Centre of Biological Engineering of the University of Minho

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Publications

Palmelloid-like phenotype in the alga Raphidocelis subcapitata exposed to pollutants: A generalized adaptive strategy to stress or a specific cellular response?

Publication . Machado, Manuela D.; Soares, Eduardo V.

This work focuses on the formation of palmelloid-like phenotype in the freshwater alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), when exposed to adverse conditions generated by the presence of organic [the antibiotic erythromycin (ERY) and the herbicide metolachlor (MET)] or inorganic [the heavy metals, cadmium (Cd) and zinc (Zn)] pollutants, at environmentally relevant concentrations. This alga in absence of stress or when exposed to ERY or Zn, up to 200 µg/L, essentially showed a single-nucleus state, although algal growth was reduced or stopped. R. subcapitata “switched” to a multinucleated state (palmelloid-like morphology) and accumulated energy-reserve compounds (neutral lipids) when stressed by 100–200 µg/L MET or 200 µg/L Cd; at these concentrations of pollutants, growth was arrested, however, the majority of the algal population (≥83 %) was alive. The formation of palmelloid-like phenotype, at sub-lethal concentrations of pollutants, was dependent on the pollutant, its concentration and exposure time. The multinucleated structure is a transitory phenotype since R. subcapitata population was able to revert to a single-nucleus state, with normal cell size, within 24–96 h (depending on the impact of the toxic in the alga), after being transferred to fresh OECD medium, without pollutants. The obtained results indicate that the formation of a palmelloid-like phenotype in R. subcapitata is dependent on the mode of action of toxics and their concentration, not constituting a generalized defense mechanism against stress. The observations here shown contribute to understanding the different strategies used by the unicellular alga R. subcapitata to cope with severe stress imposed by organic and inorganic pollutants.

2023Journal article

Restricted access

Nanocellulose- based biosensor for colorimetric detection of glucose

Publication . Neubauerova, Katrin; Carneiro, Mariana C.C.G.; Rodrigues, Lígia R.; Moreira, Felismina; Sales, Maria Goreti Ferreira

This work reports for the first time a colorimetric based biosensor using nanocellulose (NC) based supports drop-deposited onto a cellulose paper substrate for glucose detection in point-of-care. For this purpose, microcrystalline cellulose (MCC) samples were oxidized with 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO), sodium hypochlorite, and potassium bromide, to produce carboxylated NC. For the characterization, we used several methods: TEM, FTIR and conductometric titration. In all samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodium hypochlorite is added dropwise and the reaction is performed at constant pH 10. Carboxyl- NC was further casted on a cellulose substrate and used as support for glucose oxidase (GOx), horseradish peroxidase (HRP) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) reactions, aiming to yield a coloured detection system for glucose. The sensing system was generated by integrating GOx on the carboxyl- NC /cellulose substrate. Upon reaction with glucose, the enzyme produced hydrogen peroxide, which was converted into a blue-coloured product by reaction with HRP and the chromogenic reagent ABTS. The test-strip was calibrated by incubating it in different concentrations of glucose. The colours obtained were further analysed by a suitable image analysis software. Linear response for glucose ranged from 1.5 to 13.0 mM. Overall, this new test-strip used renewable material for glucose determination, which is an advantage when compared to other systems that require more complex technological approaches. Moreover, it was found that carboxyl- NC improved the colour homogeneity of the test-strip and the intrinsic linear response of concentration range.

2020Journal article

Open access

Paper-based ELISA for fast CA 15–3 detection in point-of-care

Publication . Carneiro, Mariana C.C.G.; Rodrigues, R. V.; Moreira, Felismina; Sales, Maria Goreti Ferreira

The paper-based Enzyme-Linked Immunosorbent Assay (P-ELISA) is a promising tool for diagnostic applications because the paper matrix is characterised by low cost, short analysis time, portability, and a high surface-to-volume ratio that allows the use of a small sample and reagent volume of a few microliters. In addition, colorimetric assays are suitable for low-resource areas due to their simplicity and naked-eye detectability. Although several works have been reported using paper-based colorimetric sensors for cancer biomarkers, P-ELISA for cancer antigen 15–3 (CA 15–3) has never been reported. Thus, this work reports the development of a rapid, simple and relatively inexpensive paper-based colorimetric assay for the detection of CA 15–3 as cancer biomarker. The assay was developed on a filter paper that was previously washed and chemically oxidized with periodate to generate aldehyde functional groups on the cellulose surface. After covalent binding of the first antibody, detection was performed by a colorimetric reaction based on the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by a peroxidase enzyme in the presence of H2O2. The colorimetric measurement was based on digital image acquisition analysed using ImageJ software. The linear range of the assay in buffer ranged 2 to 1100 U/mL. The performance of the assay was also successfully tested in human serum from Cormay®, offering a linear range from 2 to 200 U/mL. Thus, this P-ELISA sensor is suitable for the analysis of serum samples, since the physiological value in cancer patients is 30 U/mL. We believe that this proof-of-concept has the potential to be extended to be applied to other protein biomarkers and to be a suitable tool for cancer screening in developing countries.

2022Journal article

Restricted access

Flexible sensing devices integrating molecularly-imprinted polymers for the detection of 3-nitrotyrosine biomarker

Publication . Martins, Gabriela V.; Riveiro Rodriguez, Antonio; Chiussi, Stefano; Sales, Goreti

In recent years, the development of flexible and wearable devices for healthcare and biomedical applications has become an emerging technological goal, particularly with personalized medicine on the rise. As a response to the increasing demand for in-situ sensing platforms that fulfil some essential requirements like sensitivity, reproducibility and high stability, electrochemical sensors have boosted their way for innovative approaches. So, high-quality flexible sensing strategies are still a demand for local monitoring. Herein, a flexible three-electrode system was fabricated on transparent polymeric sheet substrate through physical deposition of gold as working, counter, and reference electrodes. Along the fabrication process, the electrochemical performance of these electrodes was assessed by means of cyclic voltammetry (CV) while gold adherence to the plastic material was continuously improved. Afterwards, a high-performance molecularly-imprinted sensing film inspired by natural recognition mechanism was assembled through electropolymerization of phenol monomer, in the presence of 3-nitrotyrosine (3-NT), directly on the gold surface. Under the optimized conditions, the flexible (bio)sensor platform was able to detect the presence of 3-NT over the concentration range 10 pg/mL – 1 μg/mL, enabling one of the lower limits of detection found in the literature (1.13 pg/mL or 24.9 pM). The obtained (bio)sensor displayed good reproducibility, stability and selectivity over the chosen interfering substances. Overall, the developed electrochemical device may serve as a flexible, miniaturized, and reliable platform, with potential to be applied in the future as wearable sensing technology.

2022Journal article

Open access

Life and death of Pseudokirchneriella subcapitata: physiological changes during chronological aging

Publication . Machado, Manuela Dias; Soares, Eduardo V.

The green alga Pseudokirchneriella subcapitata is widely used in ecotoxicity assays and has great biotechnological potential as feedstock. This work aims to characterize the physiology of this alga associated with the aging resulting from the incubation of cells for 21 days, in the OECD medium, with continuous agitation and light exposure, in a batch mode. After inoculation, cells grow exponentially during 3 days, and the culture presents a typical green color. In this phase, “young” algal cells present, predominantly, a lunate morphology with the chloroplast occupying a large part of the cell, maximum photosynthetic activity and pigments concentration, and produce starch as a reserve material. Between the 5th and the 12th days of incubation, cells are in the stationary phase. The culture becomes less green, and the cells stop dividing (≥ 99% have one nucleus) and start to age. “Old” algal cells present chloroplast shrinkage, an abrupt decline of chlorophylls content, and photosynthetic capacity (Fv/Fm and ɸPSII), accompanied by a degradation of starch and an increase of neutral lipids content. The onset of the death phase occurs after the 12th day and is characterized by the loss of cell membrane integrity of some algae (cell death). The culture stays, progressively, yellow, and the majority of the population (~93%) is composed of live cells, chronologically “old,” with a significant drop in photosynthetic activity (decay > 75% of Fv/Fm and ɸPSII) and starch content. The information here achieved can be helpful when exploring the potential of this alga in toxicity studies or in biotechnological applications.

2022Journal article

Restricted access