WO2014133451A1 - Système et procédé d'analyse de cellules non sphériques - Google Patents

Système et procédé d'analyse de cellules non sphériques Download PDF

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Publication number
WO2014133451A1
WO2014133451A1 PCT/SE2014/050251 SE2014050251W WO2014133451A1 WO 2014133451 A1 WO2014133451 A1 WO 2014133451A1 SE 2014050251 W SE2014050251 W SE 2014050251W WO 2014133451 A1 WO2014133451 A1 WO 2014133451A1
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cells
particles
channel
spherical
frequency
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PCT/SE2014/050251
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English (en)
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Ola JAKOBSSON
Carl Johan Grenvall
Thomas Laurell
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Acousort Ab
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
    • G01N2015/1415—Control of particle position
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1497—Particle shape

Definitions

  • the invention relates to a method and system to orient non-spherical cells or particles present in a suspension wherein said suspension is exposed to an acoustic force, acting in one or two dimensions, wherein the cells or particles in the suspension will orient themselves such that the net acoustic force acting on them is minimized.
  • Non-spherical but axis-symmetrical cells or particles will then be oriented such that their smallest dimension is parallel with the strongest acoustic force and wherein changing the frequency or amplitude of the acoustic actuation allows reorientation of the cells or particles 90° with respect to their previous orientation
  • Microfluidics is inherently a domain where high performance cell and particle handling has proven to be very successful.
  • Some of the ruling technology platforms, which are industrial and clinical standards for high quality cell processing, are found in the fluorescence activated cell sorter (FACS) (Fluorescent detection and sorting) and in the Coulter Counter (size distribution measurements).
  • FACS fluorescence activated cell sorter
  • Coulter Counter size distribution measurements
  • the Fluorescence Activated Cell sorter (FACS) remains a major workhorse in cell biology laboratories today. After more than 40 years of development, the FACS excels at analyzing and sorting cells at very high speed. However, some cell types are still very challenging to analyze with the conventional FACS. It was early realized in the field of cytometry that the scattered light from non-spherical cells is both a function of their relative position and orientation to the illuminating laser axis beam.
  • X and Y chromosome detection and sorting of mammalian sperm cells One of the greatest challenges in the history of flow cytometry has been X and Y chromosome detection and sorting of mammalian sperm cells.
  • the fact that there is a difference in the amount of DNA content between the X and Y chromosome bearing cells can be utilized by staining the DNA inside the cells with a fluorescent dye.
  • the intensity of fluorescent light emitted from such a labeled cell is proportional to the amount of DNA inside.
  • the emitted light can be collected and the difference in signal intensity can be measured to distinguish between the two chromosome types.
  • sperm cells are non spherical cells with a flat head and a tail
  • the problem was eventually partially solved by the invention of asymmetric sheath flow nozzles, using a hydrodynamic flow focusing technique to achieve partial orientation. Orientation efficiencies of about 60% has been reported using such nozzles and today such nozzles are being used in specialized cell sorters dedicated for sperm sorting.
  • Shape, size and morphology are properties that can be measured by taking snapshots of cells passing a camera and using image analysis software.
  • the uncertainty of the relative orientation of the cells to the optical axis of the camera may also cause artifacts in the software based image analysis, making the method unreliable for analyzing non spherical cell types.
  • erythrocytes a non-spherical but axis-symmetrical cell
  • Erythrocytes exhibit a biconcave, disc-like shape.
  • light scattering from such cells is dependent on both their position and orientation and may cause artifacts or inconsistency in the collected signal if not taken into account. While co-axial flow focusing typically is used for positioning, it does not normally control the orientation of non-spherical particles.
  • Techniques that exist for controlling the orientation of non-spherical cells in continuous flow include the use of magnetic and electric forces, non-spherical nozzle geometries, inertial focusing or fabrication of obstructions within the fluidic channels.
  • an acoustic resonance is obtained across the channel perpendicular to the flow direction where a ⁇ /2 standing wave or an integer multiple of ⁇ /2 standing waves are obtained.
  • a ⁇ /2 resonance particles or cells with a positive acoustic contrast factor ⁇ will experience an acoustic radiation force F ax according to the fundamental radiation force equation for a 1-dimensional acoustic standing wave, Eq. 1.
  • the orientation induced by the acoustic force can always be predicted and controlled.
  • a non-spherical cell/particle exposed to an acoustic force in such a resonator will be orientated so that it will orient itself such that net acoustic force acting on the cell/particle is minimized and is perpendicular to either the sidewalls or the bottom of such a channel.
  • the orientation can be chosen so that in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension of the cells or particles is either perpendicular to the sidewalls, or the top/bottom of the channel.
  • the current invention discloses an adjustable method for both alignment and orientation of non-spherical cells or particles, such as red blood cells and other non-spherical cell types or particles in a continuous flow stream.
  • the invention relates to a method to orient non-spherical cells or particles in a suspension, which comprises the steps of;
  • frequency shift keying is set so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles these will be aligned and oriented such that its smallest dimension is parallel to the strongest acoustic force along a first axis
  • Fig. 1 Schematic presentation of a microchannel on a chip.
  • FIG. 2 Schematic presentation of an essentially square/rectangular microchannel on a chip, which typically is obtained by isotropic etching of eg. glass or silicon.
  • Fig. 3 Schematic presentation of a microchannel on a chip.
  • Fig. 4 Schematic presentation of orientation of asymmetric cells or particles in a square micro- channel or capillary using one frequency. The larger arrows indicate the strongest acoustic force.
  • Fig. 5 Schematic presentation of orientation of asymmetric cells or particles in a rectangular micro-channel or capillary using multiple frequencies and transducers. The larger arrows indicate the strongest acoustic force.
  • Fig. 6 An illustration of the microfluidic chip used in the experiments.
  • Fig. 7 Classification of orientation. The cells are classified as either flat, semi tilted or upended. Care was taken to obtain images with some time apart to avoid analyzing the same cell in more than one image.
  • Fig. 8 Top left: The horizontal acoustic radiation force F horizonta
  • Fig. 9 Percentage of flat, semi tilted, and upended red blood cells of total observations.
  • Letters (x), (y) and (z) refers to the spatial position along the length (I), width (w), and the height (h) of the microchannel, respectively.
  • Letters (Q,), (v), and (p,) refers to volume flow rate, flow velocity and pressure, respectively where subscript (i) indicate multiple instances of a property.
  • suspension refers to a fluid containing solid particles or cells that are sufficiently large for sedimentation.
  • aspect ratio is intended to mean the correlation between the height:width of a cross section of the channel.
  • FSK frequency shift keying
  • transducers is intended to mean piezoelectric elements that convert electricity to vibrations.
  • the invention relates to methods to orient non-spherical cells or particles in a suspension, wherein the cells or particles are exposed to one or two dimensional acoustic forces which forces the cells or particles to be aligned and oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension of the cell/particle is parallel with the strongest acoustic force.
  • the cells or particles can then be reoriented 90 degrees, with respect to the previous orientation by altering the resonance conditions in the channel.
  • the resonance conditions are changed by altering either the amplitude, frequency or FSK rate of the actuation transducers. By such an alteration the orientation of the cells or particles can be predicted and shape of the cells can be analysed.
  • the invention relates to a method to orient non-spherical cells or particles in a suspension, which comprises the steps of;
  • the non-spherical cells or particles may be banana shaped, cubical, rod shaped, rod like flat, rod like bent, spherical with buds, or disc shaped.
  • cells including bacterial cells, as well as most of the cells derived from mammals or plants are included.
  • the cells may be any kind of eukaryotic to prokaryotic cells and examples includes both mammalian cells as well as bacterial cells. Specific examples are yeast cells, cancer cells, platelets, red blood cells, white blood cells (such as: neutrophils, eosinophils, basophils, lymphocytes, monocytes and macrophages), adipocytes, Escherichia coli and other bacteria.
  • yeast cells such as: neutrophils, eosinophils, basophils, lymphocytes, monocytes and macrophages
  • adipocytes Escherichia coli and other bacteria.
  • Another example is sperm cells, wherein there is a need to sort the X and the Y chro
  • the pressure that forces the suspension into the inlet of the channel may be induced by a pump or by a syringe as long as the pressure forces the suspension into the inlet of the channel and further into the channel.
  • the acoustic forces which force the cells or particles to be aligned and oriented may be induced by the use of one or more piezoelectric transducers as defined above.
  • the transducers may be placed at the same position, at 90° for each other or at arbitrary angles from each other depending on the purpose of the analysis.
  • the shape of the channel is either square or rectangular or essentially square or rectangular.
  • the aspect ratio may be 1:1, 1:1,5, 1:2,5 or 1:3,5.
  • 1:1 is 1:1.
  • the channel may have a width and/or height ranging from 10 ⁇ to 1000 ⁇ , 75 ⁇ to 800 ⁇ , such as from 75 ⁇ to 200 ⁇ , or ranging from 200 ⁇ to 375 ⁇ , or ranging from 300 ⁇ to 400 ⁇ , or ranging from 400 ⁇ to 700 ⁇ , or ranging from 700 ⁇ to 800 ⁇ , or being 150 ⁇ , 300 ⁇ % 188 ⁇ % 375 ⁇ or 750 ⁇ .
  • the resonance frequency for a channel with a square or rectangular cross-section is determined by the dimensions of the channel, and the speed of sound for the liquid inside.
  • the width and height of the channel may be related such that the width w divided by an integer number n equals the height h divided by an integer number m.
  • a single frequency of vibration may be chosen to fulfill a resonance condition simultaneously for the height and width dimension, such that where c is the speed of sound in the suspending fluid.
  • the resonance conditions in the channel may be controlled individually/selectively by using two separate frequencies, f x and f 2 , of vibration for width and height respectively.
  • the frequencies are chosen such that
  • n and m may be any integer number
  • the frequency of vibration may vary in a range from 1 MHz to 10 MHz and is implicitly dictated by the dimensions of the channel as mentioned above.
  • One example being when the frequency in a first step is l,88Mhz and said frequency then is altered in a next step to l,89Mhz.
  • the invention relates to a method to orient non-spherical cells or particles in a rectangular microchannel using multiple frequencies and transducer.
  • the cell or particle suspension containing non spherical cells or particles is continuously injected into the microchannel with rectangular cross-section at a given flow rate.
  • the flow rate may vary between ⁇ , ⁇ - 20mL/min per channel.
  • the cells or particles are observed through a microscope with high magnification and a high speed camera with sufficiently short shutter time to capture sharp images of cells or particles.
  • Two ultrasonic (piezoelectric) transducers are attached to the microchannel and are actuated by individual electric signals that can be sine, square, triangle or of any other periodic shape.
  • One transducer is typically actuated at a frequency that gives a vertical resonance at ⁇ /2 in the channel, but can also be multiples of this.
  • the other transducer is typically actuated at a frequency that gives a horizontal resonance at ⁇ /2 in the channel, but can also be multiples of this.
  • particles with positive acoustic contrast factor will start to migrate towards the pressure node in the system. Once an asymmetric particle reaches the pressure node, it will stop migration and orient itself according to the direction of the acoustic radiation force, F rad .
  • the particle will be oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension is parallel with the strongest acoustic radiation force. Controlling the orientation.
  • Non-spherical and axis-symmetrical cells or particles will always be oriented so that their smallest dimension is parallel with the strongest acoustic force.
  • two main acoustic, half wavelength, resonances can be found, one vertical and one horizontal.
  • the resonances can be actuated by two individual transducers and frequencies.
  • the acoustic pressure amplitude of the individual resonances is controlled by the amplitude of the electric signal actuating the individual transducer.
  • the width may be chosen such that the frequency of vibration f is
  • the channel may have a width and/or height ranging from 10 ⁇ to 1000 ⁇ , 75 ⁇ to 800 ⁇ , such as from 75 ⁇ to 200 ⁇ , or ranging from 200 ⁇ to 375 ⁇ , or ranging from 300 ⁇ to 400 ⁇ , or ranging from 400 ⁇ to 700 ⁇ , or ranging from 700 ⁇ to 800 ⁇ , or being 150 ⁇ , 300 ⁇ % 188 ⁇ % 375 ⁇ or 750 ⁇ .
  • One example is given when the frequency in a first step is l,88Mhz and said frequency then is altered in a next step to l,89Mhz.
  • the size of the microchannel constitutes an upper limit of the size of the non-spherical cells or particles to be analysed.
  • the cells or particles may be spherical, cubical, rod shaped, rod like flat, rod like bent or disc shaped
  • the cells may vary in shape and size ranging from 1 ⁇ to 50 ⁇ , such as 1-5 ⁇ % 1-25 ⁇ % 5-50 ⁇ % 5-40 ⁇ % 5-30 ⁇ % 5-25 ⁇ % 8-25 ⁇ % or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ⁇ % or from 10-20 ⁇ or 10-15 ⁇ .
  • the cells or particles may have a volume ranging from 0.0005 to 70 x 10 15 m 3 , such as 0.0005-0.003 x 10 15 0.0005-0.07 x 10 15 m 3 , 0.0005-8 x 10 15 m 3 , 0.05-0.10 x 10 15 m 3 ' 0.07-70 x 10 15 m 3 , 0.07-35 x 10 15 m 3 , 0.07-14 x 10 15 m 3 , 0.07-8 x 10 15 m 3 , 0.25-6 x 10 15 m 3 0.3-8 x 10 15 m 3 , 0.07-35 x 10 15 m 3 or 0.5-15 x 10 15 m 3 .
  • the orientation effects from the acoustic radiation force can be observed.
  • the cells will be rotated until their smallest dimension is in parallel with the strongest acoustic radiation force.
  • the acoustic energy (and thus the acoustic force) in either the vertical or horizontal resonance mode can be controlled by adjusting the driving voltage amplitude of the respectively actuating transducer. This leads the cells to be aligned and rotated into two different angles ( Figure 8).
  • a microchannel structure and holes for inlets and outlets is KOH etched in a ⁇ 100> silicon wafer of thickness 350 ⁇ and cut to the dimensions 40 mm by 3 mm.
  • a piece of borosilica glass (40 mm by 3 mm by 1 mm) is anodically bonded to seal the channel.
  • Inlets and outlets comprises of pieces of silicone tubing, which are glued to the backside of the chip to connect tubing for external fluidics.
  • the square or rectangular microchannel is made of glass, quartz, metal, ceramic or polymer.
  • the piezoceramic transducers are driven by two signal-generators equipped with signal power amplifiers, depending on the need power requirements.
  • the acoustic resonances can be controlled by tuning the frequency and voltage driving the transducers.
  • Example 1 Orientation of non-spherical cells in a rectangular or essentially rectangular silicon/glass microchannel using multiple frequencies and transducer.
  • the cell suspension containing non-spherical cells is continuously injected into the microchannel with rectangular cross-section at a given flow rate.
  • the flow rate may vary between ⁇ , ⁇ - 20mL/min per microchannel.
  • the cells are observed through a microscope with high magnification and a high speed camera with sufficiently short shutter time to capture sharp images of cells.
  • Two ultrasonic (piezoelectric) transducers are attached to the microchannel and are actuated by individual electric signals that can be sine, square, triangle shaped or a combination of sine functions of the fundamental excitation frequency and higher harmonics.
  • One transducer is typically actuated at frequency that gives a vertical resonance at ⁇ /2 in the channel, but can also be multiples of this.
  • the other transducer is typically actuated at frequency that gives a horizontal resonance at ⁇ /2 in the channel, but can also be multiples of this.
  • particles with positive acoustic contrast factor will start to migrate towards the pressure node in the system. Once a non-spherical particle reaches the pressure node, it will stop migration and orient itself according to the acoustic radiation force field. The particle will be oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles its smallest dimension becomes parallel to the acoustic radiation force.
  • Particles will always be oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles their smallest dimension becomes parallel to the strongest acoustic force.
  • a microchannel with a rectangular cross-section or essentially rectangular cross-section two main acoustic, half wavelength, resonances can be found, one vertical and one horizontal.
  • the resonances are actuated by individual transducers and frequencies.
  • the acoustic pressure amplitude of the individual resonances is controlled by the amplitude of the electric signal actuating the individual transducer. By controlling the amplitude of the individual transducers, the orientation can be changed from vertical to horizontal and vice versa.
  • Example 2 Orientation of non-spherical cells in an essentially square glass microchannel/capillary using a single frequency with minor tuning.
  • the cell suspension containing non-spherical cells is continuously injected, at a given flow rate, into the microchannel with an essentially square cross-section, having vertical and horizontal channel dimensions only differing a few percent, Channel dimensions can vary between 10-lOOOum.
  • the flow rate may vary between ⁇ , ⁇ - 20mL/min per channel.
  • the cells are observed through a microscope with high magnification and a high-speed camera with sufficiently short shutter time to capture sharp images of cells.
  • An ultrasonic (piezoelectric) transducer is attached to the microchannel and is actuated by an electric signal that can be sine, square or triangle shaped or a combination of sine functions of the fundamental excitation frequency and higher harmonics.
  • the actuation frequency is typically set to achieve a ⁇ /2 resonance in either the vertical or the horizontal direction of the channel, but can also be multiples of this.
  • the particle will be oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis- symmetrical cells/particles its smallest dimension becomes parallel to the acoustic radiation force.
  • Non-spherical and axis-symmetrical cells/particles will always be oriented so that the smallest dimension is parallel to the strongest acoustic force.
  • two acoustic resonances one vertical and one horizontal resonance
  • One of these resonances will always dominate significantly over the other mode, thus orientating the non-spherical cells/particles according to either a horizontal or vertical direction.
  • By fine tuning the frequency it is possible to find a resonance mode that excites orientation along the other axis rotated 90°, this usually occurs at 10-100 KHz from the first resonance mode depending on the vertical and horizontal dimensions of the essentially square cross-section microchannel.
  • One method to achieve this is by scanning the frequency and observe the orientation of the non-spherical cells. The frequency is scanned until the orientation angle changes 90°. Once the 2 frequencies that give vertical and horizontal orientation are determined, the orientation can be controlled as horizontal or vertical by choosing one of these frequencies as the actuation frequency. By having two resonance modes close to each other both modes are actuated although the driving frequency is chosen to have either of the modes to dominate. At the same time the weaker actuation mode assists in driving the cells/particles into the center of the channel such that cells/particles are acoustically focused in two dimensions and localized in the center of the channel and hence in the optical focal line of the imaging system. The switching between the resonance modes subsequently only decides on a horizontal or vertical orientation of the cell/particle.
  • the glass chip with channel dimensions 375 by 150 ⁇ and length 4 cm, was fabricated in borosilicate chromium blanks (Telic Company, Valencia, CA) precoated with positive photoresist and fabricated by the means of photolithography and wet etching using a mixture of HF/HN0 3 /H 2 0. Holes for inlets and outlets were drilled using a diamond glass drill and a glass lid was then thermally bonded to the chip to seal it.
  • borosilicate chromium blanks Telic Company, Valencia, CA
  • Holes for inlets and outlets were drilled using a diamond glass drill and a glass lid was then thermally bonded to the chip to seal it.
  • the chip had one trifurcation inlet and a single outlet ( Figure 6), Two piezoceramic transducers (PZT), (PZ26, Ferroperm piezoceramics, Kvistgaard, Denmark), 2 and 5 MHz respectively, were used to actuate the chip.
  • the two actuators were glued underneath the chip using cyanoacrylate glue (Loctite Super Glue, Henkel Norden AB, Sweden).
  • a dual channel function generator (AFG 3022B, Tektronix, UK Ltd., Bracknell, UK) was used to actuate the transducers and the signals were amplified using in-house built amplifiers based on a LT 1012 power amplifier (Linear Technology Corp., Milpitas, USA). The applied voltages were monitored using an oscilloscope (TDS 2120, Tektronix, UK Ltd., Bracknell, USA). Syringe pumps (Nemesys, Cetoni GmbH, Korbussen, Germany) with mounted plastic syringes (BD Plastipak, BD Bioscience, San Jose, USA) were used to control the flow rates. Images were obtained using a high-speed camera (EoSens mini MC-1370, Mikrotron GmbH, Uterschleissheim, Germany). Experiments
  • the flow rate was kept constant during all experiments at a total of 12 ⁇ min "1 distributed as 2 ⁇ min "1 for the centre inlet (blood sample) and 5 ⁇ min "1 from each side inlet (sheath flow, PBS).
  • the side inlets were used to hydrodynamically position cells in order to facilitate imaging when the actuator driving the horizontal resonance was turned off. Images of the cells were obtained at a fixed position in the channel, and care was taken so that no picture contained the same cell twice.
  • the 5M Hz transducer was driven at 10.2 Vpp with a 5.76 M Hz sine signal during all experiments. This transducer actuated a vertical resonance in the chip, forcing all cells to the vertical (cross-sectional) center of the microfluidic, putting them all in focus of the microscope lens and orientating them with their flat side orthogonal to the camera axis.
  • the 2 M Hz transducer was operated at 2.26M Hz sine, and the voltage amplitude was varied between 0-10 Vpp in order to obtain the rotation of the cells.
  • This transducer induced a horizontal resonance in the chip, driving all cells to the horizontal (cross-sectional) center of the chip when actuated
  • the orientation efficiency of the system was measured by analyzing images obtained of the cells at a fixed part of the channel.
  • the cells were classified as either flat, semi tilted, or upended, seen from above. While keeping the voltage of 5 MHz transducer constant, focusing the cells in the horizontal dimension, the voltage of the 2 MHz transducer was varied to in an interval from 0-10 V to get the cells more and more vertically oriented instead. From figure 9 three different orientation stages can be observed.
  • the maximum percentage of cells that could be horizontally oriented was 87 %. Between 2.5-3 V the cells were starting to become turned to be vertically oriented. For 2.5 V the percentage of cells that were horizontally oriented or semi tilted were about the same at 40 % and 40.8 %, respectively while the vertically oriented were 18.9 %. At 3 V this had changed in favor of the vertically oriented cells that were about the same percentage as the semi tilted (39 % and 46.3 %, respectively, while the percentage of horizontally oriented cells had decreased to 15 %. When the voltage of the transducer was varied between 3.5-10 V the vertically oriented cells were well in majority with more than 86.1 % of the cells oriented in this way. The maximum percentage of cells that could be vertically oriented was 98.1 %, while the rest 1.9 % were semi tilted.

Abstract

La présente invention concerne un procédé et un système d'orientation de cellules ou de particules non sphériques dans une suspension, ladite suspension étant exposée à une force acoustique, agissant dans une ou dans deux dimensions. Selon l'invention, les cellules ou particules de la suspension font l'objet d'une orientation de façon telle que leur dimension la plus petite est parallèle à la force acoustique la plus puissante, le changement de la fréquence ou de l'amplitude de l'activation acoustique permettant une réorientation des cellules à 90° de leur orientation précédente.
PCT/SE2014/050251 2013-02-28 2014-02-28 Système et procédé d'analyse de cellules non sphériques WO2014133451A1 (fr)

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US11426727B2 (en) 2020-04-28 2022-08-30 Siemens Healthcare Diagnostics Inc. Acoustophoretic lysis devices and methods
US11959907B2 (en) 2015-01-12 2024-04-16 Instrumentation Laboratory Company Spatial separation of particles in a particle containing solution for biomedical sensing and detection

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