Hydrazide mediated oriented coupling of antibodies on quantum dot beads for enhancing detection performance of immunochromatographic assay

Abstract

Immunochromatographic assays (ICAs) are one of the most popular on-site diagnostic tools. The bio-activity of the probe holds great promise for the detection performance of ICA. The orientation of antibodies on nanoparticle surface plays a vital role in improving the bio-activity of the probe. Hydrazide mediated oriented coupling (HDZ-O) strategy can erect the antibodies on the particle surface via the nucleophilic addition reaction between the hydrazide group of particles and the aldehyde group of the Fc region of antibodies. In this work, we synthesized the hydrazide and carboxylic group modified quantum dot beads (QBs-NH-NH2, and QBs-COOH) and compared two coupling strategies for preparing QB probes systematically. Results showed the reaction time for producing QB probe by using HDZ-O method was 20 min, and the optimal labeled antibody content was 80 μg per mg of QBs. Those for carbodiimide method were 180 min and 320 μg per mg of QBs. Moreover, the QB probe by HDZ-O showed a higher bio-activity than that by carbodiimide method under their optimal labeled antibody concentrations. Subsequently, the QB probe by HDZ-O strategy was applied further in sandwich ICA for hepatitis B surface antigen (HBsAg) detection. The results indicated that the strip has a widely dynamic linearity with HBsAg concentration ranging from 0.05 ng/mL to 3200 ng/mL. The limit of detection of the strip is 0.05 ng/mL, which is approximately fourfold lower than that of the probe prepared by carbodiimide method. In addition, the ICA strip also showed acceptable accuracy and precision for HBsAg quantitative detection in real serum samples, which have average recoveries in intra- and inter-assays that vary from 89.16% to 97.63% with the variable coefficients less than 6.98%. The proposed ICA strip showed good agreement (R2 > 0.94) with a commercial chemiluminescence kit for 40 of clinical serum samples. All in all, this study proposed a robust strategy to immobilize the antibodies with proper orientation on the surface of QBs, and the resultant QB probe can improve the detection performance of sandwich LFIA platform remarkably.