DIFERENTES ESTRATÉGIAS DE PREPARAÇÃO DE ELETRODOS A BASE DE SAM PARA O DESENVOLVIMENTO DE BIOSSENSORES ELETROQUÍMICOS

Abstract

This thesis describes the use of self-assembled monolayers (SAM) for the development of biosensors based on horseradish peroxidase enzyme - HRP. In the first chapter, the enzyme was immobilized on self-assembled monolayers of thiolactic acid (Au-TLA) and mixed SAM composed of 11-mercaptoundecanoic acid (MUA) together with the TLA (SAMmix). The steps of construction and characterization of biosensors have been carried out by electrochemical techniques and morphological ones. The enzyme immobilization method on SAM which provided higher stability was by covalent bond. The Au/TLA/HRP biosensor was used for the determination of H2O2 by chronoamperometry, yielding a detection limit (DL) of 5.46 μmol L-1 and quantification (QL) of 18 μmol L-1. Despite the good results for the H2O2 detection presented by this biosensor, the device was stable for only 6 days. Therefore, in order to increase the stability of the biosensor, SAM containing both the mercaptoundecanoic acid molecule (MUA) and TLA was obtained, to the formation of SAMmix (Au/MUA:TLA). In this system, the electron transfer rate can be considerably affected because while the TLA enables an increase of SAMmix conductivity due to formation of “islands”, the MUA provides greater stability for HRP immobilization, although it partially blocks the surface. The biosensor Au-SAMmix-HRP prepared in the ratio 0.5: 1.0 MUA/TLA showed higher sensitivity compared to other modifications ratio, with an apparent Michaelis-Menten constant 𝑲𝑴𝒂𝒑𝒑= 0.40 mmol L-1. This biosensor has been applied to the determination of hydroquinone (HQ) in the presence of a fixed amount of [H2O2] = 0.3 mmol L-1. By differential pulse voltammetry technique (DPV), the biosensor exhibited excellent electrocatalytic activity for HQ in the range 3-30 μmol L-1, with good sensitivity, DL = 1.26 μmol L-1 and QL = 4.23 μmol L -1. The SAMmix increased the stability of the biosensor for at least 15 days, which is more stable when compared to the biosensor Au/TLA/HRP. In the second chapter of this thesis, another strategy was used for immobilization of the HRP enzyme based on the formation of TLA monolayer on the top of gold nanoparticles (AuNPs) stabilized in the inorganic polymer, 3-n-propylpyridinium silsesquioxane chloride (SiPy+Cl-). The presence of AuNPs-SiPy+Cl- was confirmed by UV-Vis spectroscopy from the plasmon band at 521 nm. The AuNps showed good distribution with approximate size between 4-18 nm, evidenced by transmission electron microscopy (TEM) and by dynamic light scattering (DLS), with good stability (ζ = + 38.5 mV). The AuNPs were deposited on the glassy carbon electrode (GCE) and modified with the TLA for immobilization of HRP enzyme. The formation of this biosensor was confirmed by electrochemical impedance spectroscopy (EIS) and morphologically by field-effect scanning electron microscopy (SEM-FEG). The GCE/AuNPs/TLA/HRP biosensor was used for the detection of H2O2 with 𝑲𝑴𝒂𝒑𝒑 = 0.46 mmol L-1, which was similar to the Au-SAMmix-HRP biosensor. This biosensor has been applied to the determination of catechol (CT) in presence of [H2O2] = 0.03 mmol L-1. Using the DPV technique, the biosensor showed an excellent electrocatalytic activity for CT in the range 6-46 μmol L-1, with good sensitivity, DL = 0.852 μmol L-1 and QL = 2.84 μmol L -1. The stability of this device was approximately 25 days, being superior to other biosensors developed (TLA-HRP and SAMmix-HRP), which can be attributed to the three-dimensional immobilization of HRP molecules in this device.