Study Of Colorimetric Methods For Sensitive Detection Of Antibodies Based On Assembly Of DNAzyme

Antibody is a kind of important marker for disease diagnosis and it has become a key molecule in disease diagnosis and treatment.The antibody screening is of great significance for coping with sudden epidemics,monitoring the progress of chronic diseases,and evaluating the efficacy of antibody drugs.However,the concentration of antibody is low in the early stage of these diseases and the antibody cannot be detected during the window period.In addition,most of the current assays for antibody detection require multi-step complex operations,and sophisticated equipments.Even in some of them,immobilization of the antigen denatures the antigen,masks the epitope,and reduces the sensitivity,resulting in the delay of diagnosis and treatment.Among the various methods,colorimetry is widely applied in point-of-care antibody testing because of its inherent features of intuition,simplicity and rapidity.Therefore,according to the stable DNA and its high controllability and precise assembly characteristics,this paper proposes two new methods for detecting antibodies based on assembly of DNAzyme,which enables simple,fast,accurate and sensitive analysis of antibodies.The details are as follows:1.High-sensitivity colorimetry based on assembly of double-headed DNAzymeIn this part of the paper,we proposed a sensitive colorimetric method for one-step detection of antibodies based on catalytic hairpin assembly(CHA).We designed three hairpin structures(H1,H2,and H3),and the both ends of H1 are labeled with antigen.The experiments were carried out with Anti-DIG Ab and Anti-DNP Ab.The results show that because the distance between the two recognition sites of the antibody is ～ 120 (?),the H1 structure is destroyed,exposing the primer chain that triggers the assembly of the catalytic hairpins when the antigen binds to the antibody.Then,H2 and H3 are assembled to form many barbell-shaped structures with G-quadruplex at both ends.After adding hemin and TMB,the color of this solution changes.This sensitive colorimetric method is quick and simple.It can achieve one-step detection of antibodies,which would be used to quickly screen for epidemic antibodies.Moreover,the proposed method is versatile and can be easily adapted to the rapid and sensitive detection of other antibodies and bivalent targets in bioanalytical researches.In addition,we also use this method to achieve the joint detection of antibodies,which providing novel solutions to improve the accuracy and sensitivity of traditional antibody detection kits.2.High-sensitivity colorimetry based on assembly of tandemly repeated G-quadruplex DNAzyme sThe early detection of low abundant Anti-HCV Ab is critical for efficient diagnosis and treatment of HCV infection.In this work,a colorimetric assay method has been proposed for the sensitive detection of antibody,which applied in the assay of Anti-HCV Ab in serum sample.Within the method,we ingeniously used the rigid structure of the antibody to induce DNA strand replacement,and combined it with RCA to generate a large number of tandem repeating G-quadruplex chains on the arm of the "Y" antibody.Then,the peroxidase-mimicking DNAzmes can catalyze the oxidation of TMB to amplify the colorimetric signal after adding hemin.With the amplification of RCA reaction and the DNAzyme catalytic reaction,it demonstrates that 0.998 p M antibody can be detected by this newly developed assay.Therefore,it can serve as a useful method for the facile and sensitive detection of Anti-HCV Ab in the serum-containing sample.Additionally,this method improves the detection method previously reported based on the antibody-induced DNA conformational change,so it does not need to strictly control the length of the probe between two antigens and breaks the limit on the size of the antigen,which has wider versatility.