Surface Acoustic Wave-Driven Enhancement of Enzyme-Linked Immunosorbent Assays: ELISAW.

Enzyme-linked immunosorbent assays (ELISAs) are widely used in biology and clinical diagnosis. Relying on antigen-antibody interaction through diffusion, the standard ELISA protocol can be time-consuming, preventing its use in rapid diagnostics. We present a time-saving and more sensitive ELISA without changing the standard setup and protocol, using surface acoustic waves (SAWs) to enhance performance. Each step of the assay, from the initial antibody binding onto the walls of the well plate to the target analyte molecules' binding for detection─except, notably, for the blocking step─is improved principally via acoustic streaming-driven advection. Using SAWs, the time required for one step of an example ELISA is reduced from 60 to 15 min to achieve the same binding amount. By extending the duration of SAW exposure to 20 min, the sensitivity can be significantly improved over the 60 min, 35 °C ELISA without SAWs. It is also possible to confer beneficial improvements to bead-based ELISA by combining it with SAWs to further reduce the time required for binding to 2 min. By significantly increasing the speed of ELISA, its utility may be improved for a wide range of point-of-care diagnostics applications.

Glycocalyx transduces membrane leak in brain tumor cells exposed to sharp magnetic pulsing.

Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt ∼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 µs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This "first-physical" Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and "tunable" transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors.

Onset of Visible Capillary Waves from High-Frequency Acoustic Excitation.

Remarkably, the interface of a fluid droplet will produce visible capillary waves when exposed to acoustic waves. For example, a small (∼1 µL) sessile droplet will oscillate at a low ∼102 Hz frequency when weakly driven by acoustic waves at ∼106 Hz frequency and beyond. We measured such a droplet's interfacial response to 6.6 MHz ultrasound to gain insight into the energy transfer mechanism that spans these vastly different time scales, using high-speed microscopic digital transmission holography, a unique method to capture three-dimensional surface dynamics at nanometer space and microsecond time resolutions. We show that low-frequency capillary waves are driven into existence via a feedback mechanism between the acoustic radiation pressure and the evolving shape of the fluid interface. The acoustic pressure is distributed in the standing wave cavity of the droplet, and as the shape of the fluid interface changes in response to the distributed pressure present on the interface, the standing wave field also changes shape, feeding back to produce changes in the acoustic radiation pressure distribution in the cavity. A physical model explicitly based upon this proposed mechanism is provided, and simulations using it were verified against direct observations of both the microscale droplet interface dynamics from holography and internal pressure distributions using microparticle image velocimetry. The pressure-interface feedback model accurately predicts the vibration amplitude threshold at which capillary waves appear, the subsequent amplitude and frequency of the capillary waves, and the distribution of the standing wave pressure field within the sessile droplet responsible for the capillary waves.

Acousto-Photolithography for Programmable Shape Deformation of Composite Hydrogel Sheets.

Stimuli-responsive hydrogels with programmable shapes produced by defined patterns of particles are of great interest for the fabrication of small-scale soft actuators and robots. Patterning the particles in the hydrogels during fabrication generally requires external magnetic or electric fields, thus limiting the material choice for the particles. Acoustically driven particle manipulation, however, solely depends on the acoustic impedance difference between the particles and the surrounding fluid, making it a more versatile method to spatially control particles. Here, an approach is reported by combining direct acoustic force to align photothermal particles and photolithography to spatially immobilize these alignments within a temperature-responsive poly(N-isopropylacrylamide) hydrogel to trigger shape deformation under temperature change and light exposure. The spatial distribution of particles can be tuned by the power and frequency of the acoustic waves. Specifically, changing the spacing between the particle patterns and position alters the bending curvature and direction of this composite hydrogel sheet, respectively. Moreover, the orientation (i.e., relative angle) of the particle alignments with respect to the long axis of laser-cut hydrogel strips governs the bending behaviors and the subsequent shape deformation by external stimuli. This acousto-photolithography provides a means of spatiotemporal programming of the internal heterogeneity of composite polymeric systems.

Well-free agglomeration and on-demand three-dimensional cell cluster formation using guided surface acoustic waves through a couplant layer.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Powerful Acoustogeometric Streaming from Dynamic Geometric Nonlinearity.

Past forms of acoustic streaming, named after their progenitors Eckart (1948), Schlichting (1932), and Rayleigh (1884), serve to describe fluid and particle transport phenomena from the macro to micro-scale. Governed by the fluid viscosity, traditional acoustic streaming arises from second-order nonlinear coupling between the fluid's density and particle velocity, with the first-order acoustic wave time averaging to zero. We describe a form of acoustogeometric streaming that has a nonzero first-order contribution. Experimentally discovered in nanochannels of a height commensurate with the viscous penetration depth of the fluid in the channel, it arises from nonlinear interactions between the surrounding channel deformation and the leading order acoustic pressure field, generating flow pressures three orders of magnitude greater than any known acoustically mediated mechanism. It enables the propulsion of fluids against significant Laplace pressure, sufficient to produce 6 mm/s flow in a 130-150 nm tall nanoslit. We find quantitative agreement between theory and experiment across a variety of fluids and conditions, and identify the maximum flow rate with a channel height 1.59 times the viscous penetration depth.

Introduction to the special issue on the theory and applications of acoustofluidics.

Acoustofluidics is a burgeoning field that applies ultrasound to micro-scale to nano-scale fluidic systems. The discovery of the ability to effectively manipulate fluids and particles at small scales has yielded results that are superior to other approaches and has been built into a diverse range of research. Recasting the fundamentals of acoustics from the past to include new phenomena observed in recent years has allowed acoustical systems to impact new areas, such as drug delivery, diagnostics, and enhanced chemical processes. The contributions in this special issue address a diverse range of research topics in acoustofluidics. Topics include acoustic streaming, flows induced by bubbles, manipulation of particles using acoustic radiation forces, fluid and structural interactions, and contributions suggesting a natural limit to the particle velocity, the ability to deliver molecules to human immune T cells, and microdroplet generation via nozzle-based acoustic atomization.

An investigation of maximum particle velocity as a universal invariant-Defined by a statistical measure of failure or plastic energy loss for acoustofluidic applications.

Materials under vibration experience internal stress waves that can cause material failure or energy loss due to inelastic vibration. Traditionally, failure is defined in terms of material acceleration, yet this approach has many drawbacks, principally because it is not invariant with respect to scale, type of vibration, or material choice. Here, the likelihood of failure is instead considered in terms of the maximum vibration or particle velocity for various metals, polymers, and structural materials. The exact relationship between the maximum particle velocity and the maximum induced stress may be derived, but only if one knows the details of the vibration, material, flaws, and geometry. Statistical results with over thousands of individual trials are presented here to demonstrate a wide variety of vibrations across a sufficient variety of these choices. Failure in this context is defined as either fracture or plastic yield, the latter associated with inelastic deformation and energy loss during vibration. If the maximum permissible cyclical stress in material vibration is known, to at least an order of magnitude, the probability of this type of failure may be computed for a range of vibration velocities in each material. The results support the notion that a maximum particle velocity on the order of 1 m/s is a universal and critical limit that, upon exceeding, causes the probability of failure to become significant regardless of the details of the material, geometry, or vibration. We illustrate this in a specific example relevant to acoustofluidics, a simple surface acoustic wave device. The consequences of particle velocity limit analysis can effectively be used in materials and structural engineering to predict when dynamic material particle velocity can cause inelastic losses or failure via brittle fracture, plastic deformation, or fatigue failure.

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability.

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane "rippling" along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.

Droplet Ejection at Controlled Angles via Acoustofluidic Jetting.

We study the nozzle-free ejection of liquid droplets at controlled angles from a sessile drop actuated from two, mutually opposed directions by focused surface acoustic waves with dissimilar parameters. Previous researchers assumed that jets formed in this way are limited by the Rayleigh angle. However, when we carefully account for surface tension in addition to the driving force, acoustic streaming, we find a quantitative model that reduces to the Rayleigh angle only when inertia is dominant, and suggests larger ejection angles are possible in many practical situations. We confirm this in demonstrating ejection at more than double the Rayleigh angle. Our model explains the effects of both fluid and input parameters on experiments with a range of liquids. We extract, from this model, a dimensionless number that serves as an analog for the typical Weber number for predicting single droplet events.

Decontaminating surfaces with atomized disinfectants generated by a novel thickness-mode lithium niobate device.

We evaluated the ability of a novel lithium niobate (LN) thickness-mode device to atomize disinfectants and reduce microbial burden on model surface materials. A small-scale plastic model housed the LN thickness-mode device and circular coupon surface materials including polycarbonate, polyethylene terephthalate, stainless steel, borosilicate glass, and natural rubber. Coupon surfaces were coated with methicillin-resistant Staphylococcus aureus (MRSA) or multidrug-resistant (MDR) strains of Gram-negative bacterial pathogens (Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii), atomized with disinfectant solutions of varying viscosity (including 10% bleach, 70% ethanol (EtOH), or 25% triethylene glycol (TEG)) using the LN thickness-mode device, and assessed for surviving bacteria. The LN thickness-mode device effectively atomized disinfectants ranging from low viscosity 10% bleach solution or 70% EtOH to highly viscous 25% TEG. Coupons harboring MDR bacteria and atomized with 10% bleach solution or 70% EtOH were effectively decontaminated with ~ 100% bacterial elimination. Atomized 25% TEG effectively eliminated 100% of K. pneumoniae (CRE) from contaminated coupon surfaces but not MRSA. The enclosed small-scale plastic model established proof-of-principle that the LN thickness-mode device could atomize disinfectants of varying viscosities and decontaminate coupon surface materials harboring MDR organisms. Future studies evaluating scaled devices for patient rooms are warranted to determine their utility in hospital environmental decontamination.

Plasmon resonances of highly doped two-dimensional MoS2.

The exhibition of plasmon resonances in two-dimensional (2D) semiconductor compounds is desirable for many applications. Here, by electrochemically intercalating lithium into 2D molybdenum disulfide (MoS2) nanoflakes, plasmon resonances in the visible and near UV wavelength ranges are achieved. These plasmon resonances are controlled by the high doping level of the nanoflakes after the intercalation, producing two distinct resonance peak areas based on the crystal arrangements. The system is also benchmarked for biosensing using bovine serum albumin. This work provides a foundation for developing future 2D MoS2 based biological and optical units.

Extensional viscosity of copper nanowire suspensions in an aqueous polymer solution.

Suspensions of copper nanowires are emerging as new electronic inks for next-generation flexible electronics. Using a novel surface acoustic wave driven extensional flow technique we are able to perform currently lacking analysis of these suspensions and their complex buffer. We observe extensional viscosities from 3 mPa s (1 mPa s shear viscosity) to 37.2 Pa s via changes in the suspension concentration, thus capturing low viscosities that have been historically very challenging to measure. These changes equate to an increase in the relative extensional viscosity of nearly 12,200 times at a volume fraction of just 0.027. We also find that interactions between the wires and the necessary polymer additive affect the rheology strongly. Polymer-induced elasticity shows a reduction as the buffer relaxation time falls from 819 to 59 µs above a critical particle concentration. The results and technique presented here should aid in the future formulation of these promising nanowire suspensions and their efficient application as inks and coatings.

Motility induced changes in viscosity of suspensions of swimming microbes in extensional flows.

Suspensions of motile cells are model systems for understanding the unique mechanical properties of living materials which often consist of ensembles of self-propelled particles. We present here a quantitative comparison of theory against experiment for the rheology of such suspensions in extensional flows. The influence of motility on viscosities of cell suspensions is studied using a novel acoustically-driven microfluidic capillary-breakup extensional rheometer. Motility increases the extensional viscosity of suspensions of algal pullers, but decreases it in the case of bacterial or sperm pushers. A recent model [Saintillan, Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys., 2010, 81, 56307] for dilute active suspensions is extended to obtain predictions for higher concentrations, after independently obtaining parameters such as swimming speeds and diffusivities. We show that details of body and flagellar shape can significantly determine macroscale rheological behaviour.

Mixed mode of dissolving immersed nanodroplets at a solid-water interface.

The dissolution dynamics of microscopic oil droplets (less than 1 µm in height, i.e. nanodroplets) on a hydrophobilized silicon surface in water was experimentally studied. The lateral diameter was monitored using confocal microscopy, whereas the contact angle was measured by (disruptive) droplet polymerisation of the droplet. In general, we observed the droplets to dissolve in a mixed mode, i.e., neither in the constant contact angle mode nor in the constant contact radius mode. This means that both the lateral diameter and the contact angle of the nanodroplets decrease during the dissolution process. On average, the dissolution rate is faster for droplets with larger initial size. Droplets with the same initial size can, however, possess different dissolution rates. We ascribe the non-universal dissolution rates to chemical and geometric surface heterogeneities (that lead to contact line pinning) and cooperative effects from the mass exchange among neighbouring droplets.

Investigation of the morphological diversity of the potentially zoonotic Trypanosoma copemani in quokkas and Gilbert's potoroos.

Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, yet little is known of the pathogenicity and life-cycles of trypanosomes in native Australian mammals. Trypanosoma copemani is known to be infective to a variety of Australian marsupials and has recently been shown to be potentially zoonotic as it is resistant to normal human serum. In the present study, in vivo and in vitro examination of blood and cultures from Australian marsupials was conducted using light microscopy, immunofluorescence, scanning electron microscopy and fluorescence in situ hybridization. Promastigote, sphaeromastigote and amastigote life-cycle stages were detected in vivo and in vitro. Novel trypanosome-like stages were also detected both in vivo and in vitro representing an oval stage, an extremely thin stage, an adherent stage and a tiny round stage. The tiny round and adherent stages appeared to adhere to erythrocytes causing potential haematological damage with clinical effects similar to haemolytic anaemia. The present study shows for the first time that trypomastigotes are not the only life-cycle stages circulating within the blood stream of trypanosome infected Australian native marsupials and provides insights into possible pathogenic mechanisms of this potentially zoonotic trypanosome species.

Toward complete miniaturisation of flow injection analysis systems: microfluidic enhancement of chemiluminescent detection.

Conventional flow injection systems for aquatic environmental analysis typically comprise large laboratory benchscale equipment, which place considerable constraints for portable field use. Here, we demonstrate the use of an integrated acoustically driven microfluidic mixing scheme to enhance detection of a chemiluminescent species tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate-a common chemiluminescent reagent widely used for the analysis of a wide range of compounds such as illicit drugs, pharmaceuticals, and pesticides-such that rapid in-line quantification can be carried out with sufficient on-chip sensitivity. Specifically, we employ surface acoustic waves (SAWs) to drive intense chaotic streaming within a 100 µL chamber cast in polydimethoxylsiloxane (PDMS) atop a microfluidic chip consisting of a single crystal piezoelectric material. By optimizing the power, duration, and orientation of the SAW input, we show that the mixing intensity of the sample and reagent fed into the chamber can be increased by one to two orders of magnitude, leading to a similar enhancement in the detection sensitivity of the chemiluminescent species and thus achieving a theoretical limit of detection of 0.02 ppb (0.2 nM) of l-proline-a decade improvement over the industry gold-standard and two orders of magnitude more sensitive than that achievable with conventional systems-simply using a portable photodetector and without requiring sample preconcentration. This on-chip microfluidic mixing strategy, together with the integrated miniature photodetector and the possibility for chip-scale microfluidic actuation, then alludes to the attractive possibility of a completely miniaturized platform for portable field-use microanalytical systems.

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization.

BACKGROUND: Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. METHODS: In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. RESULTS: The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. CONCLUSION: Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.