Report of professional activity

"Biochemistry has become the foundation for understanding all biological processes. It has provided explanations for the causes of many diseases in humans, animals and plants". Science can improve people’s life. It can potentially extend our lives beyond our imagination, and also make our journey more pleasant. In particular life’s sciences are in the core of the previous statement. Life’s sciences have experienced a huge development in recent years, and it is believed that its impact in society it would be similar in size to that of the physics revolution of the last century, which have lead to some to suggest that this is the “century of biology”. This report approach all my professional career from my third year of degree in Biochemistry 2007 to the present research in 201

SERS-based biosensors for on-site determination of pancreatic cancer biomarkers

Yuselis Castaño-Guerrero acknowledges Fundação para a Ciência e a Tecnologia (FCT) her PhD Grant (SFRH/BD/145590/2019)info:eu-repo/semantics/publishedVersio

Avoiding the interference of doxorubicin with MTT measurements on the MCF-7 breast cancer cell line

Doxorubicin (DOXO) is an adjuvant chemotherapy agent and is also commonly used in cell biology research. Cytotoxic assays in cell culture are frequently used in order to stablish drug concentrations that are useful for controlling cell proliferation. One common cytotoxic method used is 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). Our present research aims to support future studies in engaging MTT assay using DOXO that exhibits a strong red coloration and fluorescence, and so it is assumed that DOXO may interfere with commonly used colorimetric assays such as MTT. The interference of DOXO in the MTT determination was evaluated in a Breast Cancer cell line Michigan Cancer Foundation-7 (MCF-7). The interference was evaluated by means of spectroscopic methods in particular spectrophometry and fluorescence spectroscopy of MTT and DOXO. We postulate that the medium and the MTT reagent itself can interfere on the metabolic activity method, so in order to achieve better results, DMEM was replaced by a neutral buffer like Phosphate-buffered saline (PBS). This protocol may be extremely useful in future studies involving DOXO.Rúben Fernandes acknowledges, on the behalf of the authors, the funding entities. Carla Luís was supported by a scholarship with the reference SAICT2016/FEDER/BIO4DIA/BTI under the supervision of Dr. Rúben Fernandes. This work was also partially supported by the grant “IBEROS—Instituto de Bioengenharia em Rede para o envelhecimento saudável”, 0245-IBEROS-1-E, cofinanced by the program INTERREG V-A POCTEP through FEDER to BIL/IBEROS-BioMark/03 developed under the supervision of Dr. Goreti Sales. This work was also supported by FCT - Fundação para a Ciência e Tecnologia (REF UID/BIM/04293/2013), by grants NORTE-01-0145-FEDER-000012, SAICT-POL/24325/2016, and SAICT-POL/24358/2016 to Drs. Goreti Sales, Raquel Soares, and Rúben Fernandes.info:eu-repo/semantics/publishedVersio

SERS and electrochemical impedance spectroscopy immunoassay for carcinoembryonic antigen

This work describes an innovative dual detection approach, combining electrochemical and surface-enhanced Raman scattering (SERS) sequential readings, on the same sensing surface. This was achieved by establishing (i) an antibody binding stage on a suitably modified screen-printed electrode (Au-SPE), to produce an electrochemical signalling system, followed by (ii) a second antibody binding stage, on the same sensing surface, comprising gold nanostars (AuNS) with a suitable Raman reporter, and acting as a second signalling system (SERS). This simple principle is applied herein to carcinoembryonic antigen (CEA) detection. The first layer of antibodies was assembled on the Au-SPE previously modified with a cysteamine layer. Binding to CEA was allowed for 30 minutes. Electrochemical impedance spectroscopy (EIS) readings followed the several stages of Au-SPE modification and generated analytical data. After, the AuNS were modified with 4-aminothiophenol (4-ATP)/Ab-CEA, and incubated on the same sensing surface, to provide SERS data. The analytical features were checked for both EIS and SERS. In EIS, the sensor showed linear response range from 0.25 to 250 ng/mL, with a linear correlation coefficient of 0.991, evaluated in serum. It also demonstrated good selectivity against creatinine and glucose. Using the SERS as signalling system, the spectra confirmed differentiated signals from the background within 0.025 ng/mL to 250 ng/mL of CEA. As expected, the Raman signal of the reporter increased with increasing CEA concentrations, and contributed to confirm the accuracy of the analytical data. Overall, this approach is simple, and may be adaptable to new multiplexing devices, also being adaptable to mass production.Authors acknowledge funding to FEDER/COMPETE 2020 and Fundação para a Ciência e Tecnologia through the projects OligoFIT (ERA-NET Cofund, JPCOFUND2/0001/2019), RamSERS (NORTE01-0247-FEDER-017834, call no. 33/SI/2015) and STRIP2SENSE (NORTE-01-0145-FEDER-024358-SAICT-POL/24358/2016). MINECOSpain CTM2017-84050-R and Xunta de Galicia (IN607A 2018/5 and Centro Singular de Investigación de Galicia, Acc. 2016-2019) are also acknowledged. YCG also acknowledge the PhD grant reference SFRH/BD/145590/2019 to Fundação para a Ciência e Tecnologia.info:eu-repo/semantics/publishedVersio

Data Employed in the Construction of a Composite Protein Database for Proteogenomic Analyses of Cephalopods Salivary Apparatus

Here we provide all datasets and details applied in the construction of a composite protein database required for the proteogenomic analyses of the article “Putative Antimicrobial Peptides of the Posterior Salivary Glands from the Cephalopod Octopus vulgaris Revealed by Exploring a Composite Protein Database”. All data, subdivided into six datasets, are deposited at the Mendeley Data repository as follows. Dataset_1 provides our composite database “All_Databases_5950827_sequences.fasta” derived from six smaller databases composed of (i) protein sequences retrieved from public databases related to cephalopods’ salivary glands, (ii) proteins identified with Proteome Discoverer software using our original data obtained by shotgun proteomic analyses of posterior salivary glands (PSGs) from three Octopus vulgaris specimens (provided as Dataset_2) and (iii) a non-redundant antimicrobial peptide (AMP) database. Dataset_3 includes the transcripts obtained by de novo assembly of 16 transcriptomes from cephalopods’ PSGs using CLC Genomics Workbench. Dataset_4 provides the proteins predicted by the TransDecoder tool from the de novo assembly of 16 transcriptomes of cephalopods’ PSGs. Further details about database construction, as well as the scripts and command lines used to construct them, are deposited within Dataset_5 and Dataset_6. The data provided in this article will assist in unravelling the role of cephalopods’ PSGs in feeding strategies, toxins and AMP productionThis study was supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by the Foundation for Science and Technology (FCT) and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/CTA-AMB/31774/2017 (POCI-01-0145-FEDER/031774/2017). We are grateful to Robert Carcasses (Full Stack developer at Kenkou GmbH, Germany) for language programming advice and assistance.info:eu-repo/semantics/publishedVersio

Breaking the classics: next-generation biosensors for the isolation, profiling and detection of extracellular vesicles

Available online 28 January 2022.Extracellular vesicles (EVs) contain biomarkers that may represent a paradigm shift in timely disease diagnosis and personalized therapeutic approaches. Despite tremendous progress in this field, the considerable complexity and heterogeneity of EVs, combined with hurdles in isolation and accurate characterization, have delayed their envisioned clinical translation. At the same time, emerging biosensor technologies are trying to overcome the limitations and provide new momentum to EVs research. In this review, the focus is given on the variety of novel approaches to improve the capture of EVs of interest from a myriad of sample types in terms of yield, purity, sensitivity, and specificity. These biosensing devices also contribute to the understanding of the content of EVs in correlation with their function. Given the pivotal role of EVs uncovered to date and the recent technological advances discussed herein, nanosensing platforms offer low-cost, fast, simple, and accurate methods that are likely to help gain more insight into the biology of EVs. Moreover, the prospect of identifying new roles and patterns will reinforce the importance of EVs and intercellular communication in health status.The authors gratefully acknowledge funding from the European Commission trough the project MindGAP (FET-Open/H2020/GA829040). The authors RV, YCG and ARC also acknowledge Fundação para a Ciência e Tecnologia their PhD grants (2020.09673.BD, SFRH/BD/145590/2019, and SFRH/BD/130107/2017, respectively).info:eu-repo/semantics/publishedVersio

Poly(thionine)-modified screen-printed electrodes for CA 19-9 detection and its properties in Raman spectroscopy

Polythionine (PTH) is an electroactive compound known for its excellent electron transfer capacity. It has stable and redox centers in its structure, and it can also be generated by electropolymerization of thionine (TH). Due to its properties, it has been used in a large number of applications, including the construction of electrochemical biosensors. In this work, PTH is explored for its ability to generate electrons, which allows it to act as an electrochemical probe in a biosensor that detects CA 19-9 on two different substrates, carbon and gold, using differential pulse voltammetry (DPV) as a reading technique in phosphate buffer (PhB). The analytical features of the resulting electrodes are given, showing linear ranges from 0.010 to 10 U/mL. The Raman spectra of PTH films on gold (substrates or nanostars) and carbon (substrates) are also presented and discussed as a potential use for SERS readings as complementary information to electrochemical data.(SFRH/BD/145590/2019)info:eu-repo/semantics/publishedVersio