Application Of Iodide Ion Synergistic Nano-mimetic Enzyme Catalysis In Colorimetric Sensing And Perylene Diimide Detection Of Various MicroRNA

Compared with natural enzymes,nano-mimetic enzymes have the advantages of cheap synthesis,tunable catalytic activity,good biocompatibility,ultra-high stability under harsh conditions,and large-scale production,making them suitable for biosensing applications.Shows huge potential application prospects.Porous materials can be used in the field of biocatalysis due to their inherent porous structure,ultra-high specific surface area,ultra-high porosity,pore tunability,and catalytic activity of peroxide nano-mimicking enzymes.In this paper,we synthesized Zn-Covalent triazine framework（Zn-CTF）materials,and fully utilized its ultra-high specific surface area,easy preparation,large porosity,good stability,good Features such as biocompatibility;Synthesis of G-quadruplex/Hemin DNase（G4/Hemin）and use of both Zn-CTF and G4/Hemin to construct a label-free visual colorimetric sensor.In addition,the quencher perylene diimide（PDI）with broad spectrum and label-free advantages was also synthesized for multi-target detection.The work of this paper is divided into the following four parts:（1）Using Zn-CTF with similar properties of peroxide nano-mimicking enzymes as the catalytic active material,a label-free visualized biological colorimetric sensing system was constructed for the quantitative detection of H₂O₂ and acetylcholinesterase activity（ACh E）.When iodide ions（I^-）were intercalated into Zn-CTF,the catalytic activity of Zn-CTF was significantly improved.I^-can synergize with Zn-CTF to catalyze 3,3’,5,5’-tetramethylbenzidine（TMB）,so that the color of the system is first oxidized from colorless to blue,and the sensitivity of the sensor is improved.The change of absorbance of oxidized TMB（Ox TMB）at 652 nm was detected by ultraviolet-visible spectrophotometer（UV-Vis）.Ox TMB,the product of TMB in the oxidized state,can be used as a photothermal probe for the conversion of photothermal energy due to its good photothermal effect,and can also be analyzed and detected by the temperature change of the system.The absorbance value and temperature change value increased with the increase of H₂O₂ concentration in the range of 1 nmol/L～2mmol/L.A signal-reduced colorimetric sensing method was established to detect ACh E.The mercaptocholine produced by the hydrolysis of mercaptoacetylcholine（ATch）can inhibit the color development of TMB.In the range of 0.01 U/L～60 U/L,the absorbance increases with the concentration of ACh E While lower,the detection limit was 2 m U/L（S/N=3）.（2）Synthesis of ochratoxin（OTA）aptamer rich in guanine base（G base）sequence,when the target OTA is added,the aptamer can self-assemble into antiparallel G-quadruplex（G4）structure,labeling hemin（Hemin）on G4 can form a peroxidase-mimicking enzyme G4/Hemin,and I^-can also enhance the activity of Hemin peroxidase nanomimetic enzyme,thus constructing a signal amplification type Visualized colorimetric sensing system for quantitative detection of OTA.I^-cooperated with G4/Hemin to catalyze H₂O₂ to generate a large number of hydroxyl radicals（·OH）,and oxidized TMB from colorless to blue.The absorbance change of Ox TMB at 652nm was detected by ultraviolet-visible spectrophotometer（UV-Vis）,and the absorbance value increased with the increase of OTA concentration in the range of 5pg/m L～180 ng/m L,The obtained detection limit was 1 pg/m L（S/N=3）.Compared with commercial enzyme-linked immunosorbent assay（ELISA）,the detection limit of this biosensor is reduced by 2 orders of magnitude.（3）Based on the previous work,G-quadruplex and Hemin can be assembled into a peroxide nano-mimetic enzyme,and the addition of I^-coordinated G4/Hemin catalyzes the oxidation of TMB by H₂O₂ to turn it from colorless to blue.In this experiment,in order to further improve the catalytic activity of G4/Hemin,an enzyme cascade reaction was used to immobilize G4/Hemin on the platform together,and the addition of mutant p53 gene could initiate hybridization chain reaction（HCR）to assemble G4 and regulate Hemin simulation Therefore,a visual colorimetric sensor based on the mutant p53 gene as a single nucleotide polymorphism（Single Nucleotide Polymophisms,SNP）model was constructed for cascade signal amplification detection,which can selectively detect mutant p53 gene fragment.The change of absorbance at652 nm was detected by ultraviolet-visible spectrophotometer（UV-Vis）.The absorbance value increased with the increase of the target mutant p53 gene concentration in the range of 10 pmol/L to 600 nmol/L.The obtained detection limit was 2 pmol/L（S/N=3）.（4）The abnormal expression of mi RNA is related to many cancers,so the detection of mi RNA occupies an extremely important position in the diagnosis of cancer.Perylene diimide（PDI）acts as a broad-spectrum quencher that can quench DNA-RNA duplex-labeled fluorophores ranging from the green fluorophore FAM to the red fluorophore Cy5.DNA modified with different fluorescent groups can specifically hybridize with various mi RNAs to form double strands.When the duplex-specific nuclease（DSN）is added,the fluorescent groups can be released.Fluorescence sensing as a quenching platform for the detection of multiple mi RNAs.The fluorescence intensities of FAM,TAMRA and Cy5 were detected by fluorescence spectrometer.When the concentrations of mi RNA-21,mi RNA-141 and mi RNA-155increased continuously,the fluorescence intensity also increased gradually.The detection range of mi RNA-21 was 2 fmol/L～20 pmol/L,and the detection limit was0.67 fmol/L（S/N=3）;the detection range of mi RNA-141 was 4 fmol/L～25 pmol/L,and the detection limit was 1.33 fmol/L（S/N=3）;the detection range of mi RNA-155is 3 fmol/L～18 pmol/L,and the detection limit is 1 fmol/L（S/N=3）.