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.

Tracheal branching in ants is area-decreasing, violating a central assumption of network transport models.

The structure of tubular transport networks is thought to underlie much of biological regularity, from individuals to ecosystems. A core assumption of transport network models is either area-preserving or area-increasing branching, such that the summed cross-sectional area of all child branches is equal to or greater than the cross-sectional area of their respective parent branch. For insects, the most diverse group of animals, the assumption of area-preserving branching of tracheae is, however, based on measurements of a single individual and an assumption of gas exchange by diffusion. Here we show that ants exhibit neither area-preserving nor area-increasing branching in their abdominal tracheal systems. We find for 20 species of ants that the sum of child tracheal cross-sectional areas is typically less than that of the parent branch (area-decreasing). The radius, rather than the area, of the parent branch is conserved across the sum of child branches. Interpretation of the tracheal system as one optimized for the release of carbon dioxide, while readily catering to oxygen demand, explains the branching pattern. Our results, together with widespread demonstration that gas exchange in insects includes, and is often dominated by, convection, indicate that for generality, network transport models must include consideration of systems with different architectures.

Self-Aligned Acoustofluidic Particle Focusing and Patterning in Microfluidic Channels from Channel-Based Acoustic Waveguides.

Acoustic fields have been widely used for manipulation of particles and cells within microfluidic systems. In this Letter, we explore a novel acoustofluidic phenomenon for particle patterning and focusing, where a periodic acoustic pressure field is produced parallel to internal channel boundaries with the imposition of either a traveling or standing surface acoustic wave (SAW). This effect results from the propagation and intersection of edge waves from the channel walls according to the Huygens-Fresnel principle and classical wave fronts from the substrate-fluid interface. We demonstrate versatile control over this effect to produce both one- and two-dimensional acoustic patterning from one-dimensional SAW fields and its utility for continuous particle focusing. Uniquely, this channel-guided acoustic focusing permits the generation of robust acoustic fields without channel resonance conditions and particle focusing positions that are difficult or impossible to produce otherwise.

Huygens-Fresnel Acoustic Interference and the Development of Robust Time-Averaged Patterns from Traveling Surface Acoustic Waves.

Periodic pattern generation using time-averaged acoustic forces conventionally requires the intersection of counterpropagating wave fields, where suspended micro-objects in a microfluidic system collect along force potential minimizing nodal or antinodal lines. Whereas this effect typically requires either multiple transducer elements or whole channel resonance, we report the generation of scalable periodic patterning positions without either of these conditions. A single propagating surface acoustic wave interacts with the proximal channel wall to produce a knife-edge effect according to the Huygens-Fresnel principle, where these cylindrically propagating waves interfere with classical wave fronts emanating from the substrate. We simulate these conditions and describe a model that accurately predicts the lateral spacing of these positions in a robust and novel approach to acoustic patterning.

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.

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.

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.

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.

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.

A star shaped acoustofluidic mixer enhances rapid malaria diagnostics via cell lysis and whole blood homogenisation in 2 seconds.

Malaria is a life-threatening disease caused by a parasite, which can be transmitted to humans through bites of infected female Anopheles mosquitoes. This disease plagues a significant population of the world, necessitating the need for better diagnostic platforms to enhance the detection sensitivity, whilst reducing processing times, sample volumes and cost. A critical step in achieving improved detection is the effective lysis of blood samples. Here, we propose the use of an acoustically actuated microfluidic mixer for enhanced blood cell lysis. Guided by numerical simulations, we experimentally demonstrate that the device is capable of lysing a 20× dilution of isolated red blood cells (RBCs) with an efficiency of ∼95% within 350 ms (0.1 mL). Further, experimental results show that the device can effectively lyse whole blood irrespective of its dilution factor. Compared to the conventional method of using water, this platform is capable of releasing a larger quantity of haemoglobin into plasma, increasing the efficiency without the need for lysis reagents. The lysis efficiency was validated with malaria infected whole blood samples, resulting in an improved sensitivity as compared to the unlysed infected samples. Partial least squares-regression (PLS-R) analysis exhibits cross-validated R2 values of 0.959 and 0.98 from unlysed and device lysed spectral datasets, respectively. Critically, as expected, the root mean square error of cross validation (RMSECV) value was significantly reduced in the acoustically lysed datasets (RMSECV of 0.97), indicating the improved quantification of parasitic infections compared to unlysed datasets (RMSECV of 1.48). High lysis efficiency and ultrafast processing of very small sample volumes makes the combined acoustofluidic/spectroscopic approach extremely attractive for point-of-care blood diagnosis, especially for detection of neonatal and congenital malaria in babies, for whom a heel prick is often the only option for blood collection.

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.

On-demand sample injection: combining acoustic actuation with a tear-drop shaped nozzle to generate droplets with precise spatial and temporal control.

An on-demand droplet injection method for controlled delivery of nanolitre-volume liquid samples to scientific instruments for subsequent analysis is presented. We employ pulsed focussed surface acoustic waves (SAW) to eject droplets from an enclosed microfluidic channel into an open environment. The 3D position of individual droplets and their time of arrival can be precisely controlled to within 61 µs in a 500 µm square target region 40 µm wide. The continuous ejection rate of 16 000 droplets per second can be tuned to produce pulsed trains of droplets from 0 up to 357 Hz. The main benefit of this technique is its ease of integration with complex microfluidic processing steps, such as droplet merging, sorting, and encapsulation, prior to sample delivery. With its ability to precisely deliver a small quantity of fluid to a pre-defined location this technology is applicable in X-ray based molecular studies, including the rapidly expanding field of X-ray free electron lasers. Fabrication procedures for this device, the underlying forcing mechanism, the role of nozzle design, and demonstration of the performance in both continuous and on-demand modes are reported.

Diffraction-based acoustic manipulation in microchannels enables continuous particle and bacteria focusing.

Acoustic fields have shown wide utility for micromanipulation, though their implementation in microfluidic devices often requires accurate alignment or highly precise channel dimensions, including in typical standing surface acoustic wave (SSAW) devices and resonant channels. In this work we investigate an approach that permits continuous microscale focusing based on diffractive acoustics, a phenomenon where a time-averaged spatially varying acoustic pressure landscape is produced by bounding a surface acoustic wave (SAW) transducer with a microchannel. By virtue of diffractive effects, this acoustic field is formed with the application of only a single travelling wave. As the field is dictated by the interplay between a propagating substrate-bound wave and a channel geometry, the pressure distribution will be identical for a given channel orientation regardless of its translation on a SAW substrate, and where small variations in channel size have no substantive effect on the pressure field magnitude or overall particle migration. Moreover, in the case of a channel with dimensions on the order of the diffractive fringe pattern spacing, the number of focusing positions will be identical for all channel orientations, with acoustic radiation forces pushing suspended particles to the channel edges. We explore this highly robust particle manipulation technique, determining two distinct sets of streaming and acoustic radiation dominant concentration positions, and show the continuous focusing of polystyrene 1 µm and 0.5 µm diameter particles and fluorescently labeled E. coli bacteria cells at flow rates exceeding those of previous microfluidic implementations for micron and submicron sized particles.

Ultrafast star-shaped acoustic micromixer for high throughput nanoparticle synthesis.

We present an acoustically actuated microfluidic mixer, which can operate at flowrates reaching 8 ml min-1, providing a 50-fold improvement in throughput compared to previously demonstrated acoustofluidic approaches. The device consists of a robust silicon based micro-mechanical oscillator, sandwiched between two polymeric channels which guide the fluids in and out of the system. The chip is actuated by application of an oscillatory electrical signal onto a piezoelectric disk coupled to the substrate by adhesive. At the optimal frequency, this acoustofluidic system can homogenise two fluids with a relative mixing efficiency of 91%, within 4.1 ms from first contact. The micromixer has been used to synthesize two different systems: Budesonide nanodrugs with an average diameter of 80 ± 22 nm, and DNA nanoparticles with an average diameter of 63.3 ± 24.7 nm.

Cell Adhesion, Morphology, and Metabolism Variation via Acoustic Exposure within Microfluidic Cell Handling Systems.

Acoustic fields are capable of manipulating biological samples contained within the enclosed and highly controlled environment of a microfluidic chip in a versatile manner. The use of acoustic streaming to alter fluid flows and radiation forces to control cell locations has important clinical and life science applications. While there have been significant advances in the fundamental implementation of these acoustic mechanisms, there is a considerable lack of understanding of the associated biological effects on cells. Typically a single, simple viability assay is used to demonstrate a high proportion of living cells. However, the findings of this study demonstrate that acoustic exposure can inhibit cell attachment, decrease cell spreading, and most intriguingly increase cellular metabolic activity, all without any impact upon viability rates. This has important implications by showing that mortality studies alone are inadequate for the assessment of biocompatibility, but further demonstrates that physical manipulation of cells can also be used to influence their biological activity.

Versatile platform for performing protocols on a chip utilizing surface acoustic wave (SAW) driven mixing.

We present and demonstrate a dextrous microfluidic device which features a reaction chamber with volume flexibility. This feature is critical for developing protocols directly on chip when the exact reaction is not yet defined, enabling bio/chemical reactions on chip to be performed without volumetric restrictions. This is achieved by the integration of single layer valves (for reagent dispensing) and surface acoustic wave excitation (for rapid reagent mixing). We show that a single layer valve can control the delivery of fluid into, an initially air-filled, mixing chamber. This chamber arrangement offers flexibility in the relative volume of reagents used, and so offers the capability to not only conduct, but also develop protocols on a chip. To enable this potential, we have integrated a SAW based mixer into the system, and characterised its mixing time based on frequency and power of excitation. Numerical simulations on the streaming pattern inside the chamber were conducted to probe the underlying physics of the experimental system. To demonstrate the on-chip protocol capability, the system was utilised to perform protein crystallization. Furthermore, the effect of rapid mixing, results in a significant increase in crystal size uniformity.

The size dependant behaviour of particles driven by a travelling surface acoustic wave (TSAW).

The use of travelling surface acoustic waves (TSAW) in a microfluidic system provides a powerful tool for the manipulation of particles and cells. In a TSAW driven system, acoustophoretic effects can cause suspended micro-objects to display three distinct responses: (1) swirling, driven by acoustic streaming forces, (2) migration, driven by acoustic radiation forces and (3) patterning in a spatially periodic manner, resulting from diffraction effects. Whilst the first two phenomena have been widely discussed in the literature, the periodic patterning induced by TSAW has only recently been reported and is yet to be fully elucidated. In particular, more in-depth understanding of the size-dependant nature of this effect and the factors involved are required. Herein, we present an experimental and numerical study of the transition in acoustophoretic behaviour of particles influenced by relative dominance of these three mechanisms and characterise it based on particle diameter, channel height, frequency and intensity of the TSAW driven microfluidic system. This study will enable better understanding of the performance of TSAW sorters and allow the development of TSAW systems for particle collection and patterning.

Surface acoustic wave diffraction driven mechanisms in microfluidic systems.

Acoustic forces arising from high-frequency surface acoustic waves (SAW) underpin an exciting range of promising techniques for non-contact manipulation of fluid and objects at micron scale. Despite increasing significance of SAW-driven technologies in microfluidics, the understanding of a broad range of phenomena occurring within an individual SAW system is limited. Acoustic effects including streaming and radiation force fields are often assumed to result from wave propagation in a simple planar fashion. The propagation patterns of a single SAW emanating from a finite-width source, however, cause a far richer range of physical effects. In this work, we seek a better understanding of the various effects arising from the incidence of a finite-width SAW beam propagating into a quiescent fluid. Through numerical and experimental verification, we present five distinct mechanisms within an individual system. These cause fluid swirling in two orthogonal planes, and particle trapping in two directions, as well as migration of particles in the direction of wave propagation. For a range of IDT aperture and channel dimensions, the relative importance of these mechanisms is evaluated.

Trapping and patterning of large particles and cells in a 1D ultrasonic standing wave.

The use of ultrasound for trapping and patterning particles or cells in microfluidic systems is usually confined to particles which are considerably smaller than the acoustic wavelength. In this regime, the primary forces result in particle clustering at certain locations in the sound field, whilst secondary forces, those arising due to particle-particle interaction forces, assist this clustering process. Using a wavelength closer to the size of the particles allows one particle to be held at each primary force minimum. However, to achieve this, the influence of secondary forces needs to be carefully studied, as inter-particle attraction is highly undesirable. Here, we study the effect of particle size and material properties on both the primary and secondary acoustic forces as the particle diameter is increased towards the wavelength of the 1-dimensional axisymmetric ultrasonic field. We show that the resonance frequencies of the solid sphere have an important role in the resulting secondary forces which leads to a narrow band of frequencies that allow the patterning of large particles in a 1-D array. Knowledge regarding the naturally existent secondary forces would allow for system designs enabling single cell studies to be conducted in a biologically safe manner.

Acoustic tweezing of particles using decaying opposing travelling surface acoustic waves (DOTSAW).

Surface acoustic waves offer a versatile and biocompatible method of manipulating the location of suspended particles or cells within microfluidic systems. The most common approach uses the interference of identical frequency, counter propagating travelling waves to generate a standing surface acoustic wave, in which particles migrate a distance less than half the acoustic wavelength to their nearest pressure node. The result is the formation of a periodic pattern of particles. Subsequent displacement of this pattern, the prerequisite for tweezing, can be achieved by translation of the standing wave, and with it the pressure nodes; this requires changing either the frequency of the pair of waves, or their relative phase. Here, in contrast, we examine the use of two counterpropagating traveling waves of different frequency. The non-linearity of the acoustic forces used to manipulate particles, means that a small frequency difference between the two waves creates a substantially different force field, which offers significant advantages. Firstly, this approach creates a much longer range force field, in which migration takes place across multiple wavelengths, and causes particles to be gathered together in a single trapping site. Secondly, the location of this single trapping site can be controlled by the relative amplitude of the two waves, requiring simply an attenuation of one of the electrical drive signals. Using this approach, we show that by controlling the powers of the opposing incoherent waves, 5 µm particles can be migrated laterally across a fluid flow to defined locations with an accuracy of ±10 µm.