Spectroscopic study of antibodies employing SERS-active surfaces for the development of immunosensors

Author(s)

Raquel Mariano de Almeida

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2016-04-15

Examining board members

Renato Sanches Freire; Giancarlo Esposito de Souza Brito; Paola Corio; Jean Claudio Santos Costa; Jorge Cesar Masini

• Advisor: Renato Sanches Freire; Cassius Vinicius Stevani

Abstract

This study used antibodies that can be key elements in the possible evelopment of immunosensors, due to its selectivity and the consequent determination of species of interest in clinical analysis, environmental, public health, among others. For this, Raman and SERS spectra studies were made of whole and/or fragmented antibodies (human IgG, anti-human IgG (H + L) and anti-human IgG (Fab2)). With this, it were known the main vibrational modes of these biomolecules, useful for the proposal to assemble immunosensors. Both techniques provide information on the secondary structure of proteins. When these biomolecules are immobilized on SERS-active surfaces, it is possible to determine their orientation. Considering that, in the SERS technique, it is possible to detect a molecule through hot spots, it is important that the immunoglobulins are free of impurities, such as albumin and transferrin in the case of antibodies from human serum. Thus, purification experiments of antibodies were made via affinity columns containing protein A, G or a mixture of A and G immobilized and also separation based on isoelectric point. It was tested the binding capacity (chemisorption) of human IgG or its F(ab)\' on gold surfaces by means of QCM-D technique. For the study of SERS-active surfaces of gold nanoparticles were synthesized (Au-NP), with the acquisition of extinction spectra in the UV-Vis and obtaining SEM images to characterize them. Also, changes were made in the preparation of nanocubes gold (Au-NC) to obtain gold nanorods (Au-NR), having in view that ends with structures favor the enhancement of Raman signal on SERS-active surfaces. Finally, considering the existence of various S-S links, particularly in the region called hinge, in the SERS spectrum of diluted hIgG and F(ab)\', it was found the display of a characteristic band C-S 679 cm-1. Therefore, a proposal was made about the possible direction of these non-fragmented antibodies to assume an orientation on the SERS-active surfaces with similar characteristics to the F(ab)\' (AU)

• FAPESP's process: 10/16238-8 - Development of immunosensor for analysis of antigens employing surface of SERS-active
• Grantee: Raquel Mariano de Almeida
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)