Toxicity screening of Diclofenac, Propranolol, Sertraline and Simvastatin using Danio rerio and Paracentrotus lividus embryo bioassays

Early life-stage bioassays have been used as an alternative to short-term adult toxicity tests since they are cost-effective. A single couple can produce hundreds or thousands of embryos and hence can be used as a simple high-throughput approach in toxicity studies. In the present study, zebrafish and sea urchin embryo bioassays were used to test the toxicity of four pharmaceuticals belonging to different therapeutic classes: diclofenac, propranolol, simvastatin and sertraline. Simvastatin was the most toxic tested compound for zebrafish embryo, followed by diclofenac. Sertraline was the most toxic drug to sea urchin embryos, inducing development abnormalities at the ng/L range. Overall, our results highlight the potential of sea urchin embryo bioassay as a promising and sensitive approach for the high-throughput methods to test the toxicity of new chemicals, including pharmaceuticals, and identify several drugs that should go through more detailed toxicity assays.

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Desenvolvimento de um método potenciométrico para deteção de salmonella typhimurium

A Salmonella é um microrganismo responsável por grande parte das doenças alimentares, podendo por em causa a saúde pública da área contaminada. Uma deteção rápida, eficiente e altamente sensível e extremamente importante, sendo um campo em franco desenvolvimento e alvo de variados e múltiplos estudos na comunidade cientifica atual. Foi desenvolvido um método potenciométrico para a deteção de Salmonellas, com elétrodos seletivos de iões, construídos em laboratório com pontas de micropipetas, fios de prata e sensores com composição otimizada. O elétrodo indicador escolhido foi um ESI seletivo a cadmio, para redução da probabilidade de interferências no método, devido a pouca abundancia do cadmio em amostras alimentares. Elétrodos seletivos a sódio, elétrodos de Ag/AgCl de simples e de dupla juncão foram também construídos e caracterizados para serem aplicados como elétrodos de referência. Adicionalmente otimizaram-se as condições operacionais para a analise potenciométrica, nomeadamente o elétrodo de referencia utilizado, condicionamento dos elétrodos, efeito do pH e volume da solução amostra. A capacidade de realizar leituras em volumes muito pequenos com limites de deteção na ordem dos micromolares por parte dos ESI de membrana polimérica, foi integrada num ensaio com um formato nao competitivo ELISA tipo sanduiche, utilizando um anticorpo primário ligado a nanopartículas de Fe@Au, permitindo a separação dos complexos anticorpo-antigénio formados dos restantes componentes em cada etapa do ensaio, pela simples aplicação de um campo magnético. O anticorpo secundário foi marcado com nanocristais de CdS, que são bastante estáveis e é fácil a transformação em Cd2+ livre, permitindo a leitura potenciométrica. Foram testadas várias concentrações de peroxido de hidrogénio e o efeito da luz para otimizar a dissolução de CdS. O método desenvolvido permitiu traçar curvas de calibração com soluções de Salmonellas incubadas em PBS (pH 4,4) em que o limite de deteção foi de 1100 CFU/mL e de 20 CFU/mL, utilizando volumes de amostra de 10 ƒÊL e 100 ƒÊL, respetivamente para o intervalo de linearidade de 10 a 108 CFU/mL. O método foi aplicado a uma amostra de leite bovino. A taxa de recuperação media obtida foi de 93,7% } 2,8 (media } desvio padrão), tendo em conta dois ensaios de recuperação efetuados (com duas replicas cada), utilizando um volume de amostra de 100 ƒÊL e concentrações de 100 e 1000 CFU/mL de Salmonella incubada.

Sol-Gel Chemistry in Biosensing Devices of Electrical Transduction: Application to CEA Cancer Biomarker

Sol-gel chemistry allows the immobilization of organic molecules of biological origin on suibtable solid supports, permitting their integration into biosensing devices widening the possibility of local applications. The present work is an application of this principle, where the link between electrical receptor platform and the antibody acting as biorecognition element is made by sol-gel chemistry. The immunosensor design was targeted for carcinoembryonic antigen (CEA), an important biomarker for screening the colorectal cancer, by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). The device displayed linear behavior to CEA in EIS and in SWV assays ranging from 0.50 to 1.5ng/mL, and 0.25 to 1.5ng/mL, respectively. The corresponding detection limits were 0.42 and 0.043 ng/mL. Raman spectroscopy was used to characterize the surface modifications on the conductive platform (FTO glass). Overall, simple sol-gel chemistry was effective at the biosensing design and the presented approach can be a potential method for screening CEA in point-of-care, due to the simplicity of fabrication, short response time and low cost. - See more at: http://www.eurekaselect.com/127192/article#sthash.m1AWhINx.dpuf

Protein-responsive polymers for point-of-care detection of cardiac biomarker

This work describes a novel use for the polymeric film, poly(o-aminophenol) (PAP) that was made responsive to a specific protein. This was achieved through templated electropolymerization of aminophenol (AP) in the presence of protein. The procedure involved adsorbing protein on the electrode surface and thereafter electroploymerizing the aminophenol. Proteins embedded at the outer surface of the polymeric film were digested by proteinase K and then washed away thereby creating vacant sites. The capacity of the template film to specifically rebind protein was tested with myoglobin (Myo), a cardiac biomarker for ischemia. The films acted as biomimetic artificial antibodies and were produced on a gold (Au) screen printed electrode (SPE), as a step towards disposable sensors to enable point-of-care applications. Raman spectroscopy was used to follow the surface modification of the Au-SPE. The ability of the material to rebind Myo was measured by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The devices displayed linear responses to Myo in EIS and SWV assays down to 4.0 and 3.5 μg/mL, respectively, with detection limits of 1.5 and 0.8 μg/mL. Good selectivity was observed in the presence of troponin T (TnT) and creatine kinase (CKMB) in SWV assays, and accurate results were obtained in applications to spiked serum. The sensor described in this work is a potential tool for screening Myo in point-of-care due to the simplicity of fabrication, disposability, short time response, low cost, good sensitivity and selectivity.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Electrochemical biosensor based on biomimetic material for myoglobin detection

A novel reusable molecularly imprinted polymer (MIP) assembled on a polymeric layer of carboxylated poly(vinyl chloride) (PVCsingle bondCOOH) for myoglobin (Myo) detection was developed. This polymer was casted on the gold working area of a screen printed electrode (Au-SPE), creating a novel disposable device relying on plastic antibodies. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and Fourier transform infrared spectroscopy (FTIR) studies confirmed the surface modification. The MIP/Au-SPE devices displayed a linear behaviour in EIS from 0.852 to 4.26 μg mL−1, of positive slope 6.50 ± 1.48 (kΩ mL μg−1). The limit of detection was 2.25 μg mL−1. Square wave voltammetric (SWV) assays were made in parallel and showed linear responses between 1.1 and 2.98 μg mL−1. A current decrease was observed against Myo concentration, producing average slopes of −0.28 ± 0.038 μA mL μg−1. MIP/Au-SPE also showed good results in terms of selectivity. The error% found for each interfering species were 7% for troponin T (TnT), 11% for bovine serum albumin (BSA) and 2% for creatine kinase MB (CKMB), respectively. Overall, the technical modification over the Au-SPE was found a suitable approach for screening Myo in biological fluids.

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-Screen Printed Electrodes

A gold screen printed electrode (Au-SPE) was modified by merging Molecular Imprinting and Self-Assembly Monolayer techniques for fast screening cardiac biomarkers in point-of-care (POC). For this purpose, Myoglobin (Myo) was selected as target analyte and its plastic antibody imprinted over a glutaraldehyde (Glu)/cysteamine (Cys) layer on the gold-surface. The imprinting effect was produced by growing a reticulated polymer of acrylamide (AAM) and N,N′-methylenebisacrylamide (NNMBA) around the Myo template, covalently attached to the biosensing surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were carried out in all chemical modification steps to confirm the surface changes in the Au-SPE. The analytical features of the resulting biosensor were studied by different electrochemical techniques, including EIS, square wave voltammetry (SWV) and potentiometry. The limits of detection ranged from 0.13 to 8 μg/mL. Only potentiometry assays showed limits of detection including the cut-off Myo levels. Quantitative information was also produced for Myo concentrations ≥0.2 μg/mL. The linear response of the biosensing device showed an anionic slope of ~70 mV per decade molar concentration up to 0.3 μg/mL. The interference of coexisting species was tested and good selectivity was observed. The biosensor was successfully applied to biological fluids.

Smart Plastic Antibody Material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to Myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.

Label-free Detection of Microcystin-LR in Waters Using Real-Time Potentiometric Biosensors Based on Single-Walled Carbon Nanotubes Imprinted Polymers

Microcystin-LR (MC-LR) is a dangerous toxin found in environmental waters, quantified by high performance liquid chromatography and/or enzyme-linked immunosorbent assays. Quick, low cost and on-site analysis is thus required to ensure human safety and wide screening programs. This work proposes label-free potentiometric sensors made of solid-contact electrodes coated with a surface imprinted polymer on the surface of Multi-Walled Carbon NanoTubes (CNTs) incorporated in a polyvinyl chloride membrane. The imprinting effect was checked by using non-imprinted materials. The MC-LR sensitive sensors were evaluated, characterized and applied successfully in spiked environmental waters. The presented method offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

The potential of molecular imprinting as a biosensing devices for monitoring the CEA cancer biomarker

6th Graduate Student Symposium on Molecular Imprinting

A novel antibody-like material for breast cancer antigen CA15-3, used to track breast cancer by potentiometric transduction

This work presents the development of a low cost sensor device for the diagnosis of breast cancer in point-of-care, made with new synthetic biomimetic materials inside plasticized poly(vinyl chloride), PVC, membranes, for subsequent potentiometric detection. This concept was applied to target a conventional biomarker in breast cancer: Breast Cancer Antigen (CA15-3). The new biomimetic material was obtained by molecularly-imprinted technology. In this, a plastic antibody was obtained by polymerizing around the biomarker that acted as an obstacle to the growth of the polymeric matrix. The imprinted polymer was specifically synthetized by electropolymerization on an FTO conductive glass, by using cyclic voltammetry, including 40 cycles within -0.2 and 1.0 V. The reaction used for the polymerization included monomer (pyrrol, 5.0×10-3 mol/L) and protein (CA15-3, 100U/mL), all prepared in phosphate buffer saline (PBS), with a pH of 7.2 and 1% of ethylene glycol. The biomarker was removed from the imprinted sites by proteolytic action of proteinase K. The biomimetic material was employed in the construction of potentiometric sensors and tested with regard to its affinity and selectivity for binding CA15-3, by checking the analytical performance of the obtained electrodes. For this purpose, the biomimetic material was dispersed in plasticized PVC membranes, including or not a lipophilic ionic additive, and applied on a solid conductive support of graphite. The analytical behaviour was evaluated in buffer and in synthetic serum, with regard to linear range, limit of detection, repeatability, and reproducibility. This antibody-like material was tested in synthetic serum, and good results were obtained. The best devices were able to detect 5 times less CA15-3 than that required in clinical use. Selectivity assays were also performed, showing that the various serum components did not interfere with this biomarker. Overall, the potentiometric-based methods showed several advantages compared to other methods reported in the literature. The analytical process was simple, providing fast responses for a reduced amount of analyte, with low cost and feasible miniaturization. It also allowed the detection of a wide range of concentrations, diminishing the required efforts in previous sample pre-treating stages.

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International Congress