The microfluidic trainer: Design, fabrication and validation of a tool for testing and improving manual skills

Microfluidic principles have been widely applied for more than 30 years to solve biological and micro-electromechanical problems. Despite the numerous advantages, microfluidic devices are difficult to manage as their handling comes with several technical challenges. We developed a new portable tool, the microfluidic trainer (MT), that assesses the operator handling skills and that may be used for maintaining or improving the ability to inject fluid in the inlet of microfluidic devices for in vitro cell culture applications. After several tests, we optimized the MT tester cell to reproduce the real technical challenges of a microfluidic device. In addition to an exercise path, we included an overfilling indicator and a correct infilling indicator at the inlet (control path). We manufactured the MT by engraving a 3 mm-high sheet of methacrylate with 60W CO2 laser plotter to create multiple capillary paths. We validated the device by enrolling 21 volunteers (median age 33) to fill both the MT and a commercial microfluidic device. The success rate obtained with MT significantly correlated with those of a commercial microfluidic culture plate, and its 30 min-continuous use for three times significantly improved the performance. Overall, our data demonstrate that MT is a valid assessment tool of individual performances in using microfluidic devices and may represent a low-cost solution to training, improve or warm up microfluidic handling skills

Development of flow focusing device for the visualization of leukocyte rolling adhesion

La microfluídica es un área de la microtecnología basada en chips de PDMS que está siendo utilizada cada vez más en multitud de aplicaciones. Una de estas aplicaciones es la investigación biomédica. La microfluídica o “Lab on a Chip” se ha convertido en una manera de realizar experimentos biomédicos y diagnósticos de una manera barata, rápida y eficaz. Cuando se realizan estudios sobre la extravasación leucocitaria utilizando chips microfluídicos, podemos observar la inconsistencia en la trayectoria de rodadura de los leucocitos debido a un flujo laminar. En este trabajo de fin de grado presentamos un método para centrar la interfaz de células en el centro de canal microfluídico. Cuando las células circulan por los sistemas microfluídicos, las células tienden a circular de manera aleatoria por los canales. Por tanto, con el sistema propuesto en este trabajo, dichas células serán redirigidas a la porción central del canal con el fin de recrear el fenómeno de rodadura presente en nuestro sistema circulatorio y así obtener información más detallada. Los resultados de este trabajo muestran la utilidad y la versatilidad de este dispositivo para experimentos relacionados

Current commercialization status of electrowetting-on-dielectric (EWOD) digital microfluidics.

The emergence of electrowetting-on-dielectric (EWOD) in the early 2000s made the once-obscure electrowetting phenomenon practical and led to numerous activities over the last two decades. As an eloquent microscale liquid handling technology that gave birth to digital microfluidics, EWOD has served as the basis for many commercial products over two major application areas: optical, such as liquid lenses and reflective displays, and biomedical, such as DNA library preparation and molecular diagnostics. A number of research or start-up companies (e.g., Phillips Research, Varioptic, Liquavista, and Advanced Liquid Logic) led the early commercialization efforts and eventually attracted major companies from various industry sectors (e.g., Corning, Amazon, and Illumina). Although not all of the pioneering products became an instant success, the persistent growth of liquid lenses and the recent FDA approvals of biomedical analyzers proved that EWOD is a powerful tool that deserves a wider recognition and more aggressive exploration. This review presents the history around major EWOD products that hit the market to show their winding paths to commercialization and summarizes the current state of product development to peek into the future. In providing the readers with a big picture of commercializing EWOD and digital microfluidics technology, our goal is to inspire further research exploration and new entrepreneurial adventures

Cancer Drug Screening Scale-up: Combining Biomimetic Microfluidic Platforms and Deep Learning Image Analysis

The development of cancer drugs is usually costly and time-consuming, mainly due to growing complexity in screening large number of candidate compounds and high failure rates in translation from preclinical trials to clinical approval. Despite the great efforts, the preclinical screening platforms combing good clinical relevance and high throughput for large-scale drug testing is still lacking. In addition, accumulating evidence suggests that cancer drug response can be altered by tumor microenvironment (TME), which includes not only cancer cells but also physical, and biochemical cues in niches. To improve the current cancer drug screening assays, it is important to mimic local TME to achieve better physiological relevance. In the first part of this dissertation, three TME-mimicking microfluidic platforms were introduced for three different in-vitro TME-mimicking tumor sphere models: spheres in matrix, self-aggregated spheres, and single-cell clonal spheres. First, a 3D gel-island chip investigated the heterogeneity of single-cell drug responses in biomimetic extracellular matrix (ECM). With 1,500 isolated single cell chambers containing ECM, it was demonstrated that ECM support was favorable for some population of cancer cells to maintain stemness and develop drug resistance. This result suggested the importance of drug screening at single-cell resolution in TME-mimicking platforms. Secondly, a drug combination screening chip enabling high-throughput and scalable combinatorial drug screening was demonstrated for the aggregated sphere model. Instead of screening a single drug on each of the tumors, this chip allows the screening of all pairwise drug combinations from eight different cancer drugs, in total 172 different treatment conditions, and 1,032 tested samples in a single microfluidic chip. The presented design approach was easily scalable to incorporate arbitrary number of drugs for large-scale drug screening. Finally, single-cell Hi-Sphere chip enabled high-throughput clonal sphere culture and selective retrieval. Combining fluorescent dye on-situ staining techniques, we identified rare cancer stem-like cell population and confirms its location at the leading edge of spheres. Advance in experimental throughput generates massive data, which demands the corresponding automatic analysis and intelligent interpretation capabilities. The second part of this dissertation focuses on the applications of computer vision and machine learning algorithms to automated biomedical data processing. Image analysis with convolutional neural network was applied for drug efficacy evaluation in a fast and label-free manner. The estimated drug efficacy is highly correlated with the experimental ground truth (R-value > 0.93), while the predicted half-maximal inhibitory concentration is within 8% error range. In addition, metastatic fast-moving cells could be identified after extracting morphological features from the microscope images and applying deep learning algorithm for image analysis, achieving over 99% accuracy for cell movement direction prediction and 91% for speed prediction. In summary, this dissertation presents high-throughput TME-mimicking microfluidics and deep learning image analysis for large-scale drug screening solutions.PHDElectrical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163039/1/zhangzx_1.pd

Fully integrated digital microfluidics platform for automated immunoassay; a versatile tool for rapid, specific detection of a wide range of pathogens

© 2018 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/.With the tangible threat posed by the release of chemical and biological warfare (CBW) agents, detection of airborne pathogens is a critical military and security concern. Recent air sampling techniques developed for biocollection take advantage of Electrowetting on Dielectric (EWOD) to recover material, producing highly concentrated droplet samples. Bespoke EWOD-based digital microfluidics platforms are very well suited to take full advantage of the microlitre concentrated droplet resulting from this recovery process. In this paper we present a free-standing, fully automated DMF platform for immunoassay. Using this system, we demonstrate the automated detection of four classes of CBW agent simulant biomolecules and organisms each representing credible threat agents. Taking advantage of the full magnetic separation process with antibody-bound microbeads, rapid and complete separation of specific target antigen can be achieved with minimal washing steps allowing for very rapid detection. Here, we report clear detection of four categories of antigens achieved with assay completion times of between six and ten minutes. Detection of HSA, Bacillus atrophaeus (BG spores), MS2 bacteriophage and Escherichia coli are demonstrated with estimated limit of detection of respectively 30 ng ml -1, 4 × 10 4 cfu ml -1, 10 6 pfu ml -1 and 2 × 10 7 cfu ml -1. The fully-integrated portable platform described in this paper is highly compatible with the next generation of electrowetting-coupled air samplers and thus shows strong potential toward future in-field deployable biodetection systems and could have key implication in life-changing sectors such as healthcare, environment or food security.Peer reviewe

Blood cells separation and sorting techniques of passive microfluidic devices: From fabrication to applications

Since the first microfluidic device was developed more than three decades ago, microfluidics is seen as a technology that exhibits unique features to provide a significant change in the way that modern biology is performed. Blood and blood cells are recognized as important biomarkers of many diseases. Taken advantage of microfluidics assets, changes on blood cell physicochemical properties can be used for fast and accurate clinical diagnosis. In this review, an overview of the microfabrication techniques is given, especially for biomedical applications, as well as a synopsis of some design considerations regarding microfluidic devices. The blood cells separation and sorting techniques were also reviewed, highlighting the main achievements and breakthroughs in the last decades.This work was supported by projects UID/EEA/04436/2019, UID/EMS/04077/2019, UID/EMS/00532/2019 from FCT; and by projects NORTE-01-0145-FEDER-028178, NORTE-01-0145-FEDER-029394, and NORTE01-0145-FEDER-030171 funded by NORTE 2020 Portugal Regional Operational Programme, under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and by Fundação para a Ciência e Tecnologia (FCT), IP. Conflicts of Interest: Th

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

A Modular Open-Technology Device to Measure and Adjust Concentration of Sperm Samples for Cryopreservation

Repositories for aquatic germplasm can safeguard the genetic diversity of species of interest to aquaculture, research, and conservation. The development of such repositories is impeded by a lack of standardization both within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as sperm concentration, that strongly affect the success and consistency of cryopreservation. The wide dissemination and use of specialized tools and devices can improve processing reliability, provide data logging, produce custom hardware to address unique problems, and save costs, time, and labor. The goal of the present work was to develop a low-cost and open-technology approach to standardize the concentration of sperm samples prior to cryopreservation. The specific objectives were to: 1) fabricate and test a peristaltic pump and optical evaluation module (POEM); 2) fabricate and test a prototype of the modular, open-technology concentration measurement and adjustment system (CMAS), which incorporated the POEM; 3) identify opportunities to extend the CMAS to microliter volumes through low-cost resin 3-D printing, and 4) identify strategies from this work that can be applied to future open fabrication efforts. The POEM and CMAS were prototyped and tested with biological samples. A relationship between optical signal and cell concentration of channel catfish (Ictalurus punctatus) sperm samples was established by linear regression. In a blind trial, cell concentrations were estimated with the POEM and correlated closely to their known concentrations (linear regression R2 = 0.9945) in a range of 1 × 108 to 4 × 109 cells/mL. The CMAS was able to estimate and adjust the concentration of a sample of the marine microalgae Tetraselmis chuii as a preparatory step for cryopreservation. To explore the possible use of the CMAS with microliter sample volumes in future work, evaluation of low-cost resin 3-D printing showed that this technology can approach conventional microfabrication techniques in feature quality and resolution. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm, invite new users, makers, and developers into the open-technology community, and increase the reach and capabilities of aquatic germplasm repositories

Micro-manufacturing : research, technology outcomes and development issues

Besides continuing effort in developing MEMS-based manufacturing techniques, latest effort in Micro-manufacturing is also in Non-MEMS-based manufacturing. Research and technological development (RTD) in this field is encouraged by the increased demand on micro-components as well as promised development in the scaling down of the traditional macro-manufacturing processes for micro-length-scale manufacturing. This paper highlights some EU funded research activities in micro/nano-manufacturing, and gives examples of the latest development in micro-manufacturing methods/techniques, process chains, hybrid-processes, manufacturing equipment and supporting technologies/device, etc., which is followed by a summary of the achievements of the EU MASMICRO project. Finally, concluding remarks are given, which raise several issues concerning further development in micro-manufacturing

Optical sensor system for monitoring the pH of cellular media: application to an organ-on-a-chip platform

Dissertação de mestrado integrado em Engenharia FísicapH is a physiological parameter that changes its value according to cellular state of a human organ. When a tumour is being developed, it is known that they have a more acid interstitial pH than normal tissues. This is mainly due to the high metabolic activity of the abnormal cells with increase of acidic sub-products and the absence of organised vasculature of tumours, that leads to poor tissue oxygenation. Indeed, in in vitro animal systems, such as static cell culture experiments or advanced microfluidic devices, the cell’s metabolic activity during incubation causes the alteration of the cell culture pH, which drops the pH from close to the physiological (7.4) to acidic ones (lower than 7.0). Because low environmental pH inhibits cell survival, proliferation and activity, cell culture media has to be consecutively replaced for fresh one. For the pH monitoring, cell culture media is, in general, complemented with a pH colorimetric indicator (e.g. phenol red). However, the colour change of the cell culture media does not quantify the pH value. For this reason, the monitoring and quantification of cell culture medium pH, especially in advanced cell culture devices, such as organ-on-a-chip (OoC), which contain healthy and/or tumour organ models, is still a challenge and a parameter of utmost importance for the maintenance of homeostasis (auto-regulation). The pH of tissues can be measured by a variety of techniques, being pH electrodes the most used. Nevertheless, other methods can be used for pH measurement, such as optical sensors. In general, this technique eliminates the tissue injury effects, but results in an integrated measurement over a long period and demanding a relatively large volume of sample. Particularly, in OoC platforms, the small size of each OoC constituent part, has triggered the development of micro(bio)sensors to be integrated in the microchambers that feed the perfusion chambers containing the organ models, which are used for monitoring the pH of the cell culture media in circulation. In this study, a literature review of pH sensors that can be miniaturised and integrated in OoC was investigated. Based on this previews literature research, and the presence of a colorimetric pH indicator (phenol red) that is commercially added to cell culture media, a miniaturization of an optical pH sensor, for real-time sensing of the cell culture medium feeding advanced microfluidic devices was investigated. This strategy can have several advantages, such as low-cost implementation and improvement of the pH reading based on the beam-splitter phenomenon. For this purpose, a microchamber, processed by micromilling in PMMA, was developed and optimised to support the pH optical sensing system, creating a prototype device that can be directly incorporated into an OoC platform. For the pH sensing experiments, buffered solutions with stablished pH and phenol red were used to test and optimize the optical sensor, by analysing their transmittance signal. In this study, the colorimetric pH indicator (phenol red) was added in the same concentration than the one used in commercial cell culture media. The results shown that the pH reading was successful achieved in intervals of 0.2 pH units, in a range between 6.0 to 8.0.O pH é um parâmetro fisiológico, cujo valor se altera de acordo com o estado celular de um órgão humano. Quando um tumor está em poliferação, este tem um pH intersticial mais ácido do que os tecidos normais. Isto deve-se principalmente à elevada atividade metabólica das células anormais com o aumento de subprodutos ácidos e a ausência de vasculatura organizada dos tumores, que leva à pobre oxigenação dos tecidos. De facto, em sistemas animais in vitro, tais como experiências de cultura celular estática ou dispositivos microfluídicos avançados, a atividade metabólica da célula durante a incubação provoca a alteração do pH da cultura celular, que baixa o pH de perto do fisiológico (7.4) para um mais ácido (inferior a 7.0). O baixo pH fisiológico inibe a sobrevivência, proliferação e atividade celular, e, em consequência, os meios de cultura celular têm de ser substituídos consecutivamente. Para a monitorização do pH, os meios de cultura celular são, em geral, complementados com um indicador colorimétrico de pH (por exemplo, vermelho de fenol). No entanto, a mudança de cor dos meios de cultura celular não quantifica o valor do pH. Por esta razão, a monitorização e quantificação do pH dos meios de cultura celular, especialmente em dispositivos avançados de cultura celular, tais como Organ-on-a-chip (OoC), que contêm modelos de órgãos saudáveis e/ou tumorais, é ainda um desafio e um parâmetro da maior importância para a manutenção da homeostase (autorregulação). O pH dos tecidos pode ser medido por uma variedade de técnicas, sendo os elétrodos de pH os mais utilizados. Contudo, outros métodos podem ser utilizados para a medição do pH, tais como os sensores óticos. Esta técnica elimina os efeitos da lesão tecidual, mas resulta numa medição integrada durante um longo período e a partir de um volume relativamente grande de amostra. Particularmente, nas plataformas de OoC, o pequeno tamanho de cada parte constituinte de OoC, desencadeou o desenvolvimento de micro(bio)sensores para serem integrados nas microcâmaras que alimentam as câmaras de perfusão, as quais contêm os modelos de órgãos, e que podem ser utilizados para monitorizar o pH dos meios de cultura celular em circulação. Neste estudo, foi investigada uma revisão bibliográfica de sensores de pH que podem ser miniaturizados e integrados na OoC. Com base nesta pesquisa bibliográfica prévia, e a presença de um indicador colorimétrico de pH (vermelho fenol) que é comercialmente adicionado aos meios de cultura celular, foi investigada uma miniaturização de um sensor ótico de pH para a deteção em tempo real do meio de cultura celular que alimenta dispositivos microfluídicos avançados. Esta estratégia pode ter várias vantagens, tais como a implementação a baixo custo e a melhoria da leitura do pH com base no fenómeno do separador de feixe. Para este efeito, foi desenvolvida e otimizada uma microcâmara, processada por micromilling em PMMA, para apoiar o sistema de deteção ótica de pH, criando um dispositivo protótipo que pode ser diretamente incorporado numa plataforma OoC. Para as experiências de deteção de pH, foram utilizadas soluções tampão com pH estabilizado e vermelho fenol para testar e otimizar o sensor ótico, através da análise do seu sinal de transmitância. Assim, o indicador colorimétrico de pH vermelho de fenol foi adicionado na mesma concentração encontrada nos meios de cultura celular comerciais. Os resultados mostram que a leitura do pH foi bem-sucedida em intervalos de 0.2 unidades de pH, num intervalo entre 6.0 a 8.0.This work results partially of the project NORTE-01-0145-FEDER-029394, RTChip4Theranostics, supported by Programa Operacional Regional do Norte - Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by Fundação para a Ciência e Tecnologia (FCT), IP, project reference PTDC/EMD-EMD/29394/2017