Desenvolvimento de genossensor eletroquímico para diagnóstico de meningite pneumocócica

Abstract

Pneumococcal meningitis are caused by the bacterium Streptococcus pneumoniae and presents a high rate of morbidity and mortality. Early diagnosis of the causative agent of meningitis is justified by its positive impact on clinical management, avoiding complications such as hearing loss, intellectual disability, motor abnormality and visual disturbances, reducing morbidity and mortality rates. The identification of microorganisms using biosensors has been widely studied and its efficiency constantly proven. Biosensors allow simple, fast, accurate and accessible tests. In this work, graphite electrodes modified with poly(4- aminophenol) was used for the immobilization of a probe sequence (Strep1) complementary for a specific target (Strep2), isolated from conserved regions of S. pneumoniae, aiming the development of a genosensor specific for pneumococcal meningitis. The electrodeposition of poly(4-aminophenol) on graphite electrode was confirmed using voltammetric techniques and scanning electron microscopy images. After surface modification with poly(4-aminophenol), the surface was functionalized with the specific probe for S. pneumoniae (Strep1), followed by its hybridization with the complementary target (Strep2). Results indicate that the presence of the polymer caused an increase in probe immobilization efficiency in almost 12%, monitoring the guanine peak when compared to the bare graphite electrode. The hybridization time between Strep1(probe): Strep2(target) was optimized and the detection was performed directly and indirectly. In the second case, ethidium bromide was used as an indicator. The platform was able to recognize the genomic DNA of the bacterium in human serum. The performance of the genosensor was analyzed against different concentrations of the solution containing genomic material of the bacteria, through differential pulse voltammetry and electrochemical impedance spectroscopy. The detection limits were 54 ng mL-1 and 28 ng mL-1 in each technique respectively. The platform presented characteristics such as specificity and selectivity. The system remain stable during 60 days stocked. Topographic analysis of the genosensor surface were evaluated by atomic force microscopy. The results presented in this work indicate that the poly(4-aminophenol)/Strep1 platform is promising for the molecular diagnosis of pneumococcal meningitis in enriched human serum.