WO2015134831A1 - Appareil et processus de commande acoustique, et fabrication de l'appareil - Google Patents

Appareil et processus de commande acoustique, et fabrication de l'appareil Download PDF

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Publication number
WO2015134831A1
WO2015134831A1 PCT/US2015/019093 US2015019093W WO2015134831A1 WO 2015134831 A1 WO2015134831 A1 WO 2015134831A1 US 2015019093 W US2015019093 W US 2015019093W WO 2015134831 A1 WO2015134831 A1 WO 2015134831A1
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Prior art keywords
control apparatus
acoustic
acoustic control
interdigital transducer
transducer arrangement
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PCT/US2015/019093
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English (en)
Inventor
Feng Guo
Peng Li
Tony Jun Huang
Stephen J. Benkovic
James R. Fick
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The Penn State Research Foundation
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Publication of WO2015134831A1 publication Critical patent/WO2015134831A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0626—Fluid handling related problems using levitated droplets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/089—Virtual walls for guiding liquids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0436—Moving fluids with specific forces or mechanical means specific forces vibrational forces acoustic forces, e.g. surface acoustic waves [SAW]

Definitions

  • the present invention is directed to acoustic control. More particularly, the present invention is directed to an acoustic control apparatus, an acoustic control process, and an acoustic control apparatus fabrication process, where the acoustic control is due to interference patterns formed from two or more acoustic waves generated by interdigital transducer arrangements.
  • Multicellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanisms and processes of cell-cell interactions is valuable to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes. Despite significant advances in this field, to further understand how cells interact and communicate with each other, a means to precisely control the spatial and temporal association of cells, and to create defined cellular assemblies is urgently needed. [0005] While several methods have been employed to pattern cells, limitations still exist for the demonstrated technologies such as optical, electrical, magnetic, hydrodynamic, contact printing and so on.
  • DEP Dielectrophoresis
  • the surface modification methods restrict the space available for the cells to grow, while the magnetic assembly method requires cells to be labeled with magnetic beads.
  • Use of optical tweezers is label-free and without contact, but it requires very high laser power to manipulate cells, leading to a high risk of cell damage.
  • an acoustic control apparatus includes a first interdigital transducer arrangement positioned to direct a first acoustic wave, a second interdigital transducer arrangement positioned to direct a second acoustic wave in a non-parallel direction relative to the first acoustic wave, and a manipulation region at least partially defined by an interference pattern at least partially formed by interaction between the first acoustic wave and the second acoustic wave.
  • an acoustic control process includes directing a first surface acoustic wave generated from a first transducer arrangement, directing a second surface acoustic wave generated from a second transducer arrangement in a non-parallel direction relative to the first surface acoustic wave, and at least partially defining a manipulation region by an interference pattern formed by the first surface acoustic wave and the second acoustic wave.
  • an acoustic control apparatus fabrication process includes depositing metal on a photoresist-patterned substrate to produce a metal deposit, performing a lift-off process on the metal deposit to produce one or more interdigital transducer arrangements, aligning one or more microfluidic channels with the one or more interdigital transducer arrangements to produce an assembly, and maintaining the assembly at a temperature range of between 0°C and 90°C to produce the acoustic control apparatus.
  • FIG. 1 is a perspective view of an embodiment of an acoustic control apparatus with a first interference pattern, according to the disclosure.
  • FIG. 2 is a perspective view of an embodiment of an acoustic control apparatus with a second interference pattern, according to the disclosure.
  • FIG. 3 is an interference pattern having a net-like array of two-dimensional pressure nodes, according to an embodiment of the disclosure.
  • FIG. 4 is an interference pattern having a dot-array like pattern of pressure nodes, according to an embodiment of the disclosure.
  • FIG. 5 is a graphical depiction of cell-cell distance over time in response to an input signal, according to the disclosure.
  • FIG. 6 shows two HEK cells with different intercellular distances of 20 micrometers, 15 micrometers, 5 micrometers, and 0 micrometers, controlled according to an embodiment of the disclosure.
  • FIG. 7 shows cells in direct contact, with fluorescence dye transferred to the neighboring cell after one hour, indicating the formation of functional gap junctions, according to the disclosure.
  • FIG. 8 shows separated cells, with no transfer of fluorescence dye after one hour, controlled according to an embodiment of the disclosure.
  • FIG. 9 shows an acoustic potential distribution with a defined acoustic potential forming an elongate ovular shape, controlled according to an embodiment of the disclosure.
  • FIG. 10 shows an acoustic potential distribution with a defined acoustic potential forming an elongate perpendicular ovular shape relative to the elongate ovular shape of FIG. 9, controlled according to an embodiment of the disclosure.
  • FIG. 11 shows an acoustic potential distribution with a defined acoustic potential forming a generally circular shape, controlled according to an embodiment of the disclosure.
  • FIG. 12 shows an acoustic potential distribution with a defined acoustic potential forming a generally circular shape compressed relative to FIG. 11, controlled according to an embodiment of the disclosure.
  • FIG. 13 shows a two-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 14 shows a three-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 15 shows a four-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 16 shows a five-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 17 shows a single-layer cluster of two rows of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 18 shows a single-layer generally triangular cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 19 shows a single-layer generally circular cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 20 shows a single-layer cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.
  • FIG. 21 shows a schematic axial view of an acoustic control apparatus, according to the disclosure.
  • FIG. 22 shows bright-field image and time-lapse fluorescence images of two cells trapped within an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye results in dye transfer between the two cells over time, controlled according to an embodiment of the disclosure.
  • FIG. 23 shows bright-field image and time-lapse fluorescence images of a three-cell system trapped by an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye results in transfer of fluorescent molecules throughout the cell assembly, controlled according to an embodiment of the disclosure.
  • FIG. 24 shows bright-field image and time-lapse fluorescence images of a linear cell assembly trapped by a linear acoustic pressure node, results in dye molecules being transferred sequentially because of the defined linear assembly, controlled according to an embodiment of the disclosure.
  • FIG. 25 shows bright-field image and time-lapse fluorescence images of a two- dimensional, multiple-cell system trapped by a spherically shaped acoustic pressure node, allowing dye transfer to occur with all neighboring cells simultaneously, controlled according to an embodiment of the disclosure.
  • FIG. 26 schematically depicts an acoustic control apparatus with linear assemblies of cells formed under the control of a tunable acoustic well, whereby upon removal of the acoustic field, the cells drop to the surface and attach, according to the disclosure.
  • FIG. 27 shows in-suspension and attachment of HEK 293T cells over time controlled by acoustic potential, according to an embodiment of the disclosure.
  • FIG. 28 shows in-suspension and attachment of HMVEC cells over time controlled by acoustic potential, according to an embodiment of the disclosure.
  • FIG. 29 shows dye transfer of HMVEC cells over time controlled by acoustic potential, according to an embodiment of the disclosure.
  • FIG. 30 shows in-suspension and attachment of HeLa S3 cells over time controlled by acoustic potential, according to an embodiment of the disclosure.
  • FIG. 31 shows dye transfer of HeLa S3 cells over time controlled by acoustic potential, according to an embodiment of the disclosure.
  • an acoustic control apparatus for example, in comparison to concepts failing to include one or more of the features disclosed herein, permit improved ability to understand the mechanisms and processes of cell-cell interactions (for example, in physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes), permit precise spatial and temporal control of cells, permit creation of defined cellular assemblies, permit control and/or separation without modification to a cell's active state, permit control and/or separation without removing or affecting nutrients and/or osmolality, permit control and/or separation without restricting space available for the cells to grow, permit control and/or separation without labeling and/or lasers, permit control and/or separation with higher precision (for example, controlling intercellular distance at micron-scale) and/or higher throughput (for example, forming arrays of cell assemblies with tunable geometric configurations), permit simultaneous control and/or separation of
  • an acoustic control apparatus 101 includes a first interdigital transducer arrangement 103 positioned to direct a first acoustic wave 105 forming a first acoustic wave pattern and a second interdigital transducer arrangement 107 positioned to direct a second acoustic wave 109 forming a second acoustic wave pattern, for example, surface acoustic waves (SAWs).
  • SAWs surface acoustic waves
  • the first acoustic wave 105 (and the first acoustic wave pattern) and the second acoustic wave pattern 109 (and the second acoustic wave pattern) at least partially define a manipulation region 1 11, for example, by forming an interference pattern 1 13 based upon being positioned in a non-parallel arrangement.
  • the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 are arranged in any suitable configuration capable of producing the interference pattern 1 13 based upon the non-parallel arrangement.
  • Suitable non-parallel arrangements include the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 being relatively positioned between 45 degrees and orthogonal (90 degrees), orthogonal or substantially orthogonal, 75 degrees, 60 degrees, 45 degrees, 30 degrees, 15 degrees, 10 degrees, 5 degrees, or any suitable combination, sub-combination, range, or subrange therein.
  • the acoustic control apparatus 101 includes or consists of more than two interdigital transducer arrangements, such as, three interdigital transducer arrangements, four interdigital transducer arrangements, five interdigital transducer arrangements, or more than five interdigital transducer arrangements.
  • the acoustic control apparatus 101 consists of two or three interdigital transducer arrangements.
  • the interference pattern 1 13 controls or manipulates particles (for example, micrometer- sized and/or nanometer-sized particles), fluids (for example, immiscible fluids), or any other material responsive to acoustic energy. For example, based upon a first input frequency and/or phase, the first acoustic wave 105 (and the first acoustic wave pattern) is generated and, based upon a second input frequency and/or phase, the second acoustic wave 109 (and the first acoustic wave pattern) is generated.
  • the input frequencies are generated by radiofrequency (RF) signals.
  • the first input frequency and/or phase differs from the second input frequency and/or phase, but the resonance frequency range is shared.
  • acoustic wells Sites within the interference pattern 1 13 are known as acoustic wells. In one embodiment, as many as 1,600 acoustic wells are defined by the interference pattern 113.
  • the interference pattern 113 is able to be finely tuned, thereby permitting manipulation of the geometry of particles, such as, cell assemblies, in response to a change in the first input frequency (and/or phase) and/or the second input frequency (and/or phase).
  • the acoustic control apparatus 101 is capable of creating isolated half-wavelength sized square pressure nodes. In response to input frequencies, cells or other particles are capable of being manipulated and/or controlled, for example, by acoustic radiation force and/or drag force driven by acoustic streaming.
  • the acoustic control apparatus 101 is capable of precisely activating and tuning the position and dimension of the particles within the interference pattern 113.
  • a cluster of cells or particles within the interference pattern 1 13 is capable of being precisely manipulated based upon quantity, orientation, and/or configuration.
  • suspended cell assemblies are capable of being allowed to settle to the surface at any time to attach and optionally spread.
  • the interference pattern 1 13 is finely tuned by adjusting any suitable property of the SAWs forming the interference pattern 1 13, including, but not limited to, the power, amplitude, angular frequency, phase constant, wavelength, frequency, waveform, space coordinate, time coordinate, wavenumber, period, of combination thereof, of one of or dependently or independently both of the SAWs forming the interference pattern 1 13.
  • the fine tuning of the interference pattern 1 13 may include, but is not limited to, increasing fringe spacing, decreasing fringe spacing, increasing depth in a three-dimensional configuration, decreasing depth in a three-dimensional configuration, and combinations thereof.
  • the geometric configuration of particles, manipulated by the fine tuning of the interference patter 1 13 is any suitable one-dimensional, two-dimensional or three dimensional geometry, simple geometry, or complex geometry, including, but not limited to a point, line segment, curve, triangle, quadrilateral, square, rectangle, rhombus, parallelogram, trapezoid, kite, pentagon, hexagon, octagon, nonagon, decagon, circle, oval, ellipse, parabola, hyperbola, sphere, spheroid, polygon, tetrahedron, pentahedron, hexahedron, heptahedron, octahedron, enneahedron, decahedron, dodecahedron, cone, symbol, written character, letter, numeral, alphanumeric, glyph, pictogram, syllabogram, logogram, or combination thereof.
  • the interference pattern 113 includes a pressure gradient capable of directing cells into the middle of the manipulation region.
  • the interference pattern 1 13 is a net-like array of two-dimensional pressure nodes.
  • the interference pattern 113 is a dot-array like pattern of pressure nodes.
  • the interference pattern 1 13 is contained within a chamber having an acoustic aperture that is larger than the dimensions of the chamber.
  • the interference pattern 113 is within a square chamber having dimensions of 6 millimeters by 6 millimeters and the acoustic aperture is designed to permit SAWs of 9 millimeters in both directions.
  • the interdigital transducer arrangements include any suitable material(s) capable of generating acoustic waves to define the interference pattern 1 13.
  • electrodes 117 for example, 30 to 50 pairs (such as, 40 pairs) are arranged on any suitable substrate 1 19, for example, with spacing gaps of between 50 micrometers and 300 micrometers, between 60 micrometers and 200 micrometers, between 60 micrometers and 100 micrometers, between 70 micrometers and 80 micrometers, or any suitable combination, sub-combination, range, or subrange therein.
  • Suitable substrates 119 include a lithium niobate substrate, a lithium tantalate substrate, lead zirconium titanate substrate, a polymer such as polyvinylidene fluoride (PVDF) or another fluoropolymer, quartz, another material, or a combination thereof. Additionally or alternatively, the substrate 1 19 is capable of being a 128° Y-cut piezoelectric substrate, a piezoelectric substrate with a 45° angle to the X-direction, or a combination thereof.
  • PVDF polyvinylidene fluoride
  • the first interdigital transducer arrangement 103 and/or the second interdigital transducer arrangement 107 include(s) a metal deposit on a photoresist-patterned substrate, for example, a titanium layer and a gold layer.
  • a metal deposit on a photoresist-patterned substrate for example, a titanium layer and a gold layer.
  • Suitable thicknesses of the titanium layer include, but are not limited to, less than 10 nanometers, less than 7 nanometers, between 1 nanometer and 10 nanometers, between 3 nanometers and 7 nanometers, between 4 nanometers and 6 nanometers, 5 nanometers, or any suitable combination, sub-combination, range, or subrange therein.
  • Suitable thicknesses of the gold layer include, but are not limited to, less than 200 nanometers, less than 120 nanometers, between 20 nanometers and 300 nanometers, between 50 nanometers and 150 nanometers, between 80 nanometers and 120 nanometers, 100 nanometers, or any suitable combination, sub-combination, range, or sub-range therein.
  • one or more microfluidic channels 1 15 extend through the manipulation region 1 1 1.
  • the microfluidic channel(s) 1 15 includes a polydimethylsiloxane material, a collagen coating along the flow path, a fibronectin coating along the flow path, or a combination thereof.
  • the acoustic control apparatus 101 is devoid of the microchannel(s) 1 15 and operates as a static well for analytical purposes.
  • the acoustic control apparatus 101 is fabricated by any suitable fabrication process.
  • One suitable process includes depositing metal on a photoresist-patterned substrate to produce a metal deposit, performing a lift-off process on the metal deposit to produce one or more interdigital transducer arrangements, aligning one or more of the microfluidic channels 1 15 with the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 as an assembly, and maintaining the assembly at a temperature range of between 0°C and 90°C (for example, between 10°C and 90°C, between 20°C and 90°C, between 30°C and 90°C, between 10°C and 70°C, between 20°C and 70°C, between 30°C and 70°C, between 30°C and 40°C, between 35°C and 40°C, or any suitable combination, sub- combination, range, or subrange therein) and/or for a duration of at least 1 second (for example, at least 12 hours, at least 24 hours, between 1 second and 4
  • the microfluidic channel(s) 1 15 are capable of being produced using standard lithography and PDMS mold replication methods.
  • the collagen coating and/or the fibronectin coating is/are applied, for example, with 1 mg/mL collagen type 1 or fibronectin overnight in a 37°C cell culture incubator, prior to rinsing with phosphate buffered saline (PBS buffer) before each use of the acoustic control apparatus 101.
  • PBS buffer phosphate buffered saline
  • the acoustic control apparatus 101 is capable of being used for separating and/or controlling the position of particles and/or fluids.
  • the acoustic control apparatus 101 is used in a biocompatible manner, such as, by modulating cell-cell interactions or interactions between cells and other objects (such as other cells, solid substrates, matrices, viruses, bacteria, or fluorescent beads), without the interference of cell-surface interactions.
  • Other illustrative examples of a particle include solid or hollow particles, and unicellular or multicellular organisms.
  • the acoustic control apparatus 101 is used to explore gap junction based intercellular communication and/or functional intercellular communication, for example, by visualizing dye coupling between cells.
  • Communication between cells is generally achieved through soluble factors (for example, paracrine, autocrine, and/or exosomes) or through contact such as gap junctions.
  • soluble factors for example, paracrine, autocrine, and/or exosomes
  • the control of intercellular distance for regulating these processes, as communication through soluble factors is highly dependent upon the distance between cells, and gap junction based communication occurs when cells are in contact with one another. Considering this, study of these processes is dependent upon the precise spatial control of cell assemblies.
  • the acoustic control apparatus 101 is able to control both cell-cell distance and cell arrangement and employs the formation of isolated pressure nodes with tunable pressure gradients.
  • the acoustic control apparatus 101 enables the label-free study of the correlation between cell-cell adhesion and cell-matrix adhesion (for example, the cross talk between cadherin and integrin).
  • the SAW microfluidic device is placed inside of a customized stage cell culture chamber (INUBTFP-WSKM-GM2000A, Prior Scientific).
  • a solution of cells is injected into the device using a syringe pump (KDS210, KD Scientific).
  • KDS210 syringe pump
  • Two independently controllable AC signals generated by a function generator (3102C, AFG) and amplified by two amplifiers (25A100A, Amplifier Research) are connected to two pairs of interdigital transducer arrangements to generate two sets of orthogonal propagated standing SAWs.
  • the power of the applied SAW is maintained at a range from 10 to 40 mW (working area of 5.8 cm ).
  • the pattern of movement shows a clear step-like shape that matches the period of the modulated input signal.
  • the results indicate that the movement of particles is fully controlled by the input signals.
  • This technique is further demonstrated by controlling the intercellular distance of HEK 293T cells.
  • Two distinct states, direct contact and non-contact with a small distance, in the context of forming cell-cell junctions for intercellular communication studies are analyzed.
  • Gap junction based intercellular communication involves direct contact between cells, while communication that relies upon soluble factors is highly dependent upon the distance between the cells sending and receiving the signals. The generation of these two states is important in order to isolate effects from the two types of intercellular communication.
  • FIG. 5 demonstrates that the distance between cells is capable of being controlled using the manner described above, with blue and red color indicating low and high acoustic potential, respectively, and arrows showing the direction of radiation force.
  • Two HEK 293T cells are able to be positioned at any distance smaller than their initial distance.
  • FIG. 6 shows two HEK cells with different intercellular distances of 20, 15, 5, and 0 ⁇ , respectively.
  • fluorescence dye when the cells are in direct contact, fluorescence dye can be transferred to the neighboring cell after one hour, indicating the formation of functional gap junctions. As shown in FIG. 8, when cells are separated, even by a very small distance (for example, 3 ⁇ ), no transfer of fluorescence is observed after the same period of time.
  • the well-defined acoustic pressure nodes created by the acoustic control apparatus 101 are also found to be suitable for manipulating a group of cells in order to form different geometric configurations.
  • a group of cells When a group of cells is present within the confines of the interference pattern 1 13, they are manipulated in concert and assembled into defined patterns using the acoustic radiation force.
  • the interference pattern 1 13 is determined to be highly tunable in terms of size and shape as indicated in both simulation and experimental results of FIGS. 9-12, which show acoustic potential distribution with different acoustic amplitudes corresponding with different geometries defined by an acoustic potential boundary 901.
  • the direction of the rectangle interference pattern 1 13 is re-oriented by 90°, as shown in FIG. 10, by switching the input powers of the interdigital transducer arrangements (30 mW and 13.45 MHz, 10 mW and 13.35 MHz, respectively). Similarly, the same amplitude (20 mW) is applied in both directions, forming a square-shaped or circular-shaped interference pattern (see FIGS. 1 1 and 12), thereby assembling single-layer clusters as shown in FIGS. 17-20, illustrating capabilities of reorienting, for example, to produce spherically and/or single layer cell assemblies.
  • the size of the interference pattern 1 13 is decreased by increasing the input power (30 mW) as shown in FIG. 12, forming three-dimensional cell spheres as a result.
  • Cells present in the acoustic field tend to be held at a fixed distance above the substrate by the acoustic radiation force and acoustic streaming induced hydrodynamic force.
  • the acoustic control apparatus 101 is found to be capable of maintaining cell assemblies in suspension.
  • the acoustic control apparatus 101 is used to initiate and investigate functional GJIC.
  • HEK 293T cells are known to form gap junctions when they are in contact and can exhibit vivid dye coupling properties.
  • Calcein-AM stained HEK 293T cells and unstained cells are combined at a ratio of 1 :2 to 1 :4 and then loaded into the acoustic control apparatus 101.
  • the cells are patterned into linear arrays and maintained in culture medium for the entire duration of the experimental period with the acoustic field as depicted in FIG. 21.
  • bright-field image and time-lapse fluorescence images of a three-cell system trapped by an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye result in transfer of fluorescent molecules throughout the cell assembly.
  • bright-field image and time-lapse fluorescence images of a linear cell assembly trapped by a linear acoustic pressure node result in dye molecules being transferred sequentially because of the defined linear assembly.
  • the cells are patterned in a linear array, their communication (as observed by the transfer of dye) occurs linearly. If cells are patterned in a cluster, their communication format is changed as well. As shown in FIG. 25, after tuning the acoustic well to assemble cells into a cluster, multiple cells receive the signal simultaneously from the donor cell, as shown by bright- field image and time-lapse fluorescence images of a two-dimensional, multiple-cell system trapped by a spherically shaped acoustic pressure node, allowing dye transfer to occur with all neighboring cells simultaneously. As a control, gap junction inhibitor 18, a-glycyrrhetinic acid, is used to exclude the possibility of dye leakage.
  • HEK 293T cells are capable of forming functional gap junction channels in suspension, without the need for adhesion to a substrate.
  • the acoustic control apparatus 101 therefore, provides a simple and rapid way to examine the formation and function of gap junction mediated intercellular communication in suspended cultures. Arranging cells with defined number and connection also simplifies the model for quantitative characterization of GJIC (for example, gap junction permeability).
  • the acoustic control apparatus 101 is capable of controlled patterning of cells, enabling the study of intercellular communication within groups of cells with varied architectures (for example, linear vs. sphere). [0079] In another experiment, a linear pattern of HEK 293T cells is created using a method corresponding to FIG.
  • FIG. 26 which schematically depicts the acoustic control apparatus 101 with linear assemblies of cells formed under the control of a tunable acoustic well, whereby upon removal of the acoustic field, the cells drop to the surface and attach.
  • the linear cell assembly is maintained in suspension for 1 hour due to the combination of acoustic radiation force and acoustic streaming induced hydrodynamic force. During this period, only cell-cell adhesion occurs, despite the presence of a receptive surface coated with collagen to facilitate cell attachment (see FIG. 27). After 1 hour, the SAW is removed, which allows the cells to drop to the surface and attach to form a cell-matrix interaction. After 40 minutes, HEK 293T cells show proper adhesion and expansion morphology on the surface.
  • Calcein-AM stained HMVEC cells are mixed with unstained HMVEC cells at a ratio of 1 :4 and loaded into the microchannel.
  • a one-dimensional standing SAW field is formed as described in the previous section and these HMVEC cells are patterned into linear assemblies. Once the linear pattern is stable, the SAW field is removed to allow cells to settle down and attach to the surface.
  • FIG. 28 shows that HMVEC cells start to spread 15 minutes after settling down. After 25 minutes, most of the cells attach and spread on the surface while the geometric configuration is still maintained. After all the cells become adherent to the substrate, time-lapse fluorescence images are taken to monitor the transfer of fluorescence molecules from stained (donor) cells to unstained (receiver) cells shown in FIG. 29.
  • Acoustic tweezers patterning cells and microparticles using standing surface acoustic waves (SSAW). Lab on a chip 9, 2890-5 (2009); Ding, X. et al. Surface acoustic wave microfluidics. Lab on a chip 13, 3626-49 (2013); Ding, X. et al. On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves. Proceedings of the National Academy of Sciences of the United States of America 109, 1 1 105-9 (2012); Kholodenko, B.N. Cell-signaling dynamics in time and space. Nature reviews. Molecular cell biology 7, 165-76 (2006); Shi, J. et al.

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne des appareils de commande acoustique, des processus de commande acoustique et des processus de fabrication d'appareils de commande acoustique. L'appareil de commande acoustique comprend un premier agencement transducteur interdigité positionné pour diriger une première onde acoustique, un second agencement transducteur interdigité positionné pour diriger une seconde onde acoustique dans une direction non parallèle relativement à la première onde acoustique, et une région de manipulation définie au moins en partie par un motif d'interférences formé au moins en partie par une interaction entre la première onde acoustique et la seconde onde acoustique. Le processus de commande acoustique consiste à diriger la première onde acoustique, à diriger la seconde onde acoustique et à définir au moins en partie la région de manipulation. Le processus de fabrication de l'appareil de commande acoustique consiste à déposer du métal sur un substrat ayant des motifs photorésistants, à réaliser un processus d'enlèvement, à aligner un ou plusieurs micro canaux fluidiques et à maintenir une plage de température entre 0 °C et 90 °C.
PCT/US2015/019093 2014-03-07 2015-03-06 Appareil et processus de commande acoustique, et fabrication de l'appareil WO2015134831A1 (fr)

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WO2017127686A1 (fr) * 2016-01-22 2017-07-27 Carnegie Mellon University Manipulation acoustique tridimensionnelle de cellules
WO2017157426A1 (fr) 2016-03-15 2017-09-21 Centre National De La Recherche Scientifique Pincettes acoustiques
WO2017202747A1 (fr) 2016-05-24 2017-11-30 Centre National De La Recherche Scientifique Pinces acoustiques
CN112517091A (zh) * 2020-10-28 2021-03-19 清华大学 生物样品中微小物质的分离方法
WO2021122479A1 (fr) 2019-12-18 2021-06-24 Université de Lille Dispositif électroacoustique
US11534761B2 (en) 2017-10-25 2022-12-27 Universite De Lille Acoustic tweezers
US11577241B2 (en) 2018-12-03 2023-02-14 Duke University Acoustofluidic systems including acoustic wave generators for manipulating fluids, droplets, and micro/nano objects within a fluid suspension and related methods
WO2023065063A1 (fr) * 2021-10-18 2023-04-27 Fudan University Appareil et système de manipulation de particules

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US6317389B1 (en) * 2000-04-21 2001-11-13 Kohji Toda Ultrasound-signal radiating device
US20080018199A1 (en) * 2006-05-08 2008-01-24 The Penn State Research Foundation High frequency ultrasound transducers
US20120083425A1 (en) * 2010-10-05 2012-04-05 George Steven C High-Throughput Platform Comprising Microtissues Perfused With Living Microvessels
WO2014028167A1 (fr) * 2012-07-18 2014-02-20 Loc Micro Dispositif pour réaliser des ondes acoustiques de volume de phase liquide et utilisation pour une phase liquide pour une détection
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127686A1 (fr) * 2016-01-22 2017-07-27 Carnegie Mellon University Manipulation acoustique tridimensionnelle de cellules
WO2017157426A1 (fr) 2016-03-15 2017-09-21 Centre National De La Recherche Scientifique Pincettes acoustiques
US11731127B2 (en) 2016-03-15 2023-08-22 Centre National De La Recherche Scientifique Acoustic tweezers
WO2017202747A1 (fr) 2016-05-24 2017-11-30 Centre National De La Recherche Scientifique Pinces acoustiques
US11534761B2 (en) 2017-10-25 2022-12-27 Universite De Lille Acoustic tweezers
US11577241B2 (en) 2018-12-03 2023-02-14 Duke University Acoustofluidic systems including acoustic wave generators for manipulating fluids, droplets, and micro/nano objects within a fluid suspension and related methods
WO2021122479A1 (fr) 2019-12-18 2021-06-24 Université de Lille Dispositif électroacoustique
CN112517091A (zh) * 2020-10-28 2021-03-19 清华大学 生物样品中微小物质的分离方法
WO2023065063A1 (fr) * 2021-10-18 2023-04-27 Fudan University Appareil et système de manipulation de particules

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