Driving nanozymes towards stereochemical recognition: application to biomolecules of interest in health

National Foundation for Science and Technology, I.P., FCT, with reference SFRH/BD/130107/2017info:eu-repo/semantics/publishedVersio

Molecularly imprinted polymer-based electrochemical biosensor for alpha-synuclein detection in Parkinson´s Disease

A novel and sensitive molecularly imprinted polymer (MIP)-based biosensor was developed for alpha synuclein synaptic neuronal protein detection, a specific Parkinson´s disease (PD) biomarker. PD is the second most common neurodegenerative disease worldwide [1]. The leading neuropathological defining hallmark of PD is the presence of Lewy bodies and Lewy neurites mainly composed of aggregates of alpha-synuclein [2]. An early disease diagnosis is essential for an effective treatment application, thus there is an urgent need for finding accurate, reproductible, sensitive, and inexpensive diagnosis tolls. MIP-based biosensors represent cost-effective and easy-to-use solutions for disease biomarker recognition [3]. In this work, methylene blue was combined with alpha-synuclein and applied in situ on the surface of standard carbon screen-printed electrodes through bulk polymerization. This work describes a MIP-based electrochemical biosensor capable of detecting the target template with excellent precision and accuracy using for the first-time methylene blue for sensing alpha-synuclein analyte. The developed MIP-based sensor showed great analytical performance in alpha-synuclein detection, with a linear range from 1 fM to 10 pM based on electrochemical impedance spectroscopy measurements. To conclude, the developed electrodes were used to detect alpha-synuclein in serum and the selectivity tests were performed against different target molecules.info:eu-repo/semantics/publishedVersio

Immunosensor for alpha-synuclein detection in Parkinson´s Disease

Parkinsons disease (PD) is a neurodegenerative disorder and one of the leading causes of death worldwide [1]. The principal neuropathological defining hallmark of PD is the presence of Lewy bodies mainly composed of aggregates of alpha-synuclein (-syn) [2]. This protein is a specific PD biomarker that, when detected early, can contribute to an effective PD treatment application. Immunosensors are analytical platforms that can detect disease biomarkers with great accuracy [3]. Thus, a selective and sensitive immunosensor to detect -syn protein based on a standard screen-printed gold electrode is proposed herein. The sensor assembly was done through three different and sequential steps: (1) incubation with 3-aminophenol (3-AMP) to promote the amination of the gold electrode surface; (2) incubation with the -syn antibody; and (3) bovine serum albumin application to avoid non-specific binding. The electrochemical characterization and surface modification was analysed through cyclic voltammetry, electrochemical impedance spectroscopy, and square wave voltammetry techniques. Finally, the performance of this immunosensor was assessed using several calibration curves with standard solutions. The final immunosensor exhibited a linear response from 7 pM to 70 nM. On balance, this immunosensor was characterized as a sensitive, selective, and reproducible platform, with great potential for real sample analysis.info:eu-repo/semantics/publishedVersio

Supercritical antisolvent precipitation of PHBV microparticles

The micronization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from organic solutions using supercritical antisolvent (SAS) technique has been successfully achieved. SASexperiments were carried out at different operational conditions and microspheres with mean diameters ranging from 3 to 9 mwere obtained. The effect of CO2 and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a dichloromethane (DCM) solution. The best process conditions for this mixture were, according to our study, 40 ◦C, 100 bar, 1mLmin−1 liquid flow and 10 L min−1 carbon dioxide flow. Experiments with polymers containing different HV percentages were carried out. The powders obtained became more spherical as the HV content decreased

Human gingival fibroblasts response to different endodontic sealers: an in vitro study

Endodontic treatment aims to eliminate infection of the root canals and fill the dental pulp space. The biocompatibility studies of the sealers used in root canals obturation are crucial since they are applied in direct contact with periradicular tissues. Objective: The aim of this study was to evaluate the cytotoxicity of three root canal sealers—AH Plus, Bio MTA+, and Bio C sealer—on immortalized human gingival fibroblasts. Methods: AH Plus, Bio MTA+, and Bio C sealers were evaluated through incubation in real-time and material-conditioned media. Cells were incubated for 24 h and 72 h, at three different concentrations (1, 10, and 100 mg/mL) of each sealer. The cytotoxic activity of the sealers was assessed by Methyl tetrazolium (MTT) and Sulforhodamine B (SRB) assays. Cell morphology and cytogenetic alterations were studied microscopically. Results: MTT and SRB assays revealed similar results within both approaches. Cell culture exposed to sealers through incubation in real-time revealed a cytotoxic effect of AH Plus at 100 mg/mL. Material- conditioned media study revealed a cytotoxic effect of Bio MTA+ and Bio C, increasing with higher compound concentration and reaching 50% with 100 mg/mL. Regarding the cell’s morphology, Bio C sealer revealed a decrease in cell confluence and several morphological changes. AH Plus and Bio MTA+ did not seem to affect the cell confluence however morphology alterations were observed. In the cytogenetic study, a severe decrease of the mitotic index and a large number of chromosomal aberrations were observed. The present study represents an advance in the understanding of the biocompatibility of AH Plus, Bio MTA+, and Bio C sealers. These sealers demonstrated some cytotoxicity, depending on the concentration used. Although more validation studies are still needed, this study brings very relevant results in terms of cytotoxicity, cell morphology, and cytogenetic alterations. Conclusions: These results could help in the selection of the most appropriate compounds to be used in clinical practice as well as to determine the maximum recommended amounts of each sealer. Clinical Relevance: This study highlights the potential cytotoxic effects of three commonly used root canal sealers on human gingival fibroblasts, with varying degrees of impact depending on the concentration used. The results emphasize the importance of careful consideration when selecting and applying these materials in clinical practice.info:eu-repo/semantics/publishedVersio

Molecular imprinting on nanozymes for sensing applications

As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.The authors gratefully acknowledge funding from the European Commission through the project MindGAP (FET-Open/H2020/GA829040). The author ARC also acknowledges funding to National Foundation for Science and Technology, I.P., through the Ph.D. Grant, SFRH/BD/130107/2017.info:eu-repo/semantics/publishedVersio

Preparation of controlled release microspheres using supercritical fluid technology for delivery of anti-inflammatory drugs

Ethylcellulose/methylcellulose blends were produced using different precipitation techniques and impregnated with naproxen, a non-steroidal anti-inflammatory drug (NSAID). Solvent-evaporation technique was used not only for the preparation of ethylcellulose/methylcellulose microspheres but also to encapsulate naproxen. Supercritical fluid (SCF) impregnation was also performed to prepare naproxen loaded microspheres. The microspheres, impregnated by the SCF technique, were prepared both by solvent-evaporation and by a supercritical antisolvent (SAS) process. In vitro release profiles at pH 7.4 and 1.2, of naproxen-loaded microspheres were evaluated and the results were modelled Fick’s law of diffusion and Power law. Miscrospheres prepared by supercritical antisolvent have a higher loading capacity and present a slower release profile. The systems studied present a release mechanism controlled by drug diffusion which complies Fick’s law of diffusion

Preparation of ethylcellulose/methylcellulose blends by supercritical antisolvent precipitation

The supercritical antisolvent (SAS) techniquewas used to prepare ethyl cellulose/methyl cellulose blends, two biocompatible polymers commonly used as drug carriers in controlled delivery systems. Ethyl cellulose is widely used as a drug carrier. The drug release of the delivery devices can be controlled to some extent by addition of a water-soluble or water swellable polymer, such as methyl cellulose. This leads to the solubility enhancement of poorly water-soluble molecules. SAS experiments were carried out at different operational conditions and microspheres with mean diameters ranging from 5 to 30 m were obtained. The effect of CO2 and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a mixture of dichloromethane (DCM) and dimethylsulfoxide (DMSO) (4:1 ratio). The best process conditions for this mixture were according to our study 40 ◦C and 80 bar

Trypanosomatid comparative genomics: Contributions to the study of parasite biology and different parasitic diseases

In 2005, draft sequences of the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, also known as the Tri-Tryp genomes, were published. These protozoan parasites are the causative agents of three distinct insect-borne diseases, namely sleeping sickness, Chagas disease and leishmaniasis, all with a worldwide distribution. Despite the large estimated evolutionary distance among them, a conserved core of ~6,200 trypanosomatid genes was found among the Tri-Tryp genomes. Extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host-parasite interactions. In this article, we review the recent advances in the comparative genomics of these three species. This analysis also includes data on additional sequences derived from other trypanosmatid species, as well as recent data on gene expression and functional genomics. In addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases, genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections

Employing bacteria machinery for antibiotic detection: using DNA gyrase for ciprofloxacin detection

This work describes a new successful approach for designing biosensors that detect antibiotics. It makes use of a biomimetic strategy, by employing the biochemical target of a given antibiotic as its biorecognition element. This principle was tested herein for quinolones, which target DNA gyrase in bacteria. Ciprofloxacin (CIPRO) was tested as a representative antibiotic from the quinolone group; the sensitivity of biosensor to this group was confirmed by checking the response to another quinolone antibiotic (norfloxacin, NOR) and to a non-quinolone antibiotic (ampicillin, AMP). The biorecognition element used was DNA gyrase attached by ionic interactions to a carbon support, on a working electrode on common screen-printed electrodes (SPEs). The response against antibiotics was tested for increasing concentrations of CIPRO, NOR or AMP, and following the subsequent electrical changes by electrochemical impedance spectroscopy. The DNAgyrase biosensor showed sensitive responses for CIPRO and NOR, for concentrations down to 3.02nM and 30.2nM, respectively, with a very wide response range for CRIPRO, up to 30.2µM. Its response was also confirmed selective for quinolones, when compared to its response against AMP. Further comparison to an immunosensor of similar design (adding antibodies instead of DNA gyrase) was made, revealing favourable features for the new biomimetic biosensor with 1.52nM of limit of detection (LOD). Overall, the new approach presented herein is simple and effective for antibiotic detection, displaying a selective response against a given antibiotic group. The use of bacterial machinery as biorecognition element in biosensors may also provide a valuable tool to study the mechanism of action in bacterial cells of new drugs. This is especially important in the development of new drugs to fight bacterial resistance.The authors acknowledge funding from project PTDC/AAG-TEC/5400/2014 funded by European funds, through FEDER (European Funding or Regional Development) via COMPETE2020 – POCI (operational program for internationalization and competitively) and by national funding through the National Foundation for Science and Technology, I.P. (FCT). ARC also acknowledge funding to National Foundation for Science and Technology, I.P., through the PhD Grant, SFRH/BD/130107/2017.info:eu-repo/semantics/publishedVersio