C. Wyatt Shields

Elastomeric microparticles for acoustic mediated bioseparations.

BackgroundAcoustophoresis has been utilized successfully in applications including cell trapping, focusing, and purification. One current limitation of acoustophoresis for cell sorting is the reliance on the inherent physical properties of cells (e.g., compressibility, density) instead of selecting cells based upon biologically relevant surface-presenting antigens. Introducing an acoustophoretic cell sorting approach that allows biochemical specificity may overcome this limitation, thus advancing the value of acoustophoresis approaches for both the basic research and clinical fields.ResultsThe results presented herein demonstrate the ability for negative acoustic contrast particles (NACPs) to specifically capture and transport positive acoustic contrast particles (PACPs) to the antinode of an ultrasound standing wave. Emulsification and post curing of pre-polymers, either polydimethylsiloxane (PDMS) or polyvinylmethylsiloxane (PVMS), within aqueous surfactant solution results in the formation of stable NACPs that focus onto pressure antinodes. We used either photochemical reactions with biotin-tetrafluorophenyl azide (biotin-TFPA) or end-functionalization of Pluronic F108 surfactant to biofunctionalize NACPs. These biotinylated NACPs bind specifically to streptavidin polystyrene microparticles (as cell surrogates) and transport them to the pressure antinode within an acoustofluidic chip.ConclusionTo the best of our knowledge, this is the first demonstration of using NACPs as carriers for transport of PACPs in an ultrasound standing wave. By using different silicones (i.e., PDMS, PVMS) and curing chemistries, we demonstrate versatility of silicone materials for NACPs and advance the understanding of useful approaches for preparing NACPs. This bioseparation scheme holds potential for applications requiring rapid, continuous separations such as sorting and analysis of cells and biomolecules.

Elastomeric negative acoustic contrast particles for capture, acoustophoretic transport, and confinement of cells in microfluidic systems.

We present a particle-based method for the immunospecific capture and confinement of cells using acoustic radiation forces. Ultrasonic standing waves in microfluidic systems have previously been used for the continuous focusing of cells in rapid screening and sorting applications. In aqueous fluids, cells typically exhibit positive acoustic contrast and are thus forced toward the pressure nodes of a standing wave. Conversely, elastomeric particles exhibit negative acoustic contrast and travel toward the pressure antinodes. We have developed a class of elastomeric particles that are synthesized in bulk using a simple nucleation and growth process, providing precise control over their size and functional properties. We demonstrate that the biofunctionalization of these particles can allow the capture and transport of cells to the pressure antinodes solely via acoustic radiation forces, which may enable new acoustics-based cell handling techniques such as the washing, labeling, and sorting of cells with minimal preparatory steps.

Translating microfluidics: Cell separation technologies and their barriers to commercialization.

Advances in microfluidic cell sorting have revolutionized the ways in which cell‐containing fluids are processed, now providing performances comparable to, or exceeding, traditional systems, but in a vastly miniaturized format. These technologies exploit a wide variety of physical phenomena to manipulate cells and fluid flow, such as magnetic traps, sound waves and flow‐altering micropatterns, and they can evaluate single cells by immobilizing them onto surfaces for chemotherapeutic assessment, encapsulate cells into picoliter droplets for toxicity screenings and examine the interactions between pairs of cells in response to new, experimental drugs. However, despite the massive surge of innovation in these high‐performance lab‐on‐a‐chip devices, few have undergone successful commercialization, and no device has been translated to a widely distributed clinical commodity to date. Persistent challenges such as an increasingly saturated patent landscape as well as complex user interfaces are among several factors that may contribute to their slowed progress. In this article, we identify several of the leading microfluidic technologies for sorting cells that are poised for clinical translation; we examine the principal barriers preventing their routine clinical use; finally, we provide a prospectus to elucidate the key criteria that must be met to overcome those barriers. Once established, these tools may soon transform how clinical labs study various ailments and diseases by separating cells for downstream sequencing and enabling other forms of advanced cellular or sub‐cellular analysis.

Magnetic separation of acoustically focused cancer cells from blood for magnetographic templating and analysis

Liquid biopsies hold enormous promise for the next generation of medical diagnoses. At the forefront of this effort, many are seeking to capture, enumerate and analyze circulating tumor cells (CTCs) as a means to prognosticate and develop individualized treatments for cancer. Capturing these rare cells, however, represents a major engineering challenge due to their low abundance, morphology and heterogeneity. A variety of microfluidic tools have been developed to isolate CTCs from drawn blood samples; however, few of these approaches offer a means to separate and analyze cells in an integrated system. We have developed a microfluidic platform comprised of three modules that offers high throughput separation of cancer cells from blood and on-chip organization of those cells for streamlined analyses. The first module uses an acoustic standing wave to rapidly align cells in a contact-free manner. The second module then separates magnetically labeled cells from unlabeled cells, offering purities exceeding 85% for cells and 90% for binary mixtures of synthetic particles. Finally, the third module contains a spatially periodic array of microwells with underlying micromagnets to capture individual cells for on-chip analyses (e.g., staining, imaging and quantification). This array is capable of capturing with accuracies exceeding 80% for magnetically labeled cells and 95% for magnetic particles. Overall, by virtue of its holistic processing of complex biological samples, this system has promise for the isolation and evaluation of rare cancer cells and can be readily extended to address a variety of applications across single cell biology and immunology.

Magnetographic array for the capture and enumeration of single cells and cell pairs

We present a simple microchip device consisting of an overlaid pattern of micromagnets and microwells capable of capturing magnetically labeled cells into well-defined compartments (with accuracies >95%). Its flexible design permits the programmable deposition of single cells for their direct enumeration and pairs of cells for the detailed analysis of cell-cell interactions. This cell arraying device requires no external power and can be operated solely with permanent magnets. Large scale image analysis of cells captured in this array can yield valuable information (e.g., regarding various immune parameters such as the CD4:CD8 ratio) in a miniaturized and portable platform.

Two-dimensional spatial manipulation of microparticles in continuous flows in acoustofluidic systems:

We report a modeling and experimental study of techniques to acoustically focus particles flowing through a microfluidic channel. Our theoretical model differs from prior works in that we solve an approximate 2-D wave transmission model that accounts for wave propagation in both the solid and fluid phases. Our simulations indicate that particles can be effectively focused at driving frequencies as high as 10% off of the resonant condition. This conclusion is supported by experiments on the acoustic focusing of particles in nearly square microchannels, which are studied for different flow rates, driving frequencies and placements of the lead zirconate titanate transducer, either underneath the microchannel or underneath a parallel trough. The relative acoustic potential energy and the resultant velocity fields for particles with positive acoustic contrast coefficients are estimated in the 2-D limit. Confocal microscopy was used to observe the spatial distribution of the flowing microparticles in three dimensions. Through these studies, we show that a single driving frequency from a single piezoelectric actuator can induce the 2-D concentration of particles in a microchannel with a nearly square cross section, and we correlate these behaviors with theoretical predictions. We also show that it is possible to control the extent of focusing of the microparticles, and that it is possible to decouple the focusing of microparticles in the vertical direction from the lateral direction in rectangular channels with anisotropic cross sections. This study provides guidelines to design and operate microchip-based acoustofluidic devices for precise control over the spatial arrangement of microparticles for applications such as flow cytometry and cellular sorting.

Nucleation and Growth Synthesis of Siloxane Gels to Form Functional, Monodisperse, and Acoustically Programmable Particles†

Nucleation and growth methods offer scalable means of synthesizing colloidal particles with precisely specified size for applications in chemical research, industry, and medicine. These methods have been used to prepare a class of silicone gel particles that display a range of programmable properties and narrow size distributions. The acoustic contrast factor of these particles in water is estimated and can be tuned such that the particles undergo acoustophoresis to either the pressure nodes or antinodes of acoustic standing waves. These particles can be synthesized to display surface functional groups that can be covalently modified for a range of bioanalytical and acoustophoretic sorting applications.

Sequence-encoded colloidal origami and microbot assemblies from patchy magnetic cubes

Sequence-encoded assembly of patchy magnetic microcubes enables making self-reconfiguring colloidal origami and “microbots.” Colloidal-scale assemblies that reconfigure on demand may serve as the next generation of soft “microbots,” artificial muscles, and other biomimetic devices. This requires the precise arrangement of particles into structures that are preprogrammed to reversibly change shape when actuated by external fields. The design and making of colloidal-scale assemblies with encoded directional particle-particle interactions remain a major challenge. We show how assemblies of metallodielectric patchy microcubes can be engineered to store energy through magnetic polarization and release it on demand by microscale reconfiguration. The dynamic pattern of folding and reconfiguration of the chain-like assemblies can be encoded in the sequence of the cube orientation. The residual polarization of the metallic facets on the microcubes leads to local interactions between the neighboring particles, which is directed by the conformational restrictions of their shape after harvesting energy from external magnetic fields. These structures can also be directionally moved, steered, and maneuvered by global forces from external magnetic fields. We illustrate these capabilities by examples of assemblies of specific sequences that can be actuated, reoriented, and spatially maneuvered to perform microscale operations such as capturing and transporting live cells, acting as prototypes of microbots, micromixers, and other active microstructures.

Assembly of hard spheres in a cylinder: a computational and experimental study

Hard spheres are an important benchmark of our understanding of natural and synthetic systems. In this work, colloidal experiments and Monte Carlo simulations examine the equilibrium and out-of-equilibrium assembly of hard spheres of diameter σ within cylinders of diameter σ≤D≤ 2.82σ. Although phase transitions formally do not exist in such systems, marked structural crossovers can nonetheless be observed. Over this range of D, we find in simulations that structural crossovers echo the structural changes in the sequence of densest packings. We also observe that the out-of-equilibrium self-assembly depends on the compression rate. Slow compression approximates equilibrium results, while fast compression can skip intermediate structures. Crossovers for which no continuous line-slip exists are found to be dynamically unfavorable, which is the main source of this difference. Results from colloidal sedimentation experiments at low diffusion rate are found to be consistent with the results of fast compressions, as long as appropriate boundary conditions are used.

Experimental verification of theoretical equations for acoustic radiation force on compressible spherical particles in traveling waves.

Acoustophoresis uses acoustic radiation force to remotely manipulate particles suspended in a host fluid for many scientific, technological, and medical applications, such as acoustic levitation, acoustic coagulation, contrast ultrasound imaging, ultrasound-assisted drug delivery, etc. To estimate the magnitude of acoustic radiation forces, equations derived for an inviscid host fluid are commonly used. However, there are theoretical predictions that, in the case of a traveling wave, viscous effects can dramatically change the magnitude of acoustic radiation forces, which make the equations obtained for an inviscid host fluid invalid for proper estimation of acoustic radiation forces. To date, experimental verification of these predictions has not been published. Experimental measurements of viscous effects on acoustic radiation forces in a traveling wave were conducted using a confocal optical and acoustic system and values were compared with available theories. Our results show that, even in a low-viscosity fluid such as water, the magnitude of acoustic radiation forces is increased manyfold by viscous effects in comparison with what follows from the equations derived for an inviscid fluid.