Aplicação de um sistema de multi-sensores para a detecção de gliadinas: discriminação semi-quantitativa entre alimentos com glúten e sem glúten

A doença celíaca caracteriza-se pela intolerância ou hipersensibilidade à ingestão de prolaminas existentes no trigo, centeio, cevada e aveia. As proteínas do glúten do trigo contêm aproximadamente 50% de prolaminas, denominadas por gliadinas. O tratamento para a doença celíaca consiste em fazer uma dieta livre de glúten. Assim sendo, torna-se imperativo dispor de metodologias analíticas capazes de detectar e quantificar o teor de glúten em alimentos, especialmente em alimentos rotulados “sem glúten”. Neste trabalho, estudou-se a aplicabilidade de um sistema de multi-sensores (etongue), com 36 membranas poliméricas de sensibilidade cruzada como técnica analítica alternativa para análise qualitativa e semi-quantitativa de produtos alimentares. O objectivo foi estabelecer uma distinção, entre alimentos “com” e “sem glúten”. A discriminação entre os alimentos analisados baseou-se na capacidade do sistema multisensor fornecer diferentes perfis potenciométricos consoante diferentes conteúdos de gliadinas presentes nos alimentos, após extracção com uma solução aquosa de etanol a 70%. Os perfis de sinais do sistema de multi-sensores em conjunto com métodos estatísticos multivariados de reconhecimento de padrões, nomeadamente a análise discriminante, foram utilizados para diferenciar alimentos “com” e “sem glúten”. Foram analisados quinze alimentos comercializados em Portugal e, adquiridos em supermercados: 8 alimentos cujo rótulo indicava a presença de glúten e 7 alimentos, com indicação no rótulo de ausência de glúten. O teor de gliadinas nos alimentos estudados foi confirmado por cromatografia líquida de alta resolução, após a sua extracção. O sistema de multi-sensores utilizado, apresentou um desempenho satisfatório na diferenciação de extractos com diferentes teores de gliadinas, permitindo discriminar alimentos “com” e “sem glúten” com sensibilidade e especificidades globais superiores a 95% nos dados originais e de 75% no processo de validação cruzada. Além disso permitiu classificar de forma semi-quantitativa um alimento em 3 grupos: alimento sem glúten (<10 ppm), alimento com teor de glúten (20-40 ppm) e alimento com teor de glúten (100-400 ppm). Nesta discriminação obteve-se sensibilidades e especificidades globais de 100% e superiores a 79% para os dados originais e procedimento de validação cruzada (predição), respectivamente. Celiac disease is characterized by intolerance or hypersensitivity to ingested prolamins, which are composites of wheat, rye, barley and oats. Gluten proteins of wheat are constituted of approximately 50% of prolamins named gliadins. The treatment for celiac disease is gluten - free diet. A multisensor potentiometric (e-tongue) with 36 cross-sensibility polymeric membranes was applied for qualitative foodstuffs analysis. The objective was to distinguish gluten-containing from gluten-free foods. The discrimination was based on the capability of the e-tongue device to detect different gliadins contents. The e-tongue signal profiles were used together with supervised multivariate statistical methods for pattern recognition. A set of 15 Portuguese foods (7 foods samples with gluten-free indicating and 8, indicating gluten-containing), purchased in commercial supermarkets were analyzed. The multisensor systems used, showed satisfactory performance in the differentiation of extracts with different levels of gliadins, allowing discriminating food "with gluten" and "gluten free" with an overall sensitivity and specificity higher than 95% in the original data and 75% for the cross-validation procedure. Moreover it allowed to semi-quantitatively classify food samples into 3 groups: gluten-free food (<10 ppm), gluten-containing foods (20-40 ppm) and gluten-containing food (100-400 ppm). This discrimination was achieved with an overall sensitivity and specificity of 100% and greater than 79% for the original data and cross-validation (prediction) procedure, respectively. The satisfactory results obtained showed that the multisensor potentiometric device developed based on lipo-polymeric membranes can be used as an effective tool in the preliminary detection of gluten in foods. Os resultados satisfatórios obtidos mostraram que o sistema potenciomérico de multi-sensores desenvolvido com base em membranas lipo-poliméricas pode ser utilizado como uma ferramenta na detecção preliminar de glúten em alimentos

Development of a novel aptamer-based multi-sensor device for the detection of osteopontin

Doctoral Dissertation for PhD degree in Chemical and Biological EngineeringBreast cancer is the most prevalent cancer in women worldwide and its mortality is closely associated with the development of metastasis of the primary tumor. It is currently the fifth cause of death from cancer (522,000 deaths), with 1.67 million new breast cancer cases estimated in 2012. An early diagnosis is crucial to improve patients survival and disease prognostic. Hence, sensitive and specific methods ought to be developed and improved towards this purpose. The use of aptamers as bioreceptors in the design of electrochemical aptasensors has offered new prospects in diagnostic assays in the areas of protein disease biomarkers detection. In this thesis, the possibility of using an electrochemical aptamer-based biosensor for the detection of osteopontin (OPN) has been studied. Aptamers are artificial oligonucleotides (DNA or RNA) that bind their targets with high affinity and specificity. OPN is a glycoprotein present in many tissues and body fluids that is considered a potential biomarker for breast cancer. Initially, an RNA aptamer against OPN, previously reported, was used as the bioreceptor element in the design of an aptasensor. The OPN-binding RNA aptamer was immobilized onto a screen printed gold electrode through a biotin-streptavidin interaction. The electrochemical RNA aptasensor showed a good sensitivity to human OPN and for thrombin (a protein commonly found in human serum). Thus, other specific bioreceptors that could enable the selective detection of human OPN were selected. A DNA aptamer was isolated through the SELEX methodology. This aptamer was characterized by fluorescence assays and presented a good affinity to OPN, thus it was used as the bioreceptor in the design of a new electrochemical DNA aptasensor. This aptasensor exhibited a better performance regarding the sensitivity and selectivity for the detection of human OPN as compared to the above mentioned RNA aptasensor that used the same electrode surface, immobilization method and voltammetry techniques. Finally, these two aptamers (DNA and RNA) were used to develop a labelfree aptasensor array that could simultaneously detect OPN using a dual-screen printed gold electrode. In conclusion, the electrochemical aptasensors herein developed can be an alternative to the standard methods currently used to detect/quantify protein disease biomarkers and could be a useful tool in the diagnosis of breast cancer disease.Este trabalho apresentado nesta tese foi apoiada financeiramente por uma bolsa de doutoramento (Ref.ª SFRH / BD / 65021/2009) da Fundação para a Ciência e a Tecnologia (FCT) e do Fundo Social Europeu (POPHQREN), o Projeto FCT Estratégico UID/BIO/04469/2013 unidade, o projeto RECI/certificação-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) e do Projeto "BioInd – “Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE- 07-0124-FEDER-000028, co-financiado pelo Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER.O cancro da mama é o mais frequente entre mulheres em todo o mundo, a sua mortalidade está associada ao desenvolvimento de metástases do tumor primário. Atualmente, é a quinta causa de morte por cancro (522.000 mortes), tendo sido diagnosticados cerca de 1,67 milhões de novos casos em 2012. O diagnóstico precoce é crucial para aumentar a taxa de sobrevivência e sucesso do prognóstico. Neste sentido, urge o desenvolvimento de métodos sensíveis e específicos para melhorar o diagnóstico precoce. O uso de aptâmeros, como elementos de reconhecimento biológico no desenho de biossensores eletroquímicos ( aptasensors), tem oferecido novas perspetivas de diagnóstico em particular nas áreas de deteção de proteínas associadas ao cancro (biomarcadores). Nesta tese, avaliou-se a possibilidade de usar um biossensor eletroquímico, baseado em aptâmeros, para a deteção da osteopontina humana (OPN). A OPN é uma glicoproteína presente em muitos tecidos e fluidos corporais, sendo considerada um potencial biomarcador do cancro da mama. Os aptâmeros são ácidos nucleicos artificiais de cadeia simples (ADN ou ARN) que reconhecem e se ligam com elevada afinidade e especificidade aos seus alvos. Inicialmente, um aptâmero de ARN específico para a OPN, descrito na literatura, foi utilizado como elemento de reconhecimento no desenvolvimento de um “ aptasensor”. O aptâmero foi imobilizado num elétrodo de ouro através de uma interação biotina-estreptavidina. O biossensor eletroquímico, baseado no aptâmero de ARN, apresentou boa sensibilidade para OPN humana e para trombina (proteína presente no soro humano). Nesse sentido, selecionou-se outro aptâmero de ADN com maior especificidade e seletividade para a OPN humana usando para o efeito a metodologia SELEX. Este aptâmero ADN foi então usado no desenvolvimento de um novo biossensor eletroquímico. Este novo biossensor apresentou melhor sensibilidade e seletividade para a OPN humana comparativamente com o anteriormente referido, utilizando a mesma superfície de elétrodo, método de imobilização e técnicas voltamétricas. Finalmente, um multi-biossensor foi desenvolvido usando os dois aptâmeros (ADN e ARN) para detetar simultaneamente a OPN humana. Em conclusão, os biossensores electroquímicos desenvolvidos podem ser uma alternativa aos métodos convencionais utilizados para detetar/quantificar biomarcadores, bem como uma ferramenta útil para o diagnóstico precoce do cancro da mama

Identification of a Phytophthora cinnamomi glucanase inhibitor protein: a molecular factor associated to infection mechanism.

The oomycete P. cinnamomi, the causal agent of Castanea sativa ink disease, is one of the most destructive species of Phytophthora genus, and has been associated with the decline of several forest, ornamental and fruit trees and shrubs, causing enormous economic losses worldwide. Phytophthora cell walls are composed of glucans and have no chitin. Production of glucanase inhibitor proteins (GIPs) by Phytophthora species are thought to provide them a counter-defense against plant hosts β-1,3-glucanases (Rose et al., 2002), that otherwise would degrade these pathogen cell walls. GIPs belongs to the chymotrypsin family of serine proteases but are catalytically nonfunctional because one or more residues of the essential catalytic triad are absent. We report the identification of the gene encoding the first known P. cinnamomi GIP, presumably involved in the pathogen infection mechanism. Total genomic DNA was obtained from strain P. cinnamomi Pr120 and polymerase chain reaction was used to amplify a 308bp fragment of the GIP gene, using degenerate oligonucleotide primers, which were designed based on homology of previous published sequences of Phytophthora sp. GIP’s from EMBL databases. Full gene sequence length (1171bp) was obtained by flanking the known sequence with asymmetric PCR. P. cinnamomi GIP gene encodes a 269 amino acids protein with 28,818.2Da and a calculated global iso-electric point value of 8.54. It shares great identity and similarity with already described GIPs of P. sojae and P. infestans (E-values from 3.4e-49 to 2.6e-38), showing the importance of these proteins as effectors in plant-pathogen infection process. The UniProtKB/TrEMBL accession number for the sequence reported in this paper is B0B0H5_PHYCI

Development of an electrochemical RNA-aptasensor to detect human osteopontin

Electrochemical aptasensors may be used to detect protein biomarkers related to tumor activity. Osteopontin (OPN), a protein present in several body fluids, has been suggested as a potential biomarker since its overexpression seems to be associated with breast cancer progression and metastasis. In this work, a simple and label-free voltammetric aptasensor for the detection of OPN, using an RNA aptamer previously reported to have affinity for human OPN as the molecular recognition element, and the ferro/ferricyanide solution as a redox probe, was developed. The RNA aptamer was synthetized and immobilized in a working microelectrode gold surface (diameter of 0.8 mm) of a screen-printed strip with a silver pseudo-reference electrode and a gold counter electrode. The electrochemical behavior of the electrode surface after each preparation step of the aptasensor was studied using cyclic voltammetry and square wave voltammetry. The resulting voltammetric aptasensor was used to detect OPN in standard solutions. Cyclic voltammetry results showed that the aptasensor has reasonable detection and quantification limits (3.7±0.6 nM and 11±2 nM, respectively). Indeed, the detection limit falls within the osteopontin levels reported in the literature for patients with metastatic breast cancer. Moreover, the aptasensor is able to selectively detect the target protein in the presence of other interfering proteins, except for thrombin. Considering the overall results, a possible application of the aptasensor for cancer prognosis may be foreseen in a near future.FCT and FEDER under Program PT2020 (Project UID/EQU/50020/2013); by the Strategic Project PEst-OE/EQB/LA0023/2013 and by the project ref. RECI/BBB-EBI/ 0179/2012 (project number FCOMP-01-0124-FEDER-027462) funded by FCTFundação para a Ciência e a Tecnologia(FCT) through the PhD grant SRFH/BD/65021/2009

Characterization of transglutaminase elicitor precursor from plants pathogen Phytophthora cinnamomi

The oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that includes some of the most notorious pathogens of plants. Among these, members of the genus Phytophthora cause enormous economic losses on crop species as well as environmental damage in natural ecosystems. Phytophthora cinnamomi is the most widely distributed Phytophthora species, with nearly 1000 host species

Cloning and expression analysis of glucanase genes from Phytophthora cinnamomi

Phytophthora cinnamomi is a soil-borne pseudofungus belonging to the Class Oomycetes or „water moulds' in the Kingdom Chromista (Figure 1). Is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species, as well as of some 900 other woody perennial plant species Associated with this oomycete is the ink disease of Castanea Sativa Mill. Glucan endo-1,3-β-D-glucosidase (EC 3.2.1.39) catalyzes de hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defense by digesting wall components of the fungal pathogen. In yeast, 1,3-- glucanases have been studied for their role in germination, sporulation, mating and cell growth since they are regulated in cell cycle dependent manner, and are differentially expressed during vegetative growth, mating and the late stages of sporulating diploid. In plant, 1,3--glucanases have been characterized for their major role in plant defence, as well as for their involvement in germination, microsporogenesis and embryogenesis. In oomycetes, glucanases have been studied on a biochemical level for their possible role in hyphal tip growth and branching where there is thought to be a delicate balance between cell wall synthesis and hydrolyses. In the present work, we obtained a fragment with 1231bp of the endo-1,3--glucanase gene by standard PCR, using conserved primers and the whole genomic sequence with 2586 bp was obtained by amplifying the previous sequence by asymmetric PCR. The gene expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production

Development of an electrochemical aptasensor for the detection of human osteopontin

Electrochemical aptasensors, an emerging technology, enables the detection of protein biomarkers, which may be indicative of tumour activity. Osteopontin is a protein present in body fluids, being a possible biomarker since its overexpression has been related with breast cancer progression. An \RNA\ aptamer, described in the literature, with affinity for human osteopontin, was synthetized, immobilized in a microelectrode gold surface and used for development electrochemical aptasensor for human \OPN\ detection in standard solutions. Cyclic voltammetry results showed that this aptasensor allowed detecting human osteopontin with a detection limit of 8 nM, showing a satisfactory selectivity towards the target in the presence of others proteins, except for thrombin

Optimization of the aptamers’ immobilization conditions for maximizing the response of a dualaptasensor for cancer biomarker detection

Osteopontin (OPN) is a protein that is present in several body fluids and has been reported as a possible cancer biomarker, being its overexpression associated with tumour progression and metastasis [1,2]. A simple and sensitive method that allows the simultaneous detection of single or multiple cancer biomarkers is envisaged and may be an important tool in cancer diagnosis. In this work, two bioreceptors specific for OPN, a RNA aptamer (OPN-R3) previously described by Mi and co-workers [3] and a DNA aptamer (C10K2) developed by our research group, were biotinylated and immobilized on a dual-screen printed gold electrode through streptavidin-biotin interaction. The voltammetric signals generated by the dual-aptasensor array, after the formation of the aptamers-protein complex, were monitored using cyclic voltammetry (CV) and square- wave voltammetry (SWV), using [Fe(CN)6]−3/−4 as a redox probe. The optimal immobilization conditions for the dual-aptasensor array were established by response surface methodology. The maximum voltammetric response was obtained for a 0.5 μM aptamer concentration after 20 min of aptamers’ immobilization and 30 min of aptamer- OPN interaction time at an incubation temperature of 4ºC. The satisfactory preliminary results obtained, although needing further confirmation for synthetic or real human samples, point out that the proposed electrochemical dual-aptasensor array could be a simple and sensitive tool for the detection of OPN, as well as for other potential cancer biomarkers and therefore, may be applied in the future for cancer disease monitoring.This work was also financially supported by Project POCI-01–0145-FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER through COMPETE2020 and by national funds through FCT, Portugal. S. Meirinho also acknowledges the research grant provided by Project UID/EQU/50020/2013.info:eu-repo/semantics/publishedVersio

Development of an electrochemical aptasensor for protein detection

The development of aptamer-based electrochemical biosensors as an emerging technology has made the detection of small and macromolecular analytes easier, faster, and more suited for early detection of protein biomarkers. Biomarkers are produced by body organs or tumors and measure antigens on cell surfaces. When detected in high amounts in blood, they can be suggestive of tumor activity 1,2. These markers are more often used to evaluate treatment effects or to assess the potential for metastatic disease in patients with established disease. Osteopontin (OPN) is a protein found in all body fluids, and constitutes a possible biomarker because its overexpression has been related with breast cancer evolution and metastasis 3–5. Currently, biomarkers are commonly used for the development of diagnostic methods, allowing the detection of the disease in its initial stages. An electrochemical aptasensor for the detection of OPN was developed using an RNA aptamer immobilized on a gold screenprinted electrode (Au/SPE). The immobilized biotin-modified aptamer on Au/SPE constitutes the biorecognition element for the target protein and the electrochemical signal generated from the interaction aptamer-target protein was evaluated by cyclic voltammetry (CV). A decrease in the current as a consequence of protein binding to the aptamer was observed through the analysis of the electron flow produced by a redox reaction between ferri- and ferrocyanide. The electrochemical aptasensor herein developed presents a high specificity for OPN as compared with other proteins commonly found in the biological fluids