Desenvolvimento de biossensores eletroquímicos visando a detecção de biomarcadores relacionados ao carcinoma de córtex adrenal pediátrico e à doença de Alzheimer

Abstract

The present thesis describes the preparation, characterization and potential applications of electrochemical biosensors based on carbonaceous and inorganic materials. Immunosensors based on L-arginine-functionalized gold nanoparticles (AuNPs-ARG) were applied in the label-free detection of dehydroepiandrosterone sulfate (DHEAS), a biomarker of adrenocortical carcinoma (ACC). ACC is a rare and aggressive malignancy which is characterized by the overproduction of DHEAS, and whose incidence among children from Southern Brazil is above the global average. The sensing platform was prepared by modifying oxidized glassy carbon electrodes with AuNPs-ARG, and the subsequent attachment of IgM anti-DHEA monoclonal antibodies. AuNPs-ARG were obtained by the reduction of [AuCl4]- precursor complex with D-glucose in the presence of ARG, and characterized by UV-VIS and FTIR spectroscopies, TEM, DLS, zeta potential determination and XRD. The immunosensors were characterized by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and their construction was optimized by factorial design and response surface methodology. DHEAS detection was performed by using EIS, considering the percentage variation in charge transfer resistance parameter (ΔRct) relative to the [Fe(CN)6]3-/4- redox marker, after immunoassays in the presence of the hormone. A linear relationship between ΔRct and DHEAS concentration was verified in the range of 10.0 to 110.0 μg dL-1, with a detection limit of 7.4 μg dL-1. Besides the good sensitivity, the developed immunosensor showed accuracy, specificity and stability for one month. The proposed device was successfully applied in the analysis of real patient plasma samples, with results that were comparable to the reference chemiluminescence assay (p > 0.05). Besides the presented immunosensors, biosensors based on gold electrodes (AuE) modified with DNA sequences were developed aiming the detection of interaction events with three apolipoprotein E isoforms (ApoE2, ApoE3, and ApoE4), which are related to the occurrence of late-onset Alzheimer’s disease (AlzD) in the elderly. The employed oligonucleotides consisted of two thiolated promoter sequences, named CLEAR (associated to the expression of proteins involved in cellular autophagy processes) and SIRT1 (related to the expression of the neuroprotective protein sirtuin-1 (Sirt1)), which were separately immobilized on the AuE as mixed self-assembled monolayers with 6-mercaptohexanol. The interaction of such promoters with ApoE4 has been considered one of the events that can contribute to the occurrence of AlzD, as a result of failures in cellular autophagic mechanisms and in the expression of Sirt1 protein, which favors the brain accumulation of toxic protein aggregates that progressively lead to dementia, such as senile plaques of amyloid-β peptide and neurofibrillary tangles of hyperphosphorylated forms of tau protein. The biosensors were characterized by using differential pulse voltammetry (DPV), CV, EIS and cronocoulometry, in the presence of [Ru(NH3)6]3+ redox marker. The interactions of ApoE isoforms with the immobilized oligonucleotides were evaluated by DPV and EIS, considering the percentage variations in the cathodic peak current and in the Rct parameter after incubating the biosensors in the presence of each ApoE isoform. It was verified that SIRT1 sequence showed a greater affinity to ApoE4 compared to ApoE2 and ApoE3, whereas CLEAR oligonucleotide interacted specifically to ApoE4. These behaviors suggested the existence of possible repressive effects of this isoform on the expression of proteins involved in cellular neuroprotective mechanisms, which can favor the onset of AlzD. Hence, the results obtained in the present thesis evidenced promising applications of the developed devices as efficient tools for clinical diagnosis of ACC and for the evaluation of protein-DNA interactions involved AlzD.