Browsing by Author "Sales, Goreti"
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- An ultrasensitive human cardiac troponin T graphene screen-printed electrode based on electropolymerized-molecularly imprinted conducting polymerPublication . Silva, Bárbara V.M.; Rodríguez, Blanca A.G.; Sales, Goreti; Sotomayor, Maria Del Pilar T.; Dutra, Rosa F.A nano-molecularly imprinted polymer (N-MIP) assembled on a screen-printed electrode for the cardiac troponin T (cTnT) was developed. The biomimetic surface was obtained by a co-polymer matrix as-sembled on the reduced graphene oxide (RGO) electrode surface. The cTnT active sites were engineered using pyrrole and carboxylated pyrrole that was one-step electropolymerized jointly with cTnT by cyclic voltammetry. The stepwise preparation of the biomimetic surface was characterized by cyclic and dif-ferential pulse voltammetries using the ferrocyanide/ferricyanide as redox probe. Structural and mor-phological characterization was also performed. The optimal relation of pyrrole and pyrrole-3-acid car-boxylic to perform the cTnT biomimetic nanosurface was obtained at 1:5 ratio. The analytical perfor-mance of cTnT N-MIP performed by differential pulse voltammetry showed a linear range from 0.01 to 0.1 ng mL-1 (r¼0.995, p«0.01), with a very low limit of detection (0.006 ng mL-1). The synergic effect of conductive polymer and graphene forming 3D structures of reactive sites resulted in a N-MIP with ex-cellent affinity to cTnT binding (KD¼7.3 10-13 mol L-1). The N-MIP proposed is based on a simple method of antibody obtaining with a large potential for point-of-care testing applications.
- Artificial antibodies for troponin T by its imprinting on the surface of multiwalled carbon nanotubes: Its use as sensory surfacesPublication . Moreira, Felismina T. C.; Dutra, Rosa A.F.; Noronha, João P. C.; Cunha, Alexandre L.; Sales, GoretiA novel artificial antibody for troponin T (TnT) was synthesized by molecular imprint (MI) on the surface of multiwalled carbon nanotubes (MWCNT). This was done by attaching TnT to the MWCNT surface, and filling the vacant spaces by polymerizing under mild conditions acrylamide (monomer) in N,N′-methylenebisacrylamide (cross-linker) and ammonium persulphate (initiator). After removing the template, the obtained biomaterial was able to rebind TnT and discriminate it among other interfering species. Stereochemical recognition of TnT was confirmed by the non-rebinding ability displayed by non-imprinted (NI) materials, obtained by imprinting without a template. SEM and FTIR analysis confirmed the surface modification of the MWCNT. The ability of this biomaterial to rebind TnT was confirmed by including it as electroactive compound in a PVC/plasticizer mixture coating a wire of silver, gold or titanium. Anionic slopes of 50 mV decade−1 were obtained for the gold wire coated with MI-based membranes dipped in HEPES buffer of pH 7. The limit of detection was 0.16 μg mL−1. Neither the NI-MWCNT nor the MWCNT showed the ability to recognize the template. Good selectivity was observed against creatinine, sucrose, fructose, myoglobin, sodium glutamate, thiamine and urea. The sensor was tested successfully on serum samples. It is expected that this work opens new horizons on the design of new artificial antibodies for complex protein structures.
- Avoiding the interference of doxorubicin with MTT measurements on the MCF-7 breast cancer cell linePublication . Luís, Carla; Castaño-Guerrero, Yuselis; Soares, Raquel; Sales, Goreti; Fernandes, RúbenDoxorubicin (DOXO) is an adjuvant chemotherapy agent and is also commonly used in cell biology research. Cytotoxic assays in cell culture are frequently used in order to stablish drug concentrations that are useful for controlling cell proliferation. One common cytotoxic method used is 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). Our present research aims to support future studies in engaging MTT assay using DOXO that exhibits a strong red coloration and fluorescence, and so it is assumed that DOXO may interfere with commonly used colorimetric assays such as MTT. The interference of DOXO in the MTT determination was evaluated in a Breast Cancer cell line Michigan Cancer Foundation-7 (MCF-7). The interference was evaluated by means of spectroscopic methods in particular spectrophometry and fluorescence spectroscopy of MTT and DOXO. We postulate that the medium and the MTT reagent itself can interfere on the metabolic activity method, so in order to achieve better results, DMEM was replaced by a neutral buffer like Phosphate-buffered saline (PBS). This protocol may be extremely useful in future studies involving DOXO.
- Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transductionPublication . Oliveira, Helena M. V.; Moreira, Felismina; Sales, GoretiA 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0mVdecade−1 and 1.0×10−5 to 2.7×10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.
- Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transductionPublication . Oliveira, Helena M.V.; Moreira, Felismina T. C.; Sales, GoretiA 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.
- Conductive Paper with Antibody- Like Film for Electrical Readings of BiomoleculesPublication . Tavares, Ana P. M.; Ferreira, Nádia S.; Truta, Liliana A. A. N. A.; Sales, GoretiThis work reports a novel way of producing an inexpensive substrate support to assemble a sensing film, designed for the electrical transduction of an intended biomolecule. The support uses cellulose paper as substrate, made hydrophobic with solid wax and covered by a home-made conductive ink having graphite as core material. The hydrophobicity of the paper was confirmed by contact angle measurements and the conductive ink composition was optimized with regard to its adhesion, conductivity, and thermal stability. This support was further modified targeting its application in quantitative analysis. Carnitine (CRT) was selected as target compound, a cancer biomarker. The recognition material consisted of an antibody-like receptor film for CRT, tailored on the support and prepared by electrically-sustained polymerization of 3,4-ethylenedioxythiophene (EDOT) or dodecylbenzenesulfonic acid (DBS). Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy analysis confirmed the presence of the polymeric film on the support, and the performance of the devices was extensively evaluated with regard to linear response ranges, selectivity, applicability, and reusability. Overall, the paper-based sensors offer simplicity of fabrication, low cost and excellent reusability features. The design could also be extended to other applications in electricalbased approaches to be used in point-of-care (POC).
- Development of a biosensor for a biomarker CA15-3 in breast cancer by electropolymerization of pyrrolePublication . Barbosa, Beatriz; Ferreira, Nádia; Sales, GoretiBreast cancer is a disease that affects millions of people in the world and a constant search for the reduction of its incidence is necessary. If a global cure is not possible, methods must be developed to allow an early diagnosis, thereby reducing the level of disease progression. In the present study, we aim to obtain a molecularly-imprinted polymer (MIP) for the detection of a breast cancer biomarker (CA15-3). The imprinted sites are generated by the electropolymerization of pyrrole, yielding a conducting polymer.
- Development of a sensitive ratiometric Imprinted hydrogel for the detection of matrix metalloproteinase 7 (MMP7) biomarkerPublication . Piloto, Ana Margarida L.; Ribeiro, David S.M.; Santos, João L.M.; Sales, GoretiA dual-emissive fluorescent probe was developed for the sensitive and selective detection of matrix metalloproteinase 7 (MMP7), a protein biomarker associated with pancreatic cancer. The ratiometric probe consisting of blue emitting carbon dots (CDs) and molecularly imprinted polymers (MIPs) previously assembled around red emitting quantum dots (QDs) was successfully combined for the detection of MMP7 protein. The concept is to use MIPs that function as the biorecognition elements, conjugated to cadmium telluride QDs, as the sensing system. The fluorescence intensity of red QDs is quenched with increasing concentrations of the analyte, acting as sensitive probes, while the fluorescence intensity of blue emitting CDs remains constant, acting as internal controls. The resultant fluorescence color changed from red to blue as a function of the MMP7 concentration, under a 365 nm UV lamp. The imprinted material MIP@QDs were successfully incorporated within a cellulose hydrogel containing CDs as reference probes. The resultant materials were designated as imprinted ratiometric hydrogels (imprinted rHGs). The fluorescence quenching of the imprinted rHGs occurred with increasing concentrations of MMP7, showing linearity in the range [1.49 × 10–11 – 1.92 × 10–9] g/mL, in 1000-fold diluted human serum. The imprinted rHGs showed an imprinting factor of 1.83 and a limit of detection of 4.11 × 10–12 g/mL. Overall, the imprinted rHGs developed in this work presented increased selectivity for the MMP7 protein over red emitting QDs nanoparticles and higher sensitivity comparatively to the non-imprinted rHGs.
- Development of an electrochemical biosensor for an Alzheimer biomarker detection in Point-of-CarePublication . Mendes, Francisca; Sales, Goreti; Moreira, FelisminaAlzheimer's disease (AD) is the dominant dementia condition of our society with great social and economic impact. One of the major pathological changes is the formation of extracellular insoluble deposits of β-amyloid protein (Aβ) in plaques. Pathological changes are often accompanied by neuroinflammation, abnormal activity of the neural network, dysfunction, degeneration and loss of neurons. Moreover, there is no current test capable of providing accurate diagnosis of AD and, besides that there is no treatment of prevention available, only a way to delay its progression, so the early diagnosis is the key in present. Research activities targeting such possibility include the identification of biomarkers in several biological fluids that may turn out an important means to diagnosis within future, mostly if these are found in peripheral blood (avoiding more invasive procedures). Thus, the main goal of this proposal is to develop novel, low cost (bio)sensing-devices with an aptamer as a biorecognition element of AD biomarker detection, Aβ42, in point-of-care.
- Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocoliticaPublication . Sande, Maria Georgina; Ferreira, Débora; Rodrigues, Joana; Melo, Luís; Linke, Dirk; Silva, Carla J.; Moreira, Felismina; Sales, Goreti; Rodrigues, LígiaNew point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.
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