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Droplet-based microfluidics is a valuable tool in interdisciplinary research fields like cell biology and diagnostics. Newtonian fluids, like aqueous-based solutions, are commonly used for droplet generation. However, non-Newtonian fluids, e.g., hydrogels, are becoming increasingly popular as the dispersed phase. In this study, we investigate the dynamics of non-Newtonian ultra-low-gelling agarose droplet formation under different conditions to evaluate stability, with an aim to better understand the underlying physics of droplet formation. We varied the agarose gel concentration, temperature (40, 50, and 60 degrees C), and the flow rate ratio (phi) between the continuous and dispersed phase and observed droplet formation dynamics in the squeezing regime (capillary number, Ca-c < 0.015) in a T-junction under different flow conditions. We experimentally investigated the droplet size ( L (D) / w ) as a function of those four parameters and found that L- D / w depends strongly on phi, the agarose concentration, and temperature (which affects the viscosity ratio, lambda), but is only weakly dependent on Ca-c . We then confirmed our experimental findings with numerical simulations, which showed good agreement across all conditions. We numerically showed that the agarose droplet formation process consists of five stages, namely, filling, necking, pinching, threading, and breakup, where threading is an additional stage with a non-Newtonian dispersed phase. Finally, with numerical simulation, we concluded that threading length (l(thread )) is directly proportional to phi and has a complex relation with agarose concentration, and temperature.

In this work, we have studied how the choice of the continuous phase oils affects droplet-internal acoustic manipulation and mapped the acoustic properties of the selected oils to evaluate their compatibility with droplet acoustofluidic methods. The selection of continuous phase included hydrocarbon, fluorinated, and silicone oils. To map the acoustic properties of the oils, we measured their speed of sound and density. We then studied the acoustic performance of each oil for droplet-internal manipulation through experiments and finite-element simulations (COMSOL Multiphysics^®, COMSOL, Stockholm, Sweden). From our results, we conclude that a match between the speed of sound of the continuous and dispersed phases is strongly correlated to the generation of a strong and uniform acoustic field inside the droplet. We demonstrate that conventionally favoured fluorinated oils in droplet microfluidics are no longer the best choice when considering droplet-internal acoustic focusing. Instead, hydrocarbon oils, especially linseed oil, are most suitable for this specific application as they generate stable and monodisperse droplets and bear the most resemblance to water in terms of acoustic properties. We believe this collection of data will serve the acoustofluidics community by providing results that aid in the selection of continuous phase in future droplet acoustofluidic studies and data for performing acoustofluidic simulations.

In healthy individuals, the intestinal epithelium forms a tight barrier to prevent gut bacteria from reaching blood circulation. To study the effect of probiotics, dietary compounds and drugs on gut barrier formation and disruption, human gut epithelial and bacterial cells can be cocultured in an in vitro model called the human microbial crosstalk (HuMiX) gut-on-a-chip system. Here, we present the design, fabrication and integration of thin-film electrodes into the HuMiX platform to measure transepithelial electrical resistance (TEER) as a direct readout on barrier tightness in real-time. As various aspects of the HuMiX platform have already been set in their design, such as multiple compressible layers, uneven surfaces and nontransparent materials, a novel fabrication method was developed whereby thin-film metal electrodes were first deposited on flexible substrates and sequentially integrated with the HuMiX system via a transfer-tape approach. Moreover, to measure localized TEER along the cell culture chamber, we integrated multiple electrodes that were connected to an impedance analyzer via a multiplexer. We further developed a dynamic normalization method because the active measurement area depends on the measured TEER levels. The fabrication process and system setup can be applicable to other barrier-on-chip systems. As a proof-of-concept, we measured the barrier formation of a cancerous Caco-2 cell line in real-time, which was mapped at four spatially separated positions along the HuMiX culture area.

With Organ-on-chip (OoC) systems being developed for the last 15 years, they have started to establish themselves as the more in-vivo like alternative to standard cell-culturing methods. With their ability to replicate mechanical and chemical stimulus more accurately, they are promised to improve research predictions and start reducing animal testing. Their more in-vivo like features although come with more settings to be adjusted, more dynamic behaviour, and more complexity in the results. Most of the OoCs produce only very little sample volume compared to the effort of running them, which makes it more difficult to track their status with conventional measurement techniques. Therefore, this thesis is focused on integrating and improving sensing methods for OoC applications. An in-line cytokine detection chip was built, using bead-based immunoassays and acoustic trapping to reduce the necessary sample volume by 97% to 1.5 µl, while reducing the assay time by 80% to 35 min and retaining a detection limit of 1.2 ng/ml. The internal temperature profile of acoustofluidic devices was characterised using integrated thin-film resistive temperature devices, with sensitivities <10mK. One chip was built to compare internal measurements to external measurements, allowing the cytokine detection chip to operate within safe margins at high acoustic energies and without the need to integrate its own temperature sensor. Another chip was built to measure the temperature gradient in an acoustophoresis channel to provide validation data for streaming simulations at high acoustic powers, with the thought to optimise the sample exposure of the cytokine detection chip. Lastly, a flexible thin-film electrode fabrication based on polyimide tape was developed to integrate electrodes into OoCs, the method was demonstrated by integrating multiple electrodes in a gut-on-chip model and performing impedance spectroscopies to determine the TEER value of the cell layer.

Droplet-based microfluidics involves the generation of discrete volumes in a continuous and immiscible phase. It is an attractive technique for biological, especially single-cell level, assays due to its ability to encapsulate particles and cells within individual reaction vessels at high throughput. Bulk-wave-acoustophoresis is a contactless and label free method to spatially localize particles. It has emerged as a suitable technique to concentrate and separate droplet contents on-chip. In traditional droplet microfluidics for biological applications fluorinated oils, e.g. Novec HFE-7500, are favored due to insolubility for most organic compounds, which prevents cross-contamination between droplets and their high gas permeability. However, a strong and uniform field cannot be formed inside aqueous droplets generated in HFE-7500 due to a mismatch in the acoustic properties of the two phases. Previously, the individual contributions of the acoustic properties, i.e. density, compressibility, and speed of sound, to the focusing quality were not fully investigated. We demonstrate in this work, that the speed of sound dominates the focusing performance. It is shown experimentally and numerically that matching the speed of sound between continuous and discrete phase is paramount to high quality acoustic focusing. To this end the acoustic focusing of polystyrene particles inside aqueous droplets immersed in different continuous phase fluids (hydrocarbon oil, fluorinated oil and silicone oil) is investigated. We demonstrate that a match in the speed of sound between the two phases results in higher droplet-internal particle focusing quality.

Droplet-based microfluidics involves the generation of discrete volumes of a dispersed phase (often water) in a continuous and immiscible phase (oil). Bulk acoustic wave acoustophoresis can be combined with droplet microfluidics for droplet handling, or handling of droplet content. Fluorinated oils, e.g. Novec HFE-7500, possess properties such as high permeability for gases and insolubility for most organic compounds, which makes them favorable for droplet-based applications. However, it has been observed that a deterioration in the average magnitude of the primary acoustic radiation force inside water droplets generated in HFE-7500 results in poor acoustophoretic focusing quality [2,3]. As a follow up to these studies, we compared the droplet internal acoustophoretic focusing performance inside aqueous droplets generated in a selection of oils that have previously been used in two-phase applications [4]. Based on these experiments, we conclude that a match in the speed of sound between the dispersed and continuous phase is paramount to high quality acoustic focusing. Here, we present simulation results of four selected cases, aqueous droplets immersed in linseed oil, light mineral oil, silicone oil 50 cSt and HFE-7500, and offer a theoretical explanation to why it is speed of sound that dominates acoustophoretic focusing performance.