Desarrollo de nuevos quimiosensores para la detección de metales en matrices multifuncionalesestudios in vitro e in vivo

Resumo

Nowadays, the search for new fluorescent chemosensors with multifunctional applications and high sensorial ability for a specific analyte is a driving force in the development in many areas, such as, environment, analytical sicences, nano-scale technologies, bio-medicine and imaging. In this dissertation, we present the synthesis of several new bio-inspired compounds, containing the cysteine, alanine and tyrosine amino acids, provided with fluorescent dyes, as coumarin or fluorescein. The compounds obtained were used for mapping of metal ions, such as, alkaline, alkaline-earth, transition and post-transition. New hybrid materials, as gold and silver nanoparticles with the synthesized compounds were also performed. Emissive silver nanoparticles permeation was observed in vivo in gold fish and in vitro in human hepatoma cells (HepG2), without lost of fluorescence properties. This PhD dissertation is divided in eight chapters; and a brief description of each chapter is presented. Chapter 1 shows a brief introduction to general fluorescent and colorimetric chemosensors and definitions, mechanisms of detection, chemosensors containing bio-inspired units, from single amino acids to peptide chains. The importance on the detection and quantification of metal ions and some aspects of biological applications with the emissive chromophores is described, as well. Finally, an introduction concerning the synthesis of gold and silver nanoparticles, their advantages, toxicology and imaging is shown. Chapter 2 summarizes of new emissive molecular probes based on amino acid moieties L1 and L2 and their fully characterization The sensing ability towards alkaline earth, transition, post-transition metal ions and acid-basic behavior were explored in absolute ethanol by absorption and fluorescence spectroscopy. System L1 shows a strong complexation constant with the soft metal ions. Very small gold nanoparticles, with sizes of 4.27±0.64 nm for L1 and of 2.69±0.96 nm for L2 (AuNPs) were obtained. The new stable Cou@AuNPs behaved as supramolecular chemosensors which have been selective for the heavy element Hg2+, with a concomitant change of color from pink to dark red/brown, as well as, the increase of size up to 100-fold. In Chapter 3 is presented the synthesis and characterization of two new Tyrosine Schiff-base ligands 1 and 2 bearing an indole or a thianaphthene moiety The sensing ability towards Ca2+, Zn2+, Cd2+, Cu2+, Ni2+, Hg2+ and Al3+ metal ions was explored in absolute ethanol. Both compounds showed colorimetric properties in the presence of Cu2+ ions, changing their colour from light yellow to purple (1) and dark pink (2). The minimal amount of Cu2+ that compounds 1 and 2 could quantify was ca. of 8 ppm. Nanomaterials, as gold and silver nanoparticles were successfully synthesized by self-reduction of compound 1 in an aqueous alkaline environment. TEM images of AgNPs and AuNPs showed sizes of 12 ± 3.5 nm and 7.50 ± 6 nm, respectively. Having in mind biological applications new fluorescent bio-inspired chemosensor 1 and 2 containing a fluorescein are presented in Chapter 4. The acid-base behavior was studied in water/ethanol (60/40) and revealed at pH=8 a strong emission fluorescence, followed by a colour change from colorless to yellow/green. Compounds 1 and 2 reveal as very sensitive to Hg2+ with colorimetric changes from yellow to pink in the presence of HEPES buffer. Very small silver nanoparticles coated with 2 of ca. 3.68 ± 0.93 nm were successfully synthesized and characterized. Toxicological studies with both compounds were also carried out. Chapter 5 describes the synthesis of emissive silver nanoparticles containing as stabilizers the compounds 1, 2 and 3, as well as, their fully characterization by UV-Vis and fluorescence emission spectroscopy, by MALDI (matrix-assisted laser desorption ionization) spectrometry, by dynamic light-scattering (DLS) and transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM). An average size of 10 ± 1.6 nm, 4.0 ± 0.9 nm and 2.8 ± 0.6 nm, were obtained from TEM images for the AgNPs with 1, 2 and 3, respectively. Nanoparticles internalization in vivo in gold fish and in vitro in human hepatoma cells (HepG2), and Oxidative stress in enzymes were also performed. Concerning the in vivo studies, the smallest nanoparticles AgNPs@3 presented larger changes in livers, whereas in cytotoxicological studies in HepG2 cells the AgNPs@1 revealed toxicity. The general conclusions of this dissertation can be found in Chapter 6.