Design And Application Of Optical Sensor Based On G-rich Sequence And Peroxidase Mimetics

Optical sensor is a receiver with molecular recognition function,which is composed of various indicators,dyes,enzymes and coenzymes,antigens and antibody nucleic acids,tissues and cells.It has the characteristics of non-destructive and high sensitivity,and have been widely used in biosensors.With the in-depth study of genes and the successful completion of the whole sequence of human genes,the application prospect of DNA,especially the G-rich sequence,in biological monitoring and the prevention and diagnosis of diseases is very promising.In recent years,great attention has been paid to the application of G-rich sequence in optical sensors to improve their performance.Natural enzymes have high catalytic activity and are widely used in various fields.However,natural enzymes are expensive and difficult to preserve,so they have strict requirements on storage environment.Compared with natural biological enzymes,simulated enzymes can be obtained by chemical method,with low cost,good stability and less harsh conservation environment than natural enzymes.3,3',5,5'-tetramethylbenzidine?TMB?is a chromogenic substrate for hydrogen peroxide.The peroxidase mimics can catalyze the oxidation of TMB by H₂O₂.Many peroxidase mimics can be designed by using G-quadruplex as the structural framework,so it is favored by scientific researchers.In this paper,three optical biosensors are designed based on the signal enhancement of G-rich sequence and the characteristics of peroxidase mimics,which have been successfully used in target detection.The main research contents are as follows:?1?Split aptamer strategy was often used to improve the sensitivity of aptasensor.However,traditional split aptamer strategy cannot be directly used to improve the label-free aptamer based Thioflavin T?Th T?displacement assay for ATP,because the split ATP aptamer display much lower enhancement effects on the fluorescence of Th T than intact aptamer.In order to address this issue,this was the first report using G-rich DNA sequence to enhance the affinity of the two split ATP aptamer halves to Th T and offer lower limit of detection?LOD?,wider linear range and higher selectivity through the enhanced molecular recognition.Compared to the intact aptamer/Th T complex,the ensemble of two G-rich split ATP aptamer fragments/Th T were higher fluorescent.ATP will displace Th T from the G-rich split ATP aptamer fragments/Th T complex,resulting in reduced fluorescence.The LOD of the proposed fluorescent ATP aptasensor was 2 n M.And the aptasensor had been successfully applied to detect ATP in 15%human serum.?2?This work reported the first example of a colorimetric H₂S sensor through and G-rich DNA.In the H₂O₂-mediated oxidation of TMB,22AG DNA can improve the catalytic capacity of Cu²⁺,where 22AG DNA as a signal amplifier.However,G4-Cu²⁺peroxidase mimetics lost their catalytic capacities after reacting with H₂S.With the increase of H₂S concentration,the catalytic capacity of G4-Cu²⁺peroxidase mimetics gradually decreased,the absorbance gradually decreased,and the color of the solution gradually became lighter and even colorless from blue.It was employed to construct a colorimetric assay of H₂S without tanglesome synthesis and instrument with LOD of 7.5 n M.The sensitivity of the sensor was also adjustable by reducing the concentration of Cu²⁺.Moreover,the developed sensor was successfully applied for quantitative determination of H₂S in human serum.?3?This work firstly reported that 22AG DNA with G-rich sequence could be induced to form a G-quadruplex in a sodium-free and potassium-free MES buffer,and the G-quadruplex had peroxidase mimetics properties.Similar to natural enzymes,they can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine?TMB?by hydrogen peroxide to produce blue products,thus achieving the colorimetric detection of H₂O₂.Based on the specific oxidation of glucose oxidase for glucose and the catalysis of G-quadruplex for H₂O₂,we designed an enzyme cascade for visual detection of glucose.The absorbance had a good linear relationship for glucose concentration in the range of 1mM?1 m M,and the LOD was 0.53mM.In addition,it was convenient for the visual detection of glucose due to the obvious color changes.The biosensor showed satisfactory recovery in human serum glucose detection.Therefore,this study provides a convenient,sensitive and promising strategy for the diagnosis and prevention of diabetes.