Ultrasound imaging often calls for the injection of contrast agents, micron-sized bubbles which echo strongly in blood and help distinguish vascularized tissue. Such microbubbles are also being augmented for targeted drug delivery and gene therapy, by the addition of surface receptors and therapeutic payloads. Unfortunately, conventional production methods yield a polydisperse population, whose nonuniform resonance and drug-loading are less than ideal. An alternative technique, microfluidic flow-focusing, is able to produce highly monodisperse microbubbles with stabilizing lipid membranes and drug-carrying oil layers. However, the published 1 kHz production rate for these uniform drug bubbles is very low compared to conventional methods, and must be improved before clinical use can be practical. In this study, flow-focusing production of oil-layered lipid microbubbles was tested up to 300 kHz, with coalescence suppressed by high lipid concentrations or inclusion of Pluronic F68 surfactant in the lipid solution. The transition between geometry-controlled and dripping production regimes was analysed, and production scaling was found to be continuous, with a power trend of exponent ~5/12 similar to literature. Unlike prior studies with this trend, however, scaling curves here were found to be pressure-dependent, particularly at lower pressure-flow equilibria (e.g. <15 psi). Adjustments in oil flow rate were observed to have a similar effect, akin to a pressure change of 1–3 psi. This analysis and characterization of high-speed dual-layer bubble generation will enable more-predictive production control, at rates practical for in vivo or clinical use.

In this study we report on a microfluidic device and droplet formation regime capable of generating clinical-scale quantities of droplet emulsions suitable in size and functionality for in vivo therapeutics. By increasing the capillary number – based on the flow rate of the continuous outer phase – in our flow-focusing device, we examine three modes of droplet breakup: geometry-controlled, dripping, and jetting. Operation of our device in the dripping regime results in the generation of highly monodisperse liquid perfluoropentane droplets in the appropriate 3–6 µm range at rates exceeding 105 droplets per second. Based on experimental results relating droplet diameter and the ratio of the continuous and dispersed phase flow rates, we derive a power series equation, valid in the dripping regime, to predict droplet size by Dd ≅ 27(QC/QD)−5/12. The volatile droplets in this study are stable for weeks at room temperature yet undergo rapid liquid-to-gas phase transition, and volume expansion, above a uniform thermal activation threshold. The opportunity exists to potentiate locoregional cancer therapies such as thermal ablation and percutaneous ethanol injection using thermal or acoustic vaporization of these monodisperse phase-change droplets to intentionally occlude the vessels of a cancer.

The production of uniform-sized and multilayer microbubbles enables promising medical applications that combine ultrasound contrast and targeted delivery of therapeutics, with improvements in the consistency of acoustic response and drug loading relative to non-uniform populations of microbubbles. Microfluidics has shown utility in the generation of such small multi-phase systems, however low production rates from individual devices limit the potential for clinical translation. We present scaled-up production of monodisperse dual-layered microbubbles in a novel multi-array microfluidic module containing four or eight hydrodynamic flow-focusing orifices. Production reached 1.34 × 105 Hz in the 8-channel configuration, and microbubble diameters in the high-speed regime (> 5 × 104 Hz) ranged between 18.6–22.3 μm with a mean pooled polydispersity index under 9 percent. Results demonstrate that microfluidic scale-up for high-output production of multilayer bubbles is possible while maintaining consistency in size production, suggesting that this method may be appropriate for future clinical applications.

Medical ultrasound imaging often employs ultrasound contrast agents (UCAs), injectable microbubbles stabilized by shells or membranes. In tissue, the compressible gas cores can strongly scatter acoustic signals, resonate, and emit harmonics. However, bubbles generated by conventional methods have nonuniform sizes, reducing the fraction that resonates with a given transducer. Microfluidic flow-focusing is an alternative production method which generates highly monodisperse bubbles with uniform constituents, enabling more-efficient contrast enhancement than current UCAs. Production size is tunable by adjusting gas pressure and solution flow rate, but solution effects on downstream stable size and lifetime have not been closely examined. This study therefore investigated several solution parameters, including the DSPC/DSPE-PEG2000 lipid ratio, concentration, viscosity, and preparation temperature to determine their effects on stabilization. It was found that bubble lifetime roughly correlated with stable size, which in turn was strongly influenced by primary-lipid-to-emulsifier ratio, analogous to its effects on conventional bubble yield and Langmuir-trough compressibility in existing studies. Raising DSPE-PEG2000 fraction in solution reduced bubble surface area in proportion to its reduction of lipid packing density at low compression in literature. In addition, the surface area was found to increase proportionately with lipid concentration above 2.1 mM. However, viscosities above or below 2.3-3.3 mPa·s seemed to reduce bubble size. Finally, lipid preparation at room temperature led to smaller bubbles compared to preparation near or above the primary lipid's phase transition point. Understanding these effects will further improve on postformation control over microfluidic bubble production, and facilitate size-tuning for optimal contrast enhancement.

Digital microfluidics (DMF) is a platform that enables highly reconfigurable and automated fluidic operations using a generic device architecture. A unique hallmark of DMF is its “flexibility”: a generic device design can be used and reused for many different, divergent fluidic operations. The flexibility of DMF is compromised when devices are permanently modified with embedded sensors. Here we introduce a solution to the “flexibility gap” between fluidic operations in digital microfluidics and embedded sensors: “plug-n-play DMF” (PnP-DMF). In PnP-DMF, devices are designed to allow for rapid and seamless exchange of sensors depending on the application needs. This paper provides “proof of concept” for PnP-DMF using commercial biosensors for glucose and β-ketone, a custom paper-based electrochemical sensor for lactate, and a generic screen-printed electroanalytical cell. We demonstrate that hot-swapping sensors between experiments allows for convenient implementation of complex processes such as automated analysis of blood samples by standard addition. Finally, we explored the suitability for using PnP sensors in tandem with other sensing modalities, combining biosensor-based electrochemical measurement of glucose with a chemiluminescent magnetic bead-based sandwich immunoassay for insulin. The latter is notable, as it constitutes the first report of an analysis of different analytes in both the supernatant and precipitate from a single sample-aliquot in a microfluidic device. The results presented here highlight the versatility of PnP-DMF, illustrating how it may be useful for a wide range of applications in diagnostics and beyond.