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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.

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.

The ability to manipulate particles or cells inside water-in-oil droplets is essential to various chemical, biological and biomedical assays performed using droplet microfluidics systems. Bulk acoustophoresis permits spatial localization of cells and particles in a label-free and non-contact manner by utilizing acoustic standing wave and is therefore a suitable particle manipulation method for integration with droplet microfluidics. Fluorinated oils, e.g., Novec HFE-7500, are most commonly used as the continuous phase in typical droplet applications as they are permeable to gases and insoluble for most organic compounds. Previous studies, however, have concluded that sufficient acoustic focusing capability cannot be achieved inside aqueous droplets generated in HFE-7500 due to a mismatch in the acoustic properties of the two phases used. The critical acoustic properties that underlie this difference in acoustic focusing capability were however not identified. This work presents a study where the suitability of a selection of oils, from three categories commonly used in droplet microfluidics (hydrocarbon oil, fluorinated oil and silicone oil), was examined for acoustic particle manipulation in water-in-oil droplets. We hypothesized that when performing acoustic focusing inside aqueous droplets confined in a microfluidic channel (i.e., plugs), due to the presence of a thin oil film between the channel wall and the droplet, the acoustic impedance between the two fluid phases must match in order to increase acoustic coupling efficiency between the two liquid phases. Through this experimental work, we demonstrate that hydrocarbon oils are most compatible with acoustic focusing in a two-phase system that generates channel-confined water-in-oil droplets due to matching acoustic properties with water. We also conclude that acoustic impedance matching between the two fluid phases is not critical for optimal droplet-internal particle manipulation. Our results suggest that a match in the speed of sound of the two phases may be more relevant in practice.

Droplet microfluidics has emerged as a powerful tool for miniaturizing biological assays. The continuous phase in droplet microfluidics often consists of fluorinated oils and fluorosurfactants. However, these fluids belong to a group of chemicals called PFASs (per-and polyfluoroalkyl substances), which are hazardous for the environment as they are not broken down but remain in the surrounding for a long time. As a result, PFAS chemicals are likely to be unavailable on the market in the near future. Consequently, there is a need to find replacements for fluorinated oils and fluorosurfactants that work well for droplet microfluidics. With this article, we aim to bring awareness to the issue and begin by reviewing the problem with using fluorinated chemicals in droplet microfluidics. Next, we compare the performance of four different oil/surfactant systems: two containing fluorinated oil and two without, to present alternatives for the droplet microfluidics community. Our results show that suitable alternative oil/surfactant systems exist that can effectively replace the conventional fluorinated solutions in droplet microfluidics.

Hydrogels are widely used for cell encapsulation and culture due to their ability to mimic extracellular matrix. Cell-laden hydrogels in the micrometer scale — microgels — offer advantages for single-cell analysis and scaffold-based 3D cell culture. However, challenges such as off-center encapsulation and asymmetric biochemical exposure lead to cellular escape and heterogeneity in cell assemblies, which compromise long-term culture and affects experimental reproducibility. Addressing these limitations requires precise three-dimensional (3D) spatial control of cells within microgels. This study presents 3D acoustic focusing of polystyrene microbeads within gelatin methacyraloyl (GelMA) hydrogel, achieved in a microwell-based acoustofluidic system that employs dual piezoelectric transducers to generate half-wavelength resonances across all three dimensions of cuboid-shaped microwells. Our findings demonstrate a versatile approach for active 3D centering of cells and microparticles in microgels, demonstrating the potential of bulk acoustic wave technologies as a biofabrication technique for biomedical research and regenerative medicine.