Desenvolvimento de sensores eletroquímicos descartáveis para detecção do biomarcador miRNA-208b visando o diagnóstico de doenças coronarianas

Abstract

DEVELOPMENT OF DISPOSABLE ELECTROCHEMICAL SENSORS FOR DETECTION OF THE BIOMARKER miRNA-208B AIMING AT THE DIAGNOSIS OF CORONARY DISEASES. Coronary diseases (CDs) are the most common type of cardiovascular disease, affecting the heart and blood vessels, and are one of the leading causes of death not only in Brazil but worldwide. The diagnosis of CD is made through electrocardiogram or monitoring of serological markers. Regardless of being the most commonly used methods for evaluating these diseases, they require specialized personnel and use nonspecific markers that take hours to be detected in the bloodstream. Due to these limitations, we propose an electrochemical method that uses magnetic particles decorated with biotin and a DNA probe (hairpin) to detect miRNA-208b, which is specific for myocardial infarction and released at the onset of cardiac necrosis. The viability of the method consists of the effective opening and annealing of the hairpin with miRNA-208b. After that, the bioconjugate is incubated with streptavidin-HRP. Thus, the temperature and denaturation time, as well as the concentration of hairpin, detection probe, and biotin, were evaluated. Moreover, the method uses a disposable device with eight independent electrochemical cells based on carbon paste, which allows for the simultaneous detection of eight different samples or targets. With the chosen conditions, the method produced an analytical range of 0,01 to 5,00x103 fmol L-1 with a detection limit of 0,22 fmol L-1. In this way, the method proved to be sensitive for the detection of miRNA-208b and specific when evaluated with other non-specific target sequences for the hairpin. Furthermore, the system's stability was monitored for 24 days, with a significant drop in the current signal only on the last day, around 69.8%. The method was applied to real blood serum samples from people with diseases that trigger heart problems without any pre-treatment steps of the samples and still corroborated with the RT-PCR results. Thus, in summary, the method proved to be sensitive, specific, low-cost, and with enormous potential to become an alternative for diagnosing coronary artery diseases.