Desenvolvimento de imunossensores eletroquímicos para detecção de biomarcadores da Malária

Abstract

Malaria is one of the most important infectious diseases that affect the population of many countries in the word, including Brazil, where it represents a serious public health problem, especially in the Amazon region. Currently, the development of simple, efficient, and low-cost methods for malaria diagnosis has intensified, and the application of biomaterials based on the use of antibodies and antigens in bioanalytical detection systems represents a promising alternative. In this work, we proposed the development of electrochemical immunosensors for detection of malaria biomarkers. The devices were fabricated on gold electrodes (disk (GE) or screen-printed (SPGE)) and using different methodologies of electrodes functionalization: polymeric matrix of dihexadecyl phosphate (DHP), conductive films (electropolymerization of Poly(pyrrole-pyrrole carboxylated), P(Py-Py3COOH)) or formation of self-assembled monolayers through the use of linkers (cysteamine (Cys)/glutaraldehyde (Glut)). The electrode modification steps were characterized by microscopic (scanning electron microscopy and atomic force microscopy), spectroscopic (Fourier-transform infrared spectroscopy, Raman spectroscopy and energy-dispersive X-ray spectroscopy) and electrochemical (cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS)) techniques. The analytical variables were optimized (antibodies concentration and incubation times in antibodies and antigens) and were performed the analytical detection of the recombinant proteins by means of DPV, EIS or enzyme-linked immunosorbent assay (ELISA). Also, tests of interfering molecules and tests with human serum samples spiked with antigen were carried out. The results showed that Ab-PfHRP2(IgG)/DHP/GE immunosensor present stability, good selectivity, and high sensitivity, detecting concentrations between 10 and 400 ng mL-1 of the target analyte, with a detection limit (LD) of 3.3 ng mL-1 by EIS, between 10 and 500 ng mL-1, with LD = 2.8 ng mL-1 by DPV, and between 15 and 125 ng mL-1, with LD = 5.5 ng mL-1 by ELISA. The BSA/Ab-PfHRP2(IgY)/P(Py-Py3COOH)/SPGE immunosensor registered a good sensitivity by EIS and ELISA in the concentration range of 100 to 1000 ng mL-1, with LD of 27.47 and 53.95 ng mL-1, respectively. The device proved to be selective for the target molecule (PfHRP2), without notable interference from the other compounds tested. The BSA/Ab-PvLDH(IgY)/Glut/Cys/GE immunosensor recorded an acceptable response by DPV from 10 to 50 µg mL-1, with LD = 455 ng mL-1, and proved to be selective for the target molecule (PvLDH). The three immunosensors exhibited a good performance for detection of the biomarkers studied and may constitute a tool for the diagnosis of malaria caused by Plasmodium falciparum and Plasmodium vivax, the two predominant species in Brazil.