RESUMEN
Exosomes are nanovesicles present in all the biological fluids, making them attractive as non-invasive biomarkers for diseases like cancer, among many others. However, exosomes are complex to separate and detect, requiring comprehensive molecular characterization for their routine use in diagnostics. This study explores the use of peptides as cost-effective and stable alternatives to antibodies for exosome binding. To achieve that, phage display technology was employed to select peptides with high specificity for target molecules in exosomes. Specifically, a selected peptide was evaluated for its ability to selectively bind breast cancer-derived exosomes. Proteomic analysis identified 38 protein candidates targeted by the peptide on exosome membranes. The binding of the peptide to breast cancer-derived exosomes was successfully demonstrated by flow cytometry and magneto-actuated immunoassays. Furthermore, an electrochemical biosensor was also tested for breast cancer-derived exosome detection and quantification. The peptide demonstrated effective binding to exosomes from aggressive cancer cell lines, offering promising results in terms of specificity and recovery. This research shows potential for developing rapid, accessible diagnostic tools for breast cancer, especially in low-resource healthcare settings.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama , Exosomas , Humanos , Femenino , Neoplasias de la Mama/diagnóstico , Exosomas/química , Biomarcadores de Tumor/análisis , Proteómica , Péptidos/metabolismoRESUMEN
In this work, a new electrochemical biosensor was developed using peptides selected by Phage Display as biorecognition phase to Breast Cancer (BC) characterization. Phage clones were selected against MCF-7 (ER-positive BC) proteins, in order to characterize patients with aggressive luminal BC. Biotin-C3 and biotin-H2 peptides were chemically synthesized and validated by flow cytometry, immunofluorescence assays, and ELISA assays, being more reactive to the MCF-7 lineage. Furthermore, a new matrix for the coupling of biomolecules on the surface of graphite electrodes was generated, through electrochemical modification with a new material derived from 3-(3-aminophenyl)propionic acid (3-3-APPA). Electrochemical and morphological characterizations were carried out, and the mechanism of electropolymerization of poly(3-3-APPA) was proposed, in which the carboxylate groups are kept in the structure of the formed polymer. Then, a biosensor was developed by immobilizing the biotin-C3 and biotin-H2 peptides in the SPE/poly(3-3-APPA)/avidin system for the detection of BC tumor markers in serological samples. Finally, peptides were validated using samples from patients with BC and Benign Breast Disease. Biotin-C3 peptide characterized luminal BC according to p53 status and to HER2 expression, being the biosensor a better strategy when compared to ELISA test. This new biosensor will open a new perspective for a rapid and electrochemical platform for the characterization of BC and its molecular subtypes.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama , Neoplasias de la Mama/diagnóstico , Técnicas Electroquímicas , Electrodos , Femenino , Humanos , Péptidos/química , PropionatosRESUMEN
Magnetic molecularly imprinted polymers (MMIPs) are constructed based on the blending of inorganic nanoparticles with molecularly imprinted polymers (MIPs). MMIPs are synthesized in a core-shell format in which inorganic nanoparticles are applied as the core part of the material while selective polymeric layers are used as the shell covering the surface of the core area. In essence, MMIPs thus reflect a combination of the best characteristics of both inorganic nanoparticles and MIPs, where the specificity of cavities imprinted on the MIP is merged with superparamagnetic properties of the nano-magnetite. The synergic combination of the two distinct materials facilitates the process of extracting analytes from complex samples. Owing to their suitable characteristics, MMIPs have become widely used in different areas of analysis.
Asunto(s)
Impresión Molecular , Adsorción , Fenómenos Magnéticos , Magnetismo , Nanopartículas de Magnetita , Polímeros Impresos Molecularmente , PolímerosRESUMEN
The present study reports the development of graphite pencil electrode modified with palladium nanoparticles (PdNPs) and its application as an electrochemical sensor for the simultaneous detection of direct yellow 50, tryptophan, carbendazim and caffeine in river water and synthetic urine samples. The combination involving the conductive surface of the graphite pencil electrode (GPE) and the enlargement of the surface area caused by the use of palladium nanoparticles (PdNPs) led to the improvement of the analytical performance of the proposed device. The surface of the GPE-PdNPs was characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The charge transfer kinetics of the electrode was evaluated based on the electrochemical analysis of the potassium ferricyanide redox probe. Using square wave voltammetry (SWV), well-defined and fully resolved anodic peaks were detected for the analytes, with peak-to-peak potential separation not less than 200 mV. Under optimised conditions, the following linear range concentrations were obtained: 0.99-9.9 µmol L-1 for direct yellow 50; 1.2-12 µmol L-1 for tryptophan; 0.20-1.6 µmol L-1 for carbendazim; and 25-190 µmol L-1 for caffeine. The sensor showed good sensitivity, repeatability, and stability. The device was successfully applied for the determination of analytes in urine and river water samples, where recovery rates close to 100% were obtained. Due to its low cost and reusability by simple polishing, the sensor has strong potential to be used as an electrochemical sensor for the determination of different analytes.