Design, Synthesis, and Evaluation of Gold Nanoparticle-Antibody-Horseradish Peroxidase Conjugates for Highly Sensitive Chemiluminescence Immunoassay (hs-CLIA)

Design and synthesis of a conjugate with high specificity and sensitivity constitutes a fundamental process for developing a highly sensitive immunoassay. In this study, we investigated the process of design, synthesis, and evaluation of gold nanoparticle (AuNP) conjugates that include both antibody and horseradish peroxidase (HRP) for application in a highly sensitive chemiluminescence immunoassay (hs-CLIA). To increase the labeling efficiency of HRP on the AuNP-based conjugates while maintaining process simplicity, two synthesis methods were suggested and evaluated using the physical adsorption-based conventional synthesis protocol. Specifically, the respective methods utilized adsorption or covalent coupling of aldehyde-activated (ald)HRP, which covalently binds to the primary amine group of a protein. The conjugates were characterized via spectroscopy and dynamic light scattering methods. Conjugate sensitivity was evaluated by not only analyzing the activity of HRP but also comparing the analytical sensitivity provided by the CL-based enzyme-linked immunosorbent assay for the detection of cardiac troponin I as a model target. Based on the results of this study, we demonstrated that the use of (ald)HRP for target labeling successfully enhanced the sensitivity of the AuNP-based conjugates; moreover, it could provide a promising labeling method for hs-CLIA.