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Droplet-microfluidic platforms enable precise processing, manipulation, and analysis of biological samples with improved throughput, cost-efficiency and reduced response times compared to conventional bulk methods. At the microscale, shifts in the relative significance of physical parameters lead to distinct emergent properties, facilitating the creation of physiologically relevant microenvironments with spatiotemporal control over chemical (e.g., growth factors) and mechanical (e.g., hydrogel scaffolds) cues. These capabilities make droplet-microfluidic systems highly suitable for biology and biomedical research, and for applications such as disease diagnostics, cell therapeutics, and regenerative medicine. A fundamental requirement for such platforms is the ability to manipulate the spatial positioning and movement of biological entities for sample enrichment, separation, localization, and patterning. Acoustic forces offer a label-free and non-invasive approach to achieve this control, making them particularly attractive in microfluidic-based devices. This thesis focuses on studying the manipulation of microparticles and cells within droplets and microgels using bulk acoustic wave, aiming to develop on-chip processes that could ultimately be integrated into various cell-related research and applications. In droplet microfluidics, understanding the effects of different fluidic environments on the acoustic manipulation of intra-droplet particles is essential, particularly given the presence of two immiscible phases. Due to biocompatibility constraints, the selection of solutions for the droplet phase is limited. Therefore, the surrounding oil phase was modified in Paper I. Results of Paper I reveal that acoustic field uniformity and strength within the droplet are optimized when the speed of sound of the aqueous droplet and the surrounding oil are closely matched. It was concluded that hydrocarbon oils are more compatible with intra-droplet acoustic focusing, as their acoustic properties more closely approximate those of water. In contrast, fluorinated oils, conventionally favored in droplet microfluidics, were found to be less suitable due to greater disparities in their acoustic properties relative to water. In a subsequent study, the environmental risks associated with fluorinated oils, classified as per- and polyfluoroalkyl substances (PFAS), were assessed. The findings indicate that viable oil/surfactant alternatives to conventional fluorinated solutions exist for specific droplet microfluidic applications, such as the mineral oil/PGPR system for bacterial culture. In Paper III, it was demonstrated that intra-droplet acoustic focusing remains effective when the droplet phase consists of more viscous hydrogel formulations. Precise spatial control of bioparticles within continuously generated hydrogel droplets was successfully achieved, followed by on-chip crosslinking to immobilize particle positions without significantly compromising cell viability. Finally, the acoustic assembly of three-dimensional polystyrene bead clusters within hydrogel matrices was demonstrated, serving as a proof-of-concept for point focusing in microgel constructs via ultrasonic techniques.

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 disciplines like toxicology and pharmacology, oxygen (O-2) respiration is a universal metric for evaluating the effects of chemicals across various model systems, including mammalian and microalgal cells. However, for these cells the common practice is to segregate populations into control and exposure groups, which assumes direct equivalence in their responses and does not take into account heterogeneity among individual cells. This lack of resolution impedes our ability to precisely investigate differences among experimental groups with small or limited sample sizes. To overcome this barrier, we introduce SlipO(2)Chip, an innovative glass microfluidic platform for precisely quantifying single-cell O-2 respiration in the coordinated absence and presence of chemical solutes. SlipO(2)Chip comprises a wet-etched fused silica channel plate on the top and a dry-etched borosilicate microwell plate at the bottom. The microwells are coated with Pt(ii) meso-tetra(pentafluorophenyl)porphine (PtTFPP), an O-2 sensing optode material and an O-2-independent reference dye. A custom 3D-printed holder facilitates the controlled horizontal movement ('slipping') of the channel plate over the microwell plate, thereby establishing or disrupting the fluid path over microwells. Collectively, these design elements enable the immobilization of single-cells in microwells, their exposure to controlled fluid flows, the coordinated opening and closing of microwells and repeated measurements of single-cell O-2 respiration. Uniquely, by sequentially executing opening and closing it becomes possible to measure single-cell respiration prior to and after exposure to chemical solutes. In a proof-of-concept application, we utilized SlipO(2)Chip to measure the impact of increasing exposures of the marine bacterial signal 2-heptyl-4-quinolone (HHQ) on the dark respiration of the diatom Ditylum brightwellii at single-cell resolution. Results revealed a concentration-dependent decrease in per-cell O-2 dark respiration, with a maximum reduction of 40.2% observed at HHQ concentrations exceeding 35.5 mu M, and a half-maximal effective concentration (EC50) of 5.8 mu M, consistent with that obtained via conventional bulk respiration methods. The ability of SlipO(2)Chip to sequentially assess the effects of chemical substances on single-cell O-2 metabolism is advantageous for research where sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies aiming to address exposure effects while accounting for cell-to-cell variability.<br />

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.

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%).

Introduction

Droplet microfluidics has become a powerful tool to generate miniaturized compartments suitable for biologicalexperiments.[1] For example, hydrogel droplets are a suitable platform to study 3D cell culture.[2] For thisapplication, it can be advantageous to prepare hydrogel constructs with an ordered cell architecture. Here, wepresent an acoustic method to align cells in hydrogel droplets and demonstrate its biocompatibility.

Experimental procedure and Results

To position the cells in a line at the center of the droplets, we use acoustophoresis. The microfluidic chip wasfabricated using deep reactive ion etching of a Si wafer to form the channels and anodic bonding of a glass waferto seal the chip. A piezoelectric transducer was glued to the chip and actuated to generate bulk acoustic waves inthe channel. The channel dimensions were 380 μm x 100 μm (width x height), which corresponds to halfwavelength lateral resonance at 2 MHz. Droplets consisting of hydrogel precursor solution (3 wt% 4-arm-PEG(10000)-acrylate, 1 wt% gelatin methacrylate and 0.1 wt% photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) were formed. The cells, 5 × 106 astrocytes/mL encapsulated in the hydrogeldroplets, were focused at the center of the droplets (pressure nodal line) by the primary acoustic radiation force,Fig 1a). By exposing the cell-laden droplets to a short UV dose, the hydrogel precursor solution was cross-linkedand the cells were “frozen” in their position. Viability of acoustically focused cells and non-focused cells (noacoustics applied) encapsulated in the cross-linked droplets were evaluated by live/dead staining, demonstratingthat acoustic focusing and on-chip cross-linking with UV is a gentle process, see Fig. 1b).[3]

Figure 1: a) The experimental procedure. b) Focused polystyrene beads in a cross-linked hydrogel droplet. c) Focusedastrocytes in a cross-linked hydrogel droplet. d) The viability of astrocytes in hydrogel droplets after 1, 3 and 7 days of culture.In blue, acoustically focused cells are shown, whereas in grey, the control group (astrocytes encapsulated in hydrogel dropletswithout acoustic focusing) is shown after 7 days of culture.

Conclusion

A microfluidic chip with an integrated piezoelectric transducer can be used to generate hydrogel dropletswhere the encapsulated cells are focused to the center line of the droplets· Acoustic focusing of cells do not decrease cell viability· The generated hydrogel droplets could be suitable cell culture scaffolds for advanced biological studies.