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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.

CoSAXS is a state-of-the-art SAXS/WAXS beamline exploiting the high brilliance of the MAX IV 3 GeV synchrotron. By coupling advances in sample environment control with fast X-ray detectors, millisecond time-resolved scattering methods can follow structural dynamics of proteins in solution. In the present work, four sample environments are discussed. A sample environment for combined SAXS with UV–vis and fluorescence spectroscopy (SUrF) enables a comprehensive understanding of the time evolution of conformation in a model protein upon acid-driven denaturation. The use of microfluidic chips with SAXS allows the mapping of concentration with very small sample volumes. For highly reproducible sequences of mixing of components, it is possible using stopped-flow and SAXS to access the initial effects of mixing at 2 millisecond timescales with good signal to noise to allow structural interpretation. The intermediate structures in a protein are explored under light and temperature perturbations by using lasers to “pump” the protein and SAXS as the “probe”. The methods described demonstrate that features at low q, corresponding to cooperative motions of the atoms in a protein, could be extracted at millisecond timescales, which results from CoSAXS being a highly-stable, low background, dedicated SAXS beamline.

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 is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.

The generation of hydrogel droplets using droplet microfluidics has emerged as a powerful tool with many applications in biology and medicine. Here, a microfluidic system to control the position of particles (beads or astrocyte cells) in hydrogel droplets using bulk acoustic standing waves is presented. The chip consisted of a droplet generator and a 380 µm wide acoustic focusing channel. Droplets comprising hydrogel precursor solution (polyethylene glycol tetraacrylate or a combination of polyethylene glycol tetraacrylate and gelatine methacrylate), photoinitiator and particles were generated. The droplets passed along the acoustic focusing channel where a half wavelength acoustic standing wave field was generated, and the particles were focused to the centre line of the droplets (i.e. the pressure nodal line) by the acoustic force. The droplets were cross-linked by exposure to UV-light, freezing the particles in their positions. With the acoustics applied, 89 ± 19% of the particles (polystyrene beads, 10 µm diameter) were positioned in an area ± 10% from the centre line. As proof-of-principle for biological particles, astrocytes were focused in hydrogel droplets using the same principle. The viability of the astrocytes after 7 days in culture was 72 ± 22% when exposed to the acoustic focusing compared with 70 ± 19% for samples not exposed to the acoustic focusing. This technology provides a platform to control the spatial position of bioparticles in hydrogel droplets, and opens up for the generation of more complex biological hydrogel structures.

We present a microfluidic system to control the position of particles (polystyrene beads or astrocyte cells) in hydrogel droplets using bulk acoustic standing waves. Droplets comprising hydrogel precursor solution, photoinitiator and (bio-)particles were generated and acoustic forces focused the beads to the droplet center line. Droplets were cross-linked by exposure to UV-light. With the acoustics applied, 89 ± 19% of the particles were positioned in the center of the hydrogel droplet. As proof-of-principle for biological applications, astrocytes were focused in hydrogel droplets. The viability of the cells after 7 days was unaffected by the acoustic focusing (72 ± 22%).

Acoustic trapping is a useful method for handling biological samples in microfluidic systems. The aim of this work is twofold: first to investigate the physics behind acoustic trapping in a glass capillary and secondly to perform binary acoustic trapping. The latter is achieved by increasing the density of the fluid in the trapping channel. The trapping device consisted of a glass capillary with a rectangular inner cross-section (height 200 µm × width 2000 µm) equipped with a small piezoelectric transducer. The piezoelectric transducer was actuated at 4 MHz to generate a localised half-wavelength acoustic standing-wave-field in the capillary, comprising of a pressure field and a velocity field. Under acoustic actuation, only particles with higher density than the fluid, i.e. having a positive dipole scattering coefficient, were trapped in the flow direction. The numerical and analytical modelling of the system show that the trapping force which retains the particles against the flow depends only on the dipole scattering coefficient in the pressure nodal plane of the acoustic field. The analytical model also reveals that the retention force is proportional to the dipole scattering coefficient, which agrees with our experimental findings. Next, we showed that in a mixture of melamine particles and polystyrene particles in a high-density fluid it is possible to selectively trap melamine particles, since melamine particles have higher density than polystyrene particles.

Acoustophoresis is a commonly used technique to manipulate microbeads suspended in single-phase fluids [1]. Fornell et al. demonstrated that acoustophoresis can also be used to manipulate microbeads in droplets when the acoustic properties of the continuous phase match with the acoustic properties of the dispersed phase [2]. Water-in-oil droplets can be considered to be ideal vials for liquid mixing and reactions, as they allow for high throughput, low cross-contamination and low reagent consumption. Droplet microfluidics has previously been applied to study enzyme reactions [3,4]. However, these approaches are limited by on-chip detection and analysis since the enzyme is mixed with the product of the reaction inside the droplets. Here, we present an acoustofluidic chip where we can start the enzymatic reaction by injecting enzyme-coupled microbeads into the droplets containing the substrate by pico-injection and then stop the enzymatic reaction by removing the enzyme-coupled microbeads using acoustophoresis [5]. The analysis can then be performed off-chip, which allows for longer exposure times compared to performing the analysis in-line.