Gold nanoparticle to antibody conjugates for diagnosis applications: molecular interactions and immunoassay development

Thesis for the Master degree in Structural and Functional Biochemistry

Bionanotechnology is an area of rapidly increasing research interest, whose development can potentially improve many fields of our society by providing several advances in biomedical, energy and environment research fields. The present work includes both applied and basic research on bionanoconjugates of gold nanoparticles (AuNP) with antibodies (Ab) referred to as AuNP@Ab. The two main themes of research are: i) development of a fluorescence immunoassay for malaria diagnosis, using a sensing methodology involving the competitive binding between a fluorophore-labeled antigen (CyAg) and the unlabeled antigen (Ag) to the AuNP@Ab conjugates. The Ag is a new biomarker of malaria infection, the heat shock protein 70 from Plasmodium falciparum (PfHsp70). ii) physico-chemical characterization of the interaction between the Ag and the AuNP@Ab using Fluorescence, Differential Centrifugal Sedimentation (DCS), and Agarose Gel Electrophoresis(AGE), intended at understanding the nature of the binding, particularly its specificity and the influence of nonspecific protein competitors. The immunoassay for malaria diagnosis was successfully established using fluorescence detection for AuNPs of different diameters (15 and 30 nm) and using AGE detection for 30 nm-AuNPs. The AuNP@Ab bionanoconjugates exhibited a specific response to the target Ag, with little, or none, non-specific binding when purified transferrin (Trf) is used. Non-specific binding of proteins from plasma was nevertheless detected. Ag binding to AuNP@Ab was assessed in competitive conditions both with plasma and with Trf. Trf inhibited the Ag binding, while plasma inhibited the Ag binding as accessed by DCS but the same conclusion could not be made for fluorescence due to the large background signal caused by plasma unspecific binding.