ISEP – BioMark – Pósteres apresentados a eventos científicos
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- Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric DetectionPublication . Rebelo, Tânia S. C. R.; Sales, M. Goreti F.; Noronha, João P.C.; Costa-Rodrigues, JoãoProstate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.
- A novel antibody-like material for breast cancer antigen CA15-3, used to track breast cancer by potentiometric transductionPublication . Santos, Alexandra R. T.; Moreira, Felismina T. C.; Sales, M. Goreti F.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.
- Host-Tailored Sensors for Carnitine Potentiometric Measurements based on Surface Molecular ImprintingPublication . Truta, Liliana A.A.N.A.; Ferreira, Nádia S.; Sales, M. Goreti F.A novel surface molecularly imprinted polymer (MIP) is presented for Carnitine (CRT), a potential biomarker of ovary cancer. It consists in a 3D polymeric network created on top of graphene layers and around the target template. The polymeric structure was obtained after radical polymerization of (vinylbenzyl)trimethylammonium chloride, 4-styrenesulfonic acid and vinyl pivalate, including in the reaction mixture ethylene glycol dimethacrylate as cross-linker and ammonium persulphate as initiator. Non-imprinted polymer (NIP) material was also produced, by excluding the template from the procedure. The imprinted material was further used for the selective determination of CRT by potentiometric transduction. A selective membrane was prepared for this purpose by using the MIP material as ionophore, and dispersing it in a plasticized poly(vinylchloride) matrix with a suitable charged lipophilic additive. All membranes were casted over a solid conductive support made of graphite and applied over the smaller end of an insulin syringe. The best membranes were also applied over conductive glass/plastic. Control membranes were also produced by replacing MIP by NIP material. The potentiometric performance of the above electrodes was evaluated against CRT solutions of increasing concentrations. Overall, the best devices displayed linear response with average slope and detection limit of 47.28 mV.decade-1 and 3.55x10-6 mol.L-1, respectively. The effect of pH upon the potentiometric response was evaluated for different buffer solutions (within 2-9) and the best performance for this sensor was obtained with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer of pH 5.2. Good selectivity was observed against albumin, ascorbic acid, glucose, creatinine and urea, tested for concentrations up to their normal physiologic levels in urine. The application of the devices to the analysis of spiked samples showed recoveries ranging from 91% (± 6.8%) to 118% (± 11.2%), with relative errors below -20%. Overall, the combination of the MIP sensory material with a suitable selective membrane and electrode design has lead to a promising tool for point-of-care applications, when applied to field monitoring of CRT in biological samples.
- Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric TransductionPublication . Moreira, Ana P. T.; Moreira, Felismina T. C.; Sales, M. Goreti F.Human hemoglobin (Hb) is a globular metalloprotein, present in the blood and involved in gas transport. Hb-associated disturbances are related to several diseases, such thalassemia, anemia, heart disease and leukemia, or to side-effects from other diseases, such as cancer. Overall, it is of great importance to know the concentration of Hb in the blood in many health-related conditions. There are many methods described in the literature for determining Hb. Most of these rely on antibody/antigen interactions, due to the high selectivity of the affinity reaction taking place between these biomolecules. However, the use of antibodies for Hb determination in routine clinical use is very expensive, due to the high cost of the material, the need for special handling and storage, and the non-reusability. These constraints may be limited by replacing natural antibodies by plastic receptors, obtained by molecular imprinting procedures. Thus, this work describes a novel smart plastic antibody material (SPAM) by surface imprinting technique for the detection of Hb and its application to design small, portable and low cost potentiometric devices. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its subsequent interaction with different vinyl monomers, of different chemical functions and ionic charges. Control materials were designed in parallel to assess the ability of establishing stereochemical recognition of Hb and the effect of the kind/charge of the monomers employed. Scanning Electron Microscopy analysis confirmed the surface modification of the silica material used for imprint. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Suitable emf variations were detected only for selective membranes having a SPAM material and a charged lipophilic anionic additive. All control materials were unable to produce a potentiometric response. Overall, good features were obtained for SPAM-based selective membranes carrying an anionic lipophilic additive. In HEPES buffer of pH 5, limits of detection were 43.8μg/mL for a linear response after 83.8μg/mL with a cationic slope of +40.4mV/decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.
- New modified electrochemical conductive paper support for BSA detectionPublication . Ferreira, Nádia S.; Moreira, Ana P. T.; Sales, M. Goreti F.Chemical sensors and biosensors are widely used to detect various kinds of protein target biomolecules. Molecularly Imprinted Polymers (MIPs) have raised great interest in this area, because these act as antibody-like recognition materials, with high affinity to the template molecule. Compared to natural antibodies, these are also of lower cost and higher stability. There are different types of supports used to carry MIP materials, mostly of these made of gold, favourably assembled on a Screen Printed Electrode (SPE) strategy. For this work a new kind of support for the sensing layer was developed: conductive paper. This support was made by modifying first cellulose paper with paraffin wax (to make it waterproof), and casting a carbon-ink on it afterwards, to turn it conductive. The SPAM approach previously reported in1 was employed herein to assemble to MIP sensing material on the conductive paper. The selected charged monomers were (vinylbenzyl) trimethlammonium chloride (positive charge) or vinylbenzoic acid (negative charge), used to generate binding positions with single-type charge (positive or negative). The non-specific binding area of the MIP layer was assembled by chronoamperometry-assisted polymerization (at 1 V, for 60, 120 or 180 seconds) of vinylbenzoate, cross-linked with ethylene glycol vinyl ether. The BSA biomolecules lying within the polymeric matrix were removed by Proteinase K action. All preparation stages of the MIP assembly were followed by FTIR, Raman spectroscopy and, electrochemical analysis. In general, the best results were obtained for longer polymerization times and positively charged binding sites (which was consistent with a negatively-charged protein under physiological pH, as BSA). Linear responses against BSA concentration ranged from 0.005 to 100 mg/mL, in PBS buffer standard solutions. The sensor was further calibrated in standard solutions that were prepared in synthetic or real urine, and the analytical response became more sensitive and stable. Compared to the literature, the detection capability of the developed device is better than most of the reported electrodes. Overall, the simplicity, low cost and good analytical performance of the BSA SPE device, prepared with positively charged binding positions, seems a suitable approach for practical application in clinical context. Further studies with real samples are required, as well as gathering with electronic-supporting devices to allow on-site readings.
- Carnitine tailored Sensors on Surface Molecular Imprinting based on Graphene layersPublication . Truta, Liliana A.A.N.A.; Ferreira, Nádia S.; Sales, M. Goreti F.A new biosensor based on surface molecularly imprinted polymer (MIP) on graphene layers was successfully developed. It consists in a 3D polymeric network created on top of surface and around the target template, Carnitine (CRT), a potential biomarker of ovary cancer. The polymeric structure was obtained after radical polymerization of (vinylbenzyl)trimethylammonium chloride, 4-styrenesulfonic acid and vinyl pivalate, including in the reaction mixture ethylene glycol dimethacrylate as cross-linker and ammonium persulphate as initiator. Non-imprinted polymer (NIP) material was also produced, by excluding the template from the procedure. The imprinted graphene structures were further used for the selective determination of CRT by potentiometric transduction. For this purpose, a selective membrane was prepared by using the MIP material as ionophore, and dispersing it in a plasticized poly(vinylchloride) matrix, that included (or not) a suitable amount of charged lipophilic additive. The membranes were casted over a solid conductive support, made of graphite or of conductive glass. Control membranes were also produced by replacing MIP by NIP material. The potentiometric performance of the above electrodes was assessed against CRT solutions of increasing concentrations. Graphite supports displayed the best analytical features, with average slope and detection limit of 40.51 mVdecade-1 and 3.55x10-6 molL-1, respectively. The effect of pH upon the potentiometric response was evaluated for different buffer solutions (within 2-9) and the best performance for this sensor was obtained with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer of pH 5.2. The interference effect of albumin, ascorbic acid, glucose, creatinine and urea in the performance of the electrochemical unit was tested for concentrations up to their normal physiologic levels in urine and good selectivity was observed. The application of the devices to the analysis of spiked samples showed recoveries ranging from 91% (± 6.8%) to 118% (± 11.2%), with relative errors below -20%. Overall, the combination of the MIP sensory material with a suitable selective membrane and electrode design showed to be a promising tool for point-of-care applications.
- A biomimetic sensor for monitoring oxidative stress biomarker in point-of-carePublication . Martins, Gabriela V.; Fortunato, Elvira; Fernandes, Helena R.; Sales, M. Goreti F.Free radicals and other reactive species are constantly generated in vivo and can cause oxidative damage to biomolecules, a process that seems to play an important role at the origin of cancer. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a major product of DNA hydroxylation and is considered a biomarker of damage caused by oxidative stress (OS). Thus, early diagnosis of OS biomarkers may be used as a fundamental tool in cancer prevention and in more efficient therapeutic strategies. For this purpose, a biomimetic sensor for 8-OHdG detection and quantification by Electrochemical Impedance Spectroscopy (EIS) is proposed herein. The biomimetic sensor was obtained by modifying a clean gold (Au) electrode with a OH-terminal thiol compound, followed by direct electropolymerization of phenol in the presence of 8-OHdG. The biomimetic/Au acted as working electrode, while glassy carbon and Ag/AgCl were used as counter and reference electrodes, respectively. Electropolymerization of phenol was performed by Cyclic Voltammetry (CV) over the potential range 0.2 to 0.9 V in pH 7.0 PBS buffer, enabling the formation of a non-conductive layer. Non-imprinted materials (NIM) were also performed by removing the template from the procedure and, then, the ability of the polymer to interact non-specifically with the template was measured. Preliminary results showed the development of a direct and label-free biomimetic sensor with good performance, stability and sensibility. In particular, only MIP material was able to rebind to the target molecule and produce a linear response against EIS on the range 0.010 to 10ng/ml. Overall, the biosensor described herein is simple, precise and may allow routine use for biological samples on-site.
- A new biomimetic sensor for detecting carnitine, a potential biomarker in ovarian cancerPublication . Tavares, Ana P. M.; Ferreira, Nádia S.; Truta, Liliana A.A.N.A.; Sales, M. Goreti F.Carnitine (CRT) displays an important role in cellular metabolism and energy production. It has actions that include the metabolites associated with glycolysis and β-oxidation of fatty acids. The change of its levels in biological fluids has been associated to the presence of ovarian cancer, making CRT a potential biomarker of the disease. Sensitive CRT determination (in low levels) becomes therefore important, for which a low cost and sensitive device would be appreciated. A biomimetic polymer is proposed herein for this purpose, produced by bulk electropolymerization around a hydrophobic paper substrate that was made conductive by casting a graphite-based ink. Materials and methods The electrode substrate was prepared by modifying cellulose paper, first with solid wax and after with carbon ink. The hydrophobicity of the paper was tested by contact angle and the ink properties evaluated by Thermogravimetry, Raman Spectroscopy and FTIR. Two different biomimetic materials were electropolymerized over the carbon conductive support: 3,4-ethylenedioxythiophene (EDOT) and dodecilbenzenesulfonic acid sodium salt (NaDBS). The polymeric film depositions were obtained by chronoamperometry at 0.9 V vs Ag/AgCl during 240 s. The obtained sensors were characterized by Electrochemical Impedance Spectroscopy (EIS), in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.0.
- Disposable immunosensor with simple antibody orientation for label-free real-time detection of a cancer biomarkerPublication . Ferreira, Nádia S.; Sales, M. Goreti F.This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2’-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The Anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented proposal favored Ab/Ag affinity. The immunosensor design was evaluated by Quartz-Crystal microbalance with Dissipation, Atomic Force Microscopy, Electrochemical Impedance Spectroscopy (EIS) and Square-Wave Voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charged transfer resistance across the electrochemical sep-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from Glucose, Urea and Creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.
- A disposable glass-based immunosensor for monitoring the cancer biomarker CEA in urinePublication . Truta, Liliana A.A.N.A.; Sales, M. Goreti F.Immunosensing is a fast and cost-effective method in clinical diagnosis, relying on antibody-antigen interaction for the quantitative detection a specific antigen. Different types of antigen species have been screened in this context, including tumor markers, assessed by enzyme-linked immunosorbent, chemiluminescence and electrochemical methods1. Tumor markers are biomolecules in tumor cells or body fluids that may be associated with cancer diseases. Carcinoembryonic antigen (CEA) is among such markers, being employed in clinical diagnosis of over 80% colorectal cancers, with relatively little expression in normal mucosa2,3. Fast determination of CEA at low cost is presently required, to enable its use over wide screening programs and application in point-of-care context. This work presents a new simple immunoassay method for CEA detection in urine using a disposable glass-based immunosensor coupled to electrochemical detection. Conductive glass covered by fluorine doped tin oxide (FTO) was used as conductive support and modified with anti-CEA by means of bottom-up approach. All stages involved in the biochemical modification were followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The analytical performance of the device was fully characterized by EIS, proving sensitive readings of CEA from 2.5x10-3 to 0.10 ng/mL. The immunosensor was applied to real urine analysis from healthy individual spiked with the antigen. Overall, the combination of the sensory material with electrode design has lead to a promising tool for point-of-care applications, when applied to field monitoring of CEA in urine samples.