Desenvolvimento de plataforma funcionalizada com poli(4-HBA) para aplicação em sensores biológicos

Abstract

In this work, the 4-hidroxtbenzoic acid electropolymerization was investigated aiming at its application as functionalized platform in biomolecule immobilization in order to develop biological sensors. The material was adsorbed over graphite electrode and electrochemically characterized by cyclic voltammetry and electrochemical impedance spectroscopy. This analysis shows formation of an electroactive material on electrode. Cyclic voltammetry was used to investigate the polymer barrier properties using the potassium ferro/ferricyanate complexes and hexamine ruthenium(II) chloride as redox probes. Due to the acid/base properties of the monomer funtional groups, the electropolymerization was carried out as function of pH (acid, neutral and basic). It was observed that electrochemically active polymeric film deposition takes place in both acid and neutral media. The films produced in these media presented two redox signal and no electrochemical activity was found when in basic media. Poly(4-HBA) film growing process studied by cyclic voltammetry, electrochemical quartz crystal microbalance and atomic force microscopy showed higher electrochemical activity, higher stability, mass deposit and roughness in acid media. Electrical properties studies performed through electrochemical impedance spectroscopy showed a charge transfer resistance (Rct) value increase with pH value increase. In the poly(4-HBA) extraction, acetonitrile was used as solvent and the polymer was chemically characterized through FTIR, DSC, EF and TG. The FTIR spectra showed that the functional groups are preserved in the polymer formation. The thermal analysis showed that around 30% of mass remains after temperature is raised to 800ºC. The fluorescence analysis showed the poly(4-HBA) photochemical activity. The electropolymerization mechanism was proposed based on the nucleophilic attack from the monomer to the cationic intermediary leading to the poly(4-HBA) formed preferentially by ring-oxygen-ring bond. The biomolecule immobilization evaluation over the functionalized surface was investigated. Oligonucleotides were immobilized for genosensor construction and the hybridization was detected through direct measurements from guanine and adenine nitrogenated bases and also, using ethidium bromide and methylene blue as intercalators. Better affinity was found with methylene blue and ssDNA and with ethidium bromide and dsDNA. The hybridization signal was detected indirectly by the intercalator electroactivity. The rK39 antigen (visceral leishmaniosis) immobilization was recognized successfully by the specific antibody and the signal transduction detected through electrochemical impedance spectroscopy. Increase in the Rct values were found on the surface containing the Antibody-Antigen complex. Such analysis showed the applicability of the functionalized platform for biomolecule immobilization, showing to be promissory for the biosensor development.