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| Author(s): |
Rafael da Fonseca Alves
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Araraquara. 2023-06-12. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara |
| Defense date: | 2023-06-02 |
| Advisor: | Maria Del Pilar Taboada Sotomayor |
| Abstract | |
Breast cancer (BC) is the most common malignant tumor in the world population. Therefore, more precise methods that can detect the disease early are needed, and innovative techniques such as Phage Display (PD) have been extensively explored. In this context, the construction of biological sensors and enzyme-linked immunosorbent assays (ELISA) based on PD-selected binding peptides emerges as a promising alternative in the development of new bioanalytical approaches, notable for their sensitivity and selectivity. This thesis, therefore, aimed to develop new bioanalytical methodologies focused on BC in line with the current demands of the oncology field. In Chapter II, a novel matrix for coupling biomolecules onto the surface of pencil graphite electrodes (PGEs) was generated through electrochemical modification using a new material derived from 3-(3-aminophenyl)propionic acid (3-3-AAFP). Electrochemical and morphological characterizations were performed, and the electro-polymerization mechanism of poly(3-3-AAFP) was proposed, in which carboxylate groups are retained in the structure of the formed polymer. A new ELISA test and an electrochemical biosensor were tested with serum samples from patients diagnosed with BC or benign breast disease (BBD) to detect circulating antigens, both using the peptide Biotein-H2 or Biotin-C3. ROC curves were plotted, and performance analysis calculations were carried out for the ELISA test and the biosensor, respectively. The peptide Biotin-H2 exhibited inferior performance (AUC of 0.677 and 0.806) compared to the peptide Biotin-C3, which maintained diagnostic potential (AUC of 0.771 and 0.951) and characterized luminal BC according to p53 status (AUC of 0.886 and 0.748) and HER2 expression (AUC of 0.748 and 0.833). The biosensor proved to be a better strategy when compared to the ELISA test. Additionally, in Chapter III, a proteomic study identified 38 potential targets for the Biotin-C3 peptide, predominantly present in the plasma membrane and cellular organelle membranes, especially mitochondria. Flow cytometry assays were conducted to detect and characterize exosomes derived from BC cell lines. For this purpose, exosomes immobilized directly on magnetic particles and indirectly on magnetic particles modified with anti-CD9, anti-CD63, and anti-CD81 antibodies were used to validate the binding capacity of the Biotin-C3 peptide to exosomes. They were also detected by magnetic ELISA and magnetic electrochemical biosensor, with lower limits of detection (LOD) for the MCF-7 cell line (LOD of 330 exosomes μL−1 for magnetic ELISA and LOD of 175 exosomes μL−1 for the biosensor) compared to other cell lines. Successful detection of exosomes derived from the Luminal cell line (MCF-7) in human serum was achieved, and the combination of anti-CD63-modified magnetic particles followed by detection based on the Biotin-C3 peptide provided promising results in terms of specificity and recovery for the ELISA test (108.8%) and the biosensor (111.9%). These new bioanalytical approaches will enable the development of rapid and innovative platforms for the characterization of BC and its molecular subtypes. (AU) | |
| FAPESP's process: | 19/10452-2 - Development of electrochemical biosensor and lateral flow device, low-cost for detection of neoplastic biomarkers |
| Grantee: | Rafael da Fonseca Alves |
| Support Opportunities: | Scholarships in Brazil - Doctorate |