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Applied Molecular Biosciences Unit
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Publications
Sensors in the Detection of Abused Substances in Forensic Contexts: A Comprehensive Review
Publication . Rosendo, Luana M.; Antunes, Mónica; Simão, Ana Y.; Brinca, Ana Teresa; Catarro, Gonçalo; Pelixo, Rodrigo; Martinho, João; Pires, Bruno; Soares, Sofia; Cascalheira, José Francisco; Passarinha, Luís; Rosado, Tiago; Barroso, Mário; Gallardo, Eugenia
orensic toxicology plays a pivotal role in elucidating the presence of drugs of abuse in both biological and solid samples, thereby aiding criminal investigations and public health initiatives. This review article explores the significance of sensor technologies in this field, focusing on diverse applications and their impact on the determination of drug abuse markers. This manuscript intends to review the transformative role of portable sensor technologies in detecting drugs of abuse in various samples. They offer precise, efficient, and real-time detection capabilities in both biological samples and solid substances. These sensors have become indispensable tools, with particular applications in various scenarios, including traffic stops, crime scenes, and workplace drug testing. The integration of portable sensor technologies in forensic toxicology is a remarkable advancement in the field. It has not only improved the speed and accuracy of drug abuse detection but has also extended the reach of forensic toxicology, making it more accessible and versatile. These advancements continue to shape forensic toxicology, ensuring swift, precise, and reliable results in criminal investigations and public health endeavours.
Stability of Cocaine, Opiates, and Metabolites in Dried Saliva Spots
Publication . Almeida, Ema; Soares, Sofia; Gonçalves, Joana; Rosado, Tiago; Fernández, Nicolás; Rodilla, Jesus M.; Passarinha, Luís A.; Barroso, Mário; Gallardo, Eugenia
Drug abuse still represents a global problem, and it is associated with an increased risk of
diseases, injuries, and deaths. Cocaine (COC) and opiates are the most abused drugs and account
for a significant number of fatalities. Therefore, it is important to develop methods capable of
effectively identifying and quantifying these substances. The present study aims to evaluate the long-
term stability of COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET),
norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM) in oral
fluid samples. The analytes of interest were isolated from the matrix (50 μL) using the dried saliva
spots (DSS) sampling approach and were subsequently analyzed by gas chromatography coupled
with tandem mass spectrometry (GC–MS/MS). The parameters that could influence the stability of
the target compounds were studied, and these were storage temperature, light, use of preservatives
(and respective concentrations), and time. The effects of each parameter were evaluated using the
design of experiments (DOE) approach. The stability of the target analytes was improved when the
DSS were stored at room temperature, in the presence of light and using 1% sodium fluoride. The
best conditions were then adopted for the DSS storage and long-term stability was assessed. COD
was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for
7 days, MOR for 14 days and BEG remained stable throughout the study (136 days). This is the first
study that evaluates the stability of these compounds in oral fluid samples after application in DSS
cards, and optimizes the conditions in order to improve their stability
Development of Dl1.72, a Novel Anti-DLL1 Antibody with Anti-Tumor Efficacy against Estrogen Receptor-Positive Breast Cancer
Publication . Silva, G; Sales-Dias, J; Casal, D; Alves, S; Domenici, G; Barreto, C; Matos, C; Lemos, AR; Matias, AT; Kucheryava, K; Ferreira, A; Moita, MR; Braga, S; Brito, C; Cabral, MG; Casalou, C; Barral, DC; Sousa, PM; Videira, PA; Bandeiras, TM; Barbas, A
Aberrant Notch signaling is implicated in several cancers, including breast cancer. However, the mechanistic details of the specific receptors and function of ligand-mediated Notch signaling that promote breast cancer remains elusive. In our studies we show that DLL1, a Notch signaling ligand, is significantly overexpressed in ERα+ luminal breast cancer. Intriguingly, DLL1 overexpression correlates with poor prognosis in ERα+ luminal breast cancer, but not in other subtypes of breast cancer. In addition, this effect is specific to DLL1, as other Notch ligands (DLL3, JAGGED1, and JAGGED2) do not influence the clinical outcome of ERα+ patients. Genetic studies show that DLL1-mediated Notch signaling in breast cancer is important for tumor cell proliferation, angiogenesis, and cancer stem cell function. Consistent with prognostic clinical data, we found the tumor-promoting function of DLL1 is exclusive to ERα+ luminal breast cancer, as loss of DLL1 inhibits both tumor growth and lung metastasis of luminal breast cancer. Importantly, we find that estrogen signaling stabilizes DLL1 protein by preventing its proteasomal and lysososmal degradations. Moreover, estrogen inhibits ubiquitination of DLL1. Together, our results highlight an unexpected and novel subtype-specific function of DLL1 in promoting luminal breast cancer that is regulated by estrogen signaling. Our studies also emphasize the critical role of assessing subtype-specific mechanisms driving tumor growth and metastasis to generate effective subtype-specific therapeutics.
Optimization and validation of a procedure using the dried saliva spots approach for the determination of tobacco markers in oral fluid
Publication . Marques, Hernâni; Rosado, Tiago; Barroso, Mário; Passarinha, Luis; Gallardo, Eugenia
Exposure to tobacco smoke is one of the most common causes of premature death worldwide and is the cause of 8 million deaths annually. We have developed, optimized, and validated a procedure for the detection of nicotine, cotinine and trans-3-hydroxycotinine (biomarkers of tobacco exposure) in oral fluid using the dried saliva spots sampling approach and gas chromatography coupled to tandem mass spectrometry, thus allowing the distinction between active and passive smokers. For optimization, four parameters were evaluated, namely extraction solvent, extraction solvent volume, extraction time and spots drying time. During method validation, the parameters selectivity, linearity, precision and accuracy, recovery, stability, and dilution factor were assessed.
Linearity was obtained for all target analytes in the concentration range of 10–200 ng/mL allowing the
quantification of compounds up to 1000 ng/mL considering the dilution factor. The method recoveries ranged
from 29.2% to 43.30% for nicotine, 66.60–89.10% for cotinine and 80.30–92.80% for trans-3-hydroxycotinine,
while achieving intra-day, inter-day and intermediate precision and accuracy values never higher than 10.37%
and ±6.62% respectively for all compounds. The herein described analytical method is the first to allow the
determination of tobacco biomarkers in oral fluid using dried saliva spots, which is considered a sensitive, simple and low-cost alternative to conventional methods.
The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry
Publication . Rosendo, Luana M.; Rosado, Tiago; Oliveira, Patrik; Simão, Ana Y.; Margalho, Cláudia; Costa, Suzel; Passarinha, Luís A.; Barroso, Mário; Gallardo, Eugenia
Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography–mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw–eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1–400 ng/mL for THC and CBD, 5–400 ng/mL for CBN and 11-OH-THC, and 10–400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6817 - DCRRNI ID
Funding Award Number
UIDB/04378/2020