Gold-coated magnetic nanoparticles (mAuNP) functionalized with antibodies are an interesting area of research. mAuNP application in bioanalysis allows to improve the analytical characteristics of immunosensors under development. Their magnetic properties are useful for separation of the analyte from a complex matrix using a magnetic field [1]. The gold layer provides biocompatibility, unique optical properties, and allows to use various methods for immobilization of antibodies on the surface of particles [2]. The orientation and loading density of immobilized antibodies has a significant influence on the analytical performance of the immunosensor, wherein antigen-binding capacity correlates with the number of accessible Fab domains [3]. Therefore, there is an ongoing effort in search of an optimal immobilization protocol. In this work, different methods of antibody immobilization were evaluated. Already existing information about mAuNP and their modification with antibodies was systematized and optimal methods were selected. To achieve this, surface plasmons resonance (SPR) spectroscopy, which is highly sensitive and reliable for the study of biomolecule interactions was used. Additionally, using a modified Bradford assay [4] and UV-vis spectroscopy the amount of immobilized antibodies was evaluated.
The main aim of this study was to apply different analytical methods for the evaluation of the loading density and orientation of antibodies immobilized onto the surface of mAuNP. Horseradish peroxidase (HRP) and antibodies against HRP were used for the design of the model system. The number of antibodies immobilized on the surface of mAuNPs was determined using the reaction between HRP, H2O2, and 3,3',5,5'-tetramethylbenzidine (TMB), which results in a change of solution color. The concentration of antibodies that were not immobilized was determined using a modified Bradford assay. SPR measurements were used for the evaluation of the interaction of the immobilized antibodies with HRP.