Hydrazide-Assisted Antibody-Oriented Strategy For The Preparation Of Gold Nanoparticles Based Immunochromatographic Strip

Gold nanoparticles（Au NPs）-based immunochromatographic assay（Au NP-ICA）is one of the most popular point-of-care（POC）diagnostic tools for the rapid detection of target analytes by the naked eye.The conjugates of antibodies and Au NPs（Au NP probes）are the crucial components in fabricating a high-performance strip because the detection sensitivity of the ICA strip is largely affected by the bioactivity of antibodies on the Au NP surface.Currently,available coupling methods for preparing Au NP probes include physical adsorption and covalent conjugation.Nevertheless,these strategies are not site-specific reaction,resulting in the random orientations of antibodies on the Au NP surface.Unfortunately,these improper orientations with one or both antigen-binding sites distributed in a sterically hindered way can decrease the bioactivity of antibodies immobilized on the Au NP surface,and thereby causing the heterogeneity of Au NP probes.Thus,a simple and fast site-specific conjugation technique is vital to maximize their bioactivities on Au NP surface for efficient target recognition.Previous studies indicate that the Fc fragment of antibodies possesses some specific groups,such as carbohydrate moieties,sulfhydryl groups,aldehyde groups,histidine residues,and so on.The aldehyde group can directly react with the hydrazide to form the covalent hydrazone bonds through the nucleophilic addition.Thus,we speculate that the residual aldehyde group in the Fc domain of antibodies can serve as a promising alternative for the site-specific directional coupling because of its high reactivity with the hydrazide-activated support surfaces.Herein,we reported a site-specific directional coupling strategy to enhance the bioactivity of Au NP probes through the specific covalent binding of the aldehyde group in the Fc domain of antibodies with the hydrazide group modified on the surface of Au NPs.Through this design,the antibodies can be erected on the Au NP surface to fully expose the Fab domain and achieve the maximized functional availability.Au NPs with the size of 100 nm was selected as a signal amplification label for the development of the ICA strip because it exhibits a higher optical signal intensity than that of the widely used 20-40 nm Au NPs.And then,the Au NPs was modified by thiolated hydrazinyl ligands and thiolated carboxyl ligands with the same mass ratio of 1:1to obtained hydrazinyl modified h Au NPs.The monoclonal antibody against the hepatitis B surface antigen（HBs Ag）was used to couple on the Au NP surface.Leveraging these Au NP probes as ICA labels,we demonstrate an improved detection of the hepatitis B surface antigen with less used amount of labeled antibody（0.2mg/pmol Au NPs）,shorter reaction time（10 min）,better antibody bioactivity,and higher detection sensitivity（2 ng/m L）compared with the carbodiimide method.The obtained strip exhibited a wide linear range for HBs Ag quantitative determination from 2 ng/m L to 1000 ng/m L,the regression equation is y=0.0026x+0.1179（R²=0.9972）.The h Au NPs-ICA showed no cross reaction to other interfering proteins including PCT,FSH,CEA,OVA,HCV,AFP,and HCG.The intra-and inter-assay recoveries for HBs Ag spiked serum samples were of 86.64%-109.64%,and the variation coefficients were 0.37%-1.61%.These findings implied that the proposed method was comparable to the commercial CLIA kit in quantifying the HBs Ag in real serum samples.In addition,h Au NPs-ICA method was also established for the quantitative detection of zearalenone in corn sample.The corresponding regression equation of the calibration curve for ZEN quantitative detection can be expressed as follows:y=-0.119ln（x）+0.7679（R²=0.9845,0.25 ng/m L<x<250 ng/m L）.The half-maximal inhibitory concentration of the proposed h Au NPs-ICA was 9.4 ng/m L,with a LOD of0.32 ng/m L.The h Au NPs-ICA also showed no cross reaction to other common mycotoxins including AFB₁,AFB₂,CIT,AFG₁,FB₁,DON and OTA.The average recoveries of intra-and inter-assays for ZEN spiked corn samples were 83.1%-118.0%,and the variation coefficients were 4.3%-13.1%.These results indicate that hydrazide-assisted directional antibody conjugation of gold nanoparticles strategy can be applied to the detection of target substances in sandwich and competitive immunochromatographic assays with improved detection performance.