Publications

Abstract Mitochondriotropic antioxidants (MC3, MC6.2, MC4 and MC7.2) based on dietary antioxidants and analogs (caffeic, hydrocaffeic, trihydroxyphenylpropanoic and trihydroxycinnamic acids) were developed. In this study, we evaluate and compare the cytotoxicity profile of novel mitochondria-targeted molecules (generally known as MitoCINs) on human HepG2 and differentiated SH-SY5Y cells with the quinone-based mitochondria-targeted antioxidants MitoQ and SkQ(1) and with two non-targeted antioxidants, resveratrol and coenzyme Q(10) (CoQ(10)). We further evaluate their effects on mitochondrial membrane potential, cellular oxygen consumption and extracellular acidification rates. Overall, MitoCINs derivatives reduced cell viability at concentrations about six times higher than those observed with MitoQ and SkQ1. A toxicity ranking for both cell lines was produced: MC4 < MC7.2 < MC3 < MC6.2. These results suggest that C-6 carbon linker and the presence of a pyrogallol group result in lower cytotoxicity. MC3 and MC6.2 affected the mitochondrial function more significantly relative to MitoQ, SkQ1, resveratrol and CoQ(10), while MC4 and MC7.2 displayed around 100-1000 times less cytotoxicity than SkQ1 and MitoQ. Based on the mitochondrial and cytotoxicity cellular data, MC4 and MC7.2 are proposed as leads that can be optimized to develop safe drug candidates with therapeutic application in mitochondrial oxidative stress-related diseases.

Abstract The electrochemical behaviour of the alkaloids berberine and palmatine at the polarized water/1,2-dichloroethane interface is studied by first time. The interface is supported on the micropores of a PET (polyethylene terephthalate) membrane by using an electrochemical cell employed in previous works. Chronoamperometry and square wave voltammetry are applied in order to determine the concentrations of the ions berberine and palmatine in water. Thus, calibration curves are built with both techniques and the resulting detection limits are discussed and compared. In addition, the chronoamperograms and square wave voltammograms recorded are used to estimate the standard transfer potentials and the aqueous diffusion coefficients for the two cations. The diffusion coefficient values are further compared with results of molecular dynamic simulations, finding a good correlation between experimental and simulated data.

Abstract The design and synthesis of artificial receptors based on molecular imprinting (MI) technology for the development of a new MIP-based biosensor for detection of the stress biomarker α-amylase in human saliva in point-of-care (PoC) applications is described in this work. The portable electrochemical devices for monitoring α-amylase consists of cost-effective and disposable gold screen-printed electrodes (AuSPEs). To build the electrochemical device, the template biomolecule was firstly immobilized directly over the working area of the gold chip previously activated with a self-assembled monolayer (SAM) of cysteamine (CA). Then, pyrrole (Py) monomer was selected as building block of a polymeric network prepared by CV electropolymerization. After the electropolymerization process, the enzyme was removed from the polymer film in order to build the specific recognition sites for the target enzyme. The MIP biosensor showed a very wide linear concentration range (between 3.0 × 10−4 to 0.60 mg mL−1 in buffer solution and between 3.0 × 10−4 to 3.0 × 10−2 mg mL−1 in human saliva) and low detection levels were achieved (LOD < 3.0 × 10−4 mg mL−1) using square wave voltammetry (SWV) as the electroanalytical technique.

Abstract This work reports the development of a simple and rapid electrochemical immunosensor for the determination of breast cancer biomarker Cancer Antigen 15-3 (CA 15-3). Disposable and cost-effective chips, consisting of gold screen-printed electrodes (AuSPEs), were used to develop the portable electrochemical devices for monitoring the biomarker in point-of-care (PoC), under clinical context. The biosensor preparation consisted of two simple steps. First, a self-assembled monolayer (SAM) of mercaptosuccinic acid (MSA) was formed at the AuSPE surface. Then, the CA 15-3 antibody was covalently bound to the carboxylic groups standing at the electrode surface using EDC/NHS chemistry. The performance of the developed immunosensor was evaluated by assessing the sensor sensitivity, linear response interval, selectivity and detection limit (LOD). The developed immunosensor provided a wide linear concentration range (from 1.0 to 1000 U mL(-1)) and low detection levels were achieved (LOD of 0.95 U mL(-1)), enabling the sensitive detection of the cancer biomarker at clinically relevant levels, using square wave voltammetry (SWV) as electroanalytical technique. Moreover, selectivity studies performed against other cancer biomarkers (CA 125 and CA 19-9) revealed that the antibody has high selectivity for CA 15-3 antigen. The immunosensor was applied to the quantification of CA 15-3 in artificial serum samples with satisfactory results.

Abstract Augmenter of liver regeneration (ALR) is a critical multi-isoform protein with its longer isoform, located in the mitochondrial intermembrane space, being part of the mitochondrial disulfide relay system (DRS). Upregulation of ALR was observed in multiple forms of cancer, among them hepatocellular carcinoma (HCC). To shed light into ALR function in HCC, we used MitoBloCK-6 to pharmacologically inhibit ALR, resulting in profound mitochondrial impairment and cancer cell proliferation deficits. These effects were mostly reversed by supplementation with bioavailable hemin b, linking ALR function to mitochondrial iron homeostasis. Since many tumor cells are known for their increased iron demand and since increased iron levels in cancer are associated with poor clinical outcome, these results help to further advance the intricate relation between iron and mitochondrial homeostasis in liver cancer.</p>

Abstract In this work, we describe an innovative methodology based on combined surface plasmon resonance (SPR) and electrochemical responses (eSPR) in the same immunoassay for screening CA 15-3 cancer biomarker with high sensitivity (and selectivity), in a very simple, label-free, accurate, and fully automated manner. Detection was achieved by performing two simple steps. In the first step, direct SPR was used to monitor CA 15-3 interaction with surface immobilized antibody. Two linear response ranges were obtained and the detection limit achieved is poor (LOD of 21 U mL(-1)). However, in the second detection step, electrochemical measurements at the SPR gold surface were performed to measure the decrease of redox probe peak current upon antigen-antibody interaction, providing a suitable amplification strategy to lower detection levels of CA 15-3 (LOD of 0.0998 U mL(-1)), without the need of additional complex and/or expensive amplification steps to enhance the sensitivity. Moreover, selectivity studies were performed against other common cancer biomarkers and the results showed that the eSPR immunosensor is selective for the CA 15-3 protein. Finally, the clinical applicability of the developed eSPR biosensing methodology was successfully applied to detect CA 15-3 in human serum samples at clinically relevant levels due to the high sensitivity of electrochemical readout. The same concept may be further extended to other proteins of interest.

Abstract In this study, Co-TiO2 nanoparticles were successfully synthesized using sol-gel and precipitation methods. The effect of Co amount on the physicochemical properties of these nanomaterials was investigated using various techniques. The obtained results showed that the structural and optical properties of the synthesized Co-TiO2 nanomaterials depended closely on the weight percent of Co added to TiO2. It was found that, for 1%Co-TiO2, a substitution of Ti4+ and Co2+/Co3+ within the lattice of TiO2 was happened. The results of the photocatalytic degradation of methyl orange (MO) experiments carried out in the presence of the as prepared nanomaterials showed that under visible light, the sample 1%Co-TiO2 exhibited the best MO conversion. The enhanced photocatalytic activity has been attributed to the efficient charge separation of electrons and holes. The mechanistic studies revealed that O-2(center dot-), h(+) and OH center dot are the major active species, and a possible mechanism degradation pathway of MO dye is proposed.

Abstract The main etiological agents in dermatophytosis of human skin and nails are Trichophyton, in particular Trichophyton rubrum (T. rubrum) and Trichophyton mentagrophytes (T. mentagrophytes). A new series of twenty-three 3- and 4-arylcoumarins was synthesized and the antifungal activities against clinical isolates of T. rubrum and T. mentagrophytes were evaluated. Sixteen out of twenty-three molecules exhibited antifungal activity against one or both fungi strains. In some cases, the activity against T. rubrum has been comparable to fluconazole, one of the standards, being 8-methoxy-3-(4'-nitrophenyl)coumarin (16) the best compound within this series (minimum inhibitory concentration, MIC=6.25 mu g/mL). The preliminary structure-activity relationship study showed that the antifungal activity depends on the position and nature of the substitution patterns. The cytotoxicity of eleven compounds on D-384 (astrocytoma), A-549 (lung cancer) and RKO (colorectal cancer) cell lines was also performed. With the aim of deeply understand the potential of these molecules as hits to develop new drugs, the theoretical absorption, distribution, metabolism and excretion (ADME) properties of the active compounds were calculated.

Abstract Central nervous system (CNS) diseases, such neurodegenerative disorders (NDs) are increasing over the last years as a consequence of a continuous growing of the life expectancy and aging of population. The molecular mechanisms underlying the pathophysiology of NDs are still not fully understood. Neurodegeneration seems to be multifactorial and associated to a complex set of events comprising oxidative stress, inflammation, protein dysfunction and aggregation, among others, that at the end lead to the demise of neurons. It has been advocated that oxidative stress induced by imbalanced redox states, involving either overproduction of reactive species or dysfunction of the antioxidant system, and the higher levels of iron in the brain has a crucial role in NDs progression. Therefore, antioxidant therapy emerged as a useful approach to modulate oxidative stress events. However, despite the promising results obtained in in vitro cell-free and cell-based assays, the pharmacokinetics/pharmacodynamics properties of the majority of antioxidants preclude their advance in pre- and clinical trials. In this chapter the nonenzymatic endogenous and exogenous antioxidants (single and combined antioxidant therapy) currently in clinical trials, as drugs or supplements, for treatment of NDs will be highlighted. From the data one can conclude that the clinical trials performed so far related with single or combined antioxidants are still in a very early stage. So, a huge research space in the antioxidant field remains to be explored that hopefully will yield new and effective neuroprotective agents in a foreseeable future.