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- Electrochemical chemically based sensors and emerging enzymatic biosensors for antidepressant drug detection: a reviewPublication . Caldevilla, Renato; Morais, Stephanie L.; Cruz, Agostinho; Delerue-Matos, Cristina; Moreira, Fernando; Pacheco, João G.; Santos, Marlene; Barroso, Maria FátimaMajor depressive disorder is a widespread condition with antidepressants as the main pharmacological treatment. However, some patients experience concerning adverse reactions or have an inadequate response to treatment. Analytical chromatographic techniques, among other techniques, are valuable tools for investigating medication complications, including those associated with antidepressants. Nevertheless, there is a growing need to address the limitations associated with these techniques. In recent years, electrochemical (bio)sensors have garnered significant attention due to their lower cost, portability, and precision. Electrochemical (bio)sensors can be used for various applications related to depression, such as monitoring the levels of antidepressants in biological and in environmental samples. They can provide accurate and rapid results, which could facilitate personalized treatment and improve patient outcomes. This state-of-the-art literature review aims to explore the latest advancements in the electrochemical detection of antidepressants. The review focuses on two types of electrochemical sensors: Chemically modified sensors and enzyme-based biosensors. The referred papers are carefully categorized according to their respective sensor type. The review examines the differences between the two sensing methods, highlights their unique features and limitations, and provides an in-depth analysis of each sensor.
- 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.
- Authenticating honey origin from Natural Park of Montesinho: innovative electrochemical genosensor devices assembled in paper-based transducersPublication . Pereira, Eduarda; Morais, Stephanie L.; Seguro, Isabel; Silva, Nádia F. D.; Santos, Marlene; Pacheco, João G.; Delerue-Matos, Cristina; Barroso, M. FátimaThe Natural Park of Montesinho (NPM) has a diverse ecosystem that is home to countless species of trees and plants characteristic of this area, including Castanea sativa. The honey produced in this region is highly appreciated for its taste and nutritional value, but it also has a wide range of health benefits. The geographical origin of the honey is a very important aspect in assessing the quality and authentication of the final product. Currently, several approaches are being explored to determinethese parameters, among which deoxyribonucleic acid (DNA)-based methods stand out.
- Enhancing the detection of Dinophysis spp. using electrochemical genosensorsPublication . Pereira, Eduarda; Silva, Aurora; Morais, Stephanie L.; Costa-Rama, Estefanía; Moreira, Patrícia R.; Fraga-Corral, M.; Torrado, Ana M.; Rodríguez, Francisco; Barros, Piedade; Cruz, Agostinho; Delerue-Matos, Cristina; Prieto, M. A.; Simal-Gandara, J.; Silva, Nádia F. D.; Santos, Marlene; Barroso, M. FátimaHarmful algal blooms (HABs) pose a significant threat to the environment and public health. These blooms are defined by an accumulation of microscopic algae in water, and they can occur inlakes, rivers, estuaries, orcoastal areas. Factors like the unregulated runoff of agricultural and industrial wastes into the aquatic environment are believed to have transformed these ecosystems into favorable habitats for algae growth and proliferation. As a result, the frequency of these blooms is rising worldwide. Although these blooms are mostly harmless, certain species, namely dinoflagellates from the genus Dinophysis, produce toxins that pose a risk for human health. Therefore, the need for technological developments towards fast and precise detection of these toxin-producing microalgae is critical to prevent socio economical damages, as well as to assess the ecological status of marine ecosystems. In this work, an analytical approach based on an electrochemical genosensor device was developed to create a low-cost platform able to detect two dinoflagellate species from the genus Dinophysis: D.acuminataand D.acuta. The design of the DNA-based sensor involved three key steps: i) Sensing phase: consisted by a mixed self-assembled monolayer composed by a linear DNA capture probe and mercaptohexanol on to the disposable screen-printed gold electrodessurface; ii) Hybridization of complementary DNA sequence by using a sandwich format assay with enzymatic labels and iii) Electrochemical detection by chronoamperometry using an enzymatic scheme to amplify the electrochemical signal. The best analytical conditions used to study the relationship between electrochemical signal and DNA target concentration, to produce the best electrochemical genosensor device. Molecular biology tools, namely Polymerase Chain Reaction (PCR), will be used for further validation of the electrochemical genosensor to confirm its reliability. These advancements in analytical technologies contribute to the on going efforts in environmental management and public health protection by providing effective means for detectingand mitigating the risks associated with HABs. Further research and widespread implementation of these methods are required to ensure the safety and sustainability of aquatic ecosystems, safeguard public health, and facilitate proactive environmental management practices.
- Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3Publication . Morais, Stephanie L.; Magalhães, Júlia M.C. S.; Domingues, Valentina F.; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaCardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015–1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.
- 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.
- 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.
- 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.
- Development of electrochemical genosensors for the CYPC*2 gene polymorphism detectionPublication . Sousa, António J. S. F.; Costa, Inês; Banegas, Rodrigo S.; Morais, Stephanie L.; Magalhães, Júlia; Rodrigues, Valentina; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaPharmacogenetic studies search for heritable genetic polymorphisms that influence responses to drug therapy. Pharmacogenetics has many possible applications in cardiovascular pharmacotherapy including screening for polymorphisms to choose agents with the greatest potential for efficacy and least risk of toxicity. Pharmacogenetics also informs dose adaptations for specific drugs in patients with aberrant metabolism. Cardiovascular diseases (CVD) are considered one of the leading causes of death worldwide. To prevent cardiovascular complications and further loss of life oral anticoagulants (e.g., warfarin) are frequently prescribed to patients. Nevertheless, warfarin therapeutic agent presents narrow therapeutic windows with well-documented health risks. Some of these dose-responses are a result of specific single-nucleotide polymorphism (SNP) genetic variations present in a patient´s DNA. Among them, determined SNP in the cytochrome P4502C9 (CYP2C9), namely the CYP2C9*2, gene has been identified as dose-response altering SNP. Therefore, the need for a rapid, selective, low-cost and in real time detection device is crucial before prescribing any anticoagulant. In this work an analytical approach based on electrochemical genosensor technique is under development to create a low-cost genotyping platform able to genotype SNPs related with the therapeutic response of warfarin. Analyzing public databases, two specific 71 bp DNA probes, one with adenine (TA) and other with guanine (TG) SNP genetic variation were selected and designed. The design of this electrochemical genosensor consists of ssDNA immobilization onto gold surfaces that act as the SNPs complementary probes. The hybridization reaction is performed in a sandwich format of the complementary ssDNA, using an enzymatic scheme to amplify the electrochemical signal. The electrochemical signal was performed by using chronoamperometric technique.
- Development of electrochemical genosensors for the detection of toxic dinoflagellate alexandrium spp.Publication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia Castro; Barroso, M. FátimaMicroalgae represent a photosynthetic microorganisms’ group that inhabit both salt and fresh waters. These microorganisms, namely several species of dinoflagellates (e.g. Alexandrium spp.)1 , are mostly harmless; however, some species produce toxins classified as unsafe to human health. The uncontrolled proliferation of such species results in a hazardous occurring event designated harmful algal blooms (HAB). The effects of these episodes can lead to severe ecological and socio-economic impacts (e.g. decrease of the local tourism, fishing and port activities, the contamination or death of the nearby wildlife, discoloration of the beach coasts). Therefore, the need for a rapid, selective and in real time detection device that can monitor the presence of these microalgae in aquaculture waters is critical to prevent human, ecological and economical losses. In this work, an analytical approach based on electrochemical genosensor techniques was developed to create a low cost platform able to detect the dinoflagellates: Alexandrium minutum and Alexandrium ostenfledii. The design of this genosensor consisted of several steps including: i) Sensing phase: Creation of a mixed self assembled monolayer (SAM) composed by a linear DNA capture probe (DNA-CP) and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE) surface; ii) Heterogenous hybridization of complementary DNA sequence (DNA target) by using a sandwich format assay with enzymatic labels and iii) Electrochemical detection by chronoamperometry using an enzymatic scheme to amplify the electrochemical signal (Figure 1). The best analytical conditions were used to study the relationship between electrochemical signal and DNA target concentration.