Methods for manipulating biological objects over the scales from micrometer to centimeter are the foundation to many biomedical applications, including the study of cell-cell interaction (Nilsson J et al., Analytica chimica acta, 649(2), 141-157; Sun J et al., Biomaterials, 35(10), 3273-3280), single-cell analysis (Wood D K et al., Proceedings of the National Academy of Sciences, 107(22), 10008-10013; Collins D J et al., Lab on a Chip, 15(17), 3439-3459), drug development (Kang L et al., Drug discovery today, 13(1-2), 1-13), point-of-care diagnostics (Gervais L et al., Advanced materials, 23(24), H151-H176; Taller D et al., Lab on a Chip, 15(7), 1656-1666; Xiao Y et al., PloS one, 11(4), e0154640), and tissue engineering (Puleo C M et al., Tissue engineering, 13(12), 2839-2854; Jamilpour N et al., ACS Biomaterials Science & Engineering, 2019). Conventional methodologies deployed using optical (Hu W et al., Lab on a Chip, 13(12), 2285-2291; Zhong M C et al., Nature communications, 4, 1768; Ashkin A et al., Nature, 330(6150), 769; Zhang H et al., Journal of the Royal Society interface, 5(24), 671-690), magnetic (Lim B et al., Nature communications, 5, 3846), and electrokinetic (Ho C T et al., Lab on a Chip, 13(18), 3578-3587; Chiang M Y et al., Science advances, 2(10), e1600964; Cheng I F et al., Biomicrofluidics, 1(2), 021503) forces are versatile, but they pose various deficiencies. Optical force can provide precise three-dimensional (3D) control of the manipulated objects but suffers from low throughput. Magnetic force is widely applied but it requires extra labeling of magnetic particles that could interfere with cell functions and downstream analyses. Other approaches based on electrokinetics, such as dielectrophoresis and electroosmosis, are simple to implement but are challenged by buffer incompatibility and electrical interference that could damage the manipulated samples. 3D printing (Chia H N et al., Journal of biological engineering, 9(1), 4; Panwar A et al., Molecules, 21(6), 685) provides another mean to form complex patterning profiles but has not been able to achieve precision control of its printed objects, thus limiting the resolution. Acoustic force, on the other hand, offers a potential avenue for noninvasive, label-free, and biocompatible manipulation.
Acoustic manipulation has attracted a lot of interests in the past for its superior biocompatibility and for its strength to control objects of sizes spanning from submicrometer to a few millimeter. Particles of different density and compressibility from the surrounding medium experience net acoustic radiation forces (ARF), incurred from non-uniform acoustic field distribution, that migrate them to either low or high potential energy regions. For particle of size much smaller than the wavelength (D<<λ), the ARF can be approximated by the following expressions (Bruus H, Lab on a Chip, 12(6), 1014-1021):
where Frad is the ARF, Urad is the acoustic potential energy, a is the radius of particle, and p and v are the first-order acoustic pressure and velocity at the particle. The material compressibility K and density p are subscripted by ‘p’ and ‘o’ for the particle and the surrounding medium, respectively. Two frequently used conventional acoustic mechanisms, bulk acoustic waves (BAWs) (Raeymaekers B et al., Journal of Applied Physics, 109(1), 014317; Leibacher I et al., Lab on a Chip, 15(13), 2896-2905; Hammarstrom B et al., Lab on a Chip, 12(21), 4296-4304; Castro A et al., Ultrasonics, 66, 166-171) and surface acoustic waves (SAWs) have been applied to generate the non-uniform acoustic field (Collins D J et al., Nature communications, 6, 8686; Ding X et al., Proceedings of the National Academy of Sciences, 109(28), 11105-11109; Guo F et al., Proceedings of the National Academy of Sciences, 113(6), 1522-1527; Tay A K et al., Lab on a Chip, 15(12), 2533-2537; Destgeer G et al., Lab on a Chip, 15(13), 2722-2738; Lin S C S et al., Lab on a Chip, 12(16), 2766-2770; Yeo L Y et al., Biomicrofluidics, 3(1), 012002; Chen Yet al., ACS nano, 7(4), 3306-3314; Ding X et al., Lab on a Chip, 12(14), 2491-2497; Bian Y et al., Microfluidics and nanofluidics, 21(8), 132; Rezk A R et al., Advanced Materials, 28(10), 2088-2088; Kang B et al., Nature communications, 9(1), 5402). In BAWs, acoustically hard structures, such as silicon or glass microfluidic chambers, are fabricated to form resonant cavities. Acoustic frequencies matching with certain acoustic modes of the cavities are chosen to excite standing waves in these structures that form the non-uniform field. However, such mechanism limits the particle patterning profile to be simple and periodic with a spatial resolution less than half of the wavelength (½λ). Although one can improve the resolution by increasing the acoustic frequencies, significant heating due to high energy attenuation can cause severe issues during manipulation of biological objects. In SAWs, standing waves can be generated by implementing pairs of interdigitated transducers (IDTs) fabricated on a piezoelectric substrate. Counter propagating SAWs leaking into the chambers can form the standing waves to create the non-uniform field. Through tuning the phases and frequencies of the electrical signals applied to IDTs, dynamic patterning can be achieved. Nevertheless, due to the nature of standing waves, SAWs face similar issue of limited patterning profiles that are typically symmetric. Furthermore, rapid attenuation of SAWs due to the energy transfer into fluid makes large area patterning difficult; a typical SAWs device cannot operate in an area greater than 1 mm×1 mm (Collins D J et al., Nature communications, 6, 8686).
Therefore, there is a need in the art for an acoustic approach able to produce high resolution, arbitrarily shaped potential energy wells across a large area. The present invention meets this unmet need.
In one aspect, the present invention relates to a compliant membrane acoustic patterning device for manipulating particles, comprising: a piezoelectric layer; a patterned layer comprising a plurality of cavities disposed on top of the piezoelectric layer, wherein each of the cavities are covered by a membrane that is flush with a top surface of the patterned layer; a fluid layer disposed on top of the patterned layer; a plurality of particles immersed in the fluid; a cover layer disposed on top of the fluid layer; and an oscillating power source configured to actuate the piezoelectric layer at an oscillation frequency.
In one embodiment, the piezoelectric layer comprises a material selected from the group consisting of: lead zirconate titate (PZT), barium titanate, and bismuth sodium titanate. In one embodiment, the piezoelectric layer has a thickness between about out 100 μm and 1000 μm. In one embodiment, the patterned layer comprises a material selected from the group consisting of: plastics, polymers, rubbers, gels, silicones, and polydimethylsiloxane (PDMS). In one embodiment, the patterned layer has a thickness between about 10 μm and 50 μm. In one embodiment, the membrane has a thickness between about 1 μm and 5 μm. In one embodiment, the membrane further comprises a coating selected from the group consisting of: a water impermeable coating, a hydrophobic coating, a hydrophilic coating, or a functionalized coating. In one embodiment, the fluid layer comprises a material selected from the group consisting of: water, cell culture media, blood, serum, and buffer solution. In one embodiment, the particle is selected from the group consisting of beads, nanoparticles, microparticles, cells, bubbles, microorganisms, nucleic acids, and proteins. In one embodiment, the cavities comprise a gas, a fluid, or air.
In one embodiment, the device further comprises a controller electrically connected to the oscillating power source and configured to modulate the oscillation frequency. In one embodiment, the device further comprises a temperature regulator and a temperature sensor, wherein the temperature regulator is configured to maintain a temperature of the device.
In another aspect, the present invention relates to a method of manipulating particles in a fluid, comprising the steps of: providing a compliant membrane acoustic patterning (CMAP) platform comprising a piezoelectric layer and a patterned layer disposed on top of the piezoelectric layer, wherein the patterned layer comprises at least one air cavity, each air cavity covered with a membrane that is flush with a top surface of the patterned layer; positioning a plurality of particles and a fluid on top of the patterned layer; positioning a cover layer on top of the fluid layer; passing an electrical signal to the piezoelectric layer that is converted into mechanical vibrations that generate acoustic waves at an oscillation frequency traveling upwards through the patterned layer, the fluid layer, and the cover layer; and forming near-field acoustic potential wells above each of the at least one air cavity by a difference in acoustic wave propagation through the patterned layer and the at least one air cavity, such that the plurality of particles accumulate on and conform to the membrane of each of the at least one air cavity.
In one embodiment, the patterned layer, air cavities, and membranes are formed by molding from a master mold, by injection molding, by stamping, by etching, or by 3D printing. In one embodiment, the electrical signal is provided by an oscillating power source electrically connected to a controller. In one embodiment, the oscillation frequency is between 1 MHz and 5 MHz. In one embodiment, the oscillation frequency is about 3 MHz.
In one embodiment, the method further comprises a step of maintaining a temperature of the platform. In one embodiment, the fluid is selected from the group consisting of: water, cell culture media, blood, serum, and buffer solution. In one embodiment, the plurality of particle is selected from the group consisting of beads, nanoparticles, microparticles, cells, bubbles, microorganisms, nucleic acids, and proteins.
The following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
(
(
The present invention relates to a near-field acoustic platform capable of synthesizing high resolution, arbitrarily shaped energy potential wells. A thin and viscoelastic membrane is utilized to modulate acoustic wavefront on a deep, sub-wavelength scale by suppressing the structural vibration selectively on the platform. This new acoustic wavefront modulation mechanism is powerful for manufacturing complex biologic products.
It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements typically found in the art. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.
Unless defined elsewhere, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.
Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments there between. This applies regardless of the breadth of the range.
Complex patterning of micro-objects in liquid is crucial to many biomedical applications. Among conventional mythologies, acoustic approaches provide superior biocompatibility but are intrinsically limited to producing periodic patterns at low resolution due to the nature of standing wave and the coupling between fluid and structure vibrations. The present invention provides a compliant membrane acoustic patterning (CMAP) platform capable of synthesizing high resolution, arbitrarily shaped energy potential wells. A thin and viscoelastic membrane is utilized to modulate acoustic wavefront on a deep, sub-wavelength scale by suppressing the structural vibration selectively on the platform. Using acoustic excitation, arbitrary patternings of microparticles and cells with a line resolution of one tenth of the wavelength of the acoustic excitation is achievable. Massively parallel patterning in areas as small as 3×3 mm2 is also possible. This new acoustic wavefront modulation mechanism is powerful for manufacturing complex biologic products.
Referring now to
Patterned layer 104 is a planar layer that is disposed on top of piezoelectric layer 102. Visible in
Fluid layer 110 is disposed on top of patterned layer 104 and membrane 108. Fluid layer 110 can comprise any suitable fluid, including but not limited to water, cell culture media, blood, serum, buffer solution, and the like. Fluid layer 110 can have any suitable height or depth, such as a height or depth between about 0.5 cm and 5 cm. Fluid layer 110 comprises a plurality of particles 112 that are desired to be patterned into shapes formed by cavities 106 in patterned layer 104. Particles 112 can comprise any desired particle, including but not limited to beads, nanoparticles, microparticles, cells, bubbles, microorganisms, nucleic acids, proteins, and the like.
Cover layer 114 is a planar layer that is disposed on top of fluid layer 110. Cover layer 114 attenuates acoustic waves to minimize wave reflection and serves to enclose fluid layer 110. Cover layer 114 can be constructed from any suitable material, including but not limited to plastics, polymers, rubbers, gels, silicones, PDMS, and the like. Cover layer 114 can have any suitable thickness. For example, cover layer 114 can have a thickness between about 0.5 cm and 5 cm.
In certain embodiments, patterned layer 104, membrane 108, and cover layer 114 are each constructed from the same material. In some embodiments, patterned layer 104, membrane 108, and cover layer 114 are each constructed from a material having an acoustic impedance substantially similar to an acoustic impedance of fluid layer 110. In some embodiments, the acoustic impedance of each of patterned layer 104, membrane 108, fluid layer 110, and cover layer 114 are within 25%, 20%, 15%, 10%, 5%, or 1% of each other.
While not pictured, it should be understood that platform 100 comprises a housing sized to fit each of the piezoelectric layer 102, patterned layer 104, fluid layer 110, and cover layer 114. The housing comprises sidewalls such that a fluid is containable within the housing to form fluid layer 110. In some embodiments, the housing comprises an internal horizontal surface area and shape matched to a horizontal surface area and shape of patterned layer 104 and cover layer 114, such that each of the patterned layer 104, and cover layer 114 sits flush within the interior of the housing. In some embodiments, platform 100 further comprises an intermediate layer 116 disposed between piezoelectric layer 102 and patterned layer 104. Intermediate layer 116 can be provided as a physical barrier between piezoelectric layer 102 and patterned layer 104 for ease of use and cleaning, such that one or more patterned layers 104 can be replaced without fouling piezoelectric layer 102. In some embodiments, a bottom surface of the housing forms intermediate layer 116. Intermediate layer 116 can be constructed from any suitable material, including but not limited to a glass, a metal, a plastic, a ceramic, and the like. Intermediate layer 116 can have any suitable thickness. For example, intermediate layer 116 can have a thickness between about 100 μm and 1000 μm.
Platform 100 is amenable to any desired modification. For example, in some embodiments platform 100 further comprises a temperature regulator and sensor, such as a thermoelectric cooler and a thermocouple, respectively. The temperature regulator can be provided to maintain the temperature of platform 100 (such as patterned layer 104 and fluid layer 110) for certain applications, and the temperature sensor can be provided to monitor the temperature of platform 100.
The present invention also provides methods of using the CMAP platform described herein to synthesize patternings of particles. Referring now to
The patterned layer can be formed using any method commonly used in the art. In various embodiments, the patterned layer with cavities and membranes can be constructed using molding (such as with a master mold), injection molding, stamping, etching, 3D printing or other forms of additive manufacturing, and the like.
The electrical signal can be provided by an oscillating power source, such as a power amplifier, connected to a controller, such as a function generator. The electrical signal can be described in terms of oscillation frequency. For example, the oscillation frequency can be between about 1 MHz and 5 MHz. In some embodiments, the oscillation frequency is about 3 MHz. In some embodiments, the method further comprises a step of maintaining a temperature of the platform. The temperature can be maintained using a temperature regulator and monitored using a temperature sensor.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
Methods that enable complex patterning of micro-objects are crucial to many biomedical applications. In recent years, acoustic manipulation has emerged as a promising approach to pattern biological samples for its superior biocompatibility. Current acoustic techniques, however, encounter a major technical barrier in forming complex patterns, and thus are limited to producing simple and periodic assembly of objects. In contrary to other physical methods, arbitrarily shaped patterns cannot be achieved using current techniques based on either surface acoustic waves (SAWs) or bulk acoustic waves (BAWs). Such barriers originate from their standing wave nature that is the underlying mechanism and the coupled fluid-structure vibrations within.
The present study demonstrates a new acoustic manipulation principle that overcomes the technical barriers of current techniques and provides, for the first time, the capability to form high-resolution, arbitrarily shaped complex patterns not feasible by existing acoustic techniques. The principle, named Compliant Membrane Acoustic Patterning (CMAP), utilizes acoustic traveling waves and air cavities embedded in an elastomer to generate near-field potential landscape for patterning. The compliant membrane formed around the cavities and the viscoelastic nature of the elastomer, combined, effectively suppress any structure vibration and eliminate high order mode patterns. As a result, arbitrarily shaped acoustic potential landscape can be realized on the surface of CMAP to create complex patterns that are nearly identical to the shape of the cavities.
The potential of CMAP in the field of acoustic manipulation, as well as in the realm of tissue engineering, is immense. CMAP is the most capable acoustic technique that enables manipulation of microscale objects, including biological cells, to form high-resolution, arbitrarily shaped complex assemblies. Furthermore, the simplicity in designing and fabricating the CMAP platform allows researchers in relevant fields to easily adapt this tool for broad impacts.
The methods and materials are now described.
Device Design and Assembly
The CMAP device,
Setup and Operation
The complete setup to using CMAP device involves a power amplifier (ENI Model 2100L), a function generator (Agilent Model 33220A), a T.E. cooler (T.E. Technology Model CP-031HT), an ultra-long working distance microscope lens (20× Mitutoyo Plan Apo), an upright microscope (Zeiss Model Axioskop 2 FS), and a mounted recording camera (Zeiss Model AxioCam mRm). Surfaces of the PZT substrate are wire-bonded and electrically connected to the power amplifier that is controlled by the function generator to feed the A.C. signals. Upon receiving the signals, the PZT transforms the sinusoidal voltages into mechanical vibrations to generate the acoustic traveling waves across the device. To prevent cell damage from excessive PZT heating, the device was operated on a T.E. cooler set at 12° C. To monitor the temperature of the device's chamber, a thermocouple (Omega OM-74) was inserted through the PDMS encapsulation and the experiment was reran with only water in the chamber; results show stabilization below the incubation temperature of 37° C., suggesting suitability for long-term operation. The entire assembly is positioned under the Mitutoyo microscope lens mounted on the Zeiss Axioskop. Patterning process is then observed through the PDMS encapsulation that allows clear visualization and is recorded using the accompanied Zeiss AxioCam.
Acoustic-Structure Interaction Simulation
Acoustic-structure module, using finite element (F.E.) solver COMSOL Multiphysics 5.3, is implemented to study the acoustic potential landscape as the result of the soft/hard, air-embedded PDMS structure interacting with the chamber fluid upon excitation.
where ω is the angular frequency in rad/s. The simulation not only allows post-processing of the acoustic potential landscape generated (
Acoustic Pressure Simulation
Acoustic pressure module, using finite element (F.E.) solver COMSOL Multiphysics 5.3, is implemented to simulate the pressure profile inside the device chamber. While the 3-D model geometry in
Thickness Measurement of the PDMS Membrane
The fabricated PDMS structures are cut to reveal the cross section of membranes, and 3 membranes are examined using SEM. The measured thicknesses are 1.09 μm, 1.14 μm, and 1.33 μm, and their average thickness is approximately 2.18 μm. For simplicity, a 2 μm membrane thickness are assumed in the simulations.
Polystyrene Beads
Both 1 μm and 10 μm fluorescent green polystyrene beads are obtained from Thermo Fisher Scientific, USA.
Microporous PDMS Beads Fabrication
Uncured PDMS using Sylgard 184 (Dow Corning Co.) with curing agent at 10:1 was mixed with the solution of dodecyl sulfate sodium salt in DI water at 1:100 mass ratio. Using a vortex mixer, mixture of the PDMS solution in water generated PDMS spherical droplets of varying sizes. Subsequently, that mixture was cured inside an oven at 70° C. for 2 hours. The solidified microporous PDMS beads were then filtered using a sterile cell strainer of 40 μm nylon mesh (Fisher Scientific).
HeLa Cell Culturing
HeLa cells (American Type Culture Collection, ATCC) were maintained in Dulbecco's modified essential medium (DMEM, Corning) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Thermo Scientific), 1% penicillin/streptomycin (Mediatech), and 1% sodium pyruvate (Corning). HeLa cells were kept in an incubator at 37° C. and 5% CO2.
The results are now described.
Operating Principle of CMAP
Compliant Membrane Acoustic Patterning (CMAP) is a device platform that allows the creation of deep sub-wavelength resolution, arbitrarily shaped acoustic potential wells near an engineered membrane. Such a potential landscape is realized by exciting acoustic traveling waves, generated using a piezoelectric ceramic PZT (lead zirconate titanate), to pass through desired shapes of air cavities sized much smaller than the wavelength and embedded in a soft, viscoelastic Polydimethylsiloxane (PDMS) structure, as illustrated in
One major challenge encountered in conventional acoustic patternings is the coupled fluid and structure vibration that complicates the design of device structure. With the CMAP platform, the effect of structure-induced vibration was minimized, otherwise it would interfere with the intended acoustic field and, ultimately, the shape of particle patterning was able to be predicted by using a simple pressure wave propagation model. This innovation can be carried out by incorporating a thin and compliant, viscoelastic PDMS membrane to interface the air cavities and the above chamber fluid. When the pressure waves propagate through the air-embedded PDMS structure, the vibration in the bulk decays within a short distance into the membrane due to two primary characteristics. One characteristic is the membrane's thinness and compliance for which it does not have sufficient stiffness to drive and move the fluid mass atop at high frequency. The second characteristic stems from material damping of the structure at high frequency that prevents the vibration energy from building up in the membrane region. Thus, the fluid pressure above the membrane region does not fluctuate much with the waves that propagate through the bulk into the fluid and remains at a relatively constant level compared to regions in the bulk. This creates a low acoustic pressure zone above the membrane and establishes a pressure gradient between the bulk and membrane regions. Since this near-field pressure zone depends on the membrane area attained from the air cavities that can be fabricated into any size and geometry, arbitrarily shaped particle patterning with a spatial resolution much smaller than the wavelength can be realized. Additionally, large area patterning can be achieved using the same actuation principle; for the fact that PZT substrate generates plane acoustic waves with uniform intensity, the maximum operating area is only limited by the PZT's available size. In short, since the acoustic potential landscape of CMAP does not rely on the formation of standing waves and since the disturbance to the landscape due to the structure-induced vibration may be minimized, the shape of potential wells simply reflects that of the air cavities.
To quantitatively understand the operation principle of CMAP, the relationship between the material properties of PDMS and their effects on structure-induced vibration was studied using numerical simulation. COMSOL acoustic-structure interaction model is implemented, as shown in
Acoustic radiation potential landscape is estimated by accounting the resulting water pressure and velocity fields near the PDMS-fluid interface into Eq. 2. For 10 μm polystyrene beads (ρp=1050 kg m−3, κp=2.4×10−1° Pa−1) (Muller P B et al., Lab on a Chip, 12(22), 4617-4627), the potential profile at 5 μm above the air-embedded PDMS structure of E′ at 100 MPa,
(shown in
Contrarily, for air-filled microporous PDMS beads that exhibit much greater compressibility than water, the contribution of the velocity term in equation 1b becomes negligible. It has been shown that sound speed in PDMS can drop rapidly from 1000 m/s to 40 m/s when porosity varies from 0 to 30% (Kovalenko A et al., Soft matter, 13(25), 4526-4532). Based on the relationship κp=1/ρc2, where c is the speed of sound, the high compressibility of porous PDMS can result in a f1 factor orders of magnitude larger than f2.
As simulated, the compliant, viscoelastic PDMS membrane effectively limits the structure-induced vibration propagating from the bulk into the membrane region. This unique feature permits membranes of sizes larger than the propagation length to be utilized for arbitrary patterning on CMAP. In
To evaluate the simulated results, the CMAP platform was fabricated using two types of PDMS of different Young's Moduli, E, to form the air-embedded, viscoelastic structures and then performed Laser Doppler Vibrometer (LDV) measurements over their surfaces. The first type was synthesized following the manufacturer's instructions using Sylgard 184 (Dow Corning Co.) to produce E of ˜1750 kPa, and the second type was synthesized as a mixture of Sylgard 527 (Dow Corning Co.) and 184 at the weight ratio of 4:1 to produce E of ˜250 kPa (Palchesko R N et al., PloS one, 7(12), e51499). Although these are static moduli, decrease in E is accompanied by decrease in both the dynamic moduli, E′ and E″ (Hanoosh W S et al., Malaysian Polymer Journal, 4(2), 52-61).Hence, the two compositions became the hard and soft, air-embedded PDMS structures representing the simulated cases of E′ at 100 MPa and 0.1 MPa, respectively. A schematic diagram representing the PDMS structures (an array of concentric rings),
Arbitrary Patterning of Microparticles
Arbitrary particle patterning has been a major complication in the field of acoustofluidics, where the patterning resolution and profile are restricted by attainable wavelength size and limited, periodic acoustic potential landscapes, respectively. Area of the patterning, too, is restrained due to weakening of wave propagation across device surface as in the case of SAWs. Alternatively, the new acoustic patterning mechanism using the CMAP platform described herein overcomes these challenges. As illustrated in
To further assess CMAP's ability in arbitrary pattering, another set of soft, air-embedded PDMS structures were fabricated consisting of numeric characters. At high concentration,
Arbitrary Patterning of Biological Cells
Similar to polystyrene beads, patterning of cells highly depends on the surface displacement of the soft, air-embedded PDMS structure, as well as the density and compressibility of the particles and their surroundings, that gives rise to the acoustic potential landscape. HeLa cells are chosen here to testify the biocompatibility of the CMAP platform. Since typical cells (ρp=1068 kg m−3, κp=3.77-10 Pa−1 as in the case of breast cells) (Hartono D et al., Lab on a Chip, 11(23), 4072-4080) in DMEM have like properties as polystyrene beads in water, their potential landscapes formed using the same soft PDMS structure should be nearly identical. As illustrated in
Numerous acoustic approaches for cell patterning have been assessed in determining the cell viability and proliferation, and prior approaches in the MHz-order acoustic fields have proven to be biocompatible (Ding X et al., Proceedings of the National Academy of Sciences, 109(28), 11105-11109; Evander M et al., Analytical chemistry, 79(7), 2984-2991; Bazou D et al., Toxicology in Vitro, 22(5), 1321-1331; Leibacher I et al., Microfluidics and Nanofluidics, 19(4), 923-933). The CMAP device platform, in the similar MHz-order of operation, provides comparable results. To prevent potential thermal damage due to heat accumulation on the CMAP device platform, the device was operated with a T.E. cooler set at 12° C. to control the chamber temperature.
The CMAP platform is a powerful tool to realize deep sub-wavelength, arbitrarily shaped patternings of microparticles and biological objects. These are achieved using a suspended, thin and compliant PDMS membrane that minimizes the effect of structure-induced vibration and that adapts to the surrounding fluid motion without offsetting the intended acoustic potential landscape. The membrane can be of any geometry, making arbitrarily shaped patterning possible. Additionally, both the PZT and the soft, air-embedded PDMS structure can be scaled up for larger area patterning based on the underlying acoustic actuation principle.
Of note here is that since the ARF in Eq. 2 includes both velocity and pressure terms that are usually coupled in practical applications, it is difficult to design a device optimized for acoustic patterning utilizing both terms. The CMAP platform is primarily designed for acoustic patterning based on the pressure term. Microparticles such as the polystyrene beads and most biological objects that have a similar density but different compressibility to water (f1>>f2) are ideal objects to be patterned on a CMAP device. For particles, such as metallic particles or air bubbles, with large density difference from water, the velocity term may dominate. Nevertheless, the patterns formed by these particles should also conform to the shape of air cavities since the cavity edges are where maximum velocity located as shown in
Although acoustic streaming force, ASF (Bruus H, Lab on a Chip, 12(1), 20-28), can be induced to counterbalance the ARF and disturb the patterning, the experimental results suggest that ARF is the driving force when the operation frequency is above 3 MHz and the particle is sized 10 μm or larger. At the onset of the operation, streaming vortices are observed only at the center of the circular membrane and extend weakly to ˜25 μm near the edge. On the other hand, the 10 μm polystyrene beads that were spread across the device migrate toward the membrane edges, where they are trapped firmly despite the later bulk movement of fluid as shown by the 1 μm beads. This strong trapping effect implies dominant strength of ARF to the patterning of 10 μm beads. The observed phenomenon of the bulk movement can be referred to as global flow, induced from the volumetric change of chamber as the upper PDMS lid expands thermally due to the heat generation from PZT. Since the upper PDMS lid (˜1 cm) is substantially thicker than the bottom soft, air-embedded PDMS structure (˜27 μm), the volumetric change should be predominately caused by the expansion of the lid. Although the 10 μm polystyrene beads and HeLa cells, respectively, outside the air cavities get drifted away, these are the excessive targets as to what the potential wells above the cavities can hold. Note that such drifts are mainly caused by the global flow because the ASF is only effective nearby the membrane edges. The drifts are favorable because they lead to overall cleaner patterning profiles without excessive targets outside the cavities. Blurring in images may be due to thermal expansion of PDMS causing structural deformation which affected microscope focusing. Besides the global flow, patternings of the 10 μm beads and HeLa cells reveal conformities to the pressure distribution simulated in
3 MHz was chosen as the operation frequency because it is a high enough value to suppress the acoustic streaming flow and a low enough value to avoid extra acoustic heating. For example, when the operation frequency is lowered to 0.5 MHz, 10 μm polystyrene beads can follow the streamlines of 1 μm beads, circulating in vortex form near the membrane edges. This leads to unstable patterning and difficulty in achieving desired profile. On the other hand, while operation at higher frequency can minimize the streaming flow, it is accompanied by larger energy attenuation in PDMS and, thus, extra heat generation that needs to be managed (Tsou J K et al., Ultrasound in medicine & biology, 34(6), 963-972).
While the CMAP platform relies on compliant, viscoelastic PDMS membrane to provide the breakthroughs in patterning, the membrane is so thin (˜2 μm) that the above fluid can penetrate through. This is evident by the fluid droplets below the membrane regions as shown in
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application claims priority to U.S. Provisional Patent Application No. 62/837,768, filed Apr. 24, 2019, the contents of which are incorporated by reference herein in its entirety.
This invention was made with government support under Grant No. 1711507 from the National Science Foundation. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2020/029747 | 4/24/2020 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62837768 | Apr 2019 | US |