP）loaded alkaline phosphatase（ALP）strategy and enzyme catalyzed in situ generation of electron donor strategy,the high sensitivity detection of biosensor was realized.Under the optimal conditions,the detection ranges of methylated RNA,METTL3/METTL14 protein and MazF protein were 0.001-50 n M,0.001-25 ng/μL and 0.001-10 U/m L,respectively.The detection limits were 0.46 p M,0.51 pg/μL and 0.42 U/μL（S/N=3）,respectively.The effects of drugs and compound pollutants on the activity of MazF protein were evaluated,which proved the applicability of the constructed method in the field of drug screening for MazF protein-related diseases.In addition,the effect of pollutants on the activity of METTL3/METTL14protein was preliminarily explored,which provided new information for the pathogenic mechanism of pollutants.（4）Se-MoSe₂-Cu₂Mo S₄ ternary composites were successfully prepared by a simple hydrothermal method and calcination method.A Z-type heterojunction was formed between Se-MoSe₂ and Cu₂Mo S₄,which greatly improved its photoelectric activity.By studying the preparation conditions and performance characterization of Se-MoSe₂-Cu₂Mo S₄,Se-MoSe₂-Cu₂Mo S₄ with a reaction temperature of 190°C and a reaction time of 8 h was selected as a photoelectric active material.A separate antibody-free photoelectrochemical biosensor was successfully prepared for the detection of methylated RNA,METTL3/METTL14 protein and MazF protein.On the surface of the electrode,Se-MoSe₂-Cu₂Mo S₄ has a strong photoelectric signal as a substrate material.After adding the light-blocking material cobalt oxyhydroxide（Co OOH）,the photoelectric signal is weakened.In the centrifuge tube,the adenine（A）on the RNA chain was methylated into m⁶A by the m⁶A methylase METTL3/METTL14 protein,and then the hybridization chain reaction（HCR）reaction was initiated by the ribonuclease MazF protein with specific recognition ability to the m⁶ACA short sequence and two biotin-carrying DNA hairpins to produce a DNA strand that captures avidin-modified alkaline phosphatase（avidin-ALP）.Avidin-ALP catalyzed the formation of ascorbic acid（AA）.The generated ascorbic acid reduced Co OOH to Co²⁺,reduced the number of Co OOH on the electrode surface,and exposed the underlying Se-MoSe₂-Cu₂Mo S₄,thereby significantly increasing the photocurrent and achieving sensitive detection of methylated RNA,METTL3/METTL14protein and MazF protein.In addition,the effects of drugs and environmental pollutants on the activity of METTL3/METTL14 protein and MazF protein were also evaluated,which proved the applicability of the constructed method in the field of drug screening for METTL3/METTL14 protein and MazF protein-related diseases.