Compartmentalised enzyme-induced phase transformations in self-assembling lipid systems.

HYPOTHESIS: Understanding the digestion of lipid-based pharmaceutical formulations and food systems is necessary for optimising drug and nutrient delivery and has been extensively studied in bulk emulsion systems using the pH-stat method [1]. However, this approach is not suitable for investigation of individual lipid droplets, in particular the interface where the lipase acts. Microfluidic approaches to study digestion at lipid-water interfaces using droplet trapping have been proposed, however the aqueous phase in that case washes over the interface presenting uncertainty over the stoichiometry of interactions [2]. The internal interface of a Janus-like droplet, containing distinct aqueous and lipid compartments, mimics the interface of a lipid droplet in aqueous solution with controlled stoichiometry [3]. Hence, it was hypothesised that the internal interface of Janus droplets can offer a precise way to study the enzymatic digestion of lipids formulations. EXPERIMENTS: Using microfluidic methods, Janus-like droplets were formed by coalescing emulsion droplets containing lipid formulation and pancreatic lipase. Polarised light microscopy (PLM) and in-situ small-angle X-ray scattering (SAXS) were used to investigate the droplets. FINDINGS: PLM revealed the growth of an aligned inverse hexagonal phase (H2), and with SAXS showed that this phase transformation and alignment resulted from enzymatic digestion. A subsequent partial transformation from H2 to inverse bicontinuous cubic phase occurred when simulated intestinal fluid was used instead of Tris buffer. Suggesting that phospholipids and bile salts could diffuse across the internal interface to locally affect their surroundings.

Rapid Concentration and Detection of Bacteria in Milk Using a Microfluidic Surface Acoustic Wave Activated Nanosieve.

Rapid detection of microbes is a key feature for monitoring food quality. Unfortunately, current detection systems rely on labor-intensive and time-consuming lab-based processes that are not suitable for point-of-interest applications and typically require several days before results are available. Here, we demonstrate a microfluidic system capable of rapidly concentrating, fluorescent staining, and detecting bacteria in unprocessed complex biological media such as milk. This concentration is done using a surface acoustic wave-driven microfluidic device which operates based on the Bjerknes force, a force generated on one particle by another in its close proximity. We exploit this effect by exciting a tightly packed bed of 50 µm polystyrene microparticles temporarily with surface acoustic waves within a microfluidic device to capture and release bacterial cells on demand. The bacterial cells are fluorescently stained during capture and then detected using fluorescence microscopy upon release. This device offers a high capturing efficiency (>80%) and a 34 Colony Forming Units (CFU)/mL limit of detection, which is 1 order of magnitude below that of plate counting at 30 CFU per standard 100 µL plate (or 300 CFU/mL). This can be attained in just 1 h of processing at 10 µL/min. With this system, we demonstrate that bacterial detection from extremely low concentration samples down to the order of ∼10 CFU/mL is possible without requiring any additional external pre- or postprocessing.

Making immotile sperm motile using high-frequency ultrasound.

Sperm motility is a natural selection with a crucial role in both natural and assisted reproduction. Common methods for increasing sperm motility are by using chemicals that cause embryotoxicity, and the multistep washing requirements of these methods lead to sperm DNA damage. We propose a rapid and noninvasive mechanotherapy approach for increasing the motility of human sperm cells by using ultrasound operating at 800 mW and 40 MHz. Single-cell analysis of sperm cells, facilitated by droplet microfluidics, shows that exposure to ultrasound leads to up to 266% boost to motility parameters of relatively immotile sperm, and as a result, 72% of these immotile sperm are graded as progressive after exposure, with a swimming velocity greater than 5 micrometer per second. These promising results offer a rapid and noninvasive clinical method for improving the motility of sperm cells in the most challenging assisted reproduction cases to replace intracytoplasmic sperm injection (ICSI) with less invasive treatments and to improve assisted reproduction outcomes.

Acousto-optofluidic 3D single cell imaging of macrophage phagocytosis of Pseudomonas Aeruginosa.

Understanding how immune cells such as monocytes or macrophages within our blood and tissue engulf and destroy foreign organisms is important for developing new therapies. The process undertaken by these cells, called phagocytosis, has yet to be observed in real-time at the single cell level. Microfluidic-based imaging platforms offer a wide range of tools for precise fluid control and biomolecule manipulation that makes regulating long term experiments and data collection possible. With the compatibility between acoustofluidics and light-sheet fluorescent microscopy (LSFM) previously demonstrated, here an acousto-optfluidic device with on-chip fluid flow direction control was developed. The standing surface acoustic waves (SSAWs) were used to trap, load and safeguard individual cells within a highly controllable fluid loop, created via the triggering of on-chip PDMS valves, to demonstrate multiple rounds of live single cell imaging. The valves allowed for the direction of the fluid flow to be changed (between forward and reverse operation) without altering the inlet flow rate, an important factor for performing reproducible and comparable imaging of samples over time. With this high-resolution imaging system, volumetric reconstructions of phagocytosed bacteria within macrophages could be resolved over a total of 9 rounds of imaging: totalling 19 reconstructed images of the cell membrane with visible intracellular bacteria.

Fully Microfabricated Surface Acoustic Wave Tweezer for Collection of Submicron Particles and Human Blood Cells.

Precise manipulation of (sub)micron particles is key for the preparation, enrichment, and quality control in many biomedical applications. Surface acoustic waves (SAW) hold tremendous promise for manipulation of (bio)particles at the micron to nanoscale ranges. In commonly used SAW tweezers, particle manipulation relies on the direct acoustic radiation effect whose superior performance fades rapidly when progressing from micron to nanoscale particles due to the increasing dominance of a second order mechanism, termed acoustic streaming. Through reproducible and high-precision realization of stiff microchannels to reliably actuate the microchannel cross-section, here we introduce an approach that allows the otherwise competing acoustic streaming to complement the acoustic radiation effect. The synergetic effect of both mechanisms markedly enhances the manipulation of nanoparticles, down to 200 nm particles, even at relatively large wavelength (300 µm). Besides spherical particles ranging from 0.1 to 3 µm, we show collections of cells mixed with different sizes and shapes inherently existing in blood including erythrocytes, leukocytes, and thrombocytes.

A novel droplet-based approach to study phase transformations in lyotropic liquid crystalline systems.

HYPOTHESIS: Lyotropic liquid crystals (LLC) and their phase transformations in response to stimuli have gathered much interest for controlled and 'on-demand' drug applications. Bulk methods of preparation impose limitations on studying the transformations, especially induced by compositional changes, such as enzymatic changes to lipid structure. Here we hypothesise that controlled microfluidic production and coalescence of dissimilar aqueous and lipid droplets emulsified in a third mutually immiscible liquid will provide a new approach to the spatio-temporal study of structure formation in lyotropic liquid crystalline materials. EXPERIMENTS: Separate lipid and aqueous droplets, dispersed in a fluorocarbon oil were generated using a microfluidic format. The chip, prepared as a hybrid polydimethylsiloxane (PDMS) and glass microfluidic device, was constructed to enable in-situ acquisition of time-resolved synchrotron small angle X-ray scattering (SAXS) and crossed polarised light microscopy of the coalesced droplets to determine the structures present during aging. FINDINGS: Janus-like droplets formed upon coalesce, with distinct lipid and aqueous portions with a gradient between the two sides of the merged droplet. SAXS and polarised light microscopy revealed a progression of mesophases as the lipid portion was hydrated by the aqueous portion via the diffusion limited interface which separated the portions. Thus demonstrating, on a droplet scale, a new approach for studying the phase transformation kinetics and identification of non-equilibrium phase in droplet-based lyotropic liquid systems.

Glass-embedded PDMS microfluidic device for enhanced concentration of nanoparticles using an ultrasonic nanosieve.

Surface acoustic wave (SAW) driven devices typically employ polymeric microfluidic channels of low acoustic impedance mismatch to the fluid in contact, to allow precise control of the wave field. Several of these applications, however, can benefit from the implementation of an acoustically reflective surface at the microfluidic channel's ceiling to increase energy retention within the fluid and hence, performance of the device. In this work, we embed a glass insert at the ceiling of the PDMS microfluidic channel used in a SAW activated nanosieve, which utilises a microparticle resonance for enrichment of nanoparticles. Due to the system's independence of performance on channel geometry and wave field pattern, the glass-inserted device allowed for a 30-fold increase in flow rate, from 0.05 µl min-1 to 1.5 µL min-1, whilst maintaining high capture efficiencies of >90%, when compared to its previously reported design. This effectively enables the system to process larger volume samples, which typically is a main limitation of these type of devices. This work demonstrates a simple way to increase the performance and throughput of SAW-based devices, especially within systems that can benefit from the energy retention.

Surface acoustic wave-driven pumpless flow for sperm rheotaxis analysis.

Sperm rheotaxis, the phenomenon where sperm cells swim against the direction of fluid flow, is one of the major guiding mechanisms for long-distance sperm migration within the female reproductive tract. However, current approaches to study this pose challenges in dealing with rare samples by continuously introducing extra buffer. Here, we developed a device utilising acoustic streaming, the steady flow driven by an acoustic perturbation, to drive a tuneable, well-regulated continuous flow with velocities ranging from 40 µm s-1 to 128 µm s-1 (corresponding to maximum shear rates of 5.6 s-1 to 24.1 s-1) in channels of interest - a range suitable for probing sperm rheotaxis behaviour. Using this device, we studied sperm rheotaxis in microchannels of distinct geometries representing the geometrical characteristics of the inner-surfaces of fallopian tubes, identified sperm dynamics with the presence of flow in channels of various widths. We found a 28% higher lateral head displacement (ALH) in sufficiently motile rheotactic sperm in a 50 µm channel in the presence of acoustically-generated flow as well as a change in migration direction and a 52% increase in curvilinear velocity (VCL) of sufficiently motile sperm in a 225 µm channel by increasing the average flow velocity from 40 µm s-1 to 130 µm s-1. These results provided insights for understanding sperm navigation strategy in the female reproductive tract, where rheotactic sperm swim near the boundaries to overcome the flow in the female reproductive tract and reach the fertilization site. This surface acoustic wave device presents a simple, pumpless alternative for studying microswimmers within in vitro models, enabling the discovery of new insights into microswimmers' migration strategies, while potentially offering opportunities for rheotaxis-based sperm selection and other flow-essential applications.

Nonequilibrium interfacial diffusion across microdroplet interface.

Increases in complexity attainable in molecular self-assembly necessitates both advanced molecular design as well as microenvironmental control. Such control is offered by microfluidics, where precise chemical compositions and gradients can be readily established. A droplet microfluidic platform combining upstream step emulsification with downstream hydrodynamic microtraps has been designed to facilitate molecular self-assembly. The step emulsification rapidly generates uniform droplets which act as reaction chambers. The hydrodynamic microtraps hold droplets against the flow ensuring they are exposed to a continuous supply of fresh fluid for constant reagent extraction and/or delivery. Additionally, the droplet immobilization permits real-time droplet characterization and reaction monitoring. Subsequently, droplets can be released from the traps through flow reversal, allowing post-process characterization. The microfluidic system was demonstrated by the phase separation of lyotropic droplets. Ethanol/water droplets were created in a continuous ambient squalene/monoolein microflow, causing the continuous extraction of ethanol from the droplets and delivery of monoolein from the ambient microflow. Unlike conventional bulk techniques and continuous microfluidics, where finite microchannel lengths necessarily impose limits to the extent to which slow processes can proceed, this approach allows extended duration reactions whilst enabling real time process monitoring.

Acoustofluidic cell micro-dispenser for single cell trajectory control.

The precise manipulation of individual cells is a key capability for the study of single cell physiological characteristics or responses to stimuli. Currently, only large cell populations can be transferred with certainty using expensive and laborious flow cytometry platforms. However, when approaching small populations of cells, this task becomes increasingly challenging. Here, we report an effective acoustofluidic micro-dispenser, utilising surface acoustic waves (SAWs), with the ability to trap and release cells on demand, which when combined with an external valve can guide the trajectory of individual cells. We demonstrate single cell trap and release with a single cell trapping effectiveness of 74%, enabling the capability of dispensing a highly controlled amount of cells without any harmful effects. This device has the potential to be easily integrated into a wide range of analytical platforms for applications such as single cell fluorescent imaging and single cell proteomic studies.

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

High-Frequency Ultrasound Boosts Bull and Human Sperm Motility.

Sperm motility is a significant predictor of male fertility potential and is directly linked to fertilization success in both natural and some forms of assisted reproduction. Sperm motility can be impaired by both genetic and environmental factors, with asthenozoospermia being a common clinical presentation. Moreover, in the setting of assisted reproductive technology clinics, there is a distinct absence of effective and noninvasive technology to increase sperm motility without detriment to the sperm cells. Here, a new method is presented to boost sperm motility by increasing the intracellular rate of metabolic activity using high frequency ultrasound. An increase of 34% in curvilinear velocity (VCL), 10% in linearity, and 32% in the number of motile sperm cells is shown by rendering immotile sperm motile, after just 20 s exposure. A similar effect with an increase of 15% in VCL treating human sperm with the same setting is also identified. This cell level mechanotherapy approach causes no significant change in cell viability or DNA fragmentation index, and, as such, has the potential to be applied to encourage natural fertilization or less invasive treatment choices such as in vitro fertilization rather than intracytoplasmic injection.

Curvature in the reproductive tract alters sperm-surface interactions.

The fallopian tube is lined with a highly complex folded epithelium surrounding a lumen that progressively narrows. To study the influence of this labyrinthine complexity on sperm behavior, we use droplet microfluidics to create soft curved interfaces over a range of curvatures corresponding to the in vivo environment. We reveal a dynamic response mechanism in sperm, switching from a progressive surface-aligned motility mode at low curvatures (larger droplets), to an aggressive surface-attacking mode at high curvatures (smaller droplets of <50 µm-radius). We show that sperm in the attacking mode swim ~33% slower, spend 1.66-fold longer at the interface and have a 66% lower beating amplitude than in the progressive mode. These findings demonstrate that surface curvature within the fallopian tube alters sperm motion from a faster surface aligned locomotion in distal regions to a prolonged physical contact with the epithelium near the site of fertilization, the latter being known to promote capacitation and fertilization competence.

Three-dimensional imaging on a chip using optofluidics light-sheet fluorescence microscopy.

Volumetric, sub-micron to micron level resolution imaging is necessary to assay phenotypes or characteristics at the sub-cellular/organelle scale. However, three-dimensional fluorescence imaging of cells is typically low throughput or compromises on the achievable resolution in space and time. Here, we capitalise on the flow control capabilities of microfluidics and combine it with microoptics to integrate light-sheet based imaging directly into a microfluidic chip. Our optofluidic system flows suspended cells through a sub-micrometer thick light-sheet formed using micro-optical components that are cast directly in polydimethylsiloxane (PDMS). This design ensures accurate alignment, drift-free operation, and easy integration with conventional microfluidics, while providing sufficient spatial resolution, optical sectioning and volumetric data acquisition. We demonstrate imaging rates of 120 ms per cell at sub-µm resolution, that allow extraction of complex cellular phenotypes, exemplified by imaging of cell clusters, receptor distribution, and the analysis of endosomal size changes.

Microfluidic enhancement of self-assembly systems.

Dynamic, kinetically-controlled, self-assembly processes are commonly observed in nature and are capable of creating intricate, functional architectures from simple precursors. However, notably, much of the research into molecular self-assembly has been performed using conventional bulk techniques where the resultant species are dictated by thermodynamic stability to yield relatively simple assemblies. Whereas, the environmental control offered by microfluidic systems offers methods to achieve non-equilibrium reaction conditions capable of increasingly sophisticated self-assembled structures. Alterations to the immediate microenvironment during the assembly of the molecules is possible, providing the basis for kinetically-controlled assembly. This review examines the key mechanism offered by microfluidic systems and the architectures required to access them. The mechanisms include diffusion-led mixing, shear gradient alignment, spatial and temporal confinement, and structural templates in multiphase systems. The works are selected and categorised in terms of the microfluidic approaches taken rather than the chemical constructs which are formed.

Bacteriophage uptake by mammalian cell layers represents a potential sink that may impact phage therapy.

It is increasingly apparent that bacteriophages, viruses that infect bacteria and more commonly referred to as simply phages, have tropisms outside their bacterial hosts. Using live tissue culture cell imaging, we demonstrate that cell type, phage size, and morphology play a major role in phage internalization. Uptake was validated under physiological conditions using a microfluidic device. Phages adhered to mammalian tissues, with adherent phages being subsequently internalized by macropinocytosis, with functional phages accumulating intracellularly. We incorporated these results into a pharmacokinetic model demonstrating the potential impact of phage accumulation by cell layers, which represents a potential sink for circulating phages in the body. During phage therapy, high doses of phages are directly administered to a patient in order to treat a bacterial infection, thereby facilitating broad interactions between phages and mammalian cells. Understanding these interactions will have important implications on innate immune responses, phage pharmacokinetics, and the efficacy of phage therapy.

Increasing the fine particle fraction of pressurised metered dose inhaler solutions with novel actuator shapes.

In this paper we demonstrate that the use of multiple orifices can improve the fine particle fraction (FPF) of pressurised metered-dose inhaler solution formulations by up to 75% when compared to a single orifice with an equivalent cross sectional area (p<0.05). While prior work has relied on metal actuator components, improvements in micro injection moulding and micro drilling now make it possible to mass produce novel orifice shapes to achieve similar FPF gains in plastic parts, with orifice diameters less than 0.2 mm. The ability to create internal features inside the actuator is also demonstrated. We show through in vitro high speed imaging that twin orifice sprays merge quickly and act as a single, modified plume. We also show for the first time that FPF and fine particle dose (FPD) are strongly correlated with the distance at which the plume velocity decays to half its initial value (R2=0.997 and 0.95 respectively). When plume velocity & FPF are increased, mouthpiece deposition decreases. This suggests that while smaller orifices produce more fine particles, higher sustained plume velocities also entrain more of the fine particles produced at the periphery of the spray due to increased shear. The effect occurs within the mouthpiece and is thus unlikely to alter the flow field in the upper airway.

High DNA integrity sperm selection using surface acoustic waves.

Male infertility is a global reproductive issue, several clinical approaches have been developed to tackle it, but their effectiveness is limited by the labour-intensive and time-consuming sperm selection procedures used. Here, we present an automated, acoustic based continuous-flow method capable of selecting high quality sperm with considerably improved motility and DNA integrity compared to the initial raw bull semen. The acoustic field translates larger sperm and guides highly motile sperm across the channel width. The result is the selection of sperm with over 50% and 60% improvement in vitality and progressive motility and more than 38% improvement in DNA integrity, respectively, while providing a clinically relevant volume and selected sperm number for the performance of in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) by selecting over 60 000 sperm in under an hour.

Pore-scale multiple-contact miscibility measurements in a microfluidic chip.

Carbon dioxide enhanced oil recovery is an interim solution as the world transitions to a cleaner energy future, extending oil production from existing fields whilst also sequestering carbon dioxide. To make this process efficient, the gas and oil need to develop miscibility over a period of time through the exchange of chemical components between the two phases, termed multiple-contact miscibility. Currently, measurements to infer the development of multiple-contact miscibility are limited to macroscopic visualization. We present a "rock-on-a-chip" measurement system that offers several potential measurements for different wetting conditions to infer the onset of multiple-contact miscibility. Here, a two-dimensional microfluidic porous medium with a stochastic distribution of pillars was created, and an analogue ternary system was used to mimic the real oil and gas multiple-contact miscibility process. Experiments were performed in two directions, imbibition and drainage, permitting study of different wetting properties of the host rock. The distinct behavior of trapped non-wetting ganglia during imbibition and the evolution of phase interfaces during drainage were observed and analyzed as the system developed miscibility. We show how these observations can be converted into rapid measurements for identifying the development of miscibility.

Exosome trapping and enrichment using a sound wave activated nano-sieve (SWANS).

Exosomes, a form of extracellular vesicle, are an important precursor in regenerative medicine. Microfluidic methods exist to capture these sub-micrometer sized objects from small quantities of sample, ideal for multiple diagnostic applications. To address the challenge of extraction from large volumes, we use the visual access offered by microfluidic techniques to probe the physical mechanisms behind a method which is compatible with future upscaling. The sound wave actuated nano-sieve uses resonant modes in a packed bed of microparticles to exert trapping forces on nanoparticles. Here, we examine the role of the microparticle size, demonstrating better performance from 15 µm particles than 7 µm particles. When applied to biological samples, we demonstrate for the first time that a packed bed of these larger particles is capable of capturing exosomes and liposomes, the captured particles being on average 20 to 40 times smaller than the pores within the trapped bed.