Abstract

The detection of biological and chemical pathogens, contaminants and other important analytes plays a crucial role in the prevention of disease spread, infections and pathologies. Immunosensors are biosensors based on the antigen-antibody interaction, which is responsible for eventually generating the actual signal. In principle, high specificity and low limit of detection are thanks to the extreme affinity that antibodies have for their antigen. For this reason, to permit the transfer of the signal it is necessary to conjugate a label molecule or a material to the antibody; the chemical groups of the antibody used for conjugations are mainly amine (-NH2), thiol (-SH), or hydroxyl (-OH) groups. The other hand, the properties of gold nanoparticles (AuNPs), such as light absorption and their excellent electroactivity, are bringing interesting immunosensing alternatives. AuNPs are so small that they exhibit characteristics that are often not observed in the bulk materials. This is due to the quantum size effect that leads to unique optical, electronic, and catalytic properties. AuNPs are also fully compatible with biomolecules, when decorated with thin organic coatings. The use of thiol groups for their functionalization is a good way to control the direction of the bond between the labeled antibody and the surface electrodic. The electroactivity of AuNPs allows the use of both electrical and electrochemical techniques for their detection, which allowed to detect low concentrations of antigens. To summarize, modified AuNPs electrodes have very large surface areas, are simple to fabricate and functionalized, retain metallic conductivity, and have facile biomolecule attachment. Considering these assumptions, the central goal of this project is the obtention of an electrode modified by labeled antibody analogues to study its response as red-ox mediator towards Corticotropin-Releasing Hormone (CRH) and cortisol, respectively. These electrodes should be interference-free with respect to sample, cheap, useful for rapid detection in aqueous solution and thus usable in various applications of interest. Consequently, using electrochemical techniques and different reaction media, flavins hetero-analogues (red-dox probes) with different substituent moieties will be studied. Thus, it will be possible to determine the behavior of a series of biologically relevant compounds, and at the same time, to determine the re-dox properties of each, allowing structure-reactivity relationships to be established. The study will be conducted in the presence of CRH y cortisol, to determine the interactions that occur between labeled antiboby with red-ox probe and their corresponding antigens. Moreover, bearing always in mind the central objective, modified electrodes will be prepared with different labeled antibodies upon various electrode surfaces. Thus, new systems, with novel electronic properties, that will enable performing a specific study on the interaction of labeled antibodies with antigens, will be investigated. To check the labeled antibody analogues morphology on the electrode surface, studies will be accomplished employing scanning electron microscopy (SEM), atomic force microscopy (AFM) or scanning tunneling microscopy (STM) to compare the likely arrangement obtained. In addition, the electrochemical behavior of the redox system in room temperature will be investigated quantitatively using an in situ electrochemical quartz crystal microbalance (EQCM) technique. Scanning electrochemical microscopy (SECM) will be used either. This will allow chemical reactivity images of the modified surfaces and quantification of the reaction rate to be attained. Furthermore, a study by electrochemical impedance spectroscopy of the modified electrodes will be done, since this is a powerful tool that makes possible the characterization of electrode surfaces: the immobilization of molecules on the surface changes the capacitance and the resistance to interfacial charge transfer of the electrode. This technique is simple, sensitive, does not require that the analyte is labeled with a redox substituent, and works well in high ionic strength media, therefore, is appropriate for biomolecular detection. To summarize, this project aims to monitor and analyze the interaction of labeled antibody with red-ox probe modified electrodes with CRH y cortisol, respectively.