ISEP – BioMark – Artigos
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- An all-in-one approach for self-powered sensing: A methanol fuel cell modified with a molecularly imprinted polymer for cancer biomarker detectionPublication . Carneiro, Liliana P.T; Pinto, Alexandra M.F.R.; Mendes, Adélio; Ferreira Sales, Maria GoretiThis work describes the development of an innovative electrochemical biosensor comprehending a passive direct methanol fuel cell (DMFC) assembly, modified by a layer of a molecularly imprinted polymer (MIP) on a carbon fabric anode electrode containing Pt/Ru nanoparticles. This MIP film was prepared from poly(3,4-ethylenedioxythiophene) (PEDOT) and polypyrrole (PPy) obtained by in situ electropolymerization of the corresponding monomers on the anode electrode surface. This MIP film is designed to detect an important cancer biomarker- carcinoembryonic antigen (CEA). This innovative, all-in-one device works in a simple way. First, CEA is incubated on the anode container of the fuel cell, then methanol is added, followed by the response evaluation (polarization curves determination). As CEA selectively interacts with the MIP film, it blocks the methanol's access to the Pt catalyst, remains specific bonded, and interferes with the subsequent polarization curves of the DMFC. Polarization curves obtained in the presence of standard solutions prepared in buffer and human serum confirmed linear responses of log CEA concentration ranging from 30 to 30 000 ng/mL in both media. The biosensor DMFC showed a sensitive response with a detection limit of 4.41 ng/mL when an aqueous 0.05 M methanol solution was used as fuel. When methanol was replaced by an ethanol solution of the same concentration (using the same setup developed for the DMFC), the lower detection limit of 3.52 ng/mL was obtained. Overall, the obtained results show that methanol/ethanol fuel cells operating without flow-through can be successfully used for the fabrication of self-powered biosensors. The novel biosensor concept presented here is simple, inexpensive, and effective, and can be further developed to meet point-of-care requirements.
- 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.
- Assessing and comparing the total antioxidant capacity of commercial beverages: application to beers, wines, waters and soft drinks using TRAP, TEAC and FRAP methods.Publication . Queirós, Raquel B.; Tafulo, Paula A. R.; Sales, M. Goreti F.This work measures and tries to compare the Antioxidant Capacity (AC) of 50 commercial beverages of different kinds: 6 wines, 12 beers, 18 soft drinks and 14 flavoured waters. Because there is no reference procedure established for this purpose, three different optical methods were used to analyse these samples: Total Radical trapping Antioxidant Parameter (TRAP), Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric ion Reducing Antioxidant Parameter (FRAP). These methods differ on the chemical background and nature of redox system. The TRAP method involves the transfer of hydrogen atoms while TEAC and FRAP involves electron transfer reactions. The AC was also assessed against three antioxidants of reference, Ascorbic acid (AA), Gallic acid (GA) and 6-hydroxy-2,5,7,8-tetramethyl- 2-carboxylic acid (Trolox). The results obtained were analyzed statistically. Anova one-way tests were applied to all results and suggested that methods and standards exhibited significant statistical differences. The possible effect of sample features in the AC, such as gas, flavours, food colouring, sweeteners, acidity regulators, preservatives, stabilizers, vitamins, juice percentage, alcohol percentage, antioxidants and the colour was also investigated. The AC levels seemed to change with brand, kind of antioxidants added, and kind of flavour, depending on the sample. In general, higher ACs were obtained for FRAP as method, and beer for kind of sample, and the standard expressing the smaller AC values was GA.
- Backside-surface imprinting as a new strategy to generate specific plastic antibody materialsPublication . Cabral-Miranda, Gustavo; Sales, M. Goreti F.; Gidlund, MagnusA backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular “tape”. The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL−1) versus charged tranfer resistance (RCT, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL−1 (RCT = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest.
- Biomimetic norfloxacin sensors made of molecularly-imprinted materials for potentiometric transductionPublication . Moreira, Felismina T. C.; Freitas, Victor De; Sales, M. Goreti F.A biomimetic sensor for norfloxacin is presented that is based on host-guest interactions and potentiometric transduction. The artificial host was imprinted into polymers made from methacrylic acid and/or 2-vinyl pyridine. The resulting particles were entrapped in a plasticized poly(vinyl chloride) (PVC) matrix. The sensors exhibit near-Nernstian response in steady state evaluations, and detection limits range from 0.40 to 1.0 μg mL−1, respectively, and are independent of pH values at between 2 and 6, and 8 and 11, respectively. Good selectivity was observed over several potential interferents. In flowing media, the sensors exhibit fast response, a sensitivity of 68.2 mV per decade, a linear range from 79 μM to 2.5 mM, a detection limit of 20 μg mL−1, and a stable baseline. The sensors were successfully applied to field monitoring of norfloxacin in fish samples, biological samples, and pharmaceutical products.
- A biomimetic photonic crystal sensor for label-free detection of urinary venous thromboembolism biomarkerPublication . Resende, Sara; Frasco, Manuela F.; Sales, Maria Goreti FerreiraThis work reports a novel label-free sensor combining photonic crystals and molecularly-imprinted polymer materials for targeting a protein in human samples within levels of clinical interest. This concept was applied to detect fibrinopeptide B (FPB) in urine, a biomarker of venous thromboembolism, which is a disease of great concern and demands innovative point-of-care devices for improved diagnosis. The molecularly-imprinted photonic polymer (MIPP) was obtained by tailoring an imprinted polymer on highly ordered silica nanoparticles assembled by vertical deposition. Owing to the hierarchical structure, the resulting MIPP exhibited optical properties that changed upon rebinding of the target analyte, FPB. Thus, the changes in reflectance intensity enabled a rapid and sensitive detection of FPB in human urine. The observed response was linear between 0.2 ng mL−1 and 22 ng mL-1 and showed a limit of detection of 0.13 ng mL-1. These features of the sensing material allow the assessment of urinary FPB at relevant clinical levels. In addition, the sensor was selective for FPB compared to the standard biomarker of venous thromboembolism, D-Dimer. The stability of the material was evidenced by the reusability study, which demonstrated reversibility of the maximum intensity after three cycles of recognition and regeneration. Moreover, the sensor showed good performance for the spiked FPB detection in control human urine. Overall, the application of such label-free sensor offers high selectivity and stability, as well as easy operation. It may constitute an alternative method for non-invasive and real-time detection of various protein biomarkers in point-of-care.
- Biomimetic Sensor Potentiometric System for Doxycycline Antibiotic Using a Molecularly Imprinted Polymer as an Artificial Recognition ElementPublication . Kamel, Ayman H.; Moreira, Felismina T. C.; Sales, M. Goreti F.Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. A Biomimetic Sensor Potentiometric System was developed for assessment of doxycycline (DOX) antibiotic. The molecularly imprinted polymer (MIP) was synthesized by using doxycycline as a template molecule, methacrylic acid (MAA) and/or acrylamide (AA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensing elements were fabricated by the inclusion of DOX imprinted polymers in polyvinyl chloride (PVC) matrix. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors under static (batch) mode of operation reveals near-Nernstian response. MIP/MAA membrane sensor was incorporated in flow-through cells and used as detectors for flow injection analysis (FIA) of DOX. The method has the requisite accuracy, sensitivity and precision to assay DOX in tablets and biological fluids.
- Biomimetic sensors of molecularly-imprinted polymers for chlorpromazine determinationPublication . Moreira, Felismina T. C.; Sales, M. Goreti F.A new man-tailored biomimetic sensor for Chlorpromazine host-guest interactions and potentiometric transduction is presented. The artificial host was imprinted within methacrylic acid, 2-vinyl pyridine and 2-acrylamido-2-methyl-1-propanesulfonic acid based polymers. Molecularly imprinted particles were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. Slopes and detection limits ranged 51–67 mV/decade and 0.46–3.9 μg/mL, respectively, in steady state conditions. Sensors were independent from the pH of test solutions within 2.0–5.5. Good selectivity was observed towards oxytetracycline, doxytetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, sulfadiazine, trimethoprim, glycine, hydroxylamine, cysteine and creatinine. Analytical features in flowing media were evaluated on a double-channel manifold, with a carrier solution of 5.0 × 10−2 mol/L phosphate buffer. Near-Nernstian response was observed over the concentration range 1.0 × 10−4 to 1.0 × 10−2 mol/L. Average slopes were about 48 mV/decade. The sensors were successfully applied to field monitoring of CPZ in fish samples, offering the advantages of simplicity, accuracy, automation feasibility and applicability to complex samples.
- A cellulose-based colour test-strip for equipment-free drug detection on-site: application to sulfadiazine in aquatic environmentPublication . Teixeira, Carla N. O.; Sales, Maria Goreti FerreiraThis work develops a simple and innovative test-strip to monitor antibiotics in aquaculture facilities by an equipment-free approach. It consists of a low-cost disposable cellulose paper that was chemically modified to produce a colour change when in contact with a given antibiotic. In brief, the cellulose substrate was subject to oxidation with periodate, followed by amination with chitosan binding and modification with Cu(II). The test strip was then dipped in the target solution and the intensity of the colour generated therein revealed the concentration of antibiotic present for concentrations higher than 0.5 mM. The higher the concentration in sulfadiazine (SDZ), the more intense the pink colour formed in the final solution, which was also turbid due to the insolubility of the formed product. This colour intensity also varied linearly with the logarithm of the SDZ concentration (from 0.5 to 5 mM), when plotted against the sum of the RGB coordinates extracted from digital pictures. The linear equation of this response was represented by (R + G + B) = − 256.1 log(SDZ, mol/L) − 362.0, with an R-squared of 0.9913. The test-strip was stable for at least 15 days and was selective in the presence of tetracycline and difloxacin, while the response to other members of the sulfadiazine family requires prior evaluation. Overall, the test-strips developed herein are inexpensive and provide valuable (semi-) quantitative data for monitoring SDZ in waters, a most valuable approach to control and reduce the level of antibiotics in fish tanks, which in turn may reduce the costs of fish production and the environmental concerns linked to this practice. Moreover, the test strip uses a cellulose substrate that has little environmental impact upon discard.