Desenvolvimento de estratégias de funcionalização em nanopartículas de ouro para aumentar a sensibilidade de testes para diagnósticos virais

Abstract

Gold nanoparticle (AuNPs) functionality has been highlighted in several areas of knowledge in recent decades, including diagnostics, where factors such as dispersion and stability are directly related to the test performance, the study of different functionalization strategies is essential. The stabilization of the nanoparticles can be conducted through polymers adsorption in their surfaces, conferring the characteristics and properties of nanoparticles. In this context, the objective of this work was to stabilize and functionalize gold nanoparticles of different dimensions (20, 40 and 80 nm). Adsorption kinetics and mathematical models allowed the determination of optimal concentrations of the heterobifunctional polymer varieties employed. However, since the preparation of the equation that predicted the nanoparticle behavior of 20 nm was not possible, for the following steps this nanoparticle size was discarded. The enzyme horseradish peroxidase (HRP) was used as an alternative to gauge the efficiency of the different strategies. Initially the enzyme horseradish peroxidase (HRP) was employed as an alternative to gauge the efficiency of different strategies. Tests that evaluated the dispersion, the capture of the enzyme and the of the stability strategies by techniques as spectroscopy in the UV and visible region, scanning electron microscopy (SEM), dynamic light scattering (DLS) and zeta potential were conducted. 40 and 80 nm gold nanoparticles were modified with heterofunctional polyethylene glycol solutions (HS-PEG-NH2 3500 and 7500 g/mol) and subjected to four different functionalisation strategies (I and III with HS-PEG-NH2 3500, II and IV with HS-PEG-NH2 7500). In strategies I and II, the polymeric solutions were placed in contact with the solution of anti-HRP antibodies, followed by addition of the colloidal solutions. While in strategies III and IV, the colloidal solutions first come into contact with the polymer solutions and then conjugated to the antibodies. Solutions with concentrations of 30, 50, 100 and 200 ng / ml HRP were added to the resulting solutions. A superior yield and efficacy of strategies I and II were observed when compared to strategies III and IV, since the former had a capture capacity of up to 5 times when stabilized for 2 h and up to 6 times for 12 h. The reduction of the average diameter of the nanoparticles to these strategies was evident when compared with those measured for the standard solutions. Furthermore, the increase of the zeta potential values for the antibody/ PEG/ nanoparticle systems, indicates that a interaction between the antibody/PEG solutions and the nanoparticles was established. Regarding stability, even considering reduction in the enzyme capture capability, strategy II presented best dispersion of particles, capture of HRP and stability during different months of the trial for both nanoparticles of 40 and 80 nm. After initial HRP capture, experiments were performed using viral diagnostic kits (Bioclin Biolisa HCV and HIV) to verify if there was gain in sensitivity with the implementation of the nanoparticles. The strategy II was evaluated for 40 nm nanoparticles and the results demonstrated the ability to detect the presence of the antigen at 1000-fold lower than the positive response presented by the kit. In addition, the HIV kit was able to intensify the responses obtained in approximately 50%. The use of functionalized nanoparticles in capture and immunodiagnostic analysis tests proved to be an excellent strategy for increasing the sensitivity of the system