Untitled

Funder

Organizational Unit

Publications

Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction

Publication . Gomes, Filipa O.; Maia, Luísa B.; Cordas, Cristina; Moura, Isabel; Delerue-Matos, Cristina; Moura, José J.G.; Morais, Simone

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors.

2019Journal article

Open access

Acetylated cashew gum-based nanoparticles for the incorporation of alkaloid epiisopiloturine

Publication . Rodrigues, Jessica do Amaral; Araújo, Alyne Rodrigues de; Pitombeira, Nadia Aline; Plácido, Alexandra; Almeida, Miguel Peixoto de; Veras, Leiz Maria Costa; Delerue-Matos, Cristina; Lima, Filipe Camargo Dalmatti Alves; Neto, Augusto Batagin; Paula, Regina Célia Monteiro de; Feitosa, Judith Pessoa Andrade; Eaton, Peter; Leite, José Roberto Souza A.; Silva, Durcilene Alves da

The natural alkaloid epiisopiloturine has recently become the focus of study for various medicinal properties, particularly for its anti-inflammatory and antischistosomal effect. The incorporation of active molecules in natural polymeric matrices has garnered increasing interest during recent decades. A new derivative of cashew gum successfully obtained by gum acetylation has shown great potential as a carrier in controlled drug release systems. In this work, epiisopiloturine was encapsulated in acetylated cashew gum nanoparticles in order to increase solubility and allow slow release, whereas the morphology results were supported by computer simulations. The particles were produced under a variety of conditions, and thoroughly characterized using light scattering and microscopic techniques. The particles were spherical and highly stable in solution, and showed drug incorporation at high levels, up to 55% efficiency. Using a dialysis-based in vitro assay, these particles were shown to release the drug via a Fickian diffusion mechanism, leading to gradual drug release over approximately 6 h. These nanoparticles show potential for the use as drug delivery system, while studies on their potential anti-inflammatory action, as well as toxicity and efficacy assays would need to be performed in the future to confirm their suitability as drug delivery candidates.

2019-05Journal article

Open access

Structure–Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian Cells

Publication . Campelo, Yuri; Ombredane, Alicia; Vasconcelos, Andreanne; Albuquerque, Lucas; Moreira, Daniel; Plácido, Alexandra; Rocha, Jefferson; Fokoue, Harold Hilarion; Yamaguchi, Lydia; Mafud, Ana; Mascarenhas, Yvonne; Delerue-Matos, Cristina; Borges, Tatiana; Joanitti, Graziella; Arcanjo, Daniel; Kato, Massuo; Kuckelhaus, Selma; Silva, Marcos; Moraes, Josué; Leite, José

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5⁻10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.

2018-06Journal article

Open access

Fluorometric method based on molecular recognition solid-phase extraction for determination of riboflavin in milk and infant formula

Publication . Osório, Marcelo V.; Marques, Sara S.; Oliveira, Hugo M.; Barreiros, Luisa; Segundo, Marcela A.

Riboflavin (vitamin B2) is involved in several biological processes, particularly in energy production, and it is acquired from food ingestion, principally from supplemented food during the first years of life. Therefore, a simple, fast and cost-effective high-throughput method for determination of riboflavin in milk and infant formula is proposed, based on selective extraction using commercially available molecularly imprinted polymers targeted to riboflavin, followed by direct fluorometric determination. Several aspects were studied, namely microplate assay conditions, the composition of eluting solution and the stability of riboflavin in the eluate. Hence, elution using 1% (v/v) acetic acid in methanol or in acetonitrile is recommended, followed by immediate analysis or solvent evaporation, with reconstitution and analysis within 24 h. The proposed method provided a LOD of 0.03 mg L−1, with working range for undiluted samples between 0.125 and 2 mg L−1, and sample throughput of 24 h−1. It was successfully applied to certified reference material NIST-1846 and also to commercial milk and infant formula samples.

2016Journal article

Open access

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum

Publication . Ramos, Inês I.; Magalhães, Luís M.; Barreiros, Luisa; Reis, Salette; Lima, José L. F. C.; Segundo, Marcela A.

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL-1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL-1 (corresponding to 5.0-60 mg mL-1 in undiluted samples), with a detection limit of 33 μg mL-1 (1.7 mg mL-1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

2017Journal article

Open access