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- Design of an electrochemical genosensor for the BDNF gene polymorphism sequence detection using an enzymatic labelled DNA probePublication . Caldevilla, Renato; Morais, Stephanie; Carvalho, Serafim; Medeiros, Rui; Delerue-Matos, Cristina; Cruz, Agostinho; Santos, Marlene; Barroso, M. FátimaThe BDNF gene is associated with high degrees of variability in antidepressant treatments. The Val66Met polymorphism is widely known as a source of this variability, warranting growing interest in genotyping patients that undergo antidepressant treatment to better suit their needs. This paper reports on an electrochemical genosensing platform, based on gold electrodes, capable of detecting this polymorphism, through the use of synthetic enzymatic labelled DNA-probes for 2 different BDNF alleles. The sensor showed promising results, and its applicability to real samples is currently being tested.
- Electrochemical genosensor for the detection of Alexandrium minutum dinoflagellatesPublication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia C.; Barroso, M. FátimaThis work addresses the development of a disposable electrochemical genosensor for the detection of the toxic dinoflagellate, Alexandrium minutum. Analyzing public databases, a specific 70 bp DNA probe, targeting A. minutum, was selected and designed. The genosensor methodology implied the immobilization of a A. minutum-specific DNA-capture probe onto screen-printed gold electrodes (SPGE). To improve both the selectivity and to avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the A. minutum gene was designed using a fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. Using this electrochemical genosensor, a oncentration range from 0.12 to 1.0 nM, a LD of 24.78 pM with a RSD <5.2% was determined. The genosensor was successfully applied to the selective analysis of the targeted A. minutum specific region denatured genomic DNA extracted from toxic dinoflagellates present in the Atlantic Ocean.
- 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.
- 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.
- Detection of alexandrium minutum dinoflagellate in environmental samples using electrochemPublication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia C.; Barroso, M. FátimaDinoflagellates are aquatic microorganisms that inhabit both salt and fresh waters. These microorganisms are mostly harmless, however, under certain conditions, some species rapidly re produce forming water blooms that not only discolor the waters but also compromise the health of every organism in the vicinity, as some dinoflagellates produce potent toxins deemed unsafe for human health (e.g. Alexandrium minutum). In this work, a disposable electrochemical genosensor for the detection of the toxic dinoflagellate Alexandrium minutum was developed. The analytical plat form methodology consisted in a sandwich format heterogeneous hybridization of complementary DNA sequences assay. The 70 bp A. minutum-specific targeting probe, the 45 bp fluorescein isothi ocyanate-labelled signaling DNA probe and the 25 bp thiolated-DNA-capture probe were designed, after analyzing public databases. To maximize the complementary DNA hybridization and to avoid the formation of strong secondary structures, a mixed mercaptohexanol (MCH) and self-assembled monolayer (SAM) A. minutum-specific DNA-capture probe was immobilized onto disposable screen-printed gold electrodes (SPGE). Using chronoamperometric measurements, the enzymatic amplification of the electrochemical signal was obtained with a concentration range from 0.12 to 1.0 nM, a LD of 24.78 pM with a RSD < 5.2 %. This electrochemical genosensor was successfully applied to the selective analysis of the targeted A. minutum specific region of denatured genomic DNA, ex tracted from toxic dinoflagellates present in the Atlantic Ocean.