AuNPs estabilizadas em silsesquioxano para a detecção eletroquímica de miRNAs associados à doença de Alzheimer

Abstract

Alzheimer's Disease (AD) is the leading cause of dementia worldwide and, thus far, has no cure. Thus, early and rapid diagnosis is crucial to minimize both neurological damage and loss of quality of life. However, early diagnosis of AD still faces many challenges, highlighting the potential contributions of fast detection through non-invasive biomarkers. In this context, the objective of this study was to develop an electrochemical biosensor for the detection of miRNAs 29a and 34a, both of which are potential non-invasive biomarkers associated with diseases such as AD. On the surface of a glassy carbon electrode (GCE) a novel nanohybrid AuNPs- Si4DMAP+Cl− was incorporated, which was synthesized using gold nanoparticles (AuNPs) stabilized by the 3-n-propyl(4-dimethylaminopyridinium) silsesquioxane chloride (Si4DMAP+Cl−). The material was characterized using FTIR, UV-Vis, SEM, TEM, and DLS techniques, demonstrating good stability (ζ = +43.0 mV), with an average hydrodynamic radius of 66.4 nm and an average particle size of 6 nm determined by particle counting. The nanohybrid was applied to the surface of two GCEs via drop-coating and electrochemical characterization was carried out in 0.1 mol L−1 PBS buffer containing [Fe(CN)6]3−/4− using Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV) and Square Wave Voltammetry (SWV). Anti-miRNA-29a and anti-miRNA-34a probes were immobilized separately on the modified GCE surfaces, followed by the immobilization of BSA molecules to finalize the biosensor architecture. These were designated as GCE/AuNPs- Si4DMAP+Cl−/probe/BSA (where probe = anti-miRNA-34a or anti-miRNA-29a). The immobilization of the anti-miRNAs was confirmed by a decrease in peak current (ip) values and a significant increase in charge transfer resistance (Rct) values, as observed through EIS and SWV analyses. These changes were attributed to the repulsion of the redox probe [Fe(CN)6]4−/3− in the presence of the immobilized biomolecules. The nanohybrid significantly enhanced the electrochemical response and charge transfer processes, proving to be effective in immobilizing both anti-miRNAs. It also demonstrated suspension stability over a period of 650 days. The developed biosensors were used for the detection of target miRNA-29a and miRNA-34a through hybridization with their complementary anti-miRNA probes, showing limits of detection (LOD) of 1.79 pmol μL-1 for miRNA-29a and 2,21 pmol μL-1 for miRNA-34a. Based on the results obtained, the proposed platform shows strong potential for the electrochemical detection of these and other miRNAs in blood samples for the diagnosis of AD and other neurodegenerative diseases.