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Droplet microfluidics has shown great potential for on-chip biological and chemical assays. However, fluid exchange in droplet microfluidics with high particle recovery is still a major bottleneck. Here, using acoustophoresis, we present for the first time a label-free method to achieve continuous background dilution in droplets containing cells with high sample recovery. The system comprises droplet generation, acoustic focusing, droplet splitting, picoinjection, and serpentine mixing on the same chip. The capacities of the picoinjection and the droplet split to dilute the background fluorescent signal in the droplets have been characterized. The sample recovery at different droplet split ratios has also been characterized. The results show a maximum of 4.3-fold background dilution with 87.7% particle recovery. We also demonstrated that the system can be used to dilute background fluorescent signal in droplets containing either polystyrene particles or endothelial cells.

We have characterised three droplet split designs for acoustic particle enrichment in water-in-oil droplets. The microfluidic channel design included a droplet generation junction, acoustic focusing channel and a trident-shaped droplet split. The microfluidic channels were dry-etched in silicon and sealed with glass lids by anodic bonding. To each microfluidic chip a piezoelectric transducer was glued, and at actuation of the transducer at the fundamental resonance frequency of the acoustic focusing channel (1.91–1.93 MHz), a half wavelength standing wave field was created between the channel walls. The acoustic force focused the encapsulated particles (3.2 μm, 4.8 μm and 9.9 μm diameter polystyrene microbeads) to the centre-line of the droplets, and when the droplets reached the droplet split the particles were directed into the centre daughter droplets. The results show that the design of the droplet split and the flow ratio between the centre and side outlet channels are the main factors that affect the particle enrichment and particle recovery in the centre daughter droplets. The highest particle enrichment was achieved in the droplet split design having the smallest centre channel (38 μm wide). Using this microfluidic chip design, we demonstrate up to 16.7-fold enrichment of 9.9 μm diameter polystyrene microbeads in the centre daughter droplets. This is almost three times higher particle enrichment than what has previously been presented using other intra-droplet particle enrichment techniques. Moreover, the acoustic technique is label-free and biocompatible.

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 emergence of droplet microfluidics as a powerful tool for on-chip biological assays has prompted the development of a variety of intra-droplet particle manipulation techniques, such as droplet acoustofluidics. Previous study has shown that the acoustic properties between the continuous and dispersed phase must match for high-quality intra-droplet particle focusing. As a follow up, this study investigates the acoustic properties, i.e., speed of sound and density, of a selection of non-polar fluids that can be used as the continuous phase in droplet microfluidic systems. Our experimental results show that within our collection, linseed oil is the non-polar phase that most closely matches the acoustic properties of water and the fluorinated oil HFE-7500 is the one that least matches the acoustic properties compared to water. We believe this collection of data will serve the community by providing results that aid in the selection of continuous phase in future droplet acoustofluidic studies and data for performing acoustofluidic simulations.

Droplet microfluidics can be used to encapsulate cells for biochemical applications, such as single-cell analysis and drug screening. However, the concentration of nutrients and growth factors decreases over time, while the concentration of catabolic byproducts increases, that make droplet hard for long-term cell culture. Here, the cells encapsulated in droplets continued to divide in the first 8 hours and then stopped to grow. We developed a droplet acoustofluidic chip that can exchange cell medium in droplets by the combination of the pico-injection and the droplet split with acoustophoresis. After running droplets through this chip, cell medium in droplets got exchanged and the cells in droplets started to grow again. By this way, long-term droplet cell culture can be achieved.