Percorrer por autor "Freitas, Maria"
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- 3D-nanostructured Au electrodes for the event-specific detection of MON810 transgenic maizePublication . Barroso, M. Fátima; Freitas, Maria; Oliveira, M. Beatriz P.P.; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; Delerue-Matos, CristinaIn the present work, the development of a genosensor for the event-specific detection of MON810 transgenic maize is proposed. Taking advantage of nanostructuration, a cost-effective three dimensional electrode was fabricated and a ternary monolayer containing a dithiol, a monothiol and the thiolated capture probe was optimized to minimize the unspecific signals. A sandwich format assay was selected as a way of precluding inefficient hybridization associated with stable secondary target structures. A comparison between the analytical performance of the Au nanostructured electrodes and commercially available screen-printed electrodes highlighted the superior performance of the nanostructured ones. Finally, the genosensor was effectively applied to detect the transgenic sequence in real samples, showing its potential for future quantitative analysis.
- Breast cancer biomarker (HER2-ECD) detection using a molecularly imprinted electrochemical sensorPublication . Pacheco, João; Rebelo, Patrícia; Freitas, Maria; Nouws, Henri; Delerue-Matos, CristinaThe extracellular domain of the human epidermal growth factor receptor 2 (HER2-ECD) is a protein breast cancer biomarker. Its quantification in peripheral blood could provide an important contribution to diagnostics and patient follow-up. In this work an electrochemical molecularly imprinted polymer (MIP) sensor for the quantification of HER2-ECD was developed. The MIP was electropolymerized by cyclic voltammetry using a solution containing phenol and HER2-ECD on a screen-printed gold electrode (AuSPE). The sensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analysis of HER2-ECD was performed by differential pulse voltammetry using ([Fe(CN)6]3−/4−as redox probe. The linear range was established in the concentration interval from 10 to 70 ng/mL HER2-ECD, with a limit of detection of 1.6 ng/L and a limit of quantification of 5.2 ng/mL. Through the analysis of other protein biomarkers, the MIP sensor was found to be selective. Furthermore, these proteins did not interfere in the analysis of the selected biomarker. The developed sensor was used for the analysis of spiked human serum samples, providing adequate recovery values and precise results. The outcomes of this study indicate that the developed MIP sensor could be useful in the non-invasive analysis of HER2-ECD in breast cancer patients.
- Electrochemical Biosensing in Cancer Diagnostics and Follow-upPublication . Freitas, Maria; Nouws, Henri; Delerue-Matos, CristinaIn cancer, screening and early detection are critical for the success of the patient's treatment and to increase the survival rate. The development of analytical tools for non‐invasive detection, through the analysis of cancer biomarkers, is imperative for disease diagnosis, treatment and follow‐up. Tumour biomarkers refer to substances or processes that, in clinical settings, are indicative of the presence of cancer in the body. These biomarkers can be detected using biosensors, that, because of their fast, accurate and point of care applicability, are prominent alternatives to the traditional methods. Moreover, the constant innovations in the biosensing field improve the determination of normal and/or elevated levels of tumour biomarkers in patients’ biological fluids (such as serum, plasma, whole blood, urine, etc.). Although several biomarkers (DNA, RNA, proteins, cells) are known, the detection of proteins and circulating tumour cells (CTCs) are the most commonly reported due to their approval as tumour biomarkers by the specialized entities and commonly accepted for diagnosis by medical and clinical teams. Therefore, electrochemical immunosensors and cytosensors are vastly described in this review, because of their fast, simple and accurate detection, the low sample volumes required, and the excellent limits of detection obtained. The biosensing strategies reported for the six most commonly diagnosed cancers (lung, breast, colorectal, prostate, liver and stomach) are summarized and the distinct phases of the sensors’ constructions (surface modification, antibody immobilization, immunochemical interactions, detection approach) and applications are discussed.
- Electrochemical Sensing Platforms for HER2-ECD Breast Cancer Biomarker DetectionPublication . Freitas, Maria; Nouws, Henri P. A.; Delerue-Matos, CristinaScreening and early diagnosis are crucial to increase the success of cancer patients’ treatments and improve the survival rate. To contribute to this success, distinct electrochemical immunosensing platforms were developed for the analysis of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2‐ECD) through sandwich assays on nanomaterial‐modified screen‐printed carbon electrodes (SPCEs). The most promising platforms showed to be SPCEs modified with (i) gold nanoparticles (AuNPs) and (ii) multiwalled carbon nanotubes combined with AuNPs. The antibody‐antigen interaction was detected using a secondary antibody labelled with alkaline phosphatase and 3‐indoxyl phosphate and silver ions as the enzymatic substrate. The electrochemical signal of the enzymatically generated metallic silver was recorded by linear sweep voltammetry. Under the optimized conditions, linear calibration plots were obtained between 7.5 and 50 ng/mL and the total assay time was 2 h 20 min, achieving LODs of 0.16 ng/mL (SPCE‐MWCNT/AuNP) and 8.5 ng/mL (SPCE‐AuNP), which are well below the established cut‐off value of 15 ng/mL for this cancer biomarker.
- Food allergen control: Tropomyosin analysis through electrochemical immunosensingPublication . Torre, Ricarda; Freitas, Maria; Costa‐Rama, Estefanía; Nouws, Henri; Delerue-Matos, CristinaRegulations of the EU obliges the indication of the presence of allergens on food labels. This work reports the development of an electrochemical immunosensor to determine tropomyosin (TPM) – a major shellfish allergen – prevailing in the muscles of crustacean species. Two linear ranges between the signal and TPM concentration were obtained: between 2.5 and 20 ng mL−1 and between 30 and 200 ng mL−1, with a lowest limit of detection of 0.47 ng mL−1. The selectivity of the optimized immunoassay, tested with other food allergens (e.g., Cyp c 1, a fish allergen), assures the effective detection of TPM, enabling successful control of foodstuff labelling. Several (12) foods, containing high and low TPM concentrations and TPM-free samples, were analysed using the sensor. A conventional ELISA kit and recovery assays were used to evaluate the accuracy of the results.
- High-performance electrochemical immunomagnetic assay for breast cancer analysisPublication . Freitas, Maria; Nouws, Henri P. A.; Keating, Elisa; Delerue-Matos, CristinaDespite the evolution of targeted therapies in oncology, some challenges such as screening and early diagnosis of cancer-related biomarkers still remain. The analysis of the Human Epidermal growth factor Receptor 2 (HER2) in biological fluids provides essential information for effective treatments. In this work we report the development of an electrochemical immunomagnetic bioassay for the analysis of the extracellular domain of HER2 (HER2-ECD) in human serum and cancer cells. Biomodified carboxylic acid functionalized magnetic beads (COOH-MBs) were used as the capture probe and an antibody labelled with alkaline phosphatase (AP) as the signalling probe. In the presence of HER2-ECD a sandwich complex was formed on the MBs, which were magnetically attracted to the surface of a screen-printed carbon electrode (SPCE). After the addition of 3-indoxyl phosphate and silver ions, used as the enzymatic substrate, the immunological interaction was detected by linear sweep voltammetry. Two linear concentration ranges were established: one between 5.0 and 50 ng/mL and another between 50 and 100 ng/mL. The developed assay provided a clinically useful detection limit (2.8 ng/mL) and has an adequate precision (Vx0 < 5%). The assay provided accurate results and was selective towards the target biomarker. Additionally, CTCs were analysed in human serum and a detection limit of 3 cells/mL was achieved for the HER+ breast cancer cell line SK-BR-3.
- Impedimetric immunosensors for the detection of Cry1Ab proteinfrom genetically modified maize seedsPublication . Freitas, Maria; Correr, Wagner; Cancino-Bernardi, Barroso; Delerue-Matos, Cristina; Zucolotto, ValtencirRegardless the controversies surrounding genetically modified organisms (GMO), their cultivation isconstantly increasing and in according to the EU legislation, labeling is mandatory for products con-taining EU-authorized-GMO higher than 0.9%. Thereby, new analytical strategies for rapid and effectivedetection of GMO on foodstuffs are required. In this work, an electrochemical immunosensor foreffective determination of Cry1Ab protein from MON810 transgenic maize (EU-authorized-GMO) isdescribed. The immunosensor was developed onto indium tin oxide (ITO) electrodes modified by3-aminopropyltrimethoxysilane (APTES) monolayer to covalently immobilize Anti-Cry1Ab polyclonalantibodies. The protein interaction with the polyclonal antibody (PAb) recognition platform was directlymonitored and measured by cyclic voltammetry and electrochemical impedance spectroscopy usingcommercially Cry1Ab protein. After the analytical features optimization a linear response from 1 to10 ng mL−1, a limit of detection (LOD) of 0.37 ng mL−1and a limit of quantification (LOQ) of 1.23 ng mL−1– which provided accurate results (RSD < 7.5%) – were achieved. The immunosensor allowed a simple andfast detection of Cry1Ab protein extracted from maize seeds with different GM maize mass percentages(0%, 0.5%, 1%, 2.5% and 5%). To crosscheck the detection of Cry1Ab protein, an enzyme-linked immunosor-bent assay (ELISA) was used. The results indicate that the immunosensor is suitable for the transgenicprotein Cry1Ab detection in GM maize representing a successfully tool to verify the compliance of theEU regulations.
- Iron oxide/gold core/shell nanomagnetic probes and CdS biolabels for amplified electrochemical immunosensing of Salmonella typhimuriumPublication . Freitas, Maria; Viswanathan, Subramanian; Nouws, Henri P. A.; Oliveira, M. Beatriz P. P.; Delerue-Matos, CristinaThere is an imminent need for rapid methods to detect and determine pathogenic bacteria in food products as alternatives to the laborious and time-consuming culture procedures. In this work, an electrochemical immunoassay using iron/gold core/shell nanoparticles (Fe@Au) conjugated with anti-Salmonella antibodies was developed. The chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles is reported. Fe@Au nanoparticles were functionalized with different self-assembled monolayers and characterized using ultraviolet-visible spectrometry, transmission electron microscopy, and voltammetric techniques. The determination of Salmonella typhimurium, on screen-printed carbon electrodes, was performed by square-wave anodic stripping voltammetry through the use of CdS nanocrystals. The calibration curve was established between 1×101 and 1×106 cells/mL and the limit of detection was 13 cells/mL. The developed method showed that it is possible to determine the bacteria in milk at low concentrations and is suitable for the rapid (less than 1 h) and sensitive detection of S. typhimurium in real samples. Therefore, the developed methodology could contribute to the improvement of the quality control of food samples.
- Magnetic dispersive micro solid-phase extraction and gas chromatography determination of organophosphorus pesticides in strawberriesPublication . Fernandes, Virgínia Cruz; Freitas, Maria; Pacheco, João; Oliveira, José Maria; Domingues, Valentina; Delerue-Matos, CristinaMagnetic nanoparticles (MNPs) with different sizes and characteristics were synthesized to be used as a QuEChERS sorbents for the determination of seven organophosphorus pesticides (OPPs) in strawberries by gas chromatography analysis with flame photometric and mass spectrometry detection. To achieve the optimum conditions of modified QuEChERS procedure several parameters affecting the cleanup efficiency including the amount of the sorbents and cleanup time were investigated. The results were compared with classical QuEChERS methodologies and the modified QuEChERS procedure using MNPs showed the better performance. Under the optimum conditions of the new methodology, three spiking levels (25, 50 and 100 μg kg-1) were evaluated in a strawberry sample. The results showed that the average recovery was 93% and the relative standard deviation was less than 12%. The enrichment factor ranged from 111 to 145%. The good linearity with coefficients of determination of 0.9904-0.9991 was obtained over the range of 25-250 μg kg-1 for 7 OPPs. It was determined that the MNPs have an excellent function as sorbent when purified even using less amount of sorbents and the magnetic properties allowed non-use of the centrifugation in cleanup step. The new methodology was applied in strawberry samples from conventional and organic farming. The new sorbents were successfully applied for extraction and determination of OPPs in strawberries.
- Molecularly imprinted electrochemical sensor for the point-of-care detection of a breast cancer biomarker (CA 15-3)Publication . Pacheco, João; Silva, Marta S.V.; Freitas, Maria; Nouws, Henri; Delerue-Matos, CristinaThe incidence of breast cancer has been increasing over the years. To control and monitor this disease several tumor biomarkers have been proposed for early diagnosis, patient follow-up and/or treatment guidance. The only serum breast cancer biomarker in current use is the cancer antigen 15-3 (CA 15-3). In this work a molecularly imprinted polymer (MIP)-based electrochemical (voltammetric) sensor to monitor breast cancer was developed, based on direct surface imprinting of CA 15-3 on a screen-printed gold electrode (Au-SPE). The imprinting was performed in two steps: (1) adsorption of CA 15-3 on the surface of the Au-SPE and (2) electropolymerization of 2-aminophenol around the adsorbed protein. After extraction of the imprinted protein voltammetric analysis was conducted using hexacyanoferrate(II/III) as redox probe, measuring the signals before and after protein binding. The sensor was characterized by voltammetric techniques and electrochemical impedance spectroscopy, and the analytical responses of imprinted and non-imprinted polymer sensors were studied. A linear relationship between the peak current intensity of the redox probe and the logarithm of CA 15-3 concentration was established between 5 and 50 U mL−1, achieving a limit of detection of 1.5 U mL−1. The prepared MIP-sensor provides fast (15 min) analysis and is cheap, easy to prepare, disposable and could easily be integrated in small portable point-of care devices.