Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório

Abstract

Respiratory syncytial virus (RSV) is an important respiratory pathogen that mainly affects children under the age of five. While RSV infections in healthy, immunocompetent adults usually result in mild uncomplicated cases, infants and the elderly often experience bronchiolitis, bronchitis, and pneumonia. Current detection methods for RSV include RTqPCR, immunochromatography, and indirect immunofluorescence assays, but they have disadvantages related to time, cost, and sensitivity. These limitations can be overcome by developing an electrochemical immunosensor, a low-cost, rapid, specific, and user-friendly device. Covalent binding is an effective methodology for antibody (Ab) immobilization, and the electrochemical modification of a graphite pencil lead working electrode (PGE) with conductive or non-conductive polymers that possess functional groups is of interest for this procedure. 3-aminophenylacetic acid (3APA) exhibits the desired characteristics and was chosen as the monomer to provide available carboxyl groups after polymerization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were employed for electropolymerization and characterization of the formed material. The CV results demonstrated the formation of poly(3APA), with 30 cycles established as the optimal condition due to the best response observed in the system. Electrochemical and pH studies in CV allowed for the determination of the number of electrons and the proton-to-electron ratio, providing insight into the proposed mechanism of 3APA electropolymerization. The immunosensor was developed on this matrix by activating the carboxylic groups of poly(3APA) in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / Nhydroxysuccinimide (NHS), followed by nucleophilic substitution reaction with the amino groups present in the antibody (Ab). After Ab immobilization, the PGE/poly(3APA) surface was blocked by glycine (Gly) to prevent non-specific interactions, followed by antigen (Ag) immobilization. Detection was performed using EIS, and the data were evaluated using equivalent circuits. The values of charge transfer resistance (Rtc) increased as the PGE was modified in the following order: PGE < PGE/poly(3APA) < PGE/poly(3APA)/Ab < PGE/poly(3APA)/Ab/Ag. The system was optimized to achieve the best detection conditions. The response variation (ΔRtc) between the complete system containing Ab/Ag and the system containing only Ab (n = 3) was utilized. For the preparation of the immunosensor, 100 ng of Ab was chosen, and the immobilization times for Ab and Gly were, respectively, 3 h and 50 min. The response time of the system, with Ag immobilization, was 30 min. Using the calibration curve of the optimized system, the calculated limit of detection (LOD) was 27.65 PFU/mL, and the limit of quantification (LOQ) was 92.15 PFU/mL, demonstrating that the proposed immunosensor is a viable alternative for the detection of RSV.