• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    METHOD AND SYSTEM FOR ORIENTATION AND / OR CLASSIFICATION OF MICROFLUID PARTICLE. The present invention relates to a system for orienting particles in a microfluidic system that includes one or more sources of radiation pressure arranged to expose the particles to radiation pressure to cause the particles to adopt a specific orientation in the fluid. A system for classifying particles in a microfluidic system includes a detection stage arranged to detect at least one difference or discriminate between particles within the fluid flow that passes through the detection stage, and one or more sources of radiation pressure passing through the particles move sequentially and a controller arranged to change the radiation energy to cause a change in the direction of movement of selected particles within the fluid flow to classify the particles. The particles can be biological particles such as sperm. The radiation pressure can be an optical pressure and can be one or more waveguides which can extend through a channel of the microfluidic system.
    公开号:BR112015001844B1
    申请号:R112015001844-0
    申请日:2013-07-29
    公开日:2021-03-02
    发明作者:Miriam Cather Simpson；Charles Alan Rohde
    申请人:Engender Technologies Limited；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The present invention relates to a method and system for guiding and / or classifying a particle in a microfluidic system. BACKGROUND
    [002] Developments in commercial biotechnology and academic medicine have directed a strong focus on methods for the classification of biological cells. The two main proposals that emerged - mass separation and single cell classification - both enrich a population of cells with a targeted subset with physicochemical characteristics (ie, size, volume, light scattering properties, etc.), immunological or functional . Mass classification generally focuses on a single discriminating cellular characteristic. Examples include cell filtration, centrifugation / sedimentation and affinity-based extraction. The main disadvantages of mass classification are lesser purity, loss of cells during the classification process, difficulty in classifying relatively rare cells, and difficulty in discriminating between subpopulations of similar cells. Bulk sorting, however, is a relatively simple method that offers high throughput. In contrast, single cell methods, the most important of which is fluorescence activated cell classification (FACS) by flow cytometry, examines each cell individually to target the desired subpopulation for isolation and then guides them to different output streams. The reduction in yield is offset by the greater advantages in classification specificity that is tunable to the desired result, a generally higher cell recovery, the ability to classify populations of rare cells or only or weakly discriminated, and the availability of classification of multiple targets based on a network of multiple cellular characteristics (that is, different types of surface receptors, each identified with a different fluorescent label). An important challenge faced by FACS flow cytometric methods is the damage incurred by some cells in the flow (shear stress) and classification processes (electric field damage). An important example is the reduced fertility of classified sperm samples that can be attributed to these disruptive physical processes.
    [003] In the agriculture sector, cell discrimination is specifically important in livestock species where artificial insemination is commonly practiced such as livestock. The use of semen of identified sex facilitates the control of the sex of the offspring for commercial benefit. The current commercially important method for sperm classification uses FACS flow cytometry, in which sperm are distinguished by their differences in DNA content. The DNA of each sperm is stained with a fluorescent dye in proportion to the content of DNA content. Since the X chromosome is larger (that is, it has more DNA) than the Y chromosome, the "female" sperm (which contains the X chromosome) will absorb a greater amount of dye than the "male" sperm (which contains the Y chromosome) and as a consequence when exposed to UV light during flow cytometry it will fluoresce with greater intensity than the sperm Y. Before detection or discrimination the sperm can be hydrodynamically oriented and the sperm can be separated into individual droplets which can then be electrically charged . After detection or discrimination, sperm are classified by electric field - interactions of charged droplets. SUMMARY OF THE INVENTION
    [004] In broad terms in one aspect the invention comprises a method for orienting particles in a microfluidic system, which includes exposing the particles to radiation pressure in a micro-fluidic system to cause at least a majority of the particles to adopt an orientation within the fluid.
    [005] In broad terms in another aspect the invention comprises a system for orienting particles in a microfluidic system, which includes one or more sources of pressure radiation arranged to expose the particles in the microfluidic system to radiation pressure to cause at least a majority of the particles adopt a specific orientation within the flow.
    [006] The particles can be biological or non-biological particles. Typically the particles are asymmetric particles. The asymmetry can be in any physical property that leads to an asymmetric interaction with the incident radiation, including but not limited to asymmetry in the physical dimensions of the particles. In some embodiments, the particles can be sperm, red blood cells, or bacteria, for example.
    [007] The radiation pressure can be an optical pressure and can be of one or more waveguides which can extend through a channel of the microfluidic system, for example, through above, below, or through the side walls of the channel , or they can touch a channel from above, below or from the side. The waveguide (s) can be one or more optical waveguides that are connected to a light source, such as a laser, to carry the light and generate the radiation pressure also referred to as the force optics, photon pressure or electromagnetic pressure. The one or more waveguides can be manufactured as part of the intrinsic manufacturing process of the microfluidic system, or can be inserted as a fiber optic unit in the construction of the final system.
    [008] A microfluidic system to orient the particles as above can also comprise a pre-stage to focus and / or singularize the particles at a particular location within the channel. This system can be hydrodynamic or based on radiation pressure.
    [009] In broad terms in another aspect the invention comprises a method for classifying particles in a microfluidic system, which includes:
    [0010] - detect at least one difference or discrimination between particles, and
    [0011] - change based on a detection or discrimination input, one or more radiation pressure sources passing through which the particles move sequentially to cause a change in the direction of movement of selected particles in the fluid flow to classify the particles .
    [0012] The particles can be directed into two or more than two different exits.
    [0013] The one or more sources of radiation pressure may be one or more waveguides, which may extend through a microfluidic system channel, for example, through above, below or through the side walls of the channel, or may run over a channel from above, below or from the side.
    [0014] The step of detecting at least one difference or discrimination between the particles can comprise an optical technique for evaluating a particle characteristic, the technique can be a fluorescence-based detection technique.
    [0015] The method may also comprise singularizing the particle flow and may also comprise focusing the particles to a specific location within the channels. The forces can be hydrodynamic or based on radiation pressure.
    [0016] The method may also comprise causing at least a majority of the particles to first adopt a specific orientation within the fluid prior to detection, where the particles are asymmetric particles. The orientation step may comprise exposing the particles to radiation pressure such as optical pressure to cause at least a majority of the particles to adhere to a specific orientation within the fluid.
    [0017] In broad terms in another aspect the invention comprises a system for classifying particles in a microfluidic system, which includes: - a detection stage arranged to detect at least one difference or discriminate between particles in the fluid flow that passes through the detection stage, and - one or more radiation pressure sources passing through which the particles move sequentially and a controller willing to change based on an input from the detection or discrimination stage, the radiation energy in one or more sources of radiation pressure to cause a change in the direction of movement of selected particles in the fluid flow to classify the particles.
    [0018] The system may be willing to change or classify the particles so that each particle is directed into one of two or more than two different exits.
    [0019] The one or more sources of radiation pressure may be one or more waveguides which may extend at least partially through a channel of the microfluidic system, for example, through above, below or through the side walls of the channel, or they can touch the channel from above, from below or from the side.
    [0020] The detection stage The detection stage can be arranged to detect or discriminate the particles by an optical technique such as a fluorescence-based detection technique.
    [0021] The particles can be biological or non-biological particles. Typically the particles are asymmetric particles. Asymmetry can be in any physical property that leads to an asymmetric interaction with the optical force, including but not limited to asymmetry in physical dimensions. In some embodiments, the particles can be sperm, red blood cells, bacteria, or nanoparticles, for example.
    [0022] A microfluidic system to orient the particles as above can also comprise a pre-stage to singularize the particle flow and can also comprise a pre-stage to focus the particles on a specific location within the channel. This system can be hydrodynamic or optical.
    [0023] The system may also comprise an orientation stage arranged to cause at least a majority of the particles to first adopt a specific orientation within the fluid, specifically where the particles are asymmetric particles. The orientation stage can comprise one or more waveguides arranged in use to expose the particles to radiation such as an optical pressure to cause at least a majority of the particles to adopt a specific orientation within the fluid.
    [0024] In broad terms, in another aspect the invention comprises a microfluidic system to identify the sex of sperm, which includes: - one or more sources of orientation radiation pressure arranged to expose the sperm to pressure to cause the sperm to the individual sperm adopts a common orientation within the fluid, - a fluorescence-based detection stage arranged to discriminate between male and female sperm in the fluid flow passing through the detection stage, and - one or more sources of change radiation pressure passing through by which the individual sperm subsequently moves, and - a controller arranged to receive input from the detection stage and control the radiation energy at one or more sources of changing radiation pressure to separately target the male and / or female sperm.
    [0025] A microfluidic system to orient the particles as above can also comprise a pre-stage to singularize the sperm flow and a pre-stage to focus the sperm in a specific location within the channel. This system can be hydrodynamic or optical.
    [0026] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include this term, the characteristics prefaced by this term in each statement do not all need to be present but other characteristics may also be present. Relative terms such as "understands" and "understood" must be interpreted in the same way. BRIEF DESCRIPTION OF THE DRAWINGS
    [0027] The invention is further described with reference to the accompanying figures in which:
    [0028] Figure 1 schematically illustrates a modality of a microfluidic system of the invention for particle classification,
    [0029] Figures 2a and 2b schematically illustrate modalities for particle orientation in which a single waveguide - Figure 2a, or multiple waveguides - Figure 2b, top the microchannel above, below or on one side of the channel,
    [0030] Figures 3a and 3b schematically illustrate modalities for particle orientation in which a single waveguide - Figure 3a, or multiple waveguides - Figure 3b, extend through the microchannel above, below or along a side wall of the canal,
    [0031] Figures 4a and 4b schematically illustrate modalities for classifying or changing selected particles in a flow stream within a microchannel, in which multiple waveguides top the channel above, below or on one side of the channel,
    [0032] Figures 5a and 5b schematically illustrate modalities for classifying or changing selected particles in a flow stream within a microchannel, in which multiple waveguides extend through the channel above, below or along the side wall of the channel,
    [0033] Figure 6a shows an embodiment of a microfluidic chip of the invention to perform sperm orientation and separation and Figure 6b shows a network of individual classification chips that can be arranged for massively parallel microfluidic implementation to classify sperm accordingly. with sex,
    [0034] Figures 7a and 7b are computational geometries used in FEM simulations referred to in the subsequent description of experimental work - Figure 7a - elliptical cylinder in water placed along the waveguide 40 μm optical axis of the waveguide terminal and Figure 7b - elliptical cylinder placed above a SU8 photo-epoxy waveguide with a small gap separating the waveguide cylinder,
    [0035] Figure 8 shows the torque on the elliptical cylinder in water over a variety of distances from the terminal of a waveguide, referred to in the subsequent description of the experimental work,
    [0036] Figure 9 shows the torque on the elliptical cylinder in water above a waveguide at 3 separation distances, referred to in the subsequent description of the experimental work,
    [0037] Figure 10 shows the forces Fx and Fy on an elliptical cylinder in water at the end of a waveguide in vertical orientation for a variety of separation distances, referred to in the subsequent description of the experimental work,
    [0038] Figure 11 shows the optical forces Fx and Fy on an elliptical cylinder in water above a waveguide for a variety of separation distances, referred to in the subsequent description of the experimental work,
    [0039] Figures 12a-c show the displacement of a symmetrical particle through an optical field as the particle flows through the end of a waveguide terminal in a microfluidic channel referred to in the subsequent description of the experimental work,
    [0040] Figures 13a and b show the displacement of the asymmetric particle by, and oriented by an optical field as the particle flows through the end of a waveguide terminal in a microfluidic channel referred to in the subsequent description of the experimental work, and
    [0041] Figure 14 shows the displacement efficiency measured as a function of particle flow velocity for symmetric particles - Figure 14a, and asymmetric particles - Figure 14b. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
    [0042] Referring to Figure 1, the microfluidic system typically provided on a microfluidic chip for particle orientation and classification comprises the focusing, orientation, detection of a discriminating characteristic which can be fluorescence, and stages of time and change sequentially over a microchannel along which particles move with the flow of fluid within the channel, from one stage to the next.
    [0043] In the modality shown, a hydrodynamic focusing and / or singularization stage 1 places the particles in a specific location within the channel.
    [0044] If the particles are asymmetrical, such as sperm, for example, the initial orientation of the particles can be random, and an orientation stage 2 orients the particles substantially all or at least a majority with a predetermined common orientation in relation to the geometry of channel. In the schematic figure the particles are shown being vertically oriented. In a preferred embodiment the particles are oriented in the orientation stage by optical forces such as an optical waveguide as will be further described. One or more waveguides can extend through the channel, for example through the top, bottom, or along the side wall of the channel, or they can touch the channel from above, from below or from the side of the channel. The waveguide can form part of the channel wall, or it can be physically separated from the microfluidic chamber.
    [0045] In this modality the detection stage 3 is a fluorescence-based detection stage and the particles are previously stained with a fluorescent dye, and the fluorescence detection stage 3 evaluates the fluorescence intensity of each particle and passes on the information of fluorescence for time and change stages 4 and 5, which change or classify particles so that each particle is directed into one of two different exits 6 and 7. Time and change stages 4 and 5 are controlled by an electronic controller 15. For example the particles can be sperm and the male sperm can be directed to exit 6 and the female sperm to exit 7 for example. Alternatively the particles can be classified to select a type of particle which is desired from another type of particle which is unwanted for the specific application, such as selecting red blood cells, for example, and in such an embodiment the desired particles can be directed to collection or further processing while unwanted particles can be directed to refuse or an outlet for refuse or some other processing.
    [0046] The particles enter the microfluidic system of the source 8. In the figure, the source 8 and the exits 6 and 7 are shown schematically as collection volumes such as chambers to contain the particles, but the particles can enter the system or the section of classification of the microfluidic system, a channel or microfluidic channels from a previous processing stage and leave the classification section for microfluidic channels loading the particles for further subsequent processing, for example.
    [0047] Figures 2a and 2b schematically illustrate modalities for particle orientation in which a single waveguide - Figure 2a, or multiple waveguides - Figure 2b, top the microchannel on one side (top, bottom or side) each side) of the channel, which can be used in the orientation stage 2 of the system in Figure 1. In Figure 2a, a radiation waveguide 9 such as an optical fiber connected to a source such as a laser touches the channel of a side of the channel. The waveguide can form part of the channel wall or can be physically separated from the microfluidic chamber. As the asymmetric particles in random orientation pass through the terminal of the waveguide 9, they are subjected to an optical force which tends to cause the asymmetric particles to orient with a common and predetermined orientation. In the embodiment of Figure 2b, the particles pass through three waveguides 9a - 9c in a series, which cumulatively orient the particles. The optical force of the first waveguide 9a can cause each particle to start rotating in the direction of a desired orientation, while the optical force of subsequent waveguides 9b and 9c continues to cause the particle to move towards the orientation desired. Figure 2a shows a single waveguide facing the channel side and Figure 2b three waveguides facing the channel side, but alternative modalities may comprise two or more than three waveguides, and the waveguides may touch the channel above, below or on each side.
    [0048] The waveguides can be manufactured as part of the device (ie, in situ) or inserted during the assembly of the device (ie, fiber optic components). Typically, waveguides can apply an optical force over an optical wavelength range from visible to near infrared (500 nm - 2 μm), and laser light sources will be the CW emission sources with less power outputs 1 W / waveguide, to minimize the optical forces applied in each interaction with the particle. The emission of the laser light can be controlled electronically, to turn it on and off as desired to generate pulses on the microsecond to millisecond time scale.
    [0049] Figures 3a and 3b schematically illustrate particle orientation modalities in which a single waveguide 10a - Figure 3a, and multiple waveguides 10a - 10d - Figure 3b, extend through the microchannel above or below the channel. The waveguide (s) extends, above, below or along a side wall of the microchannel so that particles pass through the waveguides, and in doing so are subject to the radiation emanating from the waveguides. wave and which applies a pressure of photons to orient the particles described above. Optical radiation can be supplied to the wave guide (s) of a coupling lens 12 as shown.
    [0050] An advantage of the waveguide-based guidance modalities described above, over particle orientation through hydrodynamic pressure as commonly used in sperm sex determination with conventional flow cytometry, for example, is that less force is applied in particles such as sperm to orient it, so that there is a lower likelihood of particle damage during or as a result of particle orientation. This can be specifically so for the modalities of Figures 2b and 3b which guide the particles through a series of sequential waveguides each applying a lower radiation pressure than would be required to orient the same particles with a radiation pressure of a single waveguide. This can be specifically advantageous for biological particles such as sperm, and cells, for example.
    [0051] Figures 4a and 4b show a modality to classify or change selected particles to change the direction of movement within the fluid flow in the microchannel, in which the multiple waveguides 11a - 11e top the channel on one side (or above or below) the channel. Figures 5a and 5b show a modality for classifying or changing selected particles to change the direction of movement within the fluid flow in the microchannel, in which the multiple waveguides extend through the channel above, below, or along the side of the channel . After fluorescence detection stage 3 (Figure 1), in change stage 4 (Figure 1) each particle passes through a series of waveguides 11a-11e which enter the microchannel on the side in Figures 4a and 4b, or 13a -13d which pass below or above the microchannel in the form of Figures 5a and 5b. The waveguides in Figures 4a, 4b, 5a and 5b can form part of the channel wall or can be physically separated from the microfluidic chamber.
    [0052] Referring to Figures 4a and 5a, when a desired particle is changed to a specific output 6 or 7 it passes through the change stage 5 (see Figure 1), the energy source for each of the waveguides 11a- 11e or 13a-13d is connected. As the particle passes through the first waveguide, it is deflected by the optical force, and is additionally deflected as it passes through the subsequent waveguides. All waveguides can be energized together, from a common laser source through a coupling lens 12 as shown in Figures 5a and 5b for example, or in a higher speed system the particle movement can be timed with the connection of each of the waveguides 11a- 11e or 13a-13d so that each waveguide is energized, one after the other, as the selected particle passes through each individual waveguide. Controller 6 (see Figure 1) with time stage 4 input (s) (see Figure 1) sequences the energization of the switching waveguides with the passage of the change stage of the selected particles. In the modality shown, there is a laminar flow through the channel and, referring to Figures 4a and 5a, the waveguides when energized current to deflect the selected particles from the flow on one side towards the outlet 7 through the flow limit and inward flow in the direction of exit 6. When the waveguides are not excited, there is no deflection of the particles which therefore continue to move in the direction of exit 7, as shown in Figures 4b and 5b. In the modalities shown the system is willing to change or classify the particles so that each particle is directed into one of two exits 6 and 7 but in alternative modalities the system may be willing to change or classify the particles between more than two different ones exits such as three or four exits, for example. For example, waveguides when sequentially energized can deflect selected particles from the flow on one side through a first flow limit and into a second flow and then through a second flow limit and into a third flow in the direction of a third exit. The change or classification can be based on ternary rather than binary characteristics of the particles for example. Also in alternative modalities, waveguides can operate to cause selected particles to deflect to turn into a different channel or channels instead of collection volumes, for example.
    [0053] Figure 6a shows a microfluidic chip that incorporates a modality of the invention to perform the orientation and separation of bovine sperm. The sperm sample, with the DNA already stained and rinsed, enters the chip at Sample entry, along with sheath fluid flows. The sperm sample and envelope streams are cooled by a Peltier cooling stage (not shown) under the chip, and kept at a low temperature through all the processing on the chip. The sperm is focused into the desired region of the channel in the FF region. This is then oriented in O using the radiation pressure of an FBI fiber bank. In this example, the fiber bank touches the side channel and four single-mode fibers are shown. These fibers transmit light from a laser inside the chip. After orientation, the fluorescence intensity of the sperm DNA is assessed using an L1 UV LED that illuminates the detection region below the chip. The fluorescence is coupled out of the channel using the SMF single-mode fiber and sent to a PMT photomultiplier tube detector. The output of the PMT photomultiplier tube detector is used to control the Sw shift system. If a sperm is selected to be directed to a new output channel, the laser sends light through the second fiber bank FB2 to move the sperm into the channel for a new flow of fluid. The sperm then flows through a second thermal gradient to increase the temperature in a controlled manner to a desired temperature such as room or body temperature - note that the coil path required for thermal equilibrium with the gradient is not shown for output channels for clarity. The outlet channels also include the flow outlets for the casing fluid and a refuse stream, as well as for the X- and Y chromosome sperm. A white light source L2 under the outlet channels induces the dispersion of particles that enter the individual output channels. This dispersion is detected using a third fiber bank FB3 so that the sperm change can be detected by the Si diodes PIN and sent to the controller for counting.
    [0054] Figure 6b shows how a network of individual classification chips can be used to achieve mass classification of sperm samples. The control electronics, the laser sources, the sensors, the driver for the Peltier stage (T-30 control) and the mass fluid inlet and outlet flow are external to the classification chip. In this diagram, only four chips are shown, for clarity only.
    [0055] Again an advantage of the waveguide-based particle change modalities described above, over particle change through hydrodynamic pressure, for example, is that less force is applied to the particles, and this can be specifically advantageous for the biological particles such as sperm and cells, for example. So although a waveguide-based particle change stage as described above may be preceded by a hydrodynamic pressure-based particle orientation stage (if a particle orientation stage is required), and vice versa an orientation stage particle based waveguide as described above can be followed by a hydrodynamic particle shift stage, in a preferred embodiment a system specifically for classifying asymmetric biological particles such as sperm can comprise a waveguide based orientation stage arranged to guide the sperm by radiation pressure, a detection stage such as a fluorescence-based detection stage, and a waveguide-based change stage which uses optical force to separately target the male and female sperm. The system will also have electronics such as a microprocessor-based control system. The fluorescence detection stage 3 may be arranged to irradiate the sperm previously contacted or stained with a fluorescent marker dye which adheres to the DNA of each sperm, and comprises a detector to detect the resulting fluorescence intensity. The female sperm absorbs a greater amount of dye than the male sperm, and therefore fluorescence with a higher intensity and allowing discrimination.
    [0056] Systems which are comprised of only a single waveguide are restricted in their processing speed by the limited impulse (force x time) of the optical force on the particle of interest. The interaction time is limited by the physical size of the waveguide and the speed of flow of the particles. The handling force is limited by the optical trapping potential of the waveguide. Higher forces lead to complete optical trapping, in which the particles are no longer free to move with the surrounding fluid. This determines the typical use of particle manipulation from single waveguide to low-performance, high-precision particle processing. The multiple waveguide orientation and switching modalities, as described above, offer the advantage of a continuous application of well-controlled optical force over a long time without the occurrence of optical trapping. This allows for an arbitrarily high throughput (particle flow rate) by adding in series waveguides to produce optical force by increasing the impulse applied to the particle.
    [0057] A microfluidic system of the invention for classifying sperm or particles may have at least one microchannel with a classification section in which the particles are processed as described above, or networks of such classification sections to increase throughput. The systems are preferably incorporated into a small microfluidic device or chip prepared by micromachining, polymer processing techniques or other microfabrication technologies to form microfluidic structures and comprise support pumps, valves and instrumentation. Typically the microchannel (s) can (m) have a width in the range of 10 to 500 microns, or 100 to 400 microns, and a depth in the range of 5 to 250 microns, for example. The dimensions of the microfluidic flow channel support laminar flow, with minimal turbulence. In the modalities described and illustrated in the figures, the microstructure has a flat shape with a length and width in the plane greater than the depth transversal to the plane. In alternative modalities, the depth can be greater than the length and / or width of the microchannel and reservoir and other cavities of the microsystem. In alternative modalities, the microchannels can extend in three directions, and can have curved segments as well as angles. In the modalities shown in the figures the microcavities have a rectangular or square cross section but in alternative modalities the microstructures can have a circular or oval cross section, for example, or a cross section in another way. EXPERIMENTAL
    [0058] The invention is further illustrated as an example by the following description of simulation and testing work. EXAMPLE 1
    [0059] The simulations were conducted using the finite element method (FEM) to approximate the action of optical forces on asymmetric particles. Specifically, the forces applied to elliptical particles located near waveguides such as those described above have been calculated. The annular orienting torques were calculated and the entrapment / propulsive forces were also calculated. A two-dimensional (2D) approximation of elliptical particles (a cylinder) with a larger geometric axis: less than 10 μm: 2 μm were placed at the 2D approximation terminal of a single-mode optical fiber, a plate waveguide - Figure 7a . The resulting torque applied to such an elliptical particle as a function of its orientation with respect to the waveguide optical geometric axis is shown in Figure 7b. The input power applied to the waveguide is 50 mW. Polarized waveguide modes parallel (TM) and perpendicular (TE) to the simulation plane are shown. The same 2D particle approximation was placed above a 2D approximation of an edge waveguide, as shown in Figure 8, and the resulting torque as an orientation function parallel to the waveguide optical geometric axis is shown in Figure 9 Both results show that the particle is oriented (does not have a torque applied) when its smaller geometric axis is parallel to the optical geometric axis of the waveguides. That is, when it is in the vertical orientation (annulus = 90 °). Still the graphs show that the torque is restoring (as opposed to the direction of movement) around this orientation angle. This is the equilibrium orientation of the particle due to the applied optical forces. The resulting optical force applied to the elliptical cylinder above in water when at the end of a waveguide is shown in Figure 10. The optical force applied to the same elliptical cylinder in water when above a waveguide is shown in Figure 11. These forces Optics are shown separately in a direction parallel to (Fx) and perpendicular to (Fy) waveguide optical geometric axes for a variety of particle / waveguide separations for both the TM and TE polarized waveguide modes. The particles are in the vertical / equilibrium orientation as described above. EXAMPLE 2
    [0060] The images of Figures 12a - c which show the displacement of a symmetrical particle through an optical field as a particle P moved passing through the end of a waveguide terminal W in a 100 μm rectangular microfluidic channel flow in width were collected with a 10x microscope objective with an image formed on a CMOS digital camera sensor. The particle was a spherical polystyrene bead 10 μm in diameter, and is shown flowing from top to bottom in Figure 12. Figure 12a shows the particle within the fluid flow before the waveguide. The particle interacted with the optical beam (250 mW of 532 nm coupled to a fiber in a unique way at> 50% efficiency) diverging from the waveguide terminal. This interaction generated the strong dispersion seen by saturating the image in Figure 12b. The optical force pushed the particle by displacement d as shown in Figure 12c without stopping the particle's flow along the microfluidic channel. EXAMPLE 3
    [0061] Figure 13 illustrates the orientation effects of the optical field at the end of a W waveguide adjacent to the wall of a microfluidic channel. A particle P with an asymmetric shape, specifically a bovine sperm, was loaded into a flow of fluid F within a microfluidic channel. The particle was inert and unable to move under its own propulsion. As shown in Figure 13a, the sperm initially showed a dark dispersion orientation for the imaging system. After passing through and interacting with the optical field at the terminal of the optical waveguide (outlined in Figure 13) the sperm continued to flow down the microfluidic channel with a new orientation and a shift from its initial position. The particle was moved away from the channel wall after the interaction and rotated to a new orientation, but it continued to flow down the channel. The new orientation of the sperm had a white head, indicating a rotation of 90 degrees around the long geometric axis of the particle after interacting with the optical field.
    [0062] Multiple interaction events such as those seen in Figure 12 and Figure 13 were analyzed frame by frame and the results shown in Figure 14. An image processing was used to measure the position and orientation of the particle before and after iterating with the optical field. Figure 14 shows the displacement efficiency measured as a function of particle flow velocity for symmetrical particles namely a polystyrene bead 10 μm in diameter - Figure 14a, and asymmetric particles namely a non-mobility bovine sperm- Figure 14b. For example, these particles are flowing through the end of a single waveguide with less than 200 mW of output power at a wavelength of 532 nm. The offset indicates the fiber termination distance to the edge of the microfluidic channel.
    权利要求:
    Claims (15)
    [0001]
    1. System for classifying particles in a micro-fluidic system, characterized by the fact that it comprises: at least one channel suitable for fluid flow within the channel comprising at least one input source (8) and at least two output sources ( 6, 7), and a plurality of stages arranged along the channel, a next orientation stage (2), and in the communication of the fluid with at least one input source (8), the orientation stage (2) comprising at least minus an optical waveguide (9, 9a-c, 10a, 10a-d) arranged and configured to expose the particles to radiation pressure to cause at least a majority of the particles to adopt a specific orientation within the fluid, - a detection stage (3), and in the communication of the fluid with the orientation stage (2), the detection stage (3) comprising an optical detector configured to detect at least one difference or discriminate between particles in the fluid flow that passes by det stage and a change stage (5) comprising a plurality of optical waveguides (11a-e, 13a-d) arranged so that the particle flow passes through each optical waveguide in the plurality, said optical waveguides (11a-e, 13a-d) arranged and configured to expose the particles to radiation pressure to cause a change in the direction of movement of selected particles in the fluid flow to classify the particles in one of at least two output sources (6 , 7) based on the input of the detection stage, and a controller (15).
    [0002]
    2. System according to claim 1, characterized by the fact that the optical waveguides are connected to a laser or lasers.
    [0003]
    System according to claim 1, characterized in that the waveguide (9-13d) extends through the channel or extends through the channel of the microfluidic system.
    [0004]
    4. System according to claim 1, characterized by the fact that the controller (15) is arranged to activate the radiation pressure of the optical waveguides (9-13a-d).
    [0005]
    5. System according to claim 4, characterized in that the radiation pressure deflects a particle of a flow on one side, through a flow limit or limits, and into a flow in the direction of a selected outlet (6.7).
    [0006]
    6. System according to claim 1, characterized by the fact that the optical detector is arranged to detect or discriminate particles by a fluorescence-based technique.
    [0007]
    System according to claim 1, characterized in that it also comprises, before the orientation stage (2), a focusing stage (1), in the communication of the fluid with at least one input source (6,7), the orientation stage (1) comprising an apparatus for providing a hydrodynamic pressure force, a radiation pressure force, or a combination thereof, to focus the particles to a specific location within a fluid.
    [0008]
    8. System according to claim 3, characterized in that the plurality of optical waveguides (9-13d) extends through the channel above, below or along the side walls of the channel.
    [0009]
    9. System according to claim 1, characterized by the fact that it also comprises a cooling stage capable of cooling the fluid and the particles within the fluid.
    [0010]
    10. System according to claim 1, characterized by the fact that at least one channel has a width of 10 μm to 500 μm.
    [0011]
    11. System according to claim 1, characterized by the fact that at least one channel has a depth of 5 μm to 500 μm.
    [0012]
    12. System according to claim 1, characterized by the fact that the particles are sperm.
    [0013]
    13. Method for classifying particles in a micro-fluidic system, characterized by the fact that it comprises: forcing particles to adopt a specific orientation in the fluid flow within at least one channel of the microfluidic system, contacting the particles with a produced radiation force at least one optical waveguide (9-10d); - follow the guidance, detect at least one difference, or discriminate between particles, in the fluid flow, and -cause a change in the direction of movement of selected particles in the fluid flow to classify the particles based on at least one difference or discrimination detected, in which the change in direction is caused by contacting the particles with a radiation force produced by a plurality of optical waveguides (11a-e, 13a-d).
    [0014]
    14. Method according to claim 13, characterized in that the particles are sperm.
    [0015]
    15. Method according to claim 14, characterized by the fact that the sperm is classified by sex.
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    同族专利:
    公开号 | 公开日
    EA030193B1|2018-07-31|
    EA201590285A1|2015-10-30|
    WO2014017929A1|2014-01-30|
    US20170370822A1|2017-12-28|
    EP2877832B1|2020-10-28|
    EP2877832A1|2015-06-03|
    CN104884934A|2015-09-02|
    MX2015001251A|2015-08-14|
    US20190331588A1|2019-10-31|
    ES2842552T3|2021-07-14|
    EP2877832A4|2016-04-20|
    NZ725649A|2018-05-25|
    BR112015001844A2|2018-05-29|
    IN2015KN00498A|2015-07-17|
    MX359348B|2018-09-26|
    CA2894459C|2020-07-07|
    AU2013293647B2|2017-08-31|
    EP3845885A1|2021-07-07|
    US9784663B2|2017-10-10|
    AU2013293647A1|2015-03-12|
    US20200408666A1|2020-12-31|
    UA116637C2|2018-04-25|
    US10712255B2|2020-07-14|
    CN104884934B|2018-11-02|
    US10393644B2|2019-08-27|
    US20150198517A1|2015-07-16|
    CA3079158A1|2014-01-30|
    DK2877832T3|2021-01-18|
    CA2894459A1|2014-01-30|
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    法律状态:
    2018-12-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-11-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-06-02| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-10-20| B25G| Requested change of headquarter approved|Owner name: ENGENDER TECHNOLOGIES LIMITED (NZ) |
    2020-12-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-03-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/07/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201261676391P| true| 2012-07-27|2012-07-27|
    US61/676,391|2012-07-27|
    PCT/NZ2013/000135|WO2014017929A1|2012-07-27|2013-07-29|Method and system for microfluidic particle orientation and/or sorting|
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