Study Of Novel Methods For Detection Of Antibody Based On Conformational Change Of DNAzyme

Antibody is an important kind of biomarkers in a broad range of diseases,which plays a dominant role in diagnosis of diseases,evaluation of vaccine efficacy,and development of antibody drugs.Therefore,antibody detection has a crucial significance in life science research and clinical diagnosis.Unfortunately,many of current methods for the deteciton of antibody require multiple time-consuming incubations,washing steps,or sophisticated equipments,resulting in delayed treatment.These drawbacks greatly limit their application in point-of-care(POC)diagnosis.DNAzyme is a deoxynucleotide with catalytic activity,which is obtained from in vitro screening.The high catalytic efficiency and easy-designed property make it exhibit great potential in the fields of biochemistry,analytical chemistry,biomedicine engineering and materials science.In this article,based on the principle of conformational change of DNAzyme,we reported two kinds of antibody detection methods which were simple,fast,accurate and sensitive.The major contents of the thesis were as follows:1.A Novel Method for Detection of Antibody Based on Conformational Change of G-quadruplex DNAzymeIn this section of the dissertation,we reported a simple and sensitive method for antibody detection based on protein-induced conformational change of G-quadruplex DNAzyme.Specifically,we designed a particular guanine-rich DNA strand whose terminals were modified with an antigen.In the absence of target antibody,the strand adopts a G-quadruplex conformation,and the formed G-quadruplex binds with hemin,resulting in a complex that mimics horseradish peroxidase.Because of the oxidation by activated G-quadruplex DNAzyme,the test solution gradually changes from colourless to blue.However,upon the introduction of target antibody,the rigid antibody specifically binds with two antigens at the terminals.Due to the size difference between antibody and G-quadruplex,the G-quadruplex structure undergoes a protein-induced conformational change.As a result,the G-quadruplex DNAzyme without secondary structure loses catalytic function.The test solution remains colorless.Using anti-digoxigenin antibody to demonstrate the strategy,a limit of detection(LOD)of 39 pM as well as a linear range from 0.5 nM to 1 mM could be achieved.This method is the first report using G-quadruplex DNAzyme to detect antibody.The strategy not only achieves sensitive analysis of specific antibody,but also has the advantages of simple operation and low cost,making it possess a good application prospect.2.A Novel Method for Detection of Antibody Based on Conformational Change of RNA-cleavage DNAzymeIn this work,we found that rigid antibody can tightly stretch soft,antigen-labeled RNA-cleavage DNAzyme strand,making it undergo a conformational change.Then,the hybridization reaction between RNA-cleavage DNAzyme strand and its substrate was disrupted.Based on this finding,we developed a novel strategy for sensitive and multiplexed detection of antibodies.Due to the robustness and high activity of RNA-cleavage DNAzyme,this assay could easily detect anti-digoxigenin antibody as low as 1 pM with a linear range from 0 nM to 1 nM.In addition,using a cocktail of RNA-cleavage DNAzyme,we achieved multiplexed detection of anti-digoxigenin antibody and anti-dinitrophenol antibody.This study further broadens the application of antibody detection method based on conformational change of DNAzyme,and provides a novel binding-induced signaling mechanism to detect macromolecules.It may prove to be of significant utility in clinical diagnosis and biomedical research.