Percorrer por autor "Castanheira, Michelle"
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- Challenging invasive fungal infections: development of innovative electrochemical nanogenosensors to detect Candida spp.Publication . Castanheira, Michelle; Morais, Stephanie L.; Seguro, Isabel; Santos, Marlene; Lima, Luís; Pacheco, João; Barroso, M. FátimaDespite the considerable advances in the prevention and treatment of fungal infections, invasive fungus such as Candida spp., continues to be one of the major causes of morbidity and mortality. The Global Action Fund Infections reported that, annually, more than 300 million people are infected with fungal infection, from these, about 1.5 million ends up dying. Candida albicans is the most important fungal 66 opportunistic pathogen, it can cause superficial or invasive infections. Candida, often, causes superficial infections, per example in skin or mucous membranes with simple and effective treatment, however, also can break to the bloodstream and disseminate to internal organs. It has been observed among high-risk patients such as allogeneic stem-cell transplant recipients and with acute leukemia receiving highdose chemotherapy. These patients are at a heightened risk of developing infections due to the suppression of their immune system during the transplantation process. The diagnosis of systemic fungal infections persists as a problematic issue. Therefore, the development of more efficient, sensitive and specific methods for early diagnosis is need. In this study, an easy, rapid, and accurate detection methods for fungal infections in patients undergoing hematopoietic stem cell transplantation (HSCT) was designed. To address this challenge, it was developed an electrochemical nanogenosensor for the detection of Candida albicans.This nanogenosensor was assembled in an innovative low-cost electrochemical paper based analytical devices (ePAD). A sandwich hybridization reaction was used to enhaced the sensitivity of the electrochemical signal. Preliminary results demonstrated that using this nanogenosensors it was possible to detect Candida spp., in synthetic fungus sample. Despite these results, the optimization of the nanogenosensor in terms of quantifying Candida albicans is being carried out, which will be validated in future studies.The applicability in hospital environment relatively to sensitivity, accuracy, quickness response, challenges and opportunities will be discuss in future developments.
- Design of innovative electrochemical genosensors for honey fraud and quality detection by botanical origin authenticationPublication . Morais, Stephanie L.; Castanheira, Michelle; Pereira, Eduarda; Santos, Marlene; Domingues, Valentina F.; Delerue-Matos, Cristina; Barroso, M. Fátima; Santos, MarleneEnsuring food safety has become a concern for companies, consumers and the government alike, due to the increase of fraudulent and/or adulterated food products found in the global market [1,2]. Some common fraudulent strategies include mislabelling a product's geographical origin and blending it with lower-grade substances [3]. Among the frequently adulterated food products there is honey. Honey is a highly sought-after natural food with an elevated environmental, social and commercial value due to its rich nutritional profile and numerous health benefits. However, it is also vulnerable to adulteration [4]. Therefore, it is important to develop an analytical technique that can quickly, cheaply, and easily assess the quality and safety of honeys worldwide. In this work, an electrochemical genosensor for the detection of two plant species: Erica arborea and Castanea sativa in real honey samples was designed and optimized. The first step was the construction of the genosensor. For this, DNA-target probes capable of unequivocally detecting E. arborea and C. sativa DNA were selected and designed. As a sandwich-format strategy was adopted, their complementary probes were then cut into two smaller sequences to which a thiol group (DNA-capture) and a fluorescein (DNA-signalling) were attached. Using chronoamperometry, the enzymatic amplification of the electrochemical signal was achieved with a concentration range of 0.07 to 2.00 nM. These results were then compared to the DNA from certified E. arborea and C. sativa plant leaves, amplified by PCR, and 10 commercial honeys found in local Portuguese markets. The developed genosensor was successfully applied for the detection of the DNA from the extracted plant leaves and commercial honey samples. So, the developed genosensor is a promising cost-effective and innovative analytical method to detect the botanical origin of real honeys.
- Detecting Candida spp. through an innovative genosensorPublication . Castanheira, Michelle; Morais, Stephanie; Lima, Luís; Santos, Marlene; Barroso, M. Fátima; Santos, MarleneCandida spp. is the second most common cause of invasive fungal infection in hematopoietic stem cell transplantation (HSCT) patients and are associated with high mortality rates, ranging from 40 to 90%. Timely and accurate diagnosis is crucial for successful HSCT treatment. Current diagnostic methods, relying on conventional approaches, have limitations. Molecular and serological tests, although accurate, are time demanding and require specialized equipment. Biosensors are promising tools for point-of-care applications due to their low cost, ease of use and fast results. A low-cost electrochemical genosensor was developed for C. albicans detection. The genosensor construction required a DNA oligonucleotide sequence specific to C. albicans. A 90 bp synthetic DNA fragment was selected to identify this species. The sequence was cut into two fragments: a 25 bp DNA-capture probe and a 65 bp DNAsignaling probe. Screen-printed gold electrodes (SPGE) served as the electrochemical transducer. The sensor design included pretreatment, sensing, sandwich hybridization, and electrochemical detection. SPGE were pretreated with ethanol and ultrapure water. The sandwich assay, the DNA-capture probe bonded to the target DNA, then was immobilized on the working electrode overnight. A SAM interface with capture probes and 6-mercapto-1hexanol was used to ensure probe orientation. Sandwich hybridization improved selectivity by binding the target with a fluorescein-labeled probe. The anti-fluorescein antibody was conjugated with horseradish peroxidase, and the oxidized product was detected by chronoamperometry. Preliminary results show that the sensor was able to detect the synthetic of C. albicans DNA with high selectivity and sensitivity. Further studies will be made to enhance sensitivity parameters. The developed biosensor, with high sensitivity and selectivity, could provide a portable, user-friendly, and low-cost tool for monitoring fungal infections in HSCT recipients.
- Electrochemical genosensors as a new approach on plant DNA detection and quantification for honey authenticationPublication . Morais, Stephanie; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. FátimaHoney is a natural sweet food product with multiple nutritional and medicinal properties making it a healthy alternative to processed sugars. With the consumers’ recent interest and pur-chase of dietary products the global honey market has greatly increased. To keep up with produc-tion, or simply for financial gain, some producers/companies are now blending pure honey with cheaper substances that possess similar physical characteristics. As there are no notable visible dif-ferences between the pure and adulterated honey, it is extremely difficult to determine the purity of the available honeys. In this study, an electrochemical genosensor based on the sandwich format DNA hybridization reaction between two complementary probes was developed for the detection and quantification of Erica arborea pollen DNA in real samples. Analyzing public database platforms, a 98 base-pair DNA-target probe capable of unequivocally detecting the pollen from E. arborea was selected and designed. The complementary probe to the DNA-target oligonucleotide sequence was then cut into a 28 base-pair thiolated DNA-capture probe and a 70 base-pair fluorescein isothiocya-nate-labelled DNA-signaling probe. To increase the hybridization reaction, a self-assembled mono-layer formed from mixing the DNA-capture probe with mercaptohexanol was employed. Using chronoamperometry, the enzymatic amplification of the electrochemical signal was achieved with a concentration range of 0.03 to 2.00 nM. The DNA from certified E. arborea leaves was extracted using liquid nitrogen and mechanical grinding and the targeted region amplified by PCR. The de-veloped genosensor was successfully applied for the detection and quantification of the DNA con-centration of the extracted E. arborea plant leaves. So, the developed genosensor is a promising cost-effective and innovative analytical method to detect and quantify the DNA concentration of plant DNA in real honey samples.
- Ensuring food safety: electrochemical genosensors for the authentication of plant honey originPublication . Morais, Stephanie L.; Pereira, Eduarda; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. FátimaHoney is a high-quality and natural ingredient often consumed because of its unique sweet taste and multiple therapeutic and nutritional benefits. These properties are normally intrinsically connected to the regional flora from which the plant pollen is harvested. Hence, the botanical and geographical origins of honeys play a substantial role in the end product's composition. With the recent interest in natural food products many businesses, including the honey industry, have observed a significant expansion in production and market value. However, honey is susceptible to adulteration and, as more and more adulterated honeys are being found on the global market, it is difficult to monitor the safety and quality of all honey products, making honey fraud a serious problem for both consumers and the food industry. Some of the most prevalent fraudulent acts include mislabeling the botanical and geographic origin of honeys and mixing pure honey with inferior honeys, processed sugars, syrups, and other substances. Thus, there is a need to develop an analytical tool that can quickly, cheaply, and easily guarantee the quality and safety of honey. In this study, an electrochemical genosensor, based on a sandwich format DNA hybridization reaction between two complementary probes, for the detection and quantification of two pollen producing plant species: Erica arborea and Castanea sativa were designed and optimized. Analyzing public databases, two synthetic DNA-target sequences capable of unequivocally detecting the pollen from E. arborea and C. sativa were selected and designed. Their complementary oligonucleotide probes were also designed and cut into two distinct sequences: the DNA-capture and DNA-signaling probes. In order to recognize the two plant species in real honey and pollen DNA samples and optimize the hybridization procedure, a mixed selfassembled monolayer of each plant species’ DNA-capture probe and mercaptohexanol was used. Then, the electrochemical signal was enzymatically amplified using chronoamperometric measurements. A concentration range of 0.03 to 2.00 nM for E. arborea and 0.03 to 1.00 nM for C. sativa were obtained. The developed sensors were successfully applied for the detection and quantification of the two plant species in real plant samples and, thus, indicate the botanic origin of honeys. Therefore, the developed electrochemical genosensors are a viable and affordable analytical tool to authenticate the botanical origin of honeys, ensuring honey quality and safety for consumers as well as the industries.
- Improving honey authentication: Electrochemical genosensors for the detection of Erica arborea in commercial honeyPublication . Morais, Stephanie; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. Fátima; Santos, MarleneMonofloral honeys, like heather honey, are a highly sought-after ingredient. However, food safety concerns have emerged due to the number of adulterated honeys found in the global market [1,2]. Thus, this work aimed to develop an electrochemical genosensor capable of detecting the residual DNA of Erica arborea (heather flower) in commercial honey samples. Analyzing public databases, a 98-mer E. arborea DNA-target probe and its complementary sequence were selected and designed. The developed genosensor was constructed on screen-printed gold electrodes. The electrochemical signal resulted from the sandwich-format hybridization reaction between the DNA-target and its complementary probe, previously cut into a 28-mer DNA-capture probe and a 70-mer DNA-signaling probe labeled with a fluorescein. Honeys samples were acquired from the local markets and their DNA extracted and amplified using DNA kits. Employing chronoamperometry, a linear correlation was obtained in a 0.03 to 2.00 nM concentration range. The DNA from the purchased honey samples were successfully extracted and applied to the genosensor. The developed electrochemical genosensor was able to detect the presence of E. arborea in the analyzed honey samples. This device is a promising analytical tool to combat honey fraud, facilitate honey authenticity and promote food safety.
- Plant honey origin authentication: Use of electrochemical genosensors for food safety and quality controlPublication . Morais, Stephanie; Pereira, Eduarda; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. FátimaHoney is a natural high-quality food product consumed worldwide due to its diverse nutritional profile and beneficial medical properties. These factors, along with honey’s unique sweet taste and odour, make it one of the most popular products for a healthy diet. Nevertheless, food fraud is an increasing problem with various impacts on the economy, health, and in the environment, as honey prices are established based on its botanical origin and nutritional composition. In the European Union, honey is one of the most adulterated products found in the market. Mislabeling of a honey’s geographic origin and unethical mixing with low-grade honeys, sugars, and other substances are some of the common fraudulent practices. Hence, it is imperative to develop analytical tools to quickly, cheaply, and successfully identify fraudulent products. In this work, an electrochemical genosensor for the detection of two different plant species, Calluna vulgaris (the heather flower) and Castanea sativa (the chestnut tree), was developed and optimized. Analyzing public database platforms, a 98 base pair DNA target probe for Calluna vulgaris and a 103 base pair DNA target probe for Castanea sativa were selected and designed. The developed genosensor resulted from a linear self-assembled monolayer of the DNA capture probe of each respective species immobilized onto screen-printed gold electrodes and mercaptohexanol. To improve the genosensor’s selectivity and avoid strong secondary structures, a sandwich format for both DNA target probes was designed using a complementary fluorescein isothiocyanate-labelled DNA signaling probe. Chronoamperometry measurements were performed in a 0.13 to 2.00 nM range for both species. The developed genosensor was able to detect the hybridization reaction between the synthetic strands of each plant. Therefore, electrochemical genosensors offer a promising and cost-effective analytical tool to authenticate the botanical origin of honey, guaranteeing honey safety, quality control, and authenticity for both industries and consumers and showing potential for application in combatting fraud.