Apparatus for identifying an object and related methods

Abstract

An apparatus for identifying objects and related methods are disclosed. The apparatus for identifying objects includes a microfluidic chip having a plurality of channels disposed therein, the plurality of channels including a main fluid channel into which a sample fluid mixture of objects to be identified is introduced, a plurality of sheath fluid channels into which a sheath fluid is introduced, the sheath fluid orienting the objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel, an interrogation device that detects and interrogates the oriented objects in the main fluid channel, and a focused energy device that performs an action on the objects.

Description

[0001] This invention is a divisional application of the applicant's patent application filed on June 18, 2014, entitled "Microfluidic System and Method with Focusing Energy Device", application number 2014800719520. Technical Field

[0002] This invention relates to the field of microfluidic processing technology, and in particular to devices and methods for identifying objects. Background Technology

[0003] In the separation of various particle or cellular materials (e.g., separating bovine sperm into viable and motile sperm and inactive or immobile sperm, or separating them by sex), the process is typically time-consuming and subject to strict volume constraints. Therefore, current separation technologies are unable to produce the expected yields or process large quantities of cellular material in a timely manner.

[0004] Photodamage laser systems have utilized lasers to photodamage or kill unwanted cellular objects. However, existing technologies have required the use of flow cytometers with nozzles to question and arrange individual objects in a droplet stream, and to attempt to separate and photodamage these objects as they fall into individual containers, which is difficult to achieve.

[0005] Therefore, there is a need for a method and apparatus for identifying and distinguishing target objects that is continuous, high-throughput, time- and cost-effective, and causes negligible or minimal damage to each target object. Furthermore, such an apparatus and method should also be applicable to other biological and medical fields, not only for sperm differentiation, but also for differentiating blood and other cellular materials, including viruses, organelles, spherical tissues, colloidal suspensions, and other biological materials. Summary of the Invention

[0006] This invention relates to a device and a related method for identifying objects.

[0007] In one embodiment, a device for identifying an object includes:

[0008] A microfluidic chip having multiple channels, the multiple channels including:

[0009] The main fluid channel is into which a sample fluid mixture of the object to be identified is introduced;

[0010] A ramp is provided in the main fluid channel before the first intersection where the main fluid channel and the first plurality of sheath fluid channels intersect, the ramp causing the sample fluid mixture to deviate from the central plane;

[0011] Multiple sheath fluid channels are provided, into which sheath fluid is introduced to orient the object in the main fluid channel in a predetermined direction while maintaining laminar flow in the main fluid channel; wherein the multiple sheath fluid channels hydrodynamically concentrate the object, so that the object is positioned in a confined core volume when the object flows through the main fluid channel;

[0012] The plurality of sheath fluid channels include:

[0013] The first plurality of sheath fluid channels intersect the main fluid channel at a first intersection, thereby allowing the sheath fluid to surround and compress the sample fluid mixture on all sides, thus transforming the sample fluid mixture into a relatively small, narrow flow confined by the sheath fluid, while maintaining laminar flow in the main fluid channel; and

[0014] Interrogation device, the interrogation device detecting and interrogating the oriented object in the main fluid channel; and

[0015] A focused energy device that performs an action on the object.

[0016] In one implementation, it further includes:

[0017] The interrogation device detects and interrogates the object to determine information about the object;

[0018] The information about the object determines whether the object will be targeted by the focusing energy device, wherein the object targeted by the focusing energy device is the target object;

[0019] The action of the focusing energy device is applied to the target object or the area surrounding the target object.

[0020] In one implementation, it further includes:

[0021] At least one output channel guided from the main fluid channel removes the object from the microfluidic chip, wherein the at least one output channel removes both the target object and the non-target object from the microfluidic chip.

[0022] In one implementation, it further includes:

[0023] An action chamber, in which the interrogation device interrogates the hydrodynamically focused object in the sample fluid mixture, the action chamber being disposed downstream of the first intersection in the microfluidic chip; and

[0024] A light source that emits a light beam into the actuation cavity to illuminate and excite the object in the sample fluid mixture.

[0025] In one embodiment, the light beam excites fluorescence in the object, thereby distinguishing the target object from a non-target object.

[0026] In one implementation, it further includes:

[0027] An optical signal detector that detects the light beam and converts the light beam into an electrical signal; and

[0028] A controller that analyzes the electrical signals to determine whether to target the object.

[0029] In one embodiment, the main fluid channel enters the actuation chamber in a conical shape.

[0030] In one embodiment, the main fluid channel enters the first intersection in the microfluidic chip in a conical shape at the inlet point.

[0031] In one implementation, the object is a cell.

[0032] In one embodiment, the cells to be acted upon by the focused energy device include at least one of viable or motile bovine sperm, distinguishable from inactive or immobile bovine sperm, or bovine sperm distinguishable by sex or other sex-specific variables.

[0033] In one implementation, after the inquiry of the object, the focusing energy device acts on the object for a predetermined amount of time.

[0034] In one implementation, it further includes:

[0035] Multiple microfluidic chips arranged in parallel, each microfluidic chip containing multiple sample fluid mixtures;

[0036] The single interrogation device is used for at least one of the plurality of microfluidic chips.

[0037] In one embodiment, a device for identifying an object includes:

[0038] A microfluidic chip having multiple channels, the multiple channels including:

[0039] The main fluid channel is into which a sample fluid mixture of the object to be identified is introduced;

[0040] Multiple sheath fluid channels, wherein sheath fluid is introduced into the multiple sheath fluid channels, the sheath fluid orienting the object in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel;

[0041] The plurality of sheath fluid channels intersect the main fluid channel at a first intersection, thereby compressing the sample fluid mixture on at least both sides, thus transforming the sample fluid mixture into a relatively small, narrow flow confined by the sheath fluid, while maintaining laminar flow in the main fluid channel; and

[0042] Interrogation device, the interrogation device comprising a first laser for detecting and interrogating the oriented object in the main fluid channel; and

[0043] A second laser, which provides electromagnetic energy to the object, is positioned downstream of the interrogation device.

[0044] In one implementation, it further includes:

[0045] The interrogation device detects and interrogates the object to determine information about the object;

[0046] The information about the object determines whether the object will be targeted by the second laser; and

[0047] The second laser acts on the target object or the area surrounding the target object.

[0048] In one implementation, it further includes:

[0049] At least one output channel is drawn from the main fluid channel, the at least one output channel removing the object from the microfluidic chip;

[0050] The at least one output channel removes both the target object and the non-target object from the microfluidic chip.

[0051] In one embodiment, a second sheath fluid channel compresses the sample fluid mixture from at least both sides.

[0052] In one embodiment, the plurality of sheath fluid channels hydrodynamically converge the object, causing the object to face the predetermined direction as it flows through the main fluid channel, and is positioned within a restricted core volume.

[0053] In one implementation, it further includes:

[0054] An action chamber, wherein the first laser of the interrogation device interrogates the hydrodynamically focused object in the sample fluid mixture within the action chamber, the action chamber being disposed downstream of the first intersection in the microfluidic chip, the first laser being located on one side of the main fluid channel.

[0055] In one implementation, it further includes:

[0056] An optical signal detector that detects a light beam emitted by the object and converts the light beam into an electrical signal; and

[0057] A controller that analyzes the electrical signals to determine whether to target the object.

[0058] In one implementation method

[0059] The tapered structure is located at the entrance point of the first intersection in the microfluidic chip, so that the width of the main fluid channel gradually narrows before the entrance point.

[0060] In one implementation, wherein

[0061] The main fluid channel enters the actuation cavity in a conical shape, so that the width of the main fluid channel gradually narrows before intersecting with the actuation cavity.

[0062] In one embodiment, a ramp is provided in the main fluid channel before the first intersection where the main fluid channel and the plurality of sheath fluid channels intersect, so that the width of the main fluid channel narrows before the first intersection.

[0063] In one implementation, the object is a cell.

[0064] In one embodiment, the cells to be acted upon by the second laser include at least one of viable or motile bovine sperm, distinguishable from inactive or immobile bovine sperm, or bovine sperm distinguishable by sex or other sex-specific variables.

[0065] In one implementation, after the interrogation of the object, the second laser acts on the object for a predetermined amount of time.

[0066] In one embodiment, a method for producing a non-human mammalian sperm composition includes:

[0067] Allowing sperm cells to flow through the channel;

[0068] The sperm cells were divided into an X sperm cell subset and a Y sperm cell subset;

[0069] Select the desired subgroups and eliminate the unwanted subgroups; and,

[0070] Two subpopulations of sperm cells are collected, the two subpopulations including the desired subpopulation and the unwanted subpopulation that is eliminated.

[0071] In one embodiment, the flow of sperm cell clusters through the channel includes:

[0072] Orient the sperm cells in the population to a specific location in the channel;

[0073] The sperm cells are aligned, and the sperm cells move through the channel in a single queue; and

[0074] The sperm cells are discharged through the output portion of the channel.

[0075] In one embodiment, during the steps of dividing the sperm cells into X sperm cell subpopulations and Y sperm cell subpopulations, and selecting the desired subpopulations and eliminating the unwanted subpopulations, the sperm cell populations and sheath fluid are in laminar flow.

[0076] In one embodiment, dividing the sperm cells into an X sperm cell subset and a Y sperm cell subset includes:

[0077] Irradiate the sperm cells with a light beam; and

[0078] Detect the differences between the desired subgroup and the unwanted subgroup.

[0079] In one embodiment, the light beam is split into multiple beams, each of which is configured to illuminate a different sperm cell.

[0080] Therefore, some features consistent with the invention have been outlined so that the following detailed description can be better understood and so that the present contribution to the art can be better appreciated. Of course, additional features consistent with the invention are present, which will be described below and form the subject matter of the appended claims.

[0081] In this regard, before detailing at least one embodiment of the invention, it should be understood that the invention is not limited in its application to the details of the structures and arrangements of objects presented in the following description or shown in the drawings. The methods and apparatus of the invention are capable of implementing other embodiments and can be practiced and performed in various ways. Furthermore, it should be understood that the phrases and terms used herein and in the following abstract are for descriptive purposes and should not be considered limiting.

[0082] Therefore, those skilled in the art will understand that the concepts upon which this disclosure is based can be readily used as the design basis for other structures, methods, and systems for performing the various objectives of the present invention. Therefore, it is important that the claims be deemed to include such equivalent constructions without departing from the spirit and scope of the methods and apparatus of the present invention. Attached Figure Description

[0083] The objectives, features, and advantages of the invention will be more readily understood when considered in conjunction with the accompanying drawings, taking into account the following disclosure, in which:

[0084] Figure 1A An exploded perspective view showing an illustrative embodiment of a single-layer microfluidic chip having a top “blank” layer according to one embodiment of the present invention.

[0085] Figure 1B-(a) shows an exploded perspective view of an illustrative embodiment of a two-layer microfluidic chip having functional layers on top of the bottom layer and on the bottom side of the top layer, according to another embodiment of the invention.

[0086] Figure 1B-(b) shows a top view of the bottom layer and the bottom side of the top layer of the embodiment shown in Figure 1B-(a).

[0087] Figure 1C-(a) shows an exploded perspective view of an illustrative embodiment of a three-layer microfluidic chip with three functional layers according to another embodiment of the invention, wherein the top and middle layers have functional portions below these layers.

[0088] Figure 1C-(b) shows the three-layer illustrative implementation of Figure 1C-(a) from the opposite perspective, where the layers are flipped to show the functional layers below the top and middle layers.

[0089] Figure 1D An exploded perspective view of an illustrative embodiment of a four-layer microfluidic chip according to another embodiment of the invention is shown, wherein the top layer is a “blank” layer.

[0090] Figure 2 A top view showing an illustrative embodiment of a microfluidic chip according to one embodiment of the present invention.

[0091] Figure 3A A perspective view is shown of a sample channel and a sheath or buffer channel in a dual (functional) layer microfluidic chip according to one embodiment of the present invention.

[0092] Figure 3B A perspective view is shown of a sample channel and a sheath or buffer channel in a single-layer microfluidic chip according to another embodiment of the invention.

[0093] Figure 4A A perspective view of a sample channel having a cone shape and an internal ramp, according to one embodiment of the invention, is shown, the sample channel entering the intersection of a first hydrodynamic focusing region.

[0094] Figure 4B A perspective view of a main channel with a conical shape and an internal ramp, according to one embodiment of the invention, is shown, which leads into a second hydrodynamic focusing area.

[0095] Figure 4C The ramp features in the sample channel prior to the actuation cavity are shown according to one embodiment of the invention.

[0096] Figure 5 This illustrates a light source according to an embodiment of the invention, through which current flows... Figure 1A An illustrative inquiry of a cross-sectional internal view of an object in a fluid mixture in the main channel of a microfluidic chip system.

[0097] Figure 6A An oblique schematic side view of an object according to an embodiment of the invention is shown, illustrating flow from interrogation to differentiation in the main channel of a microfluidic chip system, wherein a focusing energy device is activated after interrogation.

[0098] Figure 6B An oblique schematic side view of an object according to another embodiment of the invention is shown, illustrating the flow from interrogation to differentiation in the main channel of a microfluidic chip system, wherein a focusing energy device is activated at the outlet of the output channel.

[0099] Figure 6C An oblique schematic side view of an object according to another embodiment of the invention is shown, illustrating the flow from interrogation to differentiation in the main channel of a microfluidic chip system, wherein a focusing energy device is activated on a discontinuous droplet between the output channel outlet and the collection.

[0100] Figure 6D An oblique schematic side view of an object according to another embodiment of the invention is shown, illustrating flow from interrogation to differentiation in the main channel of a microfluidic chip system, wherein a focusing energy device is activated prior to interrogation.

[0101] Figure 7 A perspective view showing a shear force gradient across a microfluidic master channel according to one embodiment of the invention.

[0102] Figure 8A The image shows a cross-sectional view of the main channel of the microfluidic chip after the first step of hydrodynamic focusing, with sample fluid pressed against the side of the main channel and the object offset from the center of the main channel.

[0103] Figure 8BThe image shows a cross-sectional view of the main channel of the microfluidic chip after the second step of hydrodynamic focusing, where the sample fluid is compressed from above and below the main channel, so that the object is essentially located at the center of the main channel.

[0104] Figure 9 A cross-section of the main channel of a microfluidic chip according to one embodiment of the present invention is shown, wherein the object is located at the center of the main channel.

[0105] Figure 10 This is a diagram illustrating the flow velocity distribution across the main channel of a microfluidic chip according to one embodiment of the present invention.

[0106] Figure 11 A cross-section of the main channel of a microfluidic chip according to one embodiment of the present invention is shown, wherein the object is off-center.

[0107] Figure 12A The illustration shows a second hydrodynamic focusing step according to an embodiment of the invention, wherein a sheath or buffer channel enters the sample channel from above and below, parallel to the sample channel of the microfluidic chip, and from a vertical direction.

[0108] Figure 12B This illustrates another embodiment of the invention. Figure 12A The second hydrodynamic focusing step involves a microfluidic chip in which one sheath or buffer channel is smaller than the other channel.

[0109] Figure 13A A histogram showing a sperm cell in the center of the main channel of a microfluidic chip according to one embodiment of the present invention is shown.

[0110] Figure 13B A histogram of sperm cells off-center from the main channel of a microfluidic chip, according to one embodiment of the invention, is shown.

[0111] Figure 14 The illustration shows a perspective internal oblique view of an object flowing through a microfluidic chip according to one embodiment of the invention, as well as an illustrative operation of two-step hydrodynamic focusing, wherein a single sheath or buffer fluid enters from the top of the main channel.

[0112] Figure 15 A perspective view of a microfluidic chip according to one embodiment of the present invention is shown, along with an illustrative operation of two-step hydrodynamic focusing, wherein a sheath or buffer fluid channel enters vertically from the upper and lower portions of the sample channel.

[0113] Figure 16A tilted side view schematic diagram of a microfluidic chip system with interrogation and differentiation devices according to an embodiment of the present invention is shown, along with the implementation of beam optics from the opposite side of the sample channel, wherein converging optics are shared with focusing energy device optics.

[0114] Figure 17 The diagram shows a tilted side view of a microfluidic chip system with interrogation and differentiation devices according to one embodiment of the invention, and an implementation of beam optics from the same side of the sample channel, wherein the interrogation beam and the focusing energy beam are combined by the same single optics.

[0115] Figure 18 A schematic diagram is shown of a microfluidic chip system using a single interrogation device in multiple parallel configurations according to one embodiment of the present invention.

[0116] Figure 19 A schematic diagram of a flow control network for a microfluidic chip system according to one embodiment of the present invention is shown, the flow control network having external individual reservoirs for sample fluid and sheath or buffer fluid.

[0117] Figure 20 A schematic diagram of a flow control network for a microfluidic chip system according to one embodiment of the present invention is shown, the flow control network having an external single sheath or buffer reservoir and a sample reservoir.

[0118] Figure 21 A schematic diagram of a pressure regulating network with a single sheath or buffer reservoir is shown according to one embodiment of the present invention for a microfluidic chip system.

[0119] Figure 22A and Figure 22B The front and back sides of a microfluidic chip holder according to one embodiment of the present invention are shown respectively. The microfluidic chip holder has three ports for fluid to flow to a functional multilayer microfluidic chip.

[0120] Figure 23A and Figure 23B The front and back sides of a microfluidic chip holder according to one embodiment of the present invention are shown respectively. The microfluidic chip holder has four ports for fluid to flow to a single-layer microfluidic chip. Detailed Implementation

[0121] Before turning to the accompanying drawings, which illustrate illustrative embodiments in detail, it should be understood that the invention is not limited to the details and methodologies set forth in the specification or shown in the drawings. It should also be understood that terminology is for descriptive purposes only and should not be considered limiting. Efforts have been made to use the same or similar reference numerals throughout the drawings to indicate the same or similar parts.

[0122] This invention relates to a microfluidic chip having an interrogation device and a focused energy device, the interrogation device detecting and interrogating an object in a sample fluid mixture, and the focused energy device performing an action on the object or the area surrounding the object. In one embodiment, the interrogation device interrogates the object to identify the object and determine whether the object should be targeted by the focused energy device. In one embodiment, the target object is an unwanted target object.

[0123] In one embodiment, the focusing energy device is a distinguishing device that differentiates between target and non-target objects by damaging, killing, altering, disabling, or destroying the target object. The invention is implemented within a microfluidic network as a continuous flow of fluid, where objects are subjected to hydrodynamic focusing, positioning, and orientation, while allowing non-target objects to flow undisturbed through the microfluidic chip, and the focusing energy device acts on the target object, including photodamaging, killing, altering, disabling, or destroying it.

[0124] application

[0125] Various embodiments of the present invention provide methods for selecting objects in fluid mixtures, such as: selecting viable and motile bovine sperm to distinguish them from inactive and immobile bovine sperm; selecting bovine sperm by sex or other sex selection variables; selecting stem cells from a population of cells; selecting one or more labeled cells to distinguish them from unlabeled cells, distinguishing desired / undesirable characteristics; selecting cells for desired characteristics; selecting genes in the nuclear DNA of cells according to specified characteristics; selecting cells based on surface markers; selecting cells based on membrane integrity (viability), potential or expected reproductive status (fertility), cryopreservation survival, etc.; selecting cells to distinguish them from contaminants or waste; selecting healthy cells to distinguish them from damaged cells (i.e., cancer cells), such as in bone marrow extraction; selecting red blood cells to distinguish them from white blood cells and platelets in a plasma mixture; and selecting any cells to distinguish them from any other cellular objects entering a corresponding fraction; selecting damaged cells, or contaminants or waste, or any other biological material that is desired to be distinguished. The objects may be cells or beads treated with or coated with linker molecules, or embedded with one or more fluorescent or electrochromic labeling molecules. Objects can have various physical or chemical properties, such as size, shape, material, texture, etc.

[0126] In one implementation, objects from a heterogeneous population can be measured, wherein each object is examined for different amounts of additive or for different physical environments at similar amounts of additive (e.g., multiple measurements), or objects can be examined and distinguished based on tags (e.g., fluorescence), images (fluorescence or cold light emission profiles, fluorescence or cold light decay lifetimes due to size, shape, different absorption, scattering, fluorescence, cold light properties), and / or particle positions, etc.

[0127] Furthermore, the subject matter of this invention is also suitable for other medical applications. For example, the various laminar flows discussed below can be used as part of a kidney dialysis process in which waste products are removed from whole blood and the whole blood is returned to the patient. Further, various embodiments of the invention are also applicable to other biological or medical fields, such as for selecting cells, viruses, bacteria, organelles or daughter cells, spherical tissues, colloidal suspensions, fats or fat particles, colloids, immiscible particles, mitotic spheres, aggregates of cells, microorganisms, and other biological materials. For example, object selection according to the invention can include cell “washing,” in which contaminants (such as bacteria) are removed from cell suspensions, which can be particularly useful in medical and food industry applications. Further, the invention is applicable to selecting immobile cellular objects, distinguished from mobile cellular objects.

[0128] The subject matter of this invention can also be used to transfer species from one solution to another, where separation by filtration or centrifugation is impractical or undesirable. In addition to the applications discussed above, additional applications include selecting colloids of a given size from other sizes (for research or commercial applications), and washing particles such as cells, egg cells, etc. (effectively replacing the medium containing these particles and removing contaminants), or washing particles such as nanotubes from solutions of salts and surfactants, such as solutions with different salt concentrations or without surfactants.

[0129] The act of selecting a species can depend on multiple physical properties of the object, including egoactivity, self-diffusion, free fall velocity, or action under external forces (such as actuators, electromagnetic fields, or holographic traps). Selectable properties include, for example, cellular egoactivity, cell viability, object size, object mass, object density, the tendency of objects to attract or repel each other or to attract or repel other objects in a flow, object charge, object surface chemistry, and the tendency of certain other objects (i.e., molecules) to attach to the object.

[0130] Although the following discussion focuses on selecting vigorous or motile bovine sperm to distinguish from inactive or immobile bovine sperm, or selecting bovine sperm by sex and other sex selection variables, or selecting one or more labeled cells to distinguish from unlabeled cells, distinguishing desired / undesirable characteristics, etc., the apparatus, method, and system of the present invention can be extended to other types of particulate biological or cellular material that can be interrogated by fluorescence technology within a fluid stream, or that can be manipulated to one or more outlets between different fluid streams.

[0131] Sample preparation

[0132] In one embodiment, a cell counting device (such as a somatic cell counter) is used to determine the concentration of subject 160 (such as cells (i.e., untreated semen)). In one embodiment, a suitable chromosome volume is obtained (i.e., using a staining calculator worksheet), and the volume of staining TALP and subject 160 (i.e., purified semen) to be added is calculated for a predetermined cell concentration (i.e., a sperm concentration of 200 x 10⁶ / ml). For example, when the purified semen concentration = 1500 x 10⁶ / ml, a 1 ml volume of staining sample is prepared. Thus, 200 x 10⁶ / ml / 1500 x 10⁶ / ml = 0.133 ml of purified semen is added (in sequence) 0.012 ml of Hoechst 33342 (5 mg / ml stock solution) and 0.885 ml of staining TALP (pH 7.4) to equal a total chromosome volume of 1 ml at 200 x 10⁶ / ml.

[0133] In one implementation, stained TALP is prepared by filling a container (i.e., a beaker) with Milli-Q water to 2 / 3 of the total desired volume. A stir bar and a stirring pan are used to mix the solution as chemicals are added. The chemicals added in the listed order (up to the subsequently added gentamicin) include:

[0134] Table: Chemical composition of stained TALP

[0135] In one embodiment, after thorough mixing of the chemicals, the pH is adjusted to 7.4 using NaOH. Additional Milli-Q water is then used in a container (i.e., a volumetric flask) to bring the solution to the final volume. This volume is then filtered / sterilized using a sterile filter (i.e., a 0.22 sterile filter). After filtration, an antibiotic (i.e., gentamicin solution) is added, along with a volume of stained TALP stored at 5°C, and the solution can be used for 7 to 10 days.

[0136] Therefore, after staining the volume of sample with staining TALP, in one embodiment, the stained sample 120 is placed in a container (i.e., a conduit) and placed in a water bath set at 34°C to 35°C, and incubated for a predetermined time (i.e., 45 minutes). In one embodiment, after incubation, the stained sample is removed from the water bath, and an equal volume of 4.0% egg yolk TALP containing red food dye, which has been heated in the water bath set at 34°C to 35°C, is added.

[0137] To obtain a 4.0% egg yolk TALP with a red food coloring, the stained TALP was prepared as described above, and the expected volume of the final solution was determined. The volumes of stained TALP and egg yolk required to prepare the 4.0% egg yolk solution were calculated as follows:

[0138] Expected volume: For a 4% solution, (250 ml) x 0.04 = 10 ml of egg yolk is needed. Add 240 ml of staining TALP to a graduated container (i.e., a graduated cylinder) and add 10 ml of egg yolk. Add FD&C#40 food dye to this container (i.e., the graduated cylinder) to obtain a solution of 0.261 ml / 100 ml. Using the expected total volume of 250 ml, the red food dye is 0.261 ml x 250 ml / 100 = 0.653 ml. Cover the container (i.e., the graduated cylinder) with a sealing film and carefully invert it until the volume is thoroughly mixed. Then allow the volume container to stand and cool overnight in a refrigeration chamber. Then carefully pour the volume container into a sterile container, leaving any sediment at the bottom of the volume container. The volume was then filtered / sterilized through a 0.22μm cap filter, and an appropriate amount of antibiotic solution (i.e., 0.250 ml of gentamicin / 100 ml of egg yolk TALP) was added.

[0139] Therefore, after adding an equal volume of 4.0% egg yolk TALP with red food dye to the stained sample 120, the stained sample 120 is filtered by pouring it through a 20-micron filter (i.e., a CellTrics filter) and discharged into another sterile 5 ml culture container. After staining at a predetermined time (i.e., 45 minutes), an equal volume of 4.0% egg yolk TALP is added. The stained sample 120 (i.e., cells) is passed through a filter (i.e., a Partec filter with 50-micron mesh) and the sample is placed in a sample container or reservoir 233 for introduction into the microfluidic chip 100 (see See). Figures 19 to 21 ).

[0140] In one implementation, the final sperm concentration is 100 x 10⁶ / ml, and the final yolk percentage is 2%. It is anticipated that 120 new sample portions can be prepared and used every hour.

[0141] Microfluidic chip system

[0142] The various embodiments of the microfluidic chip described below utilize one or more fluid channels with multiple essentially laminar flows, allowing interrogation devices to interrogate one or more objects for identification and allowing focused energy devices to act on one or more objects, wherein the objects exit the microfluidic chip and enter one or more output ports. In one embodiment, objects not targeted by the focused energy device remain undisturbed, while the focused energy device optically damages, alters, disables, kills, or destroys the target object.

[0143] The various embodiments of the present invention thus provide selection of objects on a continuous basis (such as within a continuous, closed system) without the potential damage and contamination of prior art methods, particularly as provided in sperm separation. The continuous process of the present invention also provides significant time savings in selecting and distinguishing objects.

[0144] Although relative to Figures 1A to 2 The microfluidic chip 100 shown and Figures 20 to 21 The microfluidic chip holder 200 shown herein discusses the subject matter of the invention in detail, but it should be understood that this discussion is equally applicable to the various other embodiments or any variations thereof discussed herein.

[0145] microfluidic chip

[0146] Figure 1A This is an illustrative embodiment of the microfluidic chip 100. The microfluidic chip 100 is made of a suitable material, such as glass, or a thermoplastic (e.g., a low autofluorescence polymer), or a combination of materials, using imprinting, soft lithography, or injection molding processes well known to those skilled in the art. The microfluidic chip 100 has suitable dimensions. Each layer can have any suitable thickness, for example, in the range of approximately 300 μm to 400 μm, more preferably approximately 400 μm.

[0147] The microfluidic chip 100 includes one or more structural layers in which microchannels are disposed, serving as one or more sample input channels, one or more sheath fluid or buffer fluid channels, one or more output channels, etc. The microchannels are appropriately sized to accommodate laminar flow containing an object and can be disposed in any layer of the chip 100 at an appropriate length, as long as the objectives of the invention are achieved. In one embodiment, the dimensions of the microfluidic channels range from 50 micrometers to 500 micrometers, preferably from 100 micrometers to 300 micrometers to avoid clogging.

[0148] The expected flow rate through the microfluidic chip 100 can be controlled by: a predetermined inlet flow rate into the chip 100, by maintaining appropriate microchannel dimensions within the chip 100, by a pumping mechanism that pumps external fluid into the chip 100, and by narrowing or tapering the microchannels at various locations, and / or by providing barriers, ramps, or dividers within the microchannels (discussed further below).

[0149] More specifically, multiple input ports are provided into the microfluidic chip 100, providing access to microchannels / channels. In one embodiment, such as Figures 1A to 2 As shown, sample inlet 106 is used to sample particles or objects (i.e., cells) 160 in sample fluid mixture 120 from at least one reservoir source (see Figure 100). Figure 19 It is introduced into the main fluid channel 164 of the microfluidic chip 100.

[0150] The microfluidic chip 100 also includes at least one sheath or buffer inlet for introducing sheath fluid or buffer fluid. In one embodiment, the microfluidic chip 100 has two sheath or buffer inlets, including a sheath or buffer inlet 107 and a sheath or buffer inlet 108, both positioned close to the sample inlet 106, and both introducing sheath fluid or buffer fluid 163 into the microfluidic chip 100 (see...). Figures 1A to 3B ).

[0151] In one embodiment, three sheath or buffer inlet ports 107, 108, and 172 are provided (see [reference]). Figure 1A and Figure 3B This introduces sheath fluid or buffer fluid into channels 164 of the microfluidic chip 100. The positions of the sheath or buffer inlets 107, 108, and 172 can be varied, and they can connect to channels in the same or different structural layers within the chip 100. In one embodiment, sheath fluid or buffer fluid 163 is introduced from a common reservoir into inlets 107, 108, and 172 (see [link]). Figures 20 to 21 Alternatively, in another embodiment, sheath fluid or buffer fluid 163 is introduced from a separate reservoir into inlets 107, 108, 172 (see [reference]). Figure 19).

[0152] Sheath fluids or buffering fluids are well known in the field of microfluidics, and in one embodiment, they may contain nutrients well known in the art to maintain the viability of the object 160 (i.e., sperm cells) in the fluid mixture. Commercially available tris(hydroxymethyl)aminomethane (such as that sold by Chata Biosystems) is one example, and the sheath fluid or buffering fluid 163 may be formulated to include the following:

[0153] Water - 0.9712 L; Tris(hydroxymethyl)aminomethane - 23.88 g; Citric acid monohydrate - 11.63 g; D-fructose - 8.55 g. Adjust the pH to 6.80 ± 0.05 using hydrochloric acid, and adjust the permeability to 270 mOsm to 276 mOsm using high-purity fructose if necessary. Filter the mixture using a 0.22 micron filter.

[0154] The microfluidic chip 100 may have one or more structural layers in which microchannels are provided. These channels may be disposed within one or more layers or between layers. The following embodiments describe an bonding process, but those skilled in the art will recognize how the various features can be achieved using an injection molding process. For example, in injection molding, instead of forming two layers, two molds can be made and joined together to inject the cavity to obtain the chip of the present invention.

[0155] In one implementation, such as Figure 1A As shown, the microfluidic chip includes a structural layer 101 on which a top "blank" malleable layer 104 is disposed. This top "blank" malleable layer 104 is bonded to the functional layer 101 to form a closed microfluidic network and may have multiple holes to provide access to one or more lower layers of the chip 100. For example, the top "blank" layer 104 may have holes corresponding to input ports 106, 107, 108, 172, etc., or provide holes 145 for securing layers 101, 102, 104, etc., of the chip 100 together with pins. In one embodiment, the top layer 104 of the microfluidic chip 100 includes multiple openings configured to align with fittings on the microfluidic chip mount 200 (described further below).

[0156] In another embodiment, as shown in FIG1B-(a), the microfluidic chip 100 includes two functional structurally malleable layers 101-102 without a top "blank" layer. In this embodiment, the functional side of the top layer 102 is disposed on the underside of the layer 102, so that when these layers are placed together, channels 114, 115, 116, and 117 are formed (see FIG1B-(b)).

[0157] In another embodiment, as shown in FIG1C-(a), three functional structural layers 101, 102, and 103 are used in the microfluidic chip 100. As in FIG1B(a), layers 102 and 103 include functional sides below layers 102 and 103, wherein channels 116 and 117 are formed when layers 101 and 102 are placed together. Layer 103 has channels 114 and 115 disposed below layer 103 (see FIG1C-(b)).

[0158] In another implementation, such as Figure 1D As shown, the microfluidic chip 100 uses four structured malleable layers 101-103 and a top "blank" layer 104. In this embodiment, layer 102 includes channels 114 and 115, and layers 101 and 103 each include one of channels 116 and 117.

[0159] However, those skilled in the art will recognize that more or fewer structural layers with functional sides and with or without “blank” layers can be used, and channels can be arranged in any structural layer or different structural layers, and in any arrangement, wherein those channels are accessed through the top “blank” layer, as long as the purpose of the invention is achieved.

[0160] In one embodiment, a sample fluid mixture 120 including object 160 is introduced into sample inlet 106, and the fluid mixture 120 flows through main channel 164 to actuation chamber 129 (see...). Figures 1A to 2 In most embodiments, the sheath fluid or buffer fluid 163 is introduced into the sheath or buffer inlet 107, 108 (see [reference]). Figures 1A to 2 In another embodiment, it is introduced into the sheath or buffer inlet 107, 108 and 172 (see [reference]). Figure 1A In laminar flow, the sheath fluid or buffer fluid 163 flows through channels 114, 115, 116, and 117 before exiting through at least output channels 140 and 142, enters the main channel 164, and proceeds to the actuation chamber 129.

[0161] In one embodiment, the fluid mixture 120 from the main channel 164 merges with the sheath fluid or buffer fluid 163 from channels 114 and 115 at an intersection 161 of the microfluidic chip 100. In another embodiment, the buffer fluid 163 from channels 116 and 117 merges downstream of the second intersection 162 with the combined fluid mixture 120 and sheath fluid or buffer fluid 163 from the first intersection 161 (see...). Figures 1A to 2In one embodiment, downstream of the second intersection 162, sheath fluid or buffer fluid 163 is introduced into the main fluid channel 164 via inlet 172 (see...). Figure 1A and Figure 3B ).

[0162] In one embodiment, the dimensions of channels 114 and 115 are substantially the same as those of channels 116 and 117, as long as the expected flow rate is achieved to realize the purpose of the present invention. However, those skilled in the art will know that the dimensions can be different, as long as they achieve the expected results (discussed further below).

[0163] In one embodiment, channels 114-117 and channels 140-142 may have substantially the same dimensions; however, those skilled in the art will recognize that the dimensions of any or all channels in the microfluidic chip 100 may be varied (e.g., between 50 micrometers and 500 micrometers) as long as the desired flow rate is achieved to accomplish the purposes of the invention.

[0164] In one embodiment, channels 114, 115, 116, and 117 are disposed in the same structural layer or plane of the microfluidic chip 100, except for the layer or plane where channel 164 is disposed (see, for example, [reference]). Figure 1A Alternatively, channels 164 and sheath channels 114, 115 or sheath channels 116, 117 can be disposed between structural layers or planes of chip 100 (see, for example, FIG. 1B). Therefore, those skilled in the art will appreciate that channels 114-117, 164, and 140-142, etc., can be disposed in any layer or between any two layers. Furthermore, although channels 114-117, 164, and 140-142, etc., are described in the exemplary embodiment shown in the figures, those skilled in the art will appreciate that the specific arrangement or layout of channels on chip 100 can be any intended arrangement, as long as they implement the descriptive features of the present invention.

[0165] In one embodiment, channels 116 and 117 penetrate layer 101 (see also...) Figure 1A and Figure 2 The fluid mixture 120 in channel 164 is coupled in the same plane through holes in the penetrating layer. In one embodiment, channels 116 and 117 are substantially parallel to the input channel 164, and each channel is coupled to an intersection 161 at an angle offset from channel 164 (see...). Figure 2(See Figure 3). Sheath fluid or buffer fluid from channels 116 and 117 horizontally or laterally compresses the fluid mixture 120, thereby flattening and / or orienting the object 160 in the fluid mixture 120 along a selected or desired direction, while still maintaining laminar flow in channel 164 (i.e., the first step in the two-step hydrodynamic focusing, as further described below).

[0166] In another embodiment, channels 114 and 115 combine at intersection 162 with the fluid mixture 120 in channel 164, wherein each channel 114, 115 is offset from channel 164 at a certain angle (see...). Figure 1A The sheath fluid or buffer fluid from channels 114 and 115 flows relative to channel 164, compressing the fluid mixture 120 (see...). Figure 14 This further flattens and / or orients the object 160 in the fluid mixture 120 along a selected or desired direction, while still maintaining laminar flow in the channel 164 (i.e., the second step in the two-step hydrodynamic focusing, as further described below).

[0167] Continuing with this embodiment, a third sheath fluid or buffer fluid inlet 172 is provided downstream of the intersection 162 (see...). Figure 3B This allows a third hydrodynamic focusing step to occur, in which the sample fluid mixture 120 is compressed from above channel 164 by introducing sheath fluid or buffer fluid 163 therein.

[0168] In an alternative embodiment, after the first hydrodynamic focusing step described above, channels 114 and 115 are offset from above channel 164 by a certain angle (see [reference]). Figure 3A (and can be above and below (see below)) Figure 12A At intersection 162, the fluid mixture 120 in channel 164 is combined to compress the fluid mixture 120 in the vertical direction, thereby flattening and / or orienting the object 160 in channel 164 (i.e., the second hydrodynamic focusing step).

[0169] However, those skilled in the art will appreciate that the configuration, angle, and structural arrangement of the sheath or buffer inlet, sample inlet, and sample input channel with the sheath or buffer channel of the depicted microfluidic chip 100, as well as the hydrodynamic focusing steps, can be different, as long as they achieve the intended features of the present invention.

[0170] In one implementation, such as Figure 2As shown, channels 114, 115, 116, and 117 are depicted as partially coaxial with each other, with their center point defined by the sample input port 106. Therefore, in one embodiment, channels 114, 115, 116, and 117 are arranged in a substantially parallel configuration, wherein the distances from channels 114, 115, 116, and 117 to the main channel 164 are equidistant. However, those skilled in the art will recognize that the depicted configuration can be different, as long as it achieves the intended features of the invention.

[0171] In one embodiment, holes and pins / rods 145 are provided at various convenient locations in layers 101, 102, 103, 104, etc., to secure and align the multiple layers during the fabrication of chip 100.

[0172] In one implementation, a gasket 105 or O-ring of any desired shape may be provided to maintain a tight seal between the microfluidic chip 100 and the microfluidic chip holder 200 (see, for example, [reference needed]). Figure 1D and Figures 21 to 2 2). In the case of gasket 105, in any configuration, gasket 105 can be a single piece or multiple objects having any configuration or as intended material (i.e., rubber, silicone, etc.). In one embodiment, as Figure 1D As shown, a first pad 105 is placed at one end of the microfluidic chip 100 and is bonded or adhered to layer 104. A plurality of holes 144 are disposed in the first pad 105 and configured to align with sample inlet 106, sheath / buffer inlet 107, and sheath / buffer inlet 108.

[0173] In one embodiment, the second pad 143 may be disposed at the other end of the microfluidic chip 100 opposite to the first pad 105 (see, for example, see...). Figure 1D And it is bonded or adhered to the top structural layer 104 (using epoxy resin) (see) Figure 1D and Figures 21 to 2 2).

[0174] In one embodiment, an O-ring is used instead of a gasket to aid in sealing and to stabilize the microfluidic chip 100 in the chip holder 200.

[0175] However, those skilled in the art will recognize that one or more pads or O-rings may be applied to the outer layer of chip 100 to protect chip 100 in chip socket 200 during operation of chip 100.

[0176] In one embodiment, channels 114-117 and channels 140-142 of the microfluidic chip 100 can not only vary in dimension, but also have a tapered shape at the inlet points into other channels in the chip 100, in order to control the flow of fluid through these channels. For example, the main channel 164 can be tapered at the inlet point into intersection 161 (see...). Figure 4A (conical shape 166A), or a cone shape at the entrance point into intersection 162 (see...) Figure 4B (cone 166A) to control and accelerate the flow of sample 120 into intersection 161, and to allow sheath fluid or buffer fluid 163 from channels 116, 117 or 114, 115 to compress the sample fluid mixture 120 on at least two sides (if not all sides, depending on the location of fluid channel 164 entering intersection 161) along a first direction (i.e., horizontally or laterally) and a second direction (i.e., vertically). Figures 3A to 3B , Figures 4A to 4B (and cone-shaped body 166A).

[0177] In another embodiment, a ramp can be provided in channel 164 or channels 114-117 to control and accelerate the flow of samples through the channels. The ramp can be attached to or replace the cone.

[0178] For example, a ramp 166B may be provided in channel 164 before the sample fluid approaches intersection 161 and intersection 162, or before entering actuation chamber 129 (see...). Figure 4A and Figure 4B ).

[0179] Therefore, the sample fluid mixture 120 becomes a relatively small, narrow flow bound or surrounded by the sheath fluid or buffer fluid 163, while maintaining laminar flow in the channel 164. However, those skilled in the art will appreciate that the main channel 164 or the buffer channels 114-117 can be any physical arrangement with conical, ramp, or other internal features, such as rectangular or circular channels, as long as the purpose of the invention is achieved.

[0180] In one implementation, multiple output channels originating from main channel 164 (see...) Figure 2 It is configured to remove fluid flowing through the microfluidic chip 100, the fluid including any target or non-target object 160 and / or sheath fluid or buffer fluid 163. Figures 1A to 2In one embodiment shown, there are three output channels 140-142, including a left output channel 140, a middle output channel 141, and a right output channel 142. The left output channel 140 terminates at a first output port 111, the middle output channel 141 terminates at a second output port 112, and the right output channel 142 terminates at a third output port 113. However, there may be only one output channel 141 and one output port 112.

[0181] In one embodiment, the output channels 140-142 are deviated from the channel 164 within the cavity 129 and reach the inlet 111-113. In one embodiment, the cross-section and length of the output channels 140-142 should be maintained at a predetermined volume ratio (i.e., 2:1:2 or 1:2:1, etc.) to obtain the expected water resistance of the output channels 140-142.

[0182] In one implementation, output channels 140-142 increase in dimension from channel 164 away from cavity 129, thereby increasing the output for object 160 compared to that through associated channel 141.

[0183] In one embodiment, instead of a straight edge, multiple notches or recesses 146 may be provided at the bottom edge of the microfluidic chip 100, if necessary, to separate the output ports (i.e., output ports 111-113) and for attaching containers and external conduits (for recovering sheath fluid or buffer fluid 163, see...). Figures 19 to 21 ), etc. Through output channels 140-142 originating from the actuation cavity 129, the outputs reach the first output port 111, the second output port 112, and the third output port 113 (see...). Figure 2 ).

[0184] In one implementation, container 188 collects object 160 from second output port 112, but other containers can collect output from first output port 111 and third output port 113 (see...). Figures 6A to 6D In one embodiment, the first output port 111, the second output port 112, and the third output port 113 can be partially characterized electronically to detect the concentration of the object 160, pH measurement, cell 160 count, electrolyte concentration, etc.

[0185] In one embodiment, the focused energy device 157 acts on the target object 160 and can collect those objects 160 and non-target objects 160 as product 165 from the second output port 112.

[0186] In one implementation, the product 165 of the target object 160 and the non-target object 160 can be further processed for storage, further separation or processing, such as cryopreservation (discussed further below).

[0187] In one embodiment, the microfluidic chip 100 is disposed in a sterile state and may be filled with one or more solutions (i.e., sheath fluid or buffer fluid 163), or any fluid or material may be removed by draining the microfluidic chip 100 or by allowing sheath fluid or buffer fluid 153 or other solutions to flow through the microfluidic chip 100, according to known methods.

[0188] Action cavity

[0189] In one embodiment, downstream of intersection 162, object 160 in fluid mixture 120 flows through channel 164 into actuation chamber 129, where object 160 is questioned and acted upon. In one embodiment, channel 164 is tapered into chamber 129 (see...). Figure 4B This accelerates the flow of the fluid mixture through cavity 129. However, those skilled in the art will appreciate that channel 164 need not be tapered and can be of any dimension and size, as long as the invention is performed as intended.

[0190] In one embodiment, interrogation device 147 is used to interrogate and identify objects 160 passing through cavity 129 in the fluid mixture within channel 164. Alternatively, in one embodiment, focusing energy device 157 also functions on objects 160 passing through cavity 129.

[0191] In one embodiment, cavity 129 includes a relatively small-diameter opening or window 150 penetrating the microfluidic chip 100 and layers 101-102 (see [link]). Figure 5 When object 160 passes through channel 164, object 160 can be visualized through opening or window 150.

[0192] Additionally, a relatively large and shallow opening in layer 104 serves as the top window, and layer 101 serves as the bottom window. In one embodiment, the top window is configured to receive a first transparent cover 133, and the bottom window 152 is configured to receive a second transparent cover 132. Covers 133 and 132 can be made of any material that meets the desired transmission requirements (such as plastic, glass), or even a lens. In another embodiment, instead of windows with covers, a continuous sheet of plastic can be used. Note that although in Figure 5 The relative diameters of cover 132, cover 133 and opening 150 are shown, but these can be varied depending on design or manufacturing considerations.

[0193] In one embodiment, the first cover 133 and the second cover 132 are configured to enclose the cavity 129. (See window and covers 133, 132) Figure 5This allows the object 160 in the fluid mixture 120 within the channel 164 to be viewed through the opening 150 as it flows through the cavity 129, and to be acted upon by a suitable light source 147 and a focusing energy device 167 (discussed later).

[0194] In one implementation, due to the structure and / or configuration of the chip layer (i.e., glass), no window and / or opening is required, or the wavelength and power level of the focusing energy device 157 and the light source 147 prevent damage to the chip 100.

[0195] Interrogation device

[0196] The interrogation device of the present invention includes a light source 147 configured to emit a high-intensity light beam 148 having any wavelength that matches an excitable object in the fluid mixture 120 (see [link]). Figure 5 Although laser 147 is preferred, any other suitable light source 147 (such as light-emitting diodes (LEDs) or arc lamps) can be used to emit a beam of light to excite the object.

[0197] In one embodiment, such a high-intensity laser beam 148 from a suitable laser 147 of a preselected wavelength (e.g., a 349 nm or 355 nm continuous wave (CW) laser, or a quasi-CW pulsed laser 147) is required to excite the object 160 (i.e., sperm cells) in the fluid mixture. In another embodiment, a 532 nm green laser 147 is used.

[0198] In one embodiment, laser 147 (see Figure 5 A laser beam 148 is emitted, which passes through the cover 133 at the uppermost part of the chip 100, through the opening 150, to illuminate the object 160 flowing through the channel 164 in the cavity 129 of the chip 100, and then through the cover 132 in the layer 101 of the chip 100.

[0199] In one embodiment, the light beam 148 can be transmitted to the object 160 via an optical fiber embedded in the microfluidic chip 100 at the opening 150.

[0200] The high-intensity beam 148 interacts with the object 160 (see detailed description below) and passes through the first cover 133 to exit from the cover 132 at the bottom window, whereby the emitted light 151 induced by the beam 148 is received by the objective lens 153 or other converging optics. The objective lens 153 or other converging optics can be positioned in any suitable location relative to the microfluidic chip 100, for example, with the objective lens 153 or other converging optics parallel to the main channel and its optical axis perpendicular to the sample fluid flow 120. Because the cavity 129 is sealed by the first cover 133 and the second cover 132, the high-intensity beam 148 does not impinge on the microfluidic chip 100 and does not damage layers 101 and 104 (see detailed description below). Figure 1A Therefore, the first cover 133 and the second cover 132 help prevent damage to the microfluidic chip 100 from the high-intensity beam 148 and photon noise induced by the material (i.e., plastic) of the microfluidic chip 100.

[0201] In one embodiment, the light beam 148 passes through the chip 100, and the emitted light 151, received by the objective lens 153 or other converging optics, is detected by the detector 154 and converted into an electrical signal by an optical sensor 154 (such as a photomultiplier tube (PMT) or photodiode). This electrical signal can be digitized or processed by an analog-to-digital converter (ADC) 155 and sent to a digital signal processor (DSP)-based controller 156 or computer. The electronic controller 156 can be any electronic processor with sufficient processing power, such as a DSP, microcontroller unit (MCU), field-programmable gate array (FPGA), or even a central processing unit (CPU).

[0202] In one implementation, a DSP-based controller 156 monitors electrical signals and, based on predetermined criteria, can employ a focused energy device 157 when detecting a target object 160.

[0203] However, in another embodiment, the interrogation device only interrogates the object 160 for identification in the sample fluid flow 120 and is not connected to the focusing energy device 157 employed (see Figure 6C ).

[0204] Focusing energy device

[0205] In one implementation, in order to deliver the desired energy level to object 160, a focused energy device 157 is used to provide focused energy pulses to object 160. The focused energy device 157 may be a thermal device, electronic device, optical device, or electromagnetic device 157 having the desired wavelength and delivering high peak power to target object 160 at a very high repetition rate (or pulse frequency).

[0206] In one implementation, after being activated by controller 156, focusing energy device 157 is triggered for a predetermined time (i.e., milliseconds) (this timing is set based on the travel speed of object 160 through channel 164, which is discussed further below), and pulses are distributed to selected object or target (i.e. unwanted) object 160.

[0207] Examples of pulsed lasers 157 include mode-locked Q-switches, as well as lasers that use both mode-locking and Q-switching technologies. For example, the focused energy device 157 (such as the Avia 355-5-100 (manufactured by Coherent Corporation of Santa Clara, California)) or the Explorer XP Q-switched laser from Spectra-Physics can operate in pulse-on-demand mode and can deliver energy pulses of 15 ns or less to the target object 160 at a rate of more than 1000 pulses per second.

[0208] In one embodiment, a pulse energy level of 0.5 μJ to 8.0 μJ is used, and in a preferred embodiment, a Q-switched laser 157 in pulse-on-demand mode is used to deliver an average pulse energy of 1.8 μJ in the range of 1.3 μJ to 2.3 μJ for a single pulse. In one embodiment, the pulse width ranges from 3 nanoseconds to 1 microsecond, preferably from 5 nanoseconds to 9 nanoseconds. However, those skilled in the art will recognize that any existing or subsequently developed high-power laser with suitable high-energy pulses and pulse frequencies will be suitable for the present invention to achieve the desired target accuracy and / or effect.

[0209] In one implementation, a sealed operating area (i.e., the space between cavity 129 or chip 100 and container 188) is required to deliver pulsed energy from focusing energy device 157 to target object 160 or its surrounding area. This is important to minimize the potential impact of energy delivery from target object 160 or outside the area to other unselected objects or non-target object 160. For example, focusing energy device 157 (such as an Explorer XP355-1Q switched laser) is capable of delivering <4% rms, providing high pulse-to-pulse stability when emitting light at regular, uniform intervals.

[0210] However, for objects 160 or cells entering the action area at non-uniform intervals (i.e., the space between cavity 129 or chip 100 and container 188, see...) Figures 6A to 6DThe flow cytometry analysis and action system employs additional measures to deliver uniform pulse energy 158 to strike only the target object 160 or cells or their surrounding area. Such measures include matching laser 157 performance parameters, such as pulse length and peak power level, to achieve the desired target accuracy (i.e., in one embodiment, a hit rate of 95% or higher for photodamage or kill of the target object 160).

[0211] Furthermore, the pulse-on-demand operation and performance of the laser 157 are further tuned to achieve extremely high pulse-to-pulse stability when emitting light at non-uniform intervals, which greatly reduces spatial variability in the region affected by pulse 158. Therefore, by reducing pulse-to-pulse variability in the focusing energy device 157, unintentional actions, damage, or destruction to the non-target object 160 or cells are greatly reduced, thereby achieving, for example, a survival rate of 85% or higher for live non-target objects 160 or cells.

[0212] In one embodiment, prior to interrogation by the interrogation device 157, a focused energy device 157 (see [reference]) is used in the action region 129 (such as cavity 129) before interrogation. Figure 6D In another embodiment, after interrogation by interrogation device 157, focusing energy device 157 (see [reference]) is used in the action area (i.e., cavity 129) after interrogation. Figure 6A In yet another embodiment, after the sample fluid with object 160 leaves chip 100 and enters container 188 (before entering container 188, it is at outlet 112 or in the form of discontinuous droplets 187), the focusing energy device 157 acts on the sample fluid (see [link to documentation]). Figures 6B to 6C ).

[0213] In an embodiment where the interrogation device 147 interrogates the object 160 in the action area 129 (i.e., cavity 129), and the focusing energy device 157 acts on the object 160, based on the determination of the object 160 to be targeted, the focusing energy device 157 emits a focused energy beam 158 to act on the object 160 flowing through the channel 164 (see, for example, [reference needed]). Figure 5 and Figures 6A to 6C ).

[0214] In this embodiment, the focusing energy device 157 acts on the object 160 after interrogation in the action region 129 and after the object 160 flows through the output channel 141, but before the container 188 collects the sample fluid 120. In this embodiment, the focusing energy device 157 is used as described above, but the focusing energy device 157 is positioned to emit a beam of light between the chip 100 and the container 188. In one embodiment, the sample fluid 120 falls into the container 188 from the output port 112 through the air only in the form of droplets 187, and in another embodiment, a transparent shell may be present between the chip 100 and the container 188.

[0215] The focused energy device 157 can be configured to damage, alter, disable, kill, or destroy a target or unwanted object 160 in the sample fluid 120, or activate one of several mechanisms in the object 160 or cell, thereby causing cell damage or death.

[0216] However, depending on the intended arrangement (see further embodiments below), the target or selected object 160 may be a desired object 160, in which case the focusing energy device 157 is not activated or triggered; or the target or selected object 160 may be an unwanted object 160, where the focusing energy device 167 is activated to act on the target 160, such as damaging, altering, disabling, killing, or destroying the selected, unwanted object 160. However, these are not the only embodiments, and various embodiments are discussed further below.

[0217] In one embodiment, when the focusing energy device 157 is damaged, altered, rendered incapable, killed, or destroyed, the target 160, along with any non-target object 160, continues to flow through the main channel 164 to the intermediate output channel 141 and then to the second output port 112, entering the container 188. Sheath fluid or buffer fluid 163 flows laminarly through the output channels 140 and 142, respectively, reaching the output ports 111 and 112.

[0218] However, in one embodiment, as described above, the object 160 in channel 164 can flow out of chip 100 through output channel 141 and a single output port 112.

[0219] Therefore, in one embodiment, the method and apparatus of the present invention are capable of producing a separation product 165 of the object 160 in the container 188 (see [link]). Figures 6A to 6D (), including highly active non-target objects or desired objects 160, and target objects 160 that are light-damaged, altered, rendered incapable, destroyed, or dead.

[0220] Beam shaping and optics

[0221] To achieve satisfactory signal repetition and effective damage, alteration, incapacitation, killing, or destruction of object 160, it is advantageous to use beam-shaping optics for both the interrogation beam 148 and the focused energy beam 158 (see [reference]). Figures 16 to 17 As used herein, the phrase “beam spot” refers to the cross-section of beam 148 or beam 158.

[0222] In one embodiment, the focusing energy device 157 is located downstream of the light source 147 and on the same side as the light source 147 (see [reference]). Figure 17 However, the focusing energy device 157 can also be located downstream of the light source 147 and on the opposite side of the light source 147 (see, for example, see...). Figure 16 ).

[0223] exist Figure 16 In this embodiment, a beam-shaping optics device for interrogating the beam 148 is disposed on one side of the chip 100. The interrogating beam 148 from the light source 147 passes through the action region (i.e., cavity 129) and is received by the objective lens 153.

[0224] In one embodiment, beam 148 is expanded by beam-shaping optics 181, which may include multiple lenses arranged in a manner well known to those skilled in the art. For example, beam-shaping optics 181 may include a pair of prisms and a pair of cylindrical lenses with suitable focal lengths, or may include other lenses with or without prisms, as is available to those skilled in the art. Beam expansion achieves the final spot size at the focal point in interrogation region 129. In one embodiment, the spot of circular beam 148 is expanded using beam expander 180. Beam expansion also reduces the impact on downstream optics, thereby limiting damage and extending lifespan. However, in one embodiment, beam expanders are not utilized. Alternatively, if the source beam has an excessively large diameter, optics may be used to reduce that diameter to a suitable size.

[0225] In one embodiment, the beam-shaping optics 181 includes two perpendicular cylindrical lenses to shape the beam 148 into an ellipse perpendicular to and along the direction of the sample fluid 120 flow when focused at its center. This elliptical beam 148 spot is used to excite objects 160 passing through channels 164 of the microfluidic chip 100 and provides maximum uniform illumination at the central region of the beam 148 spot to compensate for minor fluctuations in the flow of objects 160 through channels 164. Additionally, in one embodiment, the elliptical beam shape with a wider dimension perpendicular to the sample fluid 120 flow helps reduce variations in the fluorescence signal from objects 160 (i.e., sperm cells) whose centers are not entirely within the sample fluid 120 flow. The narrow dimension keeps the beam 148 at a sufficiently high intensity to adequately excite the fluorescent dye used to interrogate the objects 160 (i.e., sperm cells). While the elliptical beam 148 spot is preferred, different beam shapes may be used in other embodiments of the invention. The power of the interrogation beam can also be adjusted to assist in interrogation and limit its impact on the interrogation target.

[0226] In one embodiment, the focused energy beam 158 is also shaped by a beam-shaping optics 180. The shape of the focused beam 158 spot of the focusing energy device 157 affects the desired target accuracy and the likelihood of affecting non-target objects 160 in the channel 164, and can be a variable beam shape depending on the application requirements. In a flow-based system, the beam width along the flow direction of the sample fluid 120 should be adjusted to be narrow enough to affect only the target object 160 and to achieve sufficient beam intensity concentration. The length of the beam 158 spot across the fluid channel 164 can be intentionally adjusted to compensate for any slight instabilities and variability in the focused flow of the sample fluid 120. Those skilled in the art can readily obtain the desired beam shaping.

[0227] In one implementation, such as Figure 16 As shown, the beam-shaping optics 180 for focusing the energy beam 158 is used to focus the beam 158 downwards to a much smaller size to increase the laser flux in the operating region 129. In one embodiment, the beam expander optics 180 expands the beam 158 (see...). Figure 16 The beam expander optics 180 may include a plurality of lenses or prisms with suitable focal lengths, as will be readily known to those skilled in the art.

[0228] In one embodiment, the beam 158 passes through a pair of deformable prisms, for example, to shape the beam 158. An optical object further focuses and compresses the beam 158 in both the horizontal and vertical directions due to the Gaussian beam properties. In one embodiment, this optical object may be, for example, a detector optics 153, such as a microscope objective or focusing lens with a short focal length. The beam spot provides a combination of energy concentration for effective action (i.e., killing, etc.) and a width sufficient to compensate for minor fluctuations in the flow of the object 160 through the microfluidic channel 164. In one embodiment, a pair of cylindrical lenses are used to extend the beam in the vertical dimension, and a spherical focusing lens or objective is used to focus the beam into an elliptical beam spot with a small diameter of 2 μm and a large diameter of 20 μm.

[0229] In alternative embodiments, different shapes and / or dimensions can be used for beam 158. Note that other large and small diameters are available to those skilled in the art and can be applied to the same process.

[0230] In another embodiment, a focusing energy device 157 is implemented from the side opposite to the interrogation beam 148, such as... Figure 17 As shown. The configuration shown is advantageous because the focusing energy device 157 is easy to implement and makes efficient use of the free space on the photodetector side of the system.

[0231] In one implementation, a dichroic mirror is used to separate specific wavelengths or to integrate specific wavelengths into the optical path. Additionally, although mirrors and dichroic mirrors / beam splitters can be used, those skilled in the art will appreciate that more than one mirror and / or dichroic mirror / beam splitter can be utilized in this system.

[0232] In one embodiment, converging optics 153 (including a microscope objective) converges fluorescence emission from object 160 in chip 100, and a dichroic mirror transmits the fluorescence emission from converging optics 153 to photodetector 154. In one embodiment, focusing energy device 157 emits a beam 158 that passes through beam-forming optics 180 (as described above), and is guided by a mirror and reflected by the dichroic mirror before reaching chip 100 via converging optics 153. Specifically, in one embodiment, the objective of converging optics 153 focuses the focused energy beam 158 entering the rear exit port of the objective into a dense spot on object 160 only slightly downstream of the interrogation / excitation point in the action region 129. However, those skilled in the art will appreciate that the focused energy beam 158 may be positioned below the output port 112 of chip 100, or slightly upstream of the interrogation / excitation region 129.

[0233] In one embodiment, the distance between the beam spot of the interrogation beam 148 and the beam spot of the focused energy beam 158 is adjustable.

[0234] In one implementation, a dichroic mirror or any beam-splitting device can separate a small point of light to camera 182 (see [reference]). Figure 5 This allows users to visually check the alignment.

[0235] In one embodiment, for general alignment purposes, camera 182 provides a visual image of the microfluidic flow environment. The camera can be used to determine the position and timing of the emission from focusing energy device 157.

[0236] In another embodiment, a focused energy beam 158 is implemented from the same side as the interrogation beam 148 (see [reference]). Figure 17 Using this method, the focusing lens for the focusing energy device 157 is not shared with the detection side, thus allowing for more flexible beam shaping and eliminating the need for a microscope objective that is rated for high power at the action (i.e., light damage, kill) wavelength, which reduces system costs.

[0237] In this embodiment, the focusing energy device 157 emits a beam 158 from the same side as the interrogation device 147, and the beam shaping optics 180 shapes the beam 158 (as described above) so that it is guided and aligned by the mirrors and dichroic mirrors to be focused onto the object 160 in the channel 164 of the chip 100. In one embodiment, the beam focusing optics 181 (as described above) is disposed between the dichroic mirrors and the chip 100 to focus the beam 158.

[0238] As described above, in this embodiment, the beam 158 is focused into a dense spot on the object 160 only slightly downstream of the interrogation / excitation point in the action region 129. However, those skilled in the art will appreciate that the focused energy beam 158 can be positioned below the output port 112 of the chip 100, or slightly upstream of the interrogation / excitation region 129 (see [link]). Figures 6A to 6D ).

[0239] Furthermore, as described above, in this embodiment, the distance between the beam spot of the interrogation beam 148 and the beam spot of the focused energy beam 158 is adjustable.

[0240] Object Focusing and Orientation

[0241] In conventional flow cytometry systems, the orientation of objects or cells (especially those with asymmetric shapes) tends to be oriented due to their proximity to a solid surface during flow. Therefore, the functionality and improvement of object or cell orientation rely on complex nozzle designs, such as directional baffles and branch structures within the nozzle. To avoid the complex design and high manufacturing costs of nozzle-based flow cytometry systems, the microfluidic chip 100 of the present invention is designed to focus, position, and orient the object 160 to optimize its resolution. Thus, in one embodiment, the object 160 (i.e., the cell) with a non-spherical shape is aligned into a confined core volume within the channel 164 when passed through the interrogation / detection beam 148 and remains in a similar and desired orientation. Consequently, more uniform scattered and detected signals are obtained, thereby helping to improve the sensitivity and stability of the system 100.

[0242] In one implementation, hydrodynamic focusing is used to orient the object 160 by positioning the sample core flow 120 off-center relative to the center of the central plane of the cross-section of the channel 164.

[0243] Two-step hydrodynamic focusing

[0244] The following describes a two-step hydrodynamic focusing process that, in one embodiment of the microfluidic chip 100, occurs during fluid flow (see Figures 1B to 100). Figure 1D ).

[0245] In one embodiment, the first hydrodynamic focusing step of the present invention is achieved by introducing a sample fluid 120 containing an object 160 (including a biological sample such as sperm cells 160) through a sample inlet 106, and introducing a sheath fluid or buffer fluid 163 through a sheath or buffer inlet 107 and a sheath or buffer inlet 108. In one embodiment, the object 160 is pre-stained with a dye (e.g., Hoechst dye) according to a known method, thereby allowing it to fluoresce and be imaged.

[0246] In one embodiment, the object 160 in the sample fluid mixture 120 flows through the main channel 164, is surrounded and shaped by the fluid flow, and has a random orientation and position (see...). Figure 3A and Figure 6AAt intersection 161, the sample mixture 120 flowing in the main channel 164 is surrounded and shaped by sheath fluid or buffer fluid 163 from channels 116 and 117, and is compressed along a first direction (i.e., at least horizontally on at least two sides of the flow (if not all sides, depending on where the main channel 164 enters intersection 161) when the sheath fluid or buffer fluid 163 encounters the sample mixture 120. This compression is referred to as hydrodynamic focusing (three-dimensional (3D)) and can be used to align the object 160 in channel 164 into a confined core volume, which can approximate a single queue configuration. Hydrodynamic focusing utilizes the significantly large sheath fluid or buffer flow in channel 164 to increase the travel velocity of the object 160 through the planar microfluidic channel 164. In one embodiment, the sample core flow 120 may also be deviated from the central plane by a ramp 166B or cone 166A structure in channel 164 before the intersection 161 of channel 164 and the first-step sheath or buffer channels 116, 117.

[0247] Therefore, object 160 is accelerated, and the spacing between objects 160 in the microfluidic channel 164 can also be increased. The velocity of object 160 depends on the flow rate of sample 120 and the ratio of that value to the total flow rate of sheath fluid or buffer fluid 163. This feature can be used to avoid clogging problems and aggregation of object 160 with highly concentrated object sample 120.

[0248] However, as Figure 8A As shown, at this stage, the core flow of sample 120 formed across the main channel 164 still shows objects 160 or cells overlapping along the depth direction or vertical axis of channel 164. In particular, the objects 160 are focused around the center of channel 164, and the objects 160 can be compressed into a thin band across the depth of channel 164. Thus, at intersection 161, the objects 160 (i.e., sperm cells) move toward the center of the width of channel 164 as the sheath fluid or buffer fluid 163 from channels 114 and 115 compresses the sample fluid 120 toward the center of channel 164.

[0249] In one embodiment, the invention includes a second focusing step in which the sheath fluid or buffer fluid 163 entering from channels 114 and 115 at intersection 162 further compresses the sample mixture 120 including the object 160 from a second direction (i.e., a vertical direction from top to bottom). Figure 14 The intersection 162 introduced into channel 164B is the second focal area. Note that although the entrances to intersection 162 from channels 114 and 115 are shown as rectangles, those skilled in the art will appreciate that any other suitable configuration (i.e., tapered, circular) can be used.

[0250] In one embodiment, channel 114 and the sheath fluid or buffer fluid 163 in channel 114 originate from the same plane (see [reference]). Figure 3A and Figure 6A Alternatively, enter channel 164 from a different plane (see...) Figure 15 Here, channels 114 and 115 are positioned above and below the main channel, respectively, thus entering channel 164 vertically, to align object 160 in both width and depth (i.e., horizontally and vertically) at the center of channel 164B as object 160 flows along channel 164B. Subsequently, the sheath flow in the second step compresses and repositions the flow formed in the main channel 164 using channels 114 and 115.

[0251] Therefore, in the second focusing step of the present invention, the vertical sheath fluid or buffer fluid 163 entering at channels 114 and 115 further compresses the sample mixture 120 and focuses the sample 120 flow at the center of the depth of channel 164, as shown. Figure 8B As shown, object 160 flows along the center of channel 164 with a restricted core volume, which can approximate a single queue construction in a specific direction.

[0252] Therefore, after these two sequential hydrodynamic focusing steps, a restricted core volume of object 160 or cell is obtained, and the position of the flow can also be adjusted along the vertical axis to the desired location (see...). Figure 8B Therefore, the object 160 introduced into the sample input port 106 undergoes a two-step hydrodynamic focusing, which allows the object 160 to move through the channel 164B in a more uniform direction (depending on the type of object 160) with a restricted core volume that can approximate a single queue construction, which allows for easier interrogation of the object 160.

[0253] Three-step hydrodynamic focus

[0254] In one embodiment, a three-step hydrodynamic focusing is performed on object 160 in chip 100. In this embodiment, as... Figure 1A and Figure 3B As shown, the first two hydrodynamic focusing steps are achieved by horizontally compressing the sample fluid flow 120 at intersections 161 and 162, and then in the third step, the sample fluid flow 120 is vertically compressed in channel 164. Sheath or buffer channels 114, 115, 116, and 117 enter channel 164 from the horizontal direction at an angle of 45 degrees or less for each channel.

[0255] More specifically, in the first hydrodynamic focusing step, sample 120 flows into the first intersection 161, and sheath fluid or buffer fluid 163 from channels 116 and 117 flows around sample 120 and immediately compresses the sample 120 flow into a thin sample 120 flow in channel 164. In one embodiment, the sample fluid channel 164 is tapered by an internal ramp before the intersection, where sheath fluid or buffer fluid channels 116 and 117 enter channel 164 (see...). Figure 3B Meanwhile, since channel 164 is shallower (i.e. smaller in dimension) than the first sheath channel 116 and the first sheath channel 117, the sheath fluid or buffer fluid 163 from channels 116 and 117 will lift the sample 120 flow to the top of the main channel 164.

[0256] In the second hydrodynamic focusing step, sheath fluid or buffer fluid 163 is introduced from channels 114 and 115 into channel 164, with channels 114 and 115 positioned near the top of the main channel 164 (see [link]). Figure 3B In one implementation, such as Figure 3B As shown, sheath or buffer channels 114 and 115 are horizontally connected to channel 164, and can have dimensions smaller than those of sheath or buffer channels 116 and 117. Therefore, since the depths of channels 114 and 115 are shallower than the main channel 164, the sheath fluid or buffer fluid 163 further compresses the sample 120 flow along the width of channel 164, thus limiting the width of the sample 120 flow. This sheath fluid or buffer fluid 163 flow significantly improves the signal measurement sensitivity.

[0257] Without a third hydrodynamic focusing step, the lack of vertical compression at intersections 161 and 162 can cause multiple objects 160 or cells to enter the detection area 129 simultaneously, thus reducing detection sensitivity or causing measurement errors, especially for high-throughput flow cytometry applications.

[0258] However, three-dimensional hydrodynamic focusing is an efficient way to align objects within a restricted core volume in channel 164, which can approximate a single queue construction. Furthermore, the consistent positioning of objects 160 in channel 164 results in minimal variability in velocity from object to object in parabolic flow.

[0259] In microfluidic-based flow cytometry, the flow rate distribution is parabolic along the rectangular cross-section of the microchannel. Figure 7 The distribution of shear force acting on the cross-section of the microfluidic channel 164 is shown. Figure 7The shear force gradient shown has a minimum shear force at the tip of the cone and a maximum shear force value in a larger region thereafter. Therefore, a hydrodynamic shear force gradient is formed across the cross-section of channel 164. The large shear force near the wall of channel 164 helps orient the asymmetric object 160.

[0260] In this embodiment, after the first two steps of hydrodynamic focusing, the sample 120 flow contracts into a very thin flow both horizontally and vertically. The ratio of independently controlled sheath fluid or buffer fluid 163 from channels 116, 117, 114, and 115 determines the size of the sample 120 flow formed in channel 164. After compression caused by the sheath fluid or buffer fluid 163 from channels 114 and 115, the object 160 may expand somewhat along the depth of channel 164, but the object 160 still follows the sample 120 flow, very close to the top liner of the main channel 164 (note: for ease of reference, the chip is described as horizontally positioned). Therefore, a third sheath fluid or buffer fluid 163 flow from channel 172 is needed to further compress the sample 120 flow vertically and to well confine the object 160 within the thin sample 120 flow.

[0261] Furthermore, since the sheath fluid or buffer fluid 163 is vertically introduced from the sheath or buffer channel 172 into the pre-confined sample 120 flow, this sheath fluid or buffer fluid 163 helps to position the sample 120 flow at a location along the cross-section of the channel 164 (i.e., to achieve...). Figure 8B (The final result shown). When object 160 passes through detection area 129, the position of object 160 can be precisely controlled by fine-tuning the flow rate of sheath fluid or buffer fluid 163 from channel 172.

[0262] In one implementation, via an external sheath conduit (see...) Figure 20 The sheath fluid or buffer fluid 163 is introduced through channel 172, rather than through microchannels penetrating the microfluidic chip 100. Therefore, an external flow controller is required to provide a constant and stable flow rate through output channel 172 (see [link to documentation]). Figures 19 to 21 ).

[0263] The design of this invention allows the core sample stream 120 to orient flat objects and position objects 160 in the channel 164 in a near-uniform physical arrangement, all of which improves the precision operation of the downstream focusing energy device 157.

[0264] Although three hydrodynamic focusing steps have been disclosed above, those skilled in the art will recognize that the configuration and number of sheaths or buffer channels can be varied relative to the orientation and focusing of the object in the sample fluid 120, as long as they achieve the desired features of the invention.

[0265] Flow control methods

[0266] To achieve the exemplary three-dimensional hydrodynamic focusing method described above, both sample fluid 120 and sheath fluid or buffer fluid 163 need to be precisely delivered so that a constant flow can be generated through the microfluidic chip 100. After being compressed by the sheath fluid or buffer fluid 163, the object 160 or cell has been accelerated, and the average spacing between the objects 160 or cells in the core flow of sample 120 is significantly elongated. The ratio of the total sheath fluid or buffer fluid 163 flow rate to the sample 120 flow rate can be adjusted between 100:1 and 1000:1. Preferably, a ratio of 200:1 to 400:1 is used in the microfluidic chip 100 of the present invention. The total flow rate of the fluid in the microfluidic chip 100 is approximately 2 ml / min to 4 ml / min. The introduced sheath fluid or buffer fluid 163 must be constant and pulsation-free to ensure a stable travel speed of the object 160 during interrogation and signal detection, and between the detection / interrogation position and the operating position of the focusing energy device 157 (see...). Figure 6A This facilitates accurate signal reading and the action of the focusing energy device 157 on the target object 160. Utilizing precise control of the fluid flowing through the main channel 164, the total flow rate variation is less than 1% of the set flow rate, and from the location where the inquiry and inspection of the target object 160 occurs to the location where the focusing energy device 157 takes effect on the target object 160, the change in the travel speed of the target object 160 for the potential action of the focusing energy device 157 is less than 1% (see [link]). Figure 6A ).

[0267] Object Orientation

[0268] One of the challenging issues in detecting flattened objects 160 (i.e., sperm cells) is that the object 160 is confined along a uniform orientation when it passes through the interrogation beam 148. Therefore, approximately uniform positioning of the object 160 and its corresponding orientation within channel 164 contribute to improved system sensitivity. Using the aforementioned hydrodynamic focusing strategy, the position of the object 160 along channel 164 can be manipulated in a controlled manner. Thus, by adjusting the ratio between the sheath fluid or buffer fluid 163 flows from channels 116, 117, and 114, and channels 115 and 172, a position of the focused sample 120 flow deviating from the center of channel 164 by approximately 5 to 20 micrometers (e.g., based on a cross-section of channel 164 with a width of 150 micrometers and a height of 100 micrometers) is preferably used for detecting flattened objects 160. Typically, the offset position of the object 160 can be adjusted from 0 to 100 micrometers.

[0269] Specifically, to align the object 160 in channel 164 to improve its orientation, the high aspect ratio of the microfluidic channel 164 is used to induce shear forces that cause the flat surface of the object 160 (i.e., sperm cell) to flip to face the wall of channel 164. Alternatively, a flow of sheath fluid or buffer fluid 163 can be actively employed to compress and position the object 160 in channel 164. These methods are described in more detail below.

[0270] i) Passive methods

[0271] In one implementation, asymmetric geometry can be used to position the focus object 160 in channel 164 by one of the following methods: a) placing an asymmetric ramp 166B in the main sample channel 164 to enhance the sample flow 120 (see above regarding...). Figure 4A (a) and b) placing the asymmetric ramp 166B in the main channel 164 before the action cavity 129 to enhance the focused sample flow 120 (see above regarding...). Figure 4B (as described above). The asymmetric features described above can be used alone or in a suitable combination of two or more. However, those skilled in the art will recognize that these features are not necessary for implementing the position of object 160 in channel 164.

[0272] In one implementation, as described above, ramps can be used in channels 114-117 to elevate the sample flow 120, but ramps are not required. The arrangement of ramps in the channels depends on the direction in which the object 160 of the sample core flow 120 needs to be decentered to improve the orientation of the object 160 or cells. However, the passive method described above offers less flexibility in changing the position of the object 160 in the main channel 164.

[0273] ii) Active methods

[0274] In alternative methods to deflect the core flow of sample 120 in channel 164, an asymmetric sheath flow 163 is introduced to adjust the position of object 160 or cell in channel 164. Several methods exist for implementing the asymmetric sheath fluid or buffered fluid flow 163, two of which are described below.

[0275] One implementation involves introducing a single sheath fluid or buffer fluid flow 163, which forms a 90-degree angle with the wall of the main channel 164, such as... Figure 3A (In the two-step hydrodynamic focusing method) or Figure 3B (As shown in the three-step hydrodynamic focusing method). In the two-step hydrodynamic focusing implementation, following the first step hydrodynamic focusing at intersection 161, the sheath fluid or buffer fluid flow 163 introduced at intersection 162 of the second step hydrodynamic focusing further compresses the sample core flow 120.

[0276] In the alternative three-step hydrodynamic focusing implementation, this compression of the sample core flow 120 occurs at the third hydrodynamic focusing intersection where channel 172 joins the main channel 164. Therefore, the final hydrodynamic focusing step positions the object 160 or cell to the desired location along the vertical axis. By controlling the ratio of the hydrodynamic focusing flow rate, the desired location of the object 160 can be obtained to achieve optimal orientation.

[0277] In the second embodiment of the two-step hydrodynamic focusing method, the two second-step sheaths or buffer channels 114, 115 merge at a certain angle relative to the wall of the main channel 164, and are parallel to the main channel 164 from above and below, as shown. Figures 12A to 12B As shown. The angle between the second-step hydrodynamic focusing channels 114 and 115 and the main channel 164 can be changed and depends on the manufacturing method. Preferably, a 90-degree angle is selected (see...). Figures 12A to 12B Sheath fluid or buffer fluid 164 at different flow rates can flow through two channels 114 and 115, which can reposition the sample core flow 120 within the main channel 164. This improves the orientation of the object 160 after it has deviated from the central plane of the channel 164. In a specific embodiment such as sperm cells 160, the sperm cells 160 tend to flip their flattened sides along the vertical axis onto the wall of the channel 164.

[0278] As from Figure 12B As can be seen from the implementation method, the dimensions of channel 114 and channel 115 are not necessarily the same (e.g. Figure 12A (as shown), so as to obtain different water resistance. Therefore, the same flow rate of the sheath fluid or buffer fluid flow 164 in the second step in channels 114 and 115 will also generate a biased fluid flow.

[0279] In summary, both the passive and active methods described above can help to optimally locate object 160 and improve its orientation in channel 164.

[0280] In one embodiment, a disc-shaped sperm cell 160 is used as an example of object 160. Due to its disc-shaped or flattened teardrop-shaped head, when the sperm cell 160 undergoes a second or third focusing step (depending on the embodiment), the sperm cell 160 will self-redirect along a predetermined direction, i.e., its flattened plane is perpendicular to the direction of beam 148 (see FIG. 6). Thus, the sperm cell 160 develops a preference for its body orientation as it passes through the hydrodynamic focusing process. Specifically, the sperm cell 160 tends to be more stable when its body is perpendicular to the compression direction. Thus, under the control of the sheath fluid or buffer fluid 163, the sperm cell 160, which started with random orientation, now achieves uniform orientation. Thus, the sperm cells 160 are not only positioned in a confined core volume at the center of channel 164B, but they also achieve uniform orientation in the most recent hydrodynamic focusing step, where the flattened plane of the sperm cell 160 is perpendicular to the compression direction.

[0281] The above methods improve the orientation of sperm cells 160 and the ability to distinguish the DNA content of X and Y sperm chromosomes (thus differentiating between X and Y sperm). Figure 13A and Figure 13B The histograms show sperm cells 160 located at the center of channel 164 and sperm cells 160 located away from the center of channel 164. Figure 13A The circles on the left and right sides of the histogram show a population of abnormally oriented sperm cells (160). The left peak of the main population causes it to lose its ability to distinguish between the X and Y sperm subpopulations and contributes to the asymmetric distribution of the X and Y sperm subpopulations.

[0282] Operation of microfluidic chip systems

[0283] Questions about the object

[0284] In one embodiment, the interrogation light source 147 is an excitation laser 147 (see [link]). Figure 16 It has a power of 350mW, a wavelength of 355nm, and a pulse width of 12ps.

[0285] In one embodiment, further downstream of the hydrodynamic focusing step, in channel 164, a light source 147 is used to detect an object 160 at opening 150 and in actuation cavity 129, passing through cover 133. The light source 147 emits a beam 148 (which may be assisted by an optical fiber), which is focused at the center of channel 164 at opening 150.

[0286] In one embodiment, object 160 is sperm cell 160, which is oriented via a hydrodynamic focusing step, such that the flat surface of sperm cell 160 faces beam 148. Furthermore, as all objects 160 or sperm cells 160 pass under beam 148, the hydrodynamic focusing step moves all objects 160 or sperm cells 160 into a confined core volume that can approximate a single queue configuration. When object 160 passes under light source 147 and is acted upon by beam 148, object 160 emits fluorescence indicating the identity of the desired object 160.

[0287] Light source 147 provides fluorescence excitation energy for detecting object 160 in action chamber 129. In an exemplary embodiment where object 160 is sperm cell 160, X chromosome cells fluoresce at a different intensity than Y chromosome cells (based on DNA content, as is well known in the art) (Note: 355 nm is chosen for the Hoescht 33342 dye used on DNA). Alternatively, in other embodiments, object 160 (which is a cell carrying one characteristic) may fluoresce at a different intensity or wavelength than cells carrying a different set of characteristics. Furthermore, object 160 can be viewed in relation to shape, size, or any other distinguishing indicator.

[0288] Therefore, in the embodiment of sperm cell 160, the illumination of the flat surface and edges of sperm cell 160 is significantly different from that of other cells. The fluorescence signal derived from the edges of sperm cell 160 is significantly stronger than that derived from the flat surface, which increases the difficulty for digital processor 156 to process the stronger signal from the edges and the conventionally weaker signals from the X and Y flat surfaces of cell 160. Therefore, flipping the flat surface of sperm cell 160 to face the laser illumination (i.e., beam 148) helps reduce orientation variability and improves the system's ability to distinguish between X sperm cell 160 and Y sperm cell 160.

[0289] In the embodiment of beam-induced fluorescence, the emitted beam 151 (in) Figure 5 The signal is then focused by objective lens 153 and subsequently converted into an electrical signal by optical sensor 154. The electrical signal is then digitized by analog-to-digital converter (ADC) 155 and sent to electronic controller 156 for signal processing.

[0290] As described above, in one embodiment, the DSP-based controller 156 monitors electrical signals, and when a specific signal is detected, the energy focusing device 157 can be used to act on the target object 160 (see [link]). Figures 6A to 6C However, in an alternative embodiment, after the focusing energy device 157 acts on the object 160, the interrogation device interrogates the object 160 (see...). Figure 6D ).

[0291] In one embodiment, interrogation of the sample 120 containing object 160 (i.e., biological material) is achieved using other methods. Therefore, portions of the microfluidic chip 100 or outputs from the microfluidic chip 100 can be examined optically or visually. Generally, methods for interrogation can include direct visual imaging, such as using a camera, and can utilize direct intense light imaging or fluorescence imaging; or, more sophisticated techniques can be used, such as spectroscopy, transmission spectroscopy, spectral imaging, or scattering spectroscopy such as dynamic light scattering or diffuse wave scattering.

[0292] In some cases, the optical interrogation region 129 can be used in conjunction with additives, such as chemicals that bind to or affect the sample mixture 120, or beads that are functionalized in the presence of certain substances or diseases to solidify and / or fluoresce. These techniques can be used to measure cell concentrations, detect diseases, or detect other parameters that characterize the subject 160.

[0293] However, in another embodiment, if fluorescence is not used, polarized backscattering can also be used. Using spectroscopic methods, the objects 160 are questioned as described above. The spectra of those objects 160 that exhibit a positive reaction and fluorescence (i.e., those objects 160 that react with the tag) are identified and selected by the focusing energy device 157.

[0294] In one implementation, the object 160 can be questioned and identified based on the reaction or combination of the object 160 with the additive or sheath fluid or buffer fluid 163, or by using the natural fluorescence of the object 160 or the fluorescence of the substance associated with the object 160 as an identity tag or background tag, or by satisfying selected size, dimension or surface characteristics, etc.

[0295] In one implementation, after the test is completed, a computer 182 (which monitors electrical signals and employs a focused energy device 157) and / or an operator can be used to select which objects 160 are discarded and which are collected.

[0296] Applications of focused energy devices

[0297] The focusing energy device 157 of the present invention can perform multiple actions on the object 160 in the channel 164 or between the chip 100 and the container 188.

[0298] In one embodiment, the focusing energy device 157 functions to light-damage or destroy the object 160 in multiple ways.

[0299] Specifically, the focused energy device 157 serves to kill the target 160 (i.e., the cell). For example, the target 160 may be an unwanted object, and after the focused energy device 167 is activated, the overheating of the intracellular environment can cause cell death. The overheating of the intracellular environment can promote, but is not limited to, protein denaturation and reduced enzyme activity.

[0300] In another method, the energy dose 158 from the focused energy device 157 is strong enough to cause the cell membrane to rupture and the cell contents to leak from the cell 160 into the surrounding environment (i.e., sheath fluid or buffer fluid 163).

[0301] In another method, the death of object 160 or cells can be caused by the formation of reactive oxygen species (ROS), which are formed due to the absorption of energy from the focused energy pulse 158 from the focused energy device 157, which, among other things, will cause DNA and protein damage.

[0302] In another embodiment, the focused energy device 157 may use a focused energy pulse 158 from the focused energy device 157 to temporarily or permanently disable a target object 160 (such as a cell 160).

[0303] For example, exposing sperm cells 160 to focused energy pulses 158 (such as those generated by a laser or LED 157) causes photoactivation within the cells 160 and results in the temporary or permanent incapacitation of the cellular mechanisms responsible for sperm motility. After incapacitating the target sperm cells 160, the resulting sample 120 contains motile sperm cells 160 and immobile (target) sperm cells 160, wherein the immobile sperm cells are unable to naturally fertilize the egg cell.

[0304] In another embodiment, it is desirable to use focused energy pulses 158 to render sperm 160 infertile through dimerization of nucleotides in DNA. When cells 160 (such as sperm cells 160) are exposed to UV light, dimerization occurs, resulting in binding between pyrimidine bases and the formation of a “crosslink” that, if not repaired, inhibits replication and transcription. Thus, although the target sperm cell 160 remains alive (as demonstrated by its motility), its fertility is greatly reduced.

[0305] In addition to sperm cells 160, a high-power focused energy source 157 (such as an LED or laser 157) can be used to photoexpose fluorescence in objects 160 (such as cells or colloids 160) that exhibit a predetermined level of fluorescence. For example, in the formation of numerous self-aggregated objects, objects 160 with a wide range of sizes that are difficult to separate from each other are formed. To produce an enhanced sample 120 of objects 160 with a desired size, all objects 160 can be fluorescently labeled using methods known in the art, and the size of the objects 160 can be determined using optical interrogation, and objects 160 exhibiting a predetermined level of fluorescence can be photoexposed.

[0306] In a specific example, semen sample 120 may contain contaminants, such as bacterial or viral cells, which are targets for photoextinction. Another example may include sperm cells 160 containing cells 160 or DNA with a given characteristic and labeled with a fluorophore for quantitative and / or qualitative measurements. Sperm cells 160 containing this characteristic can be targeted by the focusing energy device 157. In another embodiment, sperm cells 160 not containing this characteristic can be targeted for photoextinction. Specific cells 160 in other cell mixtures (such as blood) are also candidates for photoextinction treatment to reduce viability. In another embodiment, photoextincted cells / objects 160 may be undetectable downstream and therefore not subject to subsequent processing steps (i.e., subsequent processing steps can be bypassed).

[0307] In another embodiment, instead of disabling the object 160, the focused energy pulse 158 can be used to activate materials, such as trapped molecules or compounds within the object 160.

[0308] In one application, the trapped compound represents, but is not limited to, a fluorescently labeled or cell-responsive molecule. In these applications, a focused energy pulse 158 can be used to induce photoactivation of the trapped molecule or compound, which alters the cell signaling dynamics 160 used in ex vivo therapies.

[0309] In another embodiment, the focused energy pulse 158 can activate a photopolymerization event that renders the cell or colloid 160 incapable by altering the internal properties of the object 160.

[0310] In another embodiment, focused energy pulses 158 can be used to activate heat shock proteins or trigger mitochondrial biosynthesis or activation within the subject 160 or cells (including germ cells), relative to intracellular signaling pathways, to enhance cell viability and function. Additional intracellular pathways can also be activated to repair damage caused by the interrogation / inspection device 147 or multiple other factors, too numerous to list (i.e., environmental factors, thermal factors, chemical factors, etc.).

[0311] In one embodiment, those skilled in the art can generate photopolymerization using focused energy pulses 158 to temporarily or permanently encapsulate or contain target cells 160, colloids, or other objects using multi-arm polyethylene glycol acrylate / polyethylene glycol resin, etc., which facilitates the encapsulation of the target object 160 or cells. Sperm cells 160 can be encapsulated to enhance the retention of viability and fertility during commercial storage and delivery processes.

[0312] In another embodiment, a focused energy pulse 158 is used to induce a photopolymerization event on the surface of the target cell or object 160, which increases the size and density of the encapsulating material, thereby altering the size and density of the target object 160 or improving the properties and performance of the encapsulating material.

[0313] In another embodiment, a photopolymerizable sequence in the hydrophobic portion of the vesicle can be used to permanently seal the desired molecule or object 160 through encapsulation therein.

[0314] In another method, the focused energy device 157 can be used to act on the target object 160 externally or to change the environment around the target object 160.

[0315] In one method, a focused energy pulse 158 is used to heat the local environment surrounding a target cell or object 160, thereby increasing the heat sufficiently to cause toxicity to the target cell 160.

[0316] In another embodiment, a focused energy pulse 158 is used to promote the rupture of an analyte-containing delivery medium (such as a vesicle) very close to the target cell 160. The delivery medium carries molecules such as sodium fluoride (NaF) that causes the sperm cell 160 to temporarily become immobile, or heparin that promotes the acquisition of the sperm cell 160. When the concentration of the analyte or activator is locally increased, the target sperm cell 160 or other object responds to the local signal, without activating a similar response in non-target objects 160.

[0317] In another embodiment, when an object 160 or cell is attached to a surface, a focused energy pulse 158 is used to alter the surrounding environment to change the elastic modulus of the surface or to release cell-responsive chemicals from the surface of the surrounding object 160 or cell.

[0318] In one embodiment, a temperature change is caused by absorbing heat generated by light / electromagnetic (EM) waves 158, which kills the object 160 or cells.

[0319] In another embodiment, a focused energy pulse 158 is used to form or break predetermined chemical bonds in the attached material, thereby guiding the object 160 (such as stem cell 160) to differentiate into different cell lines.

[0320] In one implementation, for some applications, it is desirable to use focused energy pulses 158 to promote cell uptake or adhesion to target object 160.

[0321] In one implementation, local heating enhances cellular uptake of antibodies, cell probes, or DNA.

[0322] In one embodiment, the local heating is used to selectively promote the internalization of the object 160 into the target living cell 160, while the temperature rise is also optimized to maintain the viability of the object 160 or the cell.

[0323] In one embodiment, the object to be transported 160 is attached to an object that can induce transient microbubbles when targeted by a light source 147; for example, oligonucleotides can be conjugated with gold nanoparticles. When the gold nanoparticles are heated using a light source 147 at an optimal wavelength, microbubbles are transiently formed. Following the cavitation phenomenon of the microbubbles, the gold nanoparticles are broken apart, and multiple nanoparticles attached to the microbubbles and the object permeate through the cell membrane.

[0324] In another implementation, the temperature increase of the target object 160 is insufficient to induce microbubble formation. The localized temperature increase is optimized to maintain cell viability and selectively promote the internalization of the object 160 into the target living cells.

[0325] Similarly, in another embodiment, by using focused energy pulses 158 to attach material to a colloid or object, the geometry or properties of object 160 are altered, thus enabling additional separation techniques (such as magnetic or electric fields) to separate substances that are normally not susceptible to these forces.

[0326] Operation of focused energy device

[0327] Typically, flow cytometry analysis and action systems that use electromagnetic radiation sources (such as focused energy device 157) or lasers to act on selected objects 160 typically aim to deliver controlled energy levels to individual objects 160. In one embodiment, such a system can use focused energy device 157 to kill, alter, damage, or destroy target objects 160 or cells. In another embodiment, and among other methods, such a system can activate a target in the selected object 160 or cells, or in a fluid, medium, or matrix surrounding the selected object 160, as described above.

[0328] In the above method utilizing focused energy pulse 158, radiation can be applied through targeted emission or continuous elimination to ensure that the desired target 160 remains unaffected and to distinguish unwanted, altered, killed, destroyed, or damaged objects 160 from the sample 120. Similar considerations as described above are given when selecting the laser wavelength and laser power for targeted emission and continuous emission modes.

[0329] a. Target launch mode:

[0330] More specifically, in target firing, the focusing energy device 157 is used for the target object 160. Specifically, the object 160 in the fluid mixture of sample 120 can be evaluated for specific characteristics of the object 160 through the interrogation / detection area in cavity 129, for example, here by one or more of the methods described above.

[0331] Therefore, in a flow-based system, for example, the operating region 129 of the focused energy device 157 is downstream of the optical interrogation region interrogated using the light source 147. Alternatively, the focused energy device 157 can be utilized before interrogating a more distant downstream region. In one embodiment, the focused energy device 157 acts on the object within the operating cavity 129. The distance between the optical interrogation region and the operating region can be adjusted to accommodate different timing sequences.

[0332] The decision to hold, discard, or act on the desired object 160 is based on predetermined criteria. A trigger energy pulse 158 from the focusing energy device 157 strikes the object 160 marked for the action (see, for example). Figures 6A to 6C ).

[0333] When an object 160 or cell is not targeted, the object 160 or cell remains unaffected and flows through cavity 129, via channel 164 to output channel 141 and container 188, which collects the target object 160 and non-target object 160 as a distinguishing product 165.

[0334] In one embodiment, the laser pulse 158 has a short duration and can be selectively targeted at an individual object 160 or cell without intentionally affecting non-target objects 160 or cells that may be nearby, thereby avoiding "overspraying" of non-target objects 160 or cells. The pulse energy is selected to achieve the desired effect while avoiding unintended interference with the surrounding medium, or, for example, in a flow system, not causing unintentional cavitation or bubble formation. Various laser wavelengths can be used; however, flow requirements may vary depending on the characteristics of the target object 160, the dye, and the environment.

[0335] Laser unit 157 has limited power, especially those operating in dense models with high pulse frequencies (typically greater than 100 kHz), and it is preferable to select a laser wavelength that minimizes the required flow rate. For example, matching the wavelength of laser 157 to the absorbance of the dye, other target, or object 160 (i.e., molecules) used in the process greatly improves efficiency and effectiveness. Furthermore, pulse energy 158 is selected to deliver the desired effect while avoiding undesirable interference with the surrounding medium, or, for example, in a flow system, to prevent unintentional cavitation or bubble formation.

[0336] In a specific example concerning sperm cells 160 as object 160, a 355 nm laser 157 is used to utilize a dye (i.e., Hoechst 33342 dye) used in cell staining processes. In similar examples, a 349 nm laser 157 can be used. In these examples, pulse energy levels from 0.5 μJ to 8.0 μJ are used when unwanted sperm cells 160 are photodamaged, destroyed, or killed.

[0337] b. Continuous firing:

[0338] In continuous firing, such as in a flow-based system, focused energy pulses 158 are continuously employed and interrupted only for non-target objects 160 that do not need to be acted upon (i.e., desired objects 160 that are not to be damaged, destroyed, altered, or killed) or for the passage of waste or contaminants.

[0339] As described above, the decision to retain or discard object 160, or to act upon (including photodamage, killing, alteration, incapacitation, or destruction) object 160, is based on predetermined criteria. In an exemplary flow system, a focused energy device 157 (such as a continuous wave (CW) or fast pulsed laser or LED) delivers a continuous stream of focused energy 158 to object 160 and is used to act on each object 160 at a specific location in the flow of fluid through sample 120, including photodamage, alteration, incapacitation, destruction, or killing. When a non-target object 160 is encountered in the action area, the laser beam 158 is cut off, deflected, or otherwise interrupted for a brief period to allow the non-target object 160 (in some cases, discarded items) to pass unaffected. Target object 160 or the target object 160 flows through channel 141 into container 188.

[0340] Methods for interrupting or diffusing beam 158 include mechanical methods (masks, choppers, mirrors), optical methods (acousto-optic deflectors, acousto-optic modulators, spatial light modulators, digital micromirrors, polarization modification, liquid crystal displays), electrical methods (pulse adjustment, drop, or alteration of Q-switched lasers), or acoustic methods. Any other known or future suitable methods or techniques may be used to interrupt the focused energy beam 158.

[0341] Using either a targeted or continuous method, the energy pulse 158 has a short duration, and the focused energy device 157 can selectively target only a single object 160 without affecting other objects 160 in the fluid flow of sample 120 (i.e., limiting "collateral damage"). Depending on the user's needs, the energy pulse 158 can be selected to achieve the desired action on object 160 (i.e., damage, alter, kill, or destroy object 160). The pulsed energy 158 from the focused energy device 157 should fall within a range that will not cause interference to the fluid of sample 120 due to cavitation, bubble formation, or energy absorption methods.

[0342] Other techniques for reducing unintentional action, damage, or destruction to non-target object 160 include absorbing the majority of the pulse energy 158, changing the direction of the beam 158, or discharging excess energy into the flow stream 120 by emitting pulses 158. Specifically, these techniques may include mechanically moving a mirror or lens to defocus or deflect excess laser pulses 158 into an energy-absorbing device, electrically changing a lens to alter the propagation angle of the laser 157, and sophisticated triggering techniques that coordinate pulse energy data from the laser 157 with object-to-object timing data regarding object 160 in the current flow stream.

[0343] c. Pulse timing

[0344] The timing of the actions of the focused energy device 157 on the object 160 is not uniform and follows a Poisson distribution, with many short and very long intervals occurring. Because the laser-based motion system 157 has inherent limitations, it is preferable to include short "recharge times" between laser pulses 158. The waiting time (inherent in the focused energy device 157) plus the "charge / recharge" time is the minimum time that the focused energy device 157 can react (transmit pulses) and still provide the required energy level to the target object 160.

[0345] In one implementation, the charging time should range from 0.1 μs to 1 second, preferably from 0.1 μs to 4 ms. Pulse-to-pulse variations in the energy level affect the rate at which the desired effect is achieved on the target object 160 or cell, and the likelihood of affecting non-target objects 160 or cells. When emitted at non-uniform intervals, pulse-to-pulse stability should be high. In one example, a Q-switched laser 157 in pulse-on-demand mode is used to deliver an average pulse energy of 1.8 μJ, where individual pulses range from 1.3 μJ to 2.3 μJ.

[0346] In a flow-based system, the action region 129 can be located downstream of or upstream of the optical interrogation region in cavity 129, and the distance between the optical interrogation region and the action region can be adjusted to accommodate different timing sequences. To accommodate a sufficient charging time for the pulsed laser 157, the minimum timing sequence between interrogating the object 160 or cell and acting on the desired object 160 or cell should be no less than 1 μs.

[0347] The focused energy device 157 operates successfully over long periods of time at selectable accuracy (e.g., ranging from 75% to 95%) and at a motion rate of up to 5600 objects per second. In a system where the interval between objects 160 in a flow is controlled, the system 157 can operate at a motion rate up to the repetition rate of the laser 157.

[0348] d. Object selection

[0349] In one embodiment, a focused energy device 157 is employed prior to interrogation of object 160. However, in another embodiment, to determine which object 160 is selected to be acted upon by the focused energy device 157, as described above, a histogram or any graphical representation of the measured / calculated characteristics of object 160 after interrogation can be used to determine the group of objects 160 to act upon. In one embodiment, after interrogation is completed, a plot of the span (i.e., the transit time through the interrogated region of cavity 129) can reflect, within a particular chip 100 design, the relative size of the sample 120 core flow under different flow conditions, the distribution of objects 160 or cells across the main channel 164, and the travel velocity of objects 160, as well as the velocity variation of objects 160.

[0350] As described above, the high aspect ratio of the main channel 164 is important for the hydrodynamic focusing, migration, and orientation of the object 160 or cell. In one embodiment, an aspect ratio of less than 1 is used for the microfluidic channel 164 in this invention. Preferably, 2 / 3 of the aspect ratio is used for the main channel 164. The span value itself roughly indicates the velocity of the object 160. A large span value indicates that the object 160 passes slowly through the interrogation beam 148. A precise span size indicates that the core flow of the sample 120 is close to the central plane of the channel 164 and has a small variation in the velocity of the object 160.

[0351] In one implementation, small velocity variations of the object 160 are permitted to ensure precise targeting of the selected object 160 (whether flowing through chip 100 or exiting from output port 112), thus ensuring that the focusing energy device 157 can accurately aim at the selected object 160 or cell. Therefore, based on the positioning and orientation methods described above (i.e., active and passive methods), the object 160 is positioned close to the center of the cross-section of channel 164 to minimize velocity variations of the object 160.

[0352] In one implementation, for a flattened object 160 or cell, such as a living sperm cell 160, both directional and velocity changes need to be taken into account. Therefore, the object is pushed away from the central plane of channel 164 along the vertical axis (see...). Figure 9 B) The sperm cells 160 tend to achieve better resolution (e.g., the distinction between X sperm cells 160 and Y sperm cells 160 is greater than 50% on the histogram obtained after interrogation) and a smaller population of anomalously located cells 160. Thus, a balance is achieved between resolution and target (i.e., photodamage, killing) efficiency. In one exemplary embodiment, the core flow of sample 120 is preferably shaped to span the cross-section of the main channel 164 in the interrogation / detection region of cavity 129, approximately 10 micrometers in width, 5 to 10 micrometers in height, and offset from the central plane of the main channel 164 by approximately 2 to 10 micrometers, preferably achieved by controlling a hydrodynamic focusing step.

[0353] In one embodiment, when the object 160 is a sperm cell, the target sperm cell 160 can be a male fertile sperm cell (i.e., a sperm cell carrying the Y chromosome), and the non-target sperm cell 160 can be a female fertile sperm cell (i.e., a sperm cell carrying the X chromosome). In another embodiment, the target sperm cell 160 can be a female fertile sperm cell (i.e., a sperm cell carrying the X chromosome), and the non-target sperm cell 160 can be a male fertile sperm cell (i.e., a sperm cell carrying the Y chromosome).

[0354] In one implementation, the object 160 is acted upon prior to interrogation by utilizing localized heat shock to infuse molecules (such as DNA or other probes) that have passed through the protective outer layer and entered the object (i.e., through the protective membrane and into the cell). Figure 6D The conventional method of combining molecules using the high voltage required for successful electroporation to penetrate cell membranes can be satisfactory, as evidenced by high cell death rates. Local thermal shock represents a gentler process and is therefore more satisfactory for maintaining cell viability. A focused energy device 157 is used to generate a localized rise in temperature, thus achieving local thermal shock. Local thermal shock leads to the permeation of cells 160, thereby promoting the combination of the desired molecules. The object 160 is then detected and questioned using an interrogation device 147, which thereby determines the number or proportion of objects 160 for which molecule combination has been achieved. This method is particularly satisfactory in biosensing, cell engineering, targeted therapy, and drug / gene delivery.

[0355] e. Action Zone

[0356] In one implementation, after interrogation is performed and an acceptable histogram is obtained (i.e., with acceptable resolution and a relatively small span distribution), it is decided to use the focused energy device 157 to act on the selected object 160 or cell. One of the more important parameters is the timing setting for the pulses from the focused energy device 157 (i.e., the delay or time interval for the object 160 between the interrogation / detection beam 148 and the focused energy beam 158).

[0357] The focusing energy device 157's action zone is the area within the cross-section of the main channel 164 where it can effectively act (i.e., photodamage, alteration, incapacitation, killing, destruction, etc.) on the selected object 160 or cell, such as... Figure 16 As shown. Based on the predetermined energy level and beam shape of the focusing energy device 157, and based on the design and flow conditions of the microfluidic channel 164, the operating region can be estimated as an action percentage (e.g., less than 97%). The energy level of the focusing energy device 157 depends on the current and charging time of the focusing energy device 157. A larger overall flow velocity in the main channel 164 means that the object 160 is traveling faster. Therefore, the transit time of the object 160 through the focusing energy beam 158 is shorter. For example, for an energy level of 2.3 μJ with a specific beam shape (e.g., 2.5 μm x 15 μm) of the focusing energy device 157, and a travel velocity of approximately 7.5 m / s for the object 160, the operating region of the focusing energy device 157 is estimated to be approximately 20 μm in the Y-axis direction and approximately 16 μm in the vertical direction.

[0358] Within the action zone, the percentage of affected (i.e., damaged, altered, or killed) target objects 160 also depends on the shape and position of the sample 120 core flow. The size and position of the sample 120 core flow can be tailored to the action zone in both the horizontal and vertical directions by adjusting the flow rate of the sheath or buffered hydrodynamic focusing flow. Finally, the flow conditions for this microfluidic chip 100 can be determined at the desired energy level of the focusing energy device 157. Therefore, the shape of the sample 120 flow can preferably be limited to an example of approximately 10 micrometers in width and 5 to 10 micrometers in height.

[0359] In another embodiment, the action zone, essentially as described above, is positioned before interrogation, or after the sample fluid 120 leaves the chip 100 for use in the container 188 (see [link]). Figures 6B to 6D ).

[0360] f. Manipulation of sperm cells

[0361] As discussed above, in one exemplary embodiment using sperm cells 160 as object 160, live sperm cells 160 (i.e., bovine sperm cells with approximately 50 X chromosome cells and 50 Y chromosome cells) are introduced into sample input port 106 and, through a hydrodynamic focusing step, reach interrogation region 129. In interrogation region 129, a dye (i.e., Hoechst 33342 dye) is excited by an interrogation beam 148 from a light source 147 (such as a laser 147), which produces fluorescence in the cells 160, which is captured by a photodetector 154 after passing through objective lens 153.

[0362] Based on the characteristics of the fluorescence signal, such as differences in reflectivity, the controller 156 can individually identify and distinguish between non-target sperm cells 160 and target sperm cells 160. If sperm cell 160 is a desired sperm cell (i.e., one of X-chromosome sperm cells and Y-chromosome sperm cells), then it is determined that desired or non-target sperm cells 160 are allowed to flow unimpeded through the microfluidic channel 164 to the collection device 188. However, if sperm cell 160 is an unwanted sperm cell (i.e., the other of X-chromosome sperm cells and Y-chromosome sperm cells), then after a predetermined delay time, the focused energy device 157 is used to act on the target sperm cell 160 to allow the unwanted / target sperm cell to reach the action zone (which may be within the cavity 129 or between the output port 112 and the collection device 188).

[0363] This invention allows for jitter in the system of this invention so that the focused energy device 157 can operate more effectively in the action zone after a predetermined delay time. Depending on the travel speed of the target sperm cell 160, the target sperm cell 160 will reach the action zone in about 2 μs, and the focused energy beam 148 is most effective when aiming at the center of the target sperm cell 160. Therefore, it is preferable to limit the jitter to 1 μs or less.

[0364] As described above, the target sperm cells 160 can be altered, photodamaged, killed, modified, rendered incapable, or destroyed by the following modes of the focusing energy device 157: 1) target or "pulse-on-demand" mode, or 2) "continuous emission" mode. As described above, it is preferable to select a laser wavelength for the focusing energy device 157 that minimizes the required flux. For example, matching the laser wavelength to the absorption rate of the dye used in sperm cell staining can improve efficiency and effectiveness. For example, if Hoechst 33342 dye is used in the staining process, a laser wavelength of 355 nm is optimal for the focusing energy device 157.

[0365] In one embodiment, the target sperm cell is killed or destroyed. In another embodiment, the target sperm cell 160 is rendered sufficiently incapable, such that it is no longer able to perform its intended function. For example, the tail of the target sperm cell 160 may be rendered incapable, thus preventing the target sperm cell 160 from exhibiting forward mobility. Therefore, the targeted, incapacitated sperm cell 160 will be prevented from fertilizing the egg cell.

[0366] In one implementation, the complex software for controller 156 can be designed to meet the requirements of the high-power laser 157 described above, allowing the emission of a single laser pulse 158. Therefore, regardless of when the focusing energy device 157 issues a request, either targeted emission or "pulse-on-demand" mode delivers a constant laser pulse 158. Targeted emission mode is preferably used for samples 120 containing a majority of non-target sperm cells 160 and a relatively small number of target sperm cells 160 that need to be eliminated. However, using commercially available high-speed pulse-on-demand systems, targeted emission mode can be implemented for samples with other ratios of non-target sperm cells to target sperm cells.

[0367] In an alternative embodiment, when the number of target sperm cells 160 greatly exceeds the number of non-target sperm cells 160, the focusing energy device 157 may not be able to generate laser pulses 158 fast enough to affect (i.e., kill or disable) all target sperm cells 160. Therefore, the target emission mode becomes less efficient. In this case, the "continuous emission" mode becomes more advantageous.

[0368] In continuous emission mode, as discussed above, the focusing energy device 157 is a continuous wave (CW) or quasi-CW, or fast pulse laser 157, used to act on (i.e. kill or disable) each sperm cell 160 passing through a specific location in the microfluidic channel 164, without distinguishing between target sperm cells 160 and non-target sperm cells 160. When a group of non-target sperm cells 160 is identified, the focusing energy beam 158 is cut off, deflected, or otherwise interrupted for a brief period to allow the non-target sperm cells 160 (i.e., one of the X chromosome cells and Y chromosome cells) to pass unimpeded. This group can be any predetermined number of non-target sperm cells 160. After the non-target sperm cells 160 have passed through the action zone, the continuous emission mode is restarted.

[0369] In both the target operation mode and the continuous emission operation mode, sperm cells 160 that are too close to or overlap each other in the sample fluid 120 are killed or rendered incapable, even if one of these sperm cells 160 is a non-target sperm cell 160 rather than a target sperm cell 160. As used herein, the term "too close" means that two or more sperm cells are present within the range of the focused energy beam 158 such that both objects 160 are sufficiently acted upon by the focused energy beam 158, in which the desired action occurs in both cells. Furthermore, the focused energy beam 158 kills or renders incapable sperm cells 160 that cannot be effectively identified as either non-target or target objects 160, to ensure the overall sample purity of the differentiation product 165. The reason why the differentiation system of the present invention may fail to effectively identify non-target sperm cells 160 could be due to flow problems within the microfluidic channel 164, staining problems, or a combination of both. Because the system of the present invention makes mistakes in killing or disabling target sperm cells 160 or any sperm cells 160 that cannot be effectively identified as target or non-target, more pulses 158 can be used than the total number of actual target sperm cells 160 present in sample 129.

[0370] In one embodiment, as described above, it is preferable to include short "recharge / recharge times" between the laser pulses 158 of the focusing energy device 157. First, the spacing between two consecutive sperm cells 160 is not uniform, but rather follows a Poisson distribution. In the sample fluid flow 120, a certain percentage of sperm cells 160 are relatively close to each other. To employ the focusing energy device 157 with closer spacing (i.e., shorter elapsed times), shorter "recharge / recharge times" of the focusing energy beam 158 will be permitted. Furthermore, if two sperm cells 160 are too close to each other, causing their fluorescence signals to interfere with each other and making it impossible to clearly identify whether a sperm cell 160 is a target cell or a non-target cell, the focusing energy device 157 will have to fire multiple laser pulses 158 in a short time interval to kill or disable all unidentified sperm cells 160, thereby maximizing sample purity. Therefore, to achieve higher throughput, a short average elapsed time between pulses 158 is typically required.

[0371] As described above, after being acted upon by the focused energy device 157, the differentiated sample 120 is collected in the collection device 188. Therefore, in one embodiment, the collected product 165 comprises both non-target sperm cells 160 and target (i.e., killed, altered, damaged, or incapacitated) sperm cells 160, maintaining the same sex ratio as the original sample 120. Collection in a single container 188 does not affect the overall quality of the sample 120. Therefore, in one embodiment, the final product 165 for final fertilization comprises both non-target sperm cells 160 and target sperm cells 160. Alternatively, subsequent product 165 separation techniques (i.e., flow cytometry, electrostatic plates, holographic optical capture, etc.) can be used, for example, to separate the sperm cells 160 of the product 165 into live or dead / incapacitated sperm cells 160, or centrifugation can be used to remove unwanted waste, such as residues of killed or incapacitated target sperm cells 160.

[0372] In one embodiment, the microfluidic chip system of the present invention is used in conjunction with a separation or isolation mechanism, such as an exemplary piezoelectric actuator assembly apparatus described in U.S. Patent Application No. 13 / 943,322, filed July 16, 2013, or an optical capture system described, for example, in U.S. Patent Nos. 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927, and 8,653,442, the entire contents of which are incorporated herein by reference.

[0373] In one implementation, such as Figures 6B to 6CAs shown, the main channel 164 is shortened over the action chamber 129, thus distinguishing the object 160 from the output channel 141 and the output port 112 in the form of droplets 187 before falling towards the collection device 188. The operation of the focusing energy device 157 is the same, except that the object 160 exits the outlet of the output channel 112 (see...). Figure 6B Or during the falling of discontinuous droplets 187 (see...) Figure 6C And it is activated before entering the collection device 188.

[0374] Distinguishing the later collection of products

[0375] In one embodiment, the distinguishing object 160 is collected in a container 188 containing 20% ​​tris(hydroxymethyl)aminomethane (note: commercially available, such as those sold by ChataBiosystems), which is positioned below the output port 112 of the chip 100. In one embodiment, the contents of the container 188 are circulated at predetermined intervals to ensure mixing of the product 165 therein. In one embodiment, the container 188 can be replaced with a new container 188 when it reaches a predetermined volume (i.e., 18 ml).

[0376] In one implementation, antibiotics are added to a filled container 188 (i.e., 0.5 ml of CSS antibiotics is added to 30 ml of product 165), the time is recorded, and multiple containers 188 containing product 165 are collected in a large container and cooled. In one example, three containers 188 containing product 165 with antibiotics are placed in a 400 ml plastic container and placed in a cooling chamber at a time.

[0377] In one implementation, after cooling for a predetermined time (i.e., 2 hours), a tris(hydroxymethyl)aminomethane B extender (14% glycerol tris(hydroxymethyl)aminomethane) was added in two portions to each tube at predetermined time intervals (i.e., 15 minutes). After the latest sample 120 had been cooled for the predetermined time (i.e., 2 hours) and the B extender had been added, sample 120 was centrifuged at 850 x G for 20 minutes in a refrigerated centrifuge at 5°C. The supernatant was aspirated from each tube, leaving approximately 0.2 ml of particulate matter. All particulate matter was combined into a single pre-weighed container (i.e., tube), and the concentration of the combined particulate matter was determined using a commercially available counting protocol (i.e., SpermVision), and the final concentration was calculated to be 11.35 x 10⁶ cells / ml. The final volume was adjusted using a complete tris(hydroxymethyl)aminomethane A+B+CSS antibiotic extender (i.e., 20% egg yolk tris(hydroxymethyl)aminomethane + 14% glycerol tris(hydroxymethyl)aminomethane in a 50:50 ratio).

[0378] In an exemplary embodiment of sperm cell 160, a printed semen capillary is filled and sealed, and the capillary is frozen using liquid nitrogen vapor.

[0379] In one embodiment, post-freezing quality control measures include a bacterial infection step, wherein the frozen capillary tubes are thawed in a water bath and sterilized by wiping the thawed capillary tubes with alcohol. The capillary contents are spread onto blood agar plates (5% sheep blood) and incubated at 37°C for 24 hours to determine the bacterial contents.

[0380] In one implementation, quality control measurements of the mobility used for advancement include thawing a frozen capillary tube in a water bath and draining the capillary tube contents into smaller test tubes placed during a tube warming phase. Commercial methods, such as mobility analysis or sperm visualization, are used to determine the number of motile cells in each capillary tube.

[0381] In one implementation, quality control measures for sample purity include thawing a frozen capillary tube in a water bath and isolating live cells by treating the live cells with glass fibers. The live cell population is then subjected to FISH (fluorescence in situ hybridization) analysis to determine the sex chromosome ratio of the cattle.

[0382] Multisystem

[0383] In one implementation, multiple microfluidic chips 100, optical interrogation devices, and focusing energy devices are arranged in parallel to improve throughput.

[0384] Now refer to Figure 18 In one embodiment, a multi-system layout 400 is shown, for example, having "n" systems 401, 402, etc., each system equipped with a focusing energy device 157. In one embodiment, the multi-system layout 400 includes a single interrogation device 147 and a single interrogation beam 148 configured to provide fluorescence excitation energy for detecting a subject 160 (e.g., sperm DNA content).

[0385] In one embodiment, the multi-system layout 400 also includes beam-shaping optics 181 and a series of beam splitters 189, each beam splitter providing 50 / 50 separation of the incoming power. This makes the power of the beams 158 used for each of the multiple systems 401, 402, etc., nearly equal.

[0386] External devices

[0387] Separation device

[0388] In one embodiment, after the focusing energy device 157 acts on the object 160 flowing through the microfluidic chip 100 rather than into the output channel 141, a separation mechanism (i.e., a piezoelectric actuator assembly (external or internal), an optical trapping assembly, an electrostatic plate, or other separation mechanism well known to those skilled in the art) can separate the target object 160 from the non-target object 160 (see, for example, U.S. Patent Application No. 13 / 943,322, filed July 16, 2013, or U.S. Patent Nos. 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927, and 8,653,442, the entire contents of which are incorporated herein by reference).

[0389] Therefore, in one exemplary embodiment, a target object 160 that has been damaged, killed, rendered incapable, destroyed, or altered by the focused energy device 157 can be separated from a non-target object 160, wherein the separation mechanism separates the target object into one of the output channels 140 to 142 and separates the non-target object 160 into the others of the output channels 140 to 142.

[0390] In another embodiment, after exiting from the output port 112, the target object is separated using a separation mechanism, for example by using an electrostatic plate well known in the art.

[0391] In another embodiment, when object 160 leaves or drips from output port 112, focusing energy device 157 acts on object 160, and thereafter separation mechanism also separates target object 160 from non-target object 160 or product 165.

[0392] Pumping mechanism

[0393] like Figures 19 to 21 As shown, in one embodiment, the pumping mechanism includes a system with a pressurized gas 235 that provides pressure for pumping the sample fluid mixture 120 from the reservoir 233 (or sample tube 233) to the sample inlet 106 of the microfluidic chip 100 via a conduit 242.

[0394] Central reservoir 240 (see) Figures 20 to 21 ) or a single reservoir 241 (see Figure 19 The reservoir contains a sheath fluid or buffer fluid 163 and is connected via conduits to the respective sheath or buffer inlets 107, 108, and 172 of the microfluidic chip 100 for introducing the sheath fluid or buffer fluid 163 therein. In one embodiment, the reservoir is a removable container 237 (see...). Figures 20 to 21The microfluidic chip 100 is disposed in a pressurized container 240, and pressurized gas 235 pushes sheath fluid or buffer fluid 163 into the microfluidic chip 100. In one embodiment, the pressurized container 240 pushes sheath fluid or buffer fluid 163 into a manifold 238 having multiple different outlets (see...). Figure 21 This allows the sheath fluid or buffer fluid 163 to be delivered to the sheath or buffer inlet 107, sheath or buffer inlet 108, or sheath or buffer inlet 172 of the chip 100 via conduits 231a, 231b, or 231c, respectively. Although a three-inlet sheath fluid or buffer fluid arrangement is shown (see...), Figure 1D However, those skilled in the art will recognize that fewer or more conduits can be used to deliver the sheath fluid or buffer fluid to the microfluidic chip 100. Furthermore, although in Figures 18 to 21 The conduit is shown as entering the microfluidic chip 100, but the arrangement of the conduit relative to the input ports on the chip 100 is not shown in precise order. Furthermore, those skilled in the art will recognize that the conduit enters the chip 100 via a chip socket 200 (discussed below).

[0395] In one embodiment, each individual reservoir 241 or central reservoir 240 includes a pressure regulator 234 that regulates the pressure of the gas 235 within the reservoir 241 or reservoir 240 (see [reference]). Figures 19 to 21 In one embodiment, pressure regulator 239 regulates the pressure of gas 235 within sample container 233. Flow meter 243 and flow valve 244 control the pumping of sheath fluid or buffer fluid 163 (via seat 200) into sheath or buffer inlet 107, sheath or buffer inlet 108, or sheath or buffer inlet 172 via conduits 231a, 231b, or 231c, respectively. Thus, conduits 230, 231a, 231b, and 231c are used when initially loading sheath fluid or buffer fluid 120 into chip 100, and these conduits can be operated through chip 100 to load sample fluid 120 into sample inlet 106, or sheath or buffer inlet 107, sheath or buffer inlet 108 (and sheath or buffer inlet 172).

[0396] In one embodiment, flow meter 243 is used to provide feedback to flow valve 244 placed in the sheath flow path (see Figures 19 to 20 This allows for stable flow with constant velocity in microfluidic channels. Utilizing precise flow control, the overall flow rate variation is less than 1% of the set flow rate, and the change in the target object 160's travel speed is less than 1% during detection and between the interrogation / detection point and the action point.

[0397] In one embodiment, a sheath fluid or buffer fluid 163 is pumped through a vacuum chamber (not shown) located between a pressure vessel and a manifold to remove dissolved gases from the sheath fluid or buffer fluid. Inside the vacuum chamber, a gas-permeable conduit is positioned between an inlet port and an outlet port. As the sheath fluid or buffer fluid travels through the vacuum chamber via the gas-permeable conduit, dissolved gases permeate through the conduit walls while the liquid remains inside the conduit. The applied vacuum facilitates gas permeation through the conduit walls. In one embodiment, the gas-permeable conduit is made of a hydrophobic porous material (e.g., expanded polytetrafluoroethylene (EPTFE)) that exhibits high inlet pressure for liquids but is permeable to gases.

[0398] Computer control

[0399] In one embodiment, the user interface of the computer system 156 includes a computer screen that displays an object 160 in the field of view acquired by the CCD camera 182 on the microfluidic chip 100.

[0400] In one implementation, computer 156 or controller 156 controls any external device, such as a pump for pumping any sample fluid 120, sheath fluid, or buffer fluid 163 into the microfluidic chip 100 (i.e., ...). Figures 19 to 20 The pumping mechanism (if used) also controls any heating devices that set the temperature of the fluids 120 and 163 input into the microfluidic chip 100.

[0401] According to the illustrative implementation, any operation, step, control option, etc., can be implemented by instructions stored on a computer-readable medium (such as computer memory, database, etc.). When the instructions stored on the computer-readable medium are executed, these instructions can cause the computing device 156 to perform any operation, step, control option, etc., described herein.

[0402] The operations described in this specification can be implemented as operations performed by a data processing apparatus or processing circuitry on data stored on one or more computer-readable storage devices or received from other sources. Computer programs (also referred to as programs, software, software applications, scripts, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and computer programs can be configured in any form, including as standalone programs or as modules, objects, subroutines, objects, or other units suitable for use in a computing environment. A computer program may, but does not necessarily, correspond to a file in a file system. A program may be stored in a portion of a file that holds other programs or data (i.e., one or more scripts stored in a markup language document) in a single file dedicated to the program in question, or in multiple collaborating files (e.g., a file storing one or more modules, subroutines, or portions of code). A computer program can be configured to execute on one or more computers located in one place or distributed across multiple locations and interconnected via a communication network. Processing circuitry suitable for executing computer programs includes, for example, general-purpose microprocessors and special-purpose microprocessors, and any one or more processors of any type of digital computer.

[0403] Microfluidic chip socket

[0404] In one embodiment, the microfluidic chip 100 is mounted on a chip holder 200 (see...). Figures 22A to 23B The chip holder 200 is mounted on a translation platform (not shown) to allow the holder 200 to be positioned relative to the interrogation device and the focusing energy device. The microfluidic chip holder 200 is configured to hold the microfluidic chip 100 in a position such that the beam 148 from the interrogation device can intercept the object 160 at the opening 150 as described above, and the focusing energy device 157 can act on the object 160.

[0405] The following describes the mechanism for attaching chip 100 to socket 200 and its operation method; however, those skilled in the art will recognize that these devices can be any configuration for accommodating microfluidic chip 100, as long as the objectives of the present invention are met.

[0406] like Figures 22A to 23B As shown, in one embodiment, the microfluidic chip socket 200 is made of a suitable material (such as aluminum alloy or other suitable metal / polymer material) and includes a motherboard 201 and a mounting plate 202. The motherboard 201 and the mounting plate 202 can be of any suitable shape, but their configuration depends on the layout of the chip 100 and the requirements for accessing the chip 100.

[0407] In one implementation, the motherboard 201 has a basic L-shape, but the shape of the socket 200 depends on the layout of the chip 100 (see [reference]). Figures 22A to 23B The L-shaped mainboard 201 includes a plurality of slots configured to receive mounting screws 219 for mounting the seat 200 onto the translation platform. Any number of slots of any suitable shape and size may be included in the mainboard 201. In one embodiment, four screws 219 are used to mount the seat 200 onto the translation platform and adjust its position. However, those skilled in the art will appreciate that any number of slots and mounting screws of any size can be used.

[0408] In one embodiment, the switching valve 220 is attached to the motherboard 201 above a mounting screw 219 located on one of the pillars of the L-shaped motherboard 201 (see Figure 201). Figures 22A to 23B Furthermore, on another pillar of the L-shaped main board 201, a pair of linear actuators (such as cylinder actuators 207A and 207B) are positioned on the same side of the main board 201 as the switching valve 220 (see [link]). Figures 22A to 23B ).

[0409] In one embodiment, mounting plate 202 is attached to motherboard 201 on the other side and along another pillar of L-shaped motherboard 201 (see...). Figures 22A to 23B Assembly plate 202 includes multiple holes that accommodate multiple assemblies 204-206 (see, for example, [reference needed]). Figures 22A to 22B These assemblies 204-206 are configured to receive and engage with external conduits (see...). Figures 19 to 23B The external conduit is used to deliver fluid / samples to the microfluidic chip 100. In one embodiment, the mounting plate 202 includes three fittings 204-206 for alignment with the sheath or buffer inlet 107, the sheath or buffer inlet 108, and the sample inlet 106, respectively (see Figures 1B and 106). Figures 22A to 22B In another embodiment, four fittings 204-206 and fitting 216 are used to align with sheath or buffer inlet 107, sheath or buffer inlet 108, sample inlet 106, and sheath or buffer inlet 172, respectively (see [reference]). Figure 1A and Figures 23A to 23B However, those skilled in the art will recognize that the assemblies can be arranged in any number and location to align with that number of inlets in the microfluidic chip 100 and to allow fluid transfer from one or more reservoirs via conduits (see [link]). Figures 19 to 21 ).

[0410] In one embodiment, a pair of open washers 203A and 203B adhere the mounting plate 202 and the cylinder actuators 207A and 207B to the mainboard 201 (see Figure 201). Figures 22A to 23BTherefore, it is easy to remove the assembly board 202 from the main board 201 without removing the switching valve 220 and the cylinder actuators 207A and 207B from the main board 201.

[0411] In one embodiment, both cylinder actuators 207A and 207B include a piston (not shown) having a piston rod coupled thereto and extendable and retractable relative to the cylinder body portion. In one embodiment, air is supplied to the switching valve 208 through port 209 and discharged from cylinder actuator 207A to ports 212 and 213 through ports 210 and 211, respectively. Air is also supplied from ports 214A and 214B of cylinder actuator 207A to ports 215A and 215B of cylinder actuator 208A, respectively.

[0412] The toggle switch 220 of the switching valve 208 opens and closes to allow or prevent air from the air source from entering and reaching the cylinder actuators 207A and 207B through port 209, respectively. When air is supplied to the cylinder actuators 207A and 207B, the piston rods of the cylinder actuators 207A and 207B extend outward to the open position (not shown), and the mounting plate 202 is pushed outward from the microfluidic chip 100 to the open position to allow the user to load (or unload) the microfluidic chip 100 (see...). Figures 22A to 23B When the air supply is shut off, the piston rods of cylinder actuators 207A and 207B retract inward to the closed position (e.g., Figures 22A to 23B As shown), the assembly plate 202 is pulled toward the chip 100 and pressed onto the chip 100, forming a liquid seal between the chip 100 and the connector for the tubing used for sheath fluid or buffer fluid and sample fluid.

[0413] In one embodiment, when the microfluidic chip 100 is in the closed position, an O-ring (which is disposed on the surface of the mounting plate 202 and between the mounting plate 202 and the chip 100) forms a substantially leak-proof seal to protect the microfluidic chip 100 from damage. However, those skilled in the art will appreciate that any number and configuration of O-rings or gaskets can be used.

[0414] In one embodiment, the base 200 is positioned such that the chip 100 is at a sufficient height to accommodate at least one collection container 188 disposed below the chip 100. In another embodiment, the collection container is disposed between the output ports 111-113.

[0415] The construction and arrangement of the microfluidic chip system with focused energy devices shown in the various illustrative embodiments are merely illustrative. It should be noted that the orientation of the individual elements may vary according to other illustrative embodiments, and variations are intended to be covered by this invention. Although only some embodiments have been described in detail herein, many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described herein (e.g., variations in the size, dimensions, structure, shape, and proportions of individual elements; variations in parameter values; variations in mounting arrangements; variations in the use of materials, colors, orientations, etc.). Some elements shown as integrally formed may be composed of multiple parts or elements, the positions of elements may be reversed or otherwise changed, and the nature, number, or position of discrete elements may be altered or changed. According to alternative embodiments, the order or sequence of any process, logical algorithm, or method steps may be changed or rearranged. Other substitutions, modifications, alterations, and omissions may be made in the design, operating conditions, and arrangement of the various illustrative embodiments without departing from the scope of this invention. All such modifications and variations are included within the scope of this invention and protected by the appended claims.

Claims

1. An apparatus for identifying an object, the apparatus comprising: A microfluidic chip having multiple channels, the multiple channels including: The main fluid channel is into which a sample fluid mixture of the object to be identified is introduced; A ramp and a conical structure are provided in the main fluid channel before the intersection of the first intersection of the main fluid channel and the first plurality of sheath fluid channels, the ramp causing the sample fluid mixture to deviate from the central plane; Multiple sheath fluid channels are provided, into which sheath fluid is introduced to orient the object in the main fluid channel in a predetermined direction while maintaining laminar flow in the main fluid channel; wherein the multiple sheath fluid channels hydrodynamically concentrate the object, so that the object is positioned in a confined core volume when the object flows through the main fluid channel; The plurality of sheath fluid channels include: The first plurality of sheath fluid channels intersect the main fluid channel at a first intersection, thereby allowing the sheath fluid to surround and compress the sample fluid mixture on all sides, thus transforming the sample fluid mixture into a relatively small, narrow flow confined by the sheath fluid, while maintaining laminar flow in the main fluid channel; and Interrogation device, the interrogation device detecting and interrogating the oriented object in the main fluid channel; and A focused energy device that performs an action on the object.

2. The apparatus of claim 1, further comprising: in, The interrogation device detects and interrogates the object to determine information about the object; The information about the object determines whether the object will be targeted by the focusing energy device, wherein the object targeted by the focusing energy device is the target object; The action of the focusing energy device is applied to the target object or the area surrounding the target object.

3. The apparatus of claim 2, further comprising: At least one output channel guided from the main fluid channel removes the object from the microfluidic chip, wherein the at least one output channel removes both the target object and the non-target object from the microfluidic chip.

4. The apparatus of claim 3, further comprising: An action chamber, in which the interrogation device interrogates the hydrodynamically focused object in the sample fluid mixture, the action chamber being disposed downstream of the first intersection in the microfluidic chip; as well as A light source that emits a light beam into the actuation cavity to illuminate and excite the object in the sample fluid mixture.

5. The apparatus of claim 4, wherein, The light beam excites fluorescence in the object, thereby distinguishing the target object from non-target objects.

6. The apparatus of claim 4, further comprising: An optical signal detector that detects the light beam and converts the light beam into an electrical signal; as well as A controller that analyzes the electrical signals to determine whether to target the object.

7. The apparatus of claim 4, wherein, The main fluid channel enters the actuation chamber in a conical shape.

8. The apparatus of claim 1, wherein, The object is a cell.

9. The apparatus of claim 8, wherein, The cells to be acted upon by the focused energy device include at least one of viable or motile bovine sperm, distinguishable from inactive or immobile bovine sperm, or bovine sperm distinguishable by sex or other sex-specific variables.

10. The apparatus of claim 1, wherein, Following the inquiry into the object, the focused energy device acts on the object for a predetermined amount of time.

11. The apparatus of claim 1, further comprising: Multiple microfluidic chips arranged in parallel, each microfluidic chip containing multiple sample fluid mixtures; The single interrogation device is used for at least one of the plurality of microfluidic chips.

12. An apparatus for identifying an object, the apparatus comprising: A microfluidic chip having multiple channels, the multiple channels including: The main fluid channel is into which a sample fluid mixture of the object to be identified is introduced; A ramp and a conical structure are provided in the main fluid channel before the intersection of the first intersection of the main fluid channel and the first plurality of sheath fluid channels, the ramp causing the sample fluid mixture to deviate from the central plane; Multiple sheath fluid channels, into which sheath fluid is introduced, the sheath fluid orienting the object in the main fluid channel in a predetermined direction while maintaining laminar flow in the main fluid channel; The plurality of sheath fluid channels intersect the main fluid channel at a first intersection, thereby compressing the sample fluid mixture on at least both sides, thus transforming the sample fluid mixture into a relatively small, narrow flow confined by the sheath fluid, while maintaining laminar flow in the main fluid channel; and Interrogation device, the interrogation device comprising a first laser for detecting and interrogating the oriented object in the main fluid channel; and A second laser, which provides electromagnetic energy to the object, is positioned downstream of the interrogation device.

13. The apparatus according to claim 12, further include: in, The interrogation device detects and interrogates the object to determine information about the object; The information about the object determines whether the object will be targeted by the second laser; and The second laser acts on the target object or the area surrounding the target object.

14. The apparatus of claim 13, further comprising: At least one output channel is drawn from the main fluid channel, the at least one output channel removing the object from the microfluidic chip; The at least one output channel removes both the target object and the non-target object from the microfluidic chip.

15. The apparatus according to claim 14, wherein, The second sheath fluid channel compresses the sample fluid mixture from at least both sides.

16. The apparatus according to claim 14, wherein, The multiple sheath fluid channels hydrodynamically converge on the object, causing the object to face the predetermined direction as it flows through the main fluid channel, and to be positioned within a restricted core volume.

17. The apparatus of claim 16, further comprising: An action chamber, wherein the first laser of the interrogation device interrogates the hydrodynamically focused object in the sample fluid mixture within the action chamber, the action chamber being disposed downstream of the first intersection in the microfluidic chip, the first laser being located on one side of the main fluid channel.

18. The apparatus of claim 17, further comprising: An optical signal detector that detects a light beam emitted by the object and converts the light beam into an electrical signal; as well as A controller that analyzes the electrical signals to determine whether to target the object.

19. The apparatus according to claim 17, wherein, The tapered structure is located at the entrance point of the first intersection in the microfluidic chip, so that the width of the main fluid channel gradually narrows before the entrance point.

20. The apparatus according to claim 19, wherein, The main fluid channel enters the actuation cavity in a conical shape, so that the width of the main fluid channel gradually narrows before intersecting with the actuation cavity.

21. The apparatus according to claim 12, wherein, A ramp is provided in the main fluid channel before the first intersection where the main fluid channel and the plurality of sheath fluid channels intersect, so that the width of the main fluid channel narrows before the first intersection.

22. The apparatus according to claim 12, wherein, The object is a cell.

23. The apparatus according to claim 22, wherein, The cells to be acted upon by the second laser include at least one of viable or motile bovine sperm, distinguishable from inactive or immobile bovine sperm, or bovine sperm distinguished by sex or other sex-specific variables.

24. The apparatus according to claim 12, wherein, Following the inquiry of the object, the second laser acts on the object for a predetermined amount of time.

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