ISEP – BioMark – Pósteres apresentados a eventos científicos
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- 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.
- 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%.
- Sol-gel biomimetic material designed to target CEA cancer biomarkerPublication . Truta, Liliana A.A.N.A.; Sales, M. Goreti F.Introduction Carcinoembryonic antigen (CEA) is an important tumor marker responsible for clinical diagnosis of over 95% of all colon tumors, 50% of breast tumors, as well as tumors of the lung cancer or ovarian carcinoma [1]. The detection of CEA levels in biological samples plays an important role in the pre-diagnosis evaluation and in the follow-up examination during therapy stage [2]. The most common tool for the analysis of CEA in hospitals and clinical laboratories relies on ELISA-based procedures using antibodies as capturing probe. The overall principal offers the selectivity and sensitivity out coming from the use of antibodies, but it could be further improved by assembling the biosensors over a receptor platform and establishing a label-free measure by electrical impedance spectroscopy (EIS). Thus, the present work proposes the development of an immunosensor for CEA. Materials and Methods Electrochemical signals were measured in a Methrom Autolab potentiostat/galvanostat (Autolab PGSTAT302N) interfaced to a computer and controlled by NOVA 1.9 software. The chemical modification of the surface of the conductive glass was characterized by Raman spectroscopy with confocal microscopy (Thermo Scientific). The immunosensor was assembled by modifying conductive glass (with ITO) with an amino silane compound (APTES), activating the antibody via carbodiimide chemistry (EDAC/NHS) and binding the antibody to the amine surface over the ITO glass. The performance of the imunosensor was evaluated by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). Results and discussion The immunosensor made with an optimized composition displayed linear behavior against CEA concentration by EIS and SWV techniques. The corresponding linear ranges were 0.502-1.5 and 0.252-1.5ng/mL, with detection limits of 0.417 and 0.043 ng/mL, respectively. Overall, the obtained device may be potential method to apply for screening CEA in point-of-care due to the simplicity of fabrication, short time response, low cost and good sensitivity when compared to other analytical techniques, such as ELISA assays.
- 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.
- 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 molecularly imprinted sensor for sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative stress biomarkerPublication . Martins, Gabriela V.; Fortunato, Elvira; Fernandes, Helena R.; Sales, M. Goreti F.Early diagnosis of Oxidative Stress (OS) biomarkers can be used as a crucial tool in cancer prevention, treatment and survival. In this context, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a repair product of oxidized guanine lesions and has been acknowledged as a suitable biomarker of OS1. Under this scope, a simple and sensitive molecularly imprinted (MIP)-based sensor for detection of urinary 8-OHdG has been designed via electrochemical polymerization. The biomimetic film was assembled in-situ on the gold-modified electrode through electropolymerization of phenol monomer combined with the target molecule 8-OHdG. The electropolymerization of phenol was performed by Cyclic Voltammetry (CV) over the potential range 0.1 to 0.9 V in PBS buffer at pH 7.4, enabling the formation of a non-conductive layer. Several experimental parameters, such as, the initial concentration of the monomer and the ratio template-monomer, have been carefully optimized and the electrochemical performance of the designed MIP sensor was investigated by CV and Electrochemical Impedance Spectroscopy (EIS). In parallel, RAMAN and FTIR spectroscopies comproved the formation of polyphenol films on the electrode surface by electrochemical oxidation of phenol. Our results demonstrated that 8-OHdG molecule was successfully entrapped into the polymeric matrix, enabling a three-dimensional structure with numerous imprinted cavities sites. The developed electrochemical biosensor showed high sensitivity and selectivity towards 8-OHdG over the concentration range [0.1 - 100] pg/ml. Moreover, it was employed to detect 8-OHdG in urine samples as a non-invasive approach to assess the extent of DNA oxidative damage. Overall, this label-free biosensor constitutes a promising low-cost tool to be implemented as an easy-to-use protocol for sensitive detection of 8-OHdG in biological samples.
- 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.
- The potential of molecular imprinting as a biosensing devices for monitoring the CEA cancer biomarkerPublication . Truta, Liliana A.A.N.A.; Sales, M. Goreti F.Colorectal cancer is the third most common type of cancer and the major cause of the death throughout the world. Widely known, carcinoembryonic antigen (CEA) is an important tumour marker responsible for clinical diagnosis of 95% of all colon tumors1. The discovery of novel non-invasive biomarkers, as CEA, and its fast determination at low cost is presently required, to enable its use over wide screening programs and applications in point-of-care context, and, thus, its monitoring quite early. As a novel approach, this work proposes a novel support with molecular imprinted polymer (MIP) for CEA cancer biomarker based on carbon ink matrix linked by sol-gel chemistry on top of conductive glass covered by fluorine-doped tin oxide (FTO glass). In brief, the electrical biosensor was tailored on top of a disposable conductive glass electrode, following a bottom-up approach. The several stages of this process included the chemical modification of a homemade carbon ink layer and the assembly of a MIP or non-imprinted polymer (NIP) layer. The analytical performance of the obtained devices was followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Chemical modifications of the surface were characterized using Fourier Transform Infrared (FTIR), and Raman spectroscopy with confocal microscopy. Overall, the MIP/FTO glass-based device displayed linear responses to CEA in EIS assays from 2.5×10-3 µg.mL-1 to 1.25 µg.mL-1 in PBS buffer, with detection limits of 2.5×10-3 µg.mL-1. Successful detection of CEA was, also, achieved in spiked samples of fetal bovine serum. In conclusion, the devices developed are a promising tool for the monitoring of CEA in a point-of-care applications, due to its detection capability below the normal physiological levels expected for this cancer biomarker, simplicity of manufacture, low-cost and good sensitivity and selectivity.
- 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.
- Synthesis of an antibody-like material for the detection of AlbuminPublication . Tavares, Ana P. M.; Sales, M. Goreti F.A novel molecularly imprinted polymer (MIP) is presented for the detection of Albumin, currently a biomarker of several diseases. The material acted as an antibody for Albumin and was obtained through a bulk imprinting approach, by electropolymerizing Eriochrome blackT (EBT) around the target protein.