Desenvolvimento de imunoensaio eletroquímico para detecção de biomarcadores proteicos utilizando partículas magnéticas e arranjo de eletrodos descartáveis para o diagnóstico de câncer de próstata

Abstract

This work describes the development of electrochemical immunoassays using electrode disposable devices made from low cost materials as an alternative for detection of the biomarkers p16, GSTP1, PSA and CYFRA-21-1 for prostate cancer diagnosis. In the immunoassays developed, magnetic micro/nanoparticles were used in order to provide the capture and separation of the biomarker and the amplification of the analytical signal allowing to reach high selectivity and low limits of detection. For this propose, disposable microfluidic cell was developed and constructed containing 8 carbon-based working electrodes, an Ag|AgCl electrode as a pseudo reference electrode and a carbon electrode as an auxiliary electrode. Three different immunoassays were developed. The first work consisted in the development of an electrochemical immunoassay for the detection of the p16 biomarker. Immunoassays were performed after optimization of the parameters, such as incubation time and flow rate used in the microfluidic system. The calibration curve was prepared in calf serum with p16 concentration in the range of 1.38 to 27.7 fg mL-1. The device presented detection limit (LoD) of 0.04 fgmL-1 and sensitivity of 11.6 nA (fg/mL)-1. The second method consisted of the simultaneous detection of PSA and GSTP1 biomarkers. Peak currents obtained showed a linear response between 0.7 - 27.8 fg mL-1 for GSTP1 and 0.28-277 fg mL-1 for PSA. The sensitivities reached for GSTP1 and PSA were 22.4 nA (fg/mL)-1 for both biomarkers and the LoDs were of 0.15 fg mL-1 for GSTP1 and 0.16 fg mL-1 for the PSA. Finally, the third immunoassay consisted of the application of synthetic nanoparticles (MNPs) of Co0.25Zn0.75Fe2O4 (CoZnFeONPs) and Fe3O4 (FeONPs) used to mimic the enzyme peroxidase. The use of modified carbon work with graphene oxide (GO) and MNPs allowed the electrochemical detection of CYFRA-21-1 with LoDs of 0.30 fg mL-1 and 0.19 fg mL-1 for FeONPs and CoZnFeONPs, respectively. Finally, it was possible to test the immunosensor in serum of patients provided by the Hospital de Amor from Barretos city obtaining concordant results with ELISA method, which showed that the device is a promising choice for diagnosis of prostate cancer in relation to traditional methods as ECL and ELISA.