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Open-source tool for real-time and automated analysis of droplet-based microfluidic
Publication . Neto, Joana P.; Mota, Ana; Lopes, Gonçalo; Coelho, Beatriz J.; Frazão, João; Moura, André T.; Oliveira, Beatriz; Sieira, Bárbara; Fernandes, José; Fortunato, Elvira; Martins, Rodrigo; Igreja, Rui; Baptista, Pedro V.; Águas, Hugo; CENIMAT-i3N - Centro de Investigação de Materiais (Lab. Associado I3N); UNINOVA-Instituto de Desenvolvimento de Novas Tecnologias; DCM - Departamento de Ciência dos Materiais; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; RSC - 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.
Digital Microfluidics-Powered Real-Time Monitoring of Isothermal DNA Amplification of Cancer Biomarker
Publication . Coelho, Beatriz Jorge; Veigas, Bruno; Bettencourt, Luís; Águas, Hugo; Fortunato, Elvira; Martins, Rodrigo; Baptista, Pedro V.; Igreja, Rui; DCM - Departamento de Ciência dos Materiais; UNINOVA-Instituto de Desenvolvimento de Novas Tecnologias; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets—low frequency AC (alternating current) actuation as well as back-and-forth droplet motion—which allows for improved fluorescence readouts. Fluo-rophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BTM-SAL/31201/2017