Fast prototyping microfluidics: Integrating droplet digital lamp for absolute quantification of cancer biomarkers

UID/CTM/50025/2019 UID/Multi/04378/2019 Inn-INDIGO/0002/2015 PTDC/BTM-SAL/31201/2017 SFRH/BPD/124311/2016Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in “Shrinky-dinks” polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.publishersversionpublishe

Parylene C as a Multipurpose Material for Electronics and Microfluidics

Funding Information: This work was financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects LA/P/0037/2020, UIDP/50025/2020 and UIDB/50025/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication—i3N. Furthermore, the work received funding from FCT in the scope of projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. This work also received funding from the European Community’s H2020 program under grant agreements 716510 (ERC-2016-StG TREND), 787410 (ERC-2019-AdG DIGISMART) and 952169 (SYNERGY, H2020-WIDESPREAD-2020-5, CSA), 101008701 (EMERGE, H2020-INFRAIA-2018-2020). B. J. Coelho acknowledges FCT for the attribution of grant SFRH/BD/132904/2017 and grant COVID/BD/152453/2022. Publisher Copyright: © 2023 by the authors.Poly(p-xylylene) derivatives, widely known as Parylenes, have been considerably adopted by the scientific community for several applications, ranging from simple passive coatings to active device components. Here, we explore the thermal, structural, and electrical properties of Parylene C, and further present a variety of electronic devices featuring this polymer: transistors, capacitors, and digital microfluidic (DMF) devices. We evaluate transistors produced with Parylene C as a dielectric, substrate, and encapsulation layer, either semitransparent or fully transparent. Such transistors exhibit steep transfer curves and subthreshold slopes of 0.26 V/dec, negligible gate leak currents, and fair mobilities. Furthermore, we characterize MIM (metal–insulator–metal) structures with Parylene C as a dielectric and demonstrate the functionality of the polymer deposited in single and double layers under temperature and AC signal stimuli, mimicking the DMF stimuli. Applying temperature generally leads to a decrease in the capacitance of the dielectric layer, whereas applying an AC signal leads to an increase in said capacitance for double-layered Parylene C only. By applying the two stimuli, the capacitance seems to suffer from a balanced influence of both the separated stimuli. Lastly, we demonstrate that DMF devices with double-layered Parylene C allow for faster droplet motion and enable long nucleic acid amplification reactions.publishersversionpublishe

Paper-Based SERS Platform for One-Step Screening of Tetracycline in Milk

PTDC/CTM-NAN/2912/2014. PTDC/AAG-TEC/5400/2014. POCI-01-0145-FEDER-016637. POCI-01-0145-FEDER-007688. UID/CTM/50025/2019. FCT/MCTES SFRH/BD/115173/2016 and SFRH/BPD/124311/2016 .Throughout the last decade, the expansion of food testing has been gradually moving towards ordinary high throughput screening methods performed on-site. The demand for point-of-care testing, able to distinguish molecular signatures with high accuracy, sensitivity and specificity has been significantly increasing. This new requirement relies on the on-site detection and monitorization of molecular signatures suitable for the surveillance of food production and processing. The widespread use of antibiotics has contributed to disease control of livestock but has also created problems for the dairy industry and consumers. Its therapeutic and subtherapeutic use has increased the risk of contamination in milk in enough concentrations to cause economic losses to the dairy industry and have a health impact in highly sensitive individuals. This study focuses on the development of a simple Surface-Enhanced Raman Spectroscopy (SERS) method for fast high throughput screening of tetracycline (TET) in milk. For this, we integrate a paper-based low-cost, fully recyclable and highly stable SERS platform, with a minimal sample preparation protocol. A two-microliter sample of milk solutions spiked with TET (from 0.01 to 1000 ppm) is dried on a silver nanoparticle coated cardboard substrate and measured via a Raman spectrophotometer. The SERS substrate showed to be extremely stable with a shelf life of several months. A global spectrum principal component analysis approach was used to test all the detected vibrational modes and their correlation with TET concentration. Peak intensity ratios (455 cm−1/1280 cm−1 and 874 cm−1/1397 cm−1) were found to be correlated with TET concentrations in milk, achieving a sensitivity as low as 0.1 ppm. Results indicate that this SERS method combined with portable Raman spectrometer is a potential tool that can be used on-site for the monitoring of TET residues and other antibiotics.publishersversionpublishe

As Novas Diretrizes e a Importância do Planejamento Urbano para o Desenvolvimento de Cidades Sustentáveis

Cities are protagonists in the overall development process. Today, more than 54% of the world's population lives in cities and it is expected to reach the 60% mark by 2030. Cities have thus played a crucial role in the global sustainable development agenda. The idea of ​​urban sustainability has been disseminated through important international studies and protocols in order to overcome social, economic and environmental challenges to generate quality of life for its citizens and contribute to the development capacity of future generations. This work investigated the importance and the new directives of urban planning as a tool for the structuring of sustainable cities. A qualitative research was conducted through in - depth interviews with experts from different areas of knowledge on the theme of cities, bringing as a main result a systemic view on the role of urban planning in the development of sustainable cities. In addition, aiming at a balanced urban development, this study points out the need for a new ordering and cohesion of the different elements that compose a city and proposes ways for a new collaborative urban governance, thus contributing to the scientific development of the urban and regional planning area.Las ciudades son protagonistas en el proceso global de desarrollo. Hoy en día, más del 54% de la población mundial vive en ciudades y se espera que alcance el 60% en 2030. Las ciudades han jugado un papel crucial en la agenda global de desarrollo sostenible. La idea de la sostenibilidad urbana se ha difundido a través de importantes estudios y protocolos internacionales para superar los desafíos sociales, económicos y ambientales para generar calidad de vida para sus ciudadanos y contribuir al desarrollo de la capacidad de las generaciones futuras. Este trabajo investigó la importancia y las nuevas directivas de planificación urbana como una herramienta para la estructuración de ciudades sostenibles. Se llevó a cabo una investigación cualitativa a través de entrevistas en profundidad con expertos de diferentes áreas de conocimiento sobre el tema de las ciudades, con el resultado principal de una visión sistémica sobre el papel de la planificación urbana en el desarrollo de ciudades sostenibles. Además, con el objetivo de lograr un desarrollo urbano equilibrado, este estudio señala la necesidad de una nueva ordenación y cohesión de los diferentes elementos que componen una ciudad y propone vías para una nueva gobernanza urbana colaborativa, contribuyendo así al desarrollo científico de las ciudades y los pueblos. Área de planificación regional.As cidades são protagonistas no processo de desenvolvimento global. Atualmente mais de 54% da população mundial reside em cidades e com previsão de alcançar a marca de 60% em 2030. Assim, as cidades têm ocupado um papel crucial na agenda global de desenvolvimento sustentável. A ideia de sustentabilidade urbana vem se disseminando através de importantes estudos e protocolos internacionais, no intuito das cidades superarem desafios de ordem social, econômica e ambiental para gerar qualidade de vida aos seus cidadãos e contribuir com a capacidade de desenvolvimento das gerações futuras. Este trabalho investigou a importância e as novas diretrizes do planejamento urbano como ferramenta para a estruturação de cidades sustentáveis. Foi realizada uma pesquisa qualitativa através de entrevistas em profundidade com especialistas de diferentes áreas de conhecimento no tema de cidades, trazendo como principal resultado uma visão sistêmica sobre o papel do planejamento urbano no desenvolvimento de cidades sustentáveis. Além disso, visando um desenvolvimento urbano equilibrado, este estudo aponta a necessidade de um novo ordenamento e coesão dos diferentes elementos que compõem uma cidade e propõe caminhos para uma nova governança urbana colaborativa, contribuindo assim para o desenvolvimento científico da área de planejamento urbano e regional

Validation to Portuguese of the Scale of Student Satisfaction and Self-Confidence in Learning

Objective: translate and validate to Portuguese the Scale of Student Satisfaction and Self-Confidence in Learning. Material and Methods: methodological translation and validation study of a research tool. After following all steps of the translation process, for the validation process, the event III Workshop Brazil - Portugal: Care Delivery to Critical Patients was created, promoted by one Brazilian and another Portuguese teaching institution. Results: 103 nurses participated. As to the validity and reliability of the scale, the correlation pattern between the variables, the sampling adequacy test (Kaiser-Meyer-Olkin) and the sphericity test (Bartlett) showed good results. In the exploratory factorial analysis (Varimax), item 9 behaved better in factor 1 (Satisfaction) than in factor 2 (Self-confidence in learning). The internal consistency (Cronbach's alpha) showed coefficients of 0.86 in factor 1 with six items and 0.77 for factor 2 with 07 items. Conclusion: in Portuguese this tool was called: Escala de Satisfação de Estudantes e Autoconfiança na Aprendizagem. The results found good psychometric properties and a good potential use. The sampling size and specificity are limitations of this study, but future studies will contribute to consolidate the validity of the scale and strengthen its potential use.Objetivo: traduzir e validar para língua portuguesa a escala Student Satisfaction and Self-Confidence in Learning. Material e Métodos: estudo metodológico de tradução e validação de instrumento. Cumpridas todas as etapas do processo de tradução, para o processo de validação criou-se o evento III Workshop Brasil - Portugal: Atendimento ao Paciente Crítico, promovido por uma instituição de ensino do Brasil e outra de Portugal. Resultados: participaram 103 enfermeiros. Quanto à validade e fidelidade da escala, o padrão de correlação entre as variáveis, o teste de adequação amostral (Kaiser-Meyer-Olkin) e o teste de esfericidade (Bartlett) apresentaram bons resultados. Na análise fatorial exploratória (Varimax) o item 9 se comportou melhor no fator 1 (Satisfação) do que no fator 2 (Autoconfiança com a aprendizagem). A consistência interna (alfa de Cronbach) apresentou valores de 0,86 fator 1 com 06 itens e 0,77 para fator 2 de 07 itens. Conclusão: o presente instrumento ficou denominado em português: Escala de Satisfação de Estudantes e Autoconfiança na Aprendizagem. Os resultados constatam boas propriedades psicométricas e um bom potencial de utilização. É limitação deste trabalho o tamanho amostral e sua especificidade, porém futuros trabalhos contribuirão para consolidar a validade da escala e reforçar o seu potencial de utilização.Objetivo: traducir y validar para la lengua portuguesa la escala Student Satisfaction and Self-Confidence in Learning. Material y Métodos: estudio metodológico de traducción y validación de instrumento. Tras cumplir todas las etapas del proceso de traducción, para el proceso de validación, se creó el evento III Workshop Brasil - Portugal: Atención al Paciente Crítico, promovido por una institución de enseñanza de Brasil y otra de Portugal. Resultados: participaron 103 enfermeros. Respecto a la validez y fidelidad de la escala, el patrón de correlación entre las variables, la prueba de adecuación de la muestra (Kaiser-Meyer-Olkin) y la prueba de esfericidad (Bartlett) mostraron buenos resultados. En el análisis factorial exploratoria (Varimax), el ítem 9 se comportó mejor en el factor 1 (Satisfacción) que en el factor 2 (Autoconfianza con el aprendizaje). La consistencia interna (alfa de Cronbach) mostró valores de 0,86 para factor 1 con 06 ítems y 0,77 para factor 2 de 07 ítems. Conclusión: este instrumento fue llamado en portugués: Escala de Satisfação de Estudantes e Autoconfiança na Aprendizagem. Los resultados constatan buenas propiedades psicométricas y un buen potencial de utilización. Las limitaciones de este estudio son el tamaño de la muestra y su especificidad. Sin embargo, estudios futuros contribuirán para consolidar la validez de la escala y reforzar su potencial de utilización

Design, Fabrication and Characterization

Funding Information: This work was financed by national funds from FCT, Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects LA/P/0037/2020, UIDP/50025/2020, and UIDB/50025/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication, i3N, and also under project dPCR4FreeDNA of the same research unit, PTDC/BTM-SAL/31201/2017. Furthermore, the work received funding from FCT in the scope of projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences, UCIBIO, and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy, i4HB. B. J. Coelho acknowledges FCT for the attribution of grant SFRH/BD/132904/2017 and grant COVID/BD/152453/2022. Publisher Copyright: © 2023 by the authors.Microfluidic-based platforms have become a hallmark for chemical and biological assays, empowering micro- and nano-reaction vessels. The fusion of microfluidic technologies (digital microfluidics, continuous-flow microfluidics, and droplet microfluidics, just to name a few) presents great potential for overcoming the inherent limitations of each approach, while also elevating their respective strengths. This work exploits the combination of digital microfluidics (DMF) and droplet microfluidics (DrMF) on a single substrate, where DMF enables droplet mixing and further acts as a controlled liquid supplier for a high-throughput nano-liter droplet generator. Droplet generation is performed at a flow-focusing region, operating on dual pressure: negative pressure applied to the aqueous phase and positive pressure applied to the oil phase. We evaluate the droplets produced with our hybrid DMF–DrMF devices in terms of droplet volume, speed, and production frequency and further compare them with standalone DrMF devices. Both types of devices enable customizable droplet production (various volumes and circulation speeds), yet hybrid DMF–DrMF devices yield more controlled droplet production while achieving throughputs that are similar to standalone DrMF devices. These hybrid devices enable the production of up to four droplets per second, which reach a maximum circulation speed close to 1540 µm/s and volumes as low as 0.5 nL.publishersversionpublishe

Open-source tool for real-time and automated analysis of droplet-based microfluidic

Publisher Copyright: © 2023 The Royal Society of Chemistry.Droplet-based microfluidic technology is a powerful tool for generating large numbers of monodispersed nanoliter-sized droplets for ultra-high throughput screening of molecules or single cells. Yet further progress in the development of methods for the real-time detection and measurement of passing droplets is needed for achieving fully automated systems and ultimately scalability. Existing droplet monitoring technologies are either difficult to implement by non-experts or require complex experimentation setups. Moreover, commercially available monitoring equipment is expensive and therefore limited to a few laboratories worldwide. In this work, we validated for the first time an easy-to-use, open-source Bonsai visual programming language to accurately measure in real-time droplets generated in a microfluidic device. With this method, droplets are found and characterized from bright-field images with high processing speed. We used off-the-shelf components to achieve an optical system that allows sensitive image-based, label-free, and cost-effective monitoring. As a test of its use we present the results, in terms of droplet radius, circulation speed and production frequency, of our method and compared its performance with that of the widely-used ImageJ software. Moreover, we show that similar results are obtained regardless of the degree of expertise. Finally, our goal is to provide a robust, simple to integrate, and user-friendly tool for monitoring droplets, capable of helping researchers to get started in the laboratory immediately, even without programming experience, enabling analysis and reporting of droplet data in real-time and closed-loop experiments.publishersversionepub_ahead_of_prin

Segmentação Distribuída de Imagem de Sensoriamento Remoto a partir de Banco de Dados PostgreSQL/InterIMAGE

A abordagem de classificação baseada em objetos representa um novo paradigma no processamento de imagens de altas resoluções espaciais, espectrais e temporais, e a construção de objetos baseia-se na segmentação das imagens. A análise de imagens baseada em objetos (GEOBIA - Geographic Object-Based Image Analysis) apresenta métodos capazes de explorar, além de atributos espectrais, elementos como textura, forma ou contexto. Existem aplicações que buscam melhorar o desempenho computacional com soluções sequenciais e distribuídas, ou programas como TerraView que abordam o uso de sistemas gerenciadores de banco de dados. Este trabalho propõe explorar especificações de aplicações para integrar o Sistema Gerenciador de Banco de Dados (SGBD) PostgreSQL/PostGIS e o classificador Object-Based Image Analysis (OBIA) do InterIMAGE Desktop para processamento de grandes imagens orbitais. O método apresentado é expansível no uso da biblioteca TerraLib 5, com linguagem de programação C++. Os experimentos realizados com as representações matriciais (raster) indicaram a viabilidade das aplicações e podem se consolidar sob a forma de processos de armazenamento e processamento da segmentação no SGBD