Method and apparatus for separating biological entities

Abstract

To provide a method and a device capable of achieving magnetic separation of a high flow of cells, and dissociating the cells from an aggregate in a short period without damage to cells.SOLUTION: There is provided a device for separating a biological entity in a fluid sample, comprising: a putting channel having an outlet; a first discharge channel having a first inlet which is fluid coupled to the outlet and a second discharge channel having a second inlet; an optical detector; an actuator comprising a selector gate and a voice coil; and a magnetic field indicating a reverse magnetic pole which traverses the voice coil and is applied. Before the fluid sample passes the putting channel, and the first entity passes the outlet, the optical detector detects the first entity, a current is applied to the voice coil for actuating the voice coil for moving the actuator to a first selection position, the selector gate blocks the second inlet, and the first entity passes the outlet and the first inlet and advances to a first discharge passage.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention generally relates to methods and devices for separating biological entities, including cells, bacteria, and molecules, from human blood, body tissues, body fluids, and other human-related biological samples. The disclosed methods and devices can also be utilized to separate biological entities from animal and plant samples. More specifically, the present invention relates to methods and devices for achieving separation of biological entities using, individually or in combination, one or more microfluidic separation devices / chips ("UFL") and one or more magnetic separation devices ("MAG"). For illustrative purposes, "cells" will be primarily used hereinafter as a typical representative of biological entities in general. However, it will be understood that the methods and devices as disclosed in the present invention can be readily applied to other biological entities without limitation. Background technology

[0002] Separation of a biological entity from a fluid-based solution, such as the separation of a specific type of white blood cell from human blood, generally involves a first step of identifying a specific target biological entity, followed by a second step of physically extracting the identified target biological entity from the fluid-based solution. In human blood, various types of biological cells may have various types of surface antigens or surface receptors, which are also referred to as surface markers in the present invention. Certain surface markers on a certain type of cell may be unique to that type of cell and can be used to specifically identify that type of cell from a blood sample.

[0003] Figures 1A-1C show examples of identifying or labeling a target cell 1 using a superparamagnetic label 2 ("SPL") in Figure 1A, an optical fluorescent label 3 ("OFL") in Figure 1B, and both SPL2 and OFL3 together in Figure 1C.

[0004] In FIG. 1A, cell 1 has a surface marker 11. SPL2 is conjugated to a surface antibody or ligand, also referred to as a "probe" 21, which specifically binds to the surface marker 11 on cell 1. A large amount of SPL2 carrying probe 21 is placed in a solution containing cell 1. After incubation step 9, probe 21 selectively binds to surface marker 11 due to its specificity, and multiple SPL2 bind to the surface of cell 1. Thus, cell 1 is magnetically identified or labeled with SPL2, i.e., becomes magnetically labeled cell 10. A magnetic field with a sufficient magnetic field gradient can be applied to cell 10 to generate a physical force on SPL2 attached to the surface of cell 10. With sufficient strength, the physical force acting through SPL2 on cell 10 can be used to separate and physically remove cell 10 from the solution.

[0005] In FIG. 1B, cell 1 has a surface marker 12. OFL3 is conjugated to probe 22, which specifically binds to surface marker 12 on cell 1. A large amount of OFL3 carrying probe 23 is added to a solution containing cell 1. After incubation step 9, probe 22 selectively binds to surface marker 12 with specificity, resulting in multiple OFL3 molecules attached to the surface of cell 1. Thus, cell 1 is optically identified or labeled with OFL3, i.e., optically labeled cell 20. Using an optical-based cell separation system, cell 1 can be separated from its solution based on the optical signal generated by OFL3 under excitation light. One type of such optical-based cell separation system is a flow cytometer, in which the solution flows continuously through a conduit within the flow cytometer. At least one excitation light source generates a light spot at a first optical wavelength on the liquid flow through the conduit. If OFL3 is present at the light spot, it is excited by the first wavelength and emits light at a second wavelength. When cells 1 bound to OFL3 pass through the light spot in the flow, the OFL3 bound to the cells 1 emits an optical signal at a second wavelength. The intensity of this optical signal and the duration of the cell 1's passage through the light spot can be used by the flow cytometer to identify the presence of the cells 1. The cells 1 are then diverted into a second liquid flow path or mechanically removed from the liquid flow, thus separating them from the fluid base. In practice, the OFL3 bound to the cells 1 can be various types of fluorescent dyes or quantum dots, producing excitation light at multiple wavelengths. Multiple excitation light sources can also be used in the same flow cytometer system to produce excitation light spots at different positions in the liquid flow with different excitation light wavelengths. Combining various wavelengths produced by OFL3 on the same cells 1 can be used to increase the specificity of cell separation, especially when a combination of various types of surface markers 12 is required to specifically distinguish a subcategory target cell 1 population from the main category of cells of the same type, such as CD4 T cells from other white blood cells.

[0006] In FIG. 1C, cell 1 has both surface markers 11 and 12. Both SPL2 conjugated to probe 21 and OFL3 conjugated to probe 22 bind to the cell 1 surface after incubation step 9, forming magnetically and optically labeled cell 30. Cell 30 can be separated using a combination of magnetic and optically based cell separation systems, where magnetic separation via SPL2 can provide a fast, first-stage separation of a cell category containing cell 30, while optical separation via OFL3 can provide a second-stage separation of cell 30 after magnetic separation with higher specificity. Alternatively, cell 30 can be separated via OFL3 in the first stage and via SPL2 in the second stage. In either case, SPL2 and OFL3 together can serve to increase the speed, efficiency, and specificity of cell 1 separation compared to FIGS. 1A and 1B.

[0007] FIG. 2A shows an example of conventional magnetic separation via SPL2. In a vessel 5, a liquid solution 6 contains cells 10 (FIG. 1A) or cells 30 (FIG. 1C) that bind to multiple SPL2 on the cell surface. A magnet 4, preferably a permanent magnet, is positioned adjacent to the wall of the vessel 5. The magnet 4 has a magnetization represented by arrows 41 showing north ("N") and south ("S") poles on the top and bottom surfaces of the magnet 4. The magnetic field generated by the magnetization 41 in the solution 6 is higher at the vessel 5 wall directly opposite the north face of the magnet 4 and lower at locations within the solution 6 further away from the magnet 4, thus creating a magnetic field gradient pointing toward the magnet 4 within the solution 6. The SPL2 bound to the cells 10 / 30 are superparamagnetic, meaning that they are effectively nonmagnetic in the absence of a magnetic field but gain a magnetic moment in the presence of the magnetic field generated by the magnet 4. Due to the magnetic moment of the SPL2 and the magnetic field gradient from the magnet 4, the cells 10 / 30 are attracted by a force generated by the magnetic field from the magnet 4 toward the magnet 4. After a sufficient time 7, the cells 10 / 30 may deplete from the solution 6 and form clusters on the inner surface of the wall of the container 5 opposite the magnet 4. In conventional practice, the solution 6 may be removed from the container 5 while maintaining the position of the magnet 4 relative to the container 5, thus holding the cells 10 / 30 as clusters against the inner surface of the container 5. The magnet 4 may then be removed from the container 5. In the absence of a magnetic field, the clusters of cells 10 / 30, along with any unbound free SPL in the clusters, will self-degass and become non-magnetic over time, allowing the cells 10 / 30 to be removed from the container 5 as individual cells 10 / 30.

[0008] Conventional methods such as those shown in Figure 2A have limitations in practical application. While SPL2 is superparamagnetic, the size of the underlying superparamagnetic particles ("SPNs") contained in SPL2, e.g., iron oxide particles, ranges from 10 nm (nanometers) to 30 nm. The smaller the particle size, the more efficiently the particle becomes superparamagnetic, but it is more difficult to obtain a magnetic moment in the presence of a magnetic field. The larger the particle size, the more difficult it is to make the particle nonmagnetic when the magnetic field is removed. SPL2 generally consists of SPNs dispersed in a nonmagnetic matrix. For example, some SPL2 are solid spheres formed by uniformly mixing SPNs in a polymer base, typically larger than 1 μm (μm). In other cases, SPL2 are solid beads formed by mixing SPNs in an oxide or nitride base, e.g., iron oxide nanoparticles in a silicon oxide base, and can be hundreds or tens of nanometers in size. For the cells in Figure 2A to be suitable for further cell processing, including cell culture and cell analysis, the SPL2 size should be smaller than the cells themselves, typically a few micrometers. Therefore, submicrometer (<1 μm) SPL2 sizes are desirable. SPL2 sizes less than 500 nm are more preferable. SPL2 sizes less than 200 nm are most preferable. However, the smaller the average SPL2 size, the greater the statistical variability in SPL2 size. Figure 2B shows an example schematic of a single SPL2 magnetic moment in the presence of an applied magnetic field. The solid curve 22 indicates that the SPL2 has the nominal, or average, size of the population, and the SPL2 magnetic moment increases with increasing magnetic field. As the magnetic field strength increases from 0 to Hs, the nominal-sized SPL magnetic moment initially increases linearly with magnetic field strength until it reaches a saturation region where the magnetic moment reaches a plateau at Ms, which is determined by the saturation moment of the SPN material within the SPL2. For SPL2s smaller than the nominal size, curve 23 shows that at the same magnetic field strength, the smaller the SPL2 size, the lower the resulting moment and therefore the lower the magnetic force, and a higher magnetic field is required to reach the saturation magnetic moment Ms.In the case of SPL2 having a size larger than the nominal size, curve 24 shows that at the same magnetic field strength, the larger the size of SPL2, the easier it is to saturate to Ms at a lower field and obtain a higher moment.

[0009] Referring back to FIG. 2A , for sub-micron-sized SPL2, which is appropriate for cell separation and processing, the conventional method of FIG. 2A is limited by its inability to generate high magnetic field strength and strong magnetic field gradients in solution 6 at positions further away from the wall of container 5 facing the north face of magnet 4. Therefore, smaller SPL2, shown in curve 23 of FIG. 2B, at the far end of container 5 from magnet 4 may be difficult to magnetize by the magnetic field of magnet 4, resulting in a smaller force for moving cells 10 / 30 toward magnet 4. It may take a significant amount of time to reach complete depletion of cells 10 / 30 in solution 6 within container 5. Meanwhile, the volume of container 5 is also limited by the fact that the magnetic field strength from magnet 4 may not be sufficient to magnetize smaller SPL2, shown in curve 23 of FIG. 2B, at larger container 5 sizes. Besides the slow overall process, another drawback of the conventional method of Figure 2A is that the procedure as described in Figure 2A typically involves exposing the cell 10 / 30 mass to the atmosphere during the steps of solution removal and subsequent removal of the cells 10 / 30 from the container 5. Such exposure to the atmosphere poses challenges in achieving sterile isolation of the cells 10 / 30 for clinical purposes as well as the risk of cell 10 / 30 damage or death, which negatively impacts further processing of the cells 10 / 30.

[0010] FIG. 3A shows another example of magnetic separation of cells 10 / 30 using SPL2 in the prior art. In FIG. 3A, solution 6 containing cells 10 / 30 is passed through column 31 packed with ferromagnetic or ferromagnetic spheres 36. By applying a magnetic field across the column with magnets 32 and 33 (dotted line 34 indicates the direction of the applied magnetic field), the spheres 36 can be magnetized by the magnetic field, generating a localized magnetic field in the gaps between adjacent spheres 36. Such a localized magnetic field and the magnetic field gradient across the gaps between spheres 36 can be strong due to the small size of the gaps. As indicated by arrow 35, when SPL2 in solution 6 pass through the gaps between spheres 35 during the downward flow of solution 6, SPL2 of all sizes are effectively magnetized, and the SPL2 are attracted to the surfaces of various spheres 36 and separated from solution 6. The prior art of FIG. 3A can effectively avoid the problem of air exposure in FIG. 2A and can maintain a higher separation rate of cells 10 / 30 during flow 35 than FIG. 2A. However, an inherent problem with the method of FIG. 3A is that if the spheres 36 are ferromagnetic, or if they are ferromagnetic and much larger than the cells 10 / 30, magnetic domains will exist within the spheres 36 even after the magnets 32 and 33 are removed from the column 31. Such magnetic domains, and the magnetic domain walls between the magnetic domains, inevitably generate localized magnetic fields around the surface of the spheres 36, which continue to magnetize the SPL2 on the cells 10 / 30 and strongly attract the cells 10 / 30 once the magnets 32 and 33 are removed. Therefore, the cells 10 / 30 are inherently more difficult to remove from the column 31 of FIG. 3A than those of FIG. 2A. Losses of the cells 10 / 30 due to incomplete removal from the column 31 after separation are inherently high. In some prior art methods, pressurized, high-velocity buffer flow may be used to forcefully wash the cells 10 / 30 from the spheres in the column 36. However, such forced flow inevitably causes mechanical damage to the cells and still leaves a significant percentage of cells 10 / 30 in column 31 due to the strong domain wall magnetic field of spheres 36. Besides the loss of cells 10 / 30, another inherent problem with the method of Figure 3A is the introduction of spheres 36 as foreign objects into the flow of solution 6, which is undesirable for aseptic processes required for clinical applications.

[0011] FIG. 3B shows another prior art method similar to that of FIG. 3A, except that a mesh 37 made of ferromagnetic or ferromagnetic wires is introduced into column 31 instead of spheres or blocks 36. When a magnetic field 34 is applied by magnets 32 and 33, the wires of mesh 37 are magnetized, and adjacent wires of mesh 37 generate a local magnetic field around the wires. The gaps between the wires of the mesh allow fluid 6 to flow in direction 35 within the column. When cells 10 / 30 are close to the wires of mesh 37, they can be attracted to the wire surface due to the local magnetic field and magnetic field gradient generated by the wires of mesh 37. Compared to the prior art method of FIG. 3A, FIG. 3B allows the size of the wires and the size of the gaps in mesh 37 to be adjusted to balance the separation speed of cells 10 / 30 and cell loss in the column. However, because the gap between spheres 36 is actually much smaller than the gap size in mesh 37, the cell 10 / 30 separation rate in FIG. 3B is slower than in FIG. 3A. However, FIG. 3B still has the same cell loss problem as FIG. 3A. The magnetic domains in the wires of mesh 37 maintain the SPL2 magnetic moment after magnets 32 and 33 are removed and cells 10 / 30 are attracted to the wires by the magnetic domains and domain walls. In FIG. 3B, there is also loss of cells 10 / 30 due to the magnetic domains in the wires of mesh 37. Furthermore, FIG. 3B is the same as FIG. 3A in that it introduces mesh 37 as a foreign object in the flow of solution 6, which is undesirable for aseptic processing.

[0012] FIG. 3C shows another prior art technique in which magnets 32 and 33 are each mounted with a soft magnetic flux guide 38 having an apex. The flux guide 38 generates a localized magnetic field between the apexes of the guide 38, with a high field strength and gradient near the apex. FIG. 3C shows a cross-section of a conduit 39, which is essentially a circular tube, but a solution 6 containing cells 10 / 30 flows along the length of the tube 39 in a direction perpendicular to the cross-section. The tube 39 is positioned on one side of a gap at the apex. The magnetic field lines 34 exhibit a higher density closer to the gap, thereby exhibiting both a higher field strength and a higher field gradient toward the gap. The magnetic field 34 generates an effective force on the cells 10 / 30 in the solution 6, pulling the cells 10 / 30 from the solution 6 toward the inner wall of the tube 39 closest to the apex of the guide 38. Compared with the prior art of Figures 3A and 3B, the prior art of Figure 3C has the following advantages: (1) it does not introduce foreign matter into the flow path; (2) when magnets 32 and 33 are removed from the tube together with guide 38, there are no non-ferromagnetic or ferromagnetic spheres 36 or mesh 37 in the tube, thus avoiding magnetic domain structures associated with cell 10 / 30 loss.

[0013] However, the prior art of FIG. 3C also has inherent drawbacks. The first drawback is that the flow rate or volume of solution 6 in the conduit 39 is limited by the design of the prior art of FIG. 3C. The separation speed of cells 10 / 30 in the prior art, such as that of FIG. 3C, is insufficient for many applications. The circular conduit 39 shown in FIG. 3C experiences a high magnetic field and a high magnetic field gradient at the bottom of the conduit 39, where cells 10 / 30 closer to the bottom of the conduit 39 may experience a large force, pulling them toward the inner surface of the lower wall of the conduit 39 more quickly. However, for cells 10 / 30 closer to the top of the conduit 39, the magnetic field and gradient are significantly lower than at the bottom due to the narrow wedge gap and the position of the conduit 39 on one side of the gap. Therefore, cells 10 / 30 closer to the top of the conduit 39 experience a much smaller force and move toward the bottom of the conduit 39 at a much slower rate. Because of the limited length of the tube 39 perpendicular to its cross-section, all 10 / 30 of the cells in the fluid 6 flowing through the tube 39 must separate from the solution 6 and form aggregates on the inner surface of the tube near its apex before the solution 6 exits the tube 39. Because the velocity of the cells 10 / 30 moving from the top of the tube 39 is slower, the flow rate of the solution 6 must be slowed to allow sufficient time for all the cells 10 / 30 near the top of the tube 39 to be drawn into the aggregates. If the solution 6 flowed through the tube 39 at a higher velocity, the separation of the cells 10 / 30 from the solution would be incomplete. This limitation on flow rate due to the circular design of the tube 39 cannot be remedied by a smaller tube 39 size if the top of the tube were further away from the apex of the high magnetic field and high gradient. A circular tube 39 with a smaller cross-sectional size would bring the top of the tube 39 closer to the wedge gap. However, due to the smaller cross-sectional size, the volume of solution 6 flowing through tube 39 in a unit time frame, i.e., the flow rate of solution 6, decreases if the flow rate of solution 6 is maintained. To maintain the same flow rate as in the larger tube 39, the flow rate of solution 6 must be increased, thereby allowing less time for the cells at the top of the smaller sized tube 39 to migrate to the aggregation site, offsetting the effect of the smaller size of tube 39.

[0014] A second drawback of the prior art of FIG. 3C is that, in practical applications, the individual cells 10 / 30 cannot be dissociated from the cell 10 / 30 aggregate and unbound free SPL2 because the aggregate does not easily self-demagnetize after magnets 32 and 33, together with guide 38, are removed from tube 39. Demagnetization of SPL2 relies on the efficient dissociation of SPNs within the SPL2 into nanoparticles. However, as the aggregate forms an effectively larger mass of superparamagnetic material, the SPNs within the SPL2 are subjected to static magnetic fields from the numerous, closely packed SPNs from neighboring SPL2 in the aggregate, thereby reducing the superparamagnetic properties of the SPNs. In some cases, it takes a long time for the SPL2 of the cells 10 / 30 within the aggregate to self-demagnetize, which is impractical for many applications. In other cases, the aggregate does not self-demagnetize because the SPNs are more ferromagnetic in the aggregate form, which is undesirable. Because most of the interior area of ​​the circular tube 39 is occupied by empty space, while the aggregates are compressed at the bottom end of the tube 39, high-pressure water flow as utilized in Figure 3A is ineffective in Figure 3C; such water flows primarily through the top of the tube 39 without generating sufficient frictional force against the aggregates of cells 10 / 30 to remove them from the bottom wall of the tube 39. This shortcoming of the prior art of Figure 3C limits its utility, as the prior art does not provide an effective method for dissociating the aggregates and removing the cells 10 / 30 from the tube 39.

[0015] Prior art techniques are limited either in terms of causing cell loss and introducing foreign matter into the flow path, or in the flow rate of Solution 6 and the ability to extract separated cells from aggregates in an effective dissociation manner.

[0016] It would be desirable to have a method and apparatus that can achieve high flow rate magnetic separation of cells 10 / 30 without introducing foreign matter into the flow path of a biological solution, and that can dissociate cells 10 / 30 from aggregates in a substantially short time without damaging the cells. Summary of the Invention

[0017] The present invention describes methods and devices that are capable of (1) separating biological entities based on their physical properties, including but not limited to size, density, and compressibility; (2) separating biological entities bound to SPLs from biological solutions; (3) analyzing biological entities based on optical signals emitted by fluorescent molecules bound to surface receptors or antigens of the biological entities; and (4) sorting or separating specific biological entities based on optical signals emitted by fluorescent molecules bound to surface receptors or antigens of the biological entities by a microactuator mechanism integrated into a flow path through which the biological entities pass within a fluid sample.

[0018] The methods, components and devices as disclosed by the present invention can be utilized to separate biological entities including cells, bacteria and molecules from human blood, human body tissue, human bone, human body fluids, human hair, other human-related biological samples, as well as animal and plant samples, without limitation. [Brief explanation of the drawings]

[0019] FIG. 1A shows superparamagnetic labels (SPLs) binding to cells.

[0020] FIG. 1B illustrates optical fluorescent labels (OFLs) binding to cells.

[0021] FIG. 1C shows SPL and OFL binding to cells.

[0022] FIG. 2A shows cells bound to SPL being separated by a magnet.

[0023] FIG. 2B is a plot of SPL magnetization versus field strength for various SPL sizes.

[0024] FIG. 3A is a cross-sectional view of a prior art magnetic cell separation device.

[0025] FIG. 3B is a cross-sectional view of a prior art magnetic cell separator.

[0026] FIG. 3C is a cross-sectional view of a prior art magnetic cell separator.

[0027] FIG. 4 is a cross-sectional view of a first embodiment of a magnetic separation device ("MAG") having a "C" shaped rigid channel.

[0028] FIG. 5 is a cross-sectional view of a first embodiment of a MAG having a "C" shaped rigid channel in a detached position.

[0029] FIG. 6 is a cross-sectional view of a first embodiment of a MAG having a "C" shaped rigid channel at the separation location where cells are separated.

[0030] FIG. 7 is a side view of FIG.

[0031] FIG. 8A is a cross-sectional view of a second embodiment of a MAG.

[0032] FIG. 8B is a cross-sectional view of a third embodiment of the MAG.

[0033] FIG. 9 is a cross-sectional view of a first embodiment of a MAG having a flexible channel.

[0034] FIG. 10 shows a first embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0035] FIG. 11 shows a first embodiment of a MAG with flexible channels in a raised position after cell separation.

[0036] FIG. 12 shows a cross-sectional view of a fourth embodiment of a MAG having a "D" shaped rigid channel in a detached position.

[0037] FIG. 13 shows a cross-sectional view of a fourth embodiment of a MAG having a flexible channel.

[0038] FIG. 14 shows a cross-sectional view of a fourth embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0039] FIG. 15A shows a cross-sectional view of a fifth embodiment of the MAG.

[0040] FIG. 15B shows a cross-sectional view of a sixth embodiment of the MAG.

[0041] FIG. 15C shows a cross-sectional view of a seventh embodiment of the MAG.

[0042] FIG. 16 shows a cross-sectional view of a third embodiment of a twin MAG having a pair of flexible channels on a single channel holder.

[0043] FIG. 17 shows a cross-sectional view of a third embodiment of a pair of MAGs having a pair of flexible channels on a single channel holder in a separated position.

[0044] FIG. 18 shows four cross-sectional views of a fifth embodiment of a MAG having four flexible channels on a single channel holder.

[0045] FIG. 19 shows four cross-sectional views of a fifth embodiment of a MAG having four flexible channels on a single channel holder in the isolated position.

[0046] FIG. 20A shows a cross-sectional view of an eighth embodiment of a MAG having a rotating "D" shaped rigid channel in a detached position.

[0047] FIG. 20B shows a cross-sectional view of an eighth embodiment of a MAG having a flexible channel.

[0048] FIG. 20C shows a cross-sectional view of an eighth embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0049] FIG. 21A shows a cross-sectional view of a ninth embodiment of a MAG having a "V" shaped rigid channel in a detached position.

[0050] FIG. 21B shows a cross-sectional view of a ninth embodiment of a MAG having a flexible channel.

[0051] FIG. 21C shows a cross-sectional view of a ninth embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0052] FIG. 22A shows a third embodiment of a MAG having a flexible channel at the separation location where cells are separated and a demagnetizing ("DMAG") magnet positioned above and spaced above the MAG.

[0053] FIG. 22B shows the flexible channel of FIG. 22A moved away from the MAG to a position where the flexible channel holder is in close proximity to or in contact with the DMAG magnet.

[0054] FIG. 22C shows cells in the flexible channel of FIG. 22B being dissociated from aggregates by a DMAG magnet.

[0055] FIG. 22D shows the flexible channel of FIG. 22C moved to a low field position between the MAG and DMAG magnets.

[0056] FIG. 23A shows that after the cells are magnetically separated inside the flexible channel, mechanical vibration is applied to the flexible channel holder by a motor.

[0057] FIG. 23B shows that after the cells are magnetically separated inside the flexible channel, ultrasonic vibrations are applied to the flexible channel holder by a piezoelectric transducer (“PZT”).

[0058] FIG. 23C shows that after the cells are magnetically separated inside the flexible channel, mechanical vibrations are applied to the flexible channel by a motor.

[0059] FIG. 23D shows that after the cells are magnetically separated inside the flexible channel, ultrasonic vibrations are applied to the flexible channel by the PZT.

[0060] FIG. 23E is a side view of the flexible channel of FIG. 22D.

[0061] FIG. 24A shows a third embodiment of a MAG having a flexible channel holder that is in close proximity to or in contact with the DMAG magnet after the cells have been magnetically separated by the MAG, with the DMAG magnet positioned to the side or away from the MAG.

[0062] FIG. 24B shows the flexible channel of FIG. 24A rotated to a low field position between the MAG and DMAG magnets.

[0063] FIG. 25A shows the flexible channel holder in the demagnetized position, where the DMAG magnets are permanent magnets.

[0064] FIG. 25B shows the flexible channel holder in the demagnetized position, where the DMAG magnet is a permanent magnet with soft magnetic poles attached.

[0065] FIG. 25C shows the flexible channel holder in the demagnetized position, where the DMAG magnet is a permanent magnet with a pair of soft magnetic poles attached.

[0066] FIG. 25D shows the flexible channel holder in the demagnetized position, where the DMAG magnet is an electromagnet.

[0067] FIG. 25E shows the flexible channel holder in the demagnetization position, with mechanical vibration applied to the DMAG magnet by the motor.

[0068] FIG. 25F shows the flexible channel holder in the demagnetized position, with ultrasonic vibrations applied to the DMAG magnet by the PZT.

[0069] FIG. 26A shows a third embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0070] FIG. 26B shows the flexible channel of FIG. 26A rotated away from the MAG.

[0071] FIG. 26C shows that the aggregate of separated cells in the flexible channel of FIG. 26B is rotated to the top of the flexible channel.

[0072] FIG. 26D shows the flexible channel of FIG. 26C moved to a demagnetized position.

[0073] FIG. 27A shows a third embodiment of a MAG having a flexible channel at the separation location where cells are separated.

[0074] FIG. 27B shows the flexible channel and its holder of FIG. 27A moving away from the MAG.

[0075] FIG. 27C shows that mechanical vibration is applied to the channel holder by a motor.

[0076] FIG. 27D shows ultrasonic vibrations being applied to the channel holder by the PZT.

[0077] FIG. 28A shows a side view of a flexible channel in which the flexible channel is mechanically stretched.

[0078] FIG. 28B shows that the cells are dissociated from the aggregates after the external force of FIG. 28A is removed.

[0079] FIG. 29A shows a side view of the flexible channel where the flexible channel is mechanically compressed.

[0080] FIG. 29B shows that the cells are dissociated from the aggregates after the external force of FIG. 29A is removed.

[0081] FIG. 30A shows a side view of a flexible channel in which the flexible channel is mechanically twisted.

[0082] FIG. 30B shows that the cells are dissociated from the aggregates after the external force in FIG. 30A is removed.

[0083] FIG. 31 is a schematic diagram illustrating a method for magnetically separating biological entities from a fluid solution using MAG.

[0084] FIG. 32 shows a method for adjusting the position of the wedge of the flexible channel MAG of the MAG device.

[0085] FIG. 33A shows a flexible channel attached to the outlet port of a peristaltic pump, with a flow restrictor attached to the flexible channel to reduce flow pulsations.

[0086] FIG. 33B shows a top view of the internal structure of the first type of flow restrictor.

[0087] FIG. 33C shows a side view of a second type of flow restrictor.

[0088] FIG. 34A shows the flow restrictor of FIG. 33A removed from the flexible channel.

[0089] FIG. 34B is a schematic diagram of a fluid flow rate with large pulsations.

[0090] FIG. 35A shows the flow restrictor of FIG. 33A mated to a flexible channel.

[0091] FIG. 35B is a schematic illustration of a fluid flow rate with reduced pulsation.

[0092] FIG. 36A shows the flow restrictor of FIG. 33A increasing the pressure at the fluid inlet end of the flow restrictor.

[0093] FIG. 36B shows the flow restrictor of FIG. 36A being removed, creating a high velocity fluid pulse that pushes the dissociated cells of FIG. 36A out of the channel.

[0094] FIG. 37 is a schematic illustration of a fluid flow pulse resulting from the process of transitioning from FIG. 36A to FIG. 36B where the flow restrictor is removed.

[0095] FIG. 38A is a top view of a microfluidic chip ("UFL").

[0096] FIG. 38B is a cross-sectional view of a portion of the UFL of FIG. 38A, including the solid fluid inlet, the buffer inlet, and a portion of the UFL.

[0097] FIG. 38C is a schematic diagram illustrating a single fluid pressure node created between two sidewalls of the UFL of FIG. 38A by ultrasonic vibrations generated by a PZT.

[0098] FIG. 38D is a schematic diagram showing the hydroacoustic waves of FIG. 38C moving larger sized entities around the center of the UFL.

[0099] FIG. 39 is a schematic diagram showing a method for separating biological entities of different sizes using UFL.

[0100] FIG. 40A is a cross-sectional view of a portion of a UFL of a first embodiment including a uniformly formed soft magnetic layer.

[0101] FIG. 40B is a schematic diagram showing separation of hydroacoustic waves and entities in the UFL of FIG. 40A in the presence of a magnetic field.

[0102] FIG. 40C is a schematic diagram showing a protective layer that is placed conformally around the periphery of the UFL surface before the cap is attached.

[0103] FIG. 41A is a top view of a second embodiment of a UFL including sequentially arranged wide and narrow channels.

[0104] FIG. 41B is a cross-sectional view of the UFL of the second embodiment across a wide channel.

[0105] FIG. 41C is a cross-sectional view of the UFL of the second embodiment traversing a narrow channel.

[0106] FIG. 42A is a top view of a third embodiment of a UFL, including a wide channel and a narrow channel, and a side channel at the transition from the wide channel to the narrow channel.

[0107] FIG. 42B is a cross-sectional view of the UFL of the third embodiment across a wide channel.

[0108] FIG. 42C is a cross-sectional view of the UFL of the third embodiment traversing the narrow channel and the side channel.

[0109] FIG. 43 is a top view of a UFL according to a fourth embodiment in which the channel width narrows in three stages along the flow direction of the channel, and a side channel from the transition section.

[0110] FIG. 44A shows a first type of sample processing method involving UFL and MAG, where a first type of flow connector connects the UFL large solid outlet and the MAG inlet.

[0111] FIG. 44B shows the first type of sample processing method, where a second type of flow connector connects the UFL large solid outlet and the MAG inlet.

[0112] FIG. 44C shows the first type of sample processing method, where a third type of flow connector connects the UFL large solid outlet and the MAG inlet.

[0113] FIG. 45A shows a second type of sample processing method involving UFL and MAG, where a first type of flow connector connects the UFL small entity outlet and the MAG inlet.

[0114] FIG. 45B shows a second type of sample processing method, in which a second type of flow connector connects the UFL small entity outlet and the MAG inlet.

[0115] FIG. 45C shows a second type of sample processing method, in which a third type of flow connector connects the UFL small entity outlet and the MAG inlet.

[0116] FIG. 46A shows a third type of sample processing method involving MAG and dUFL, where a first type of flow connector connects the MAG outlet and the UFL entity fluid inlet.

[0117] FIG. 46B shows a third type of sample processing method, where a second type of flow connector connects the MAG outlet and the UFL entity fluid inlet.

[0118] FIG. 46C shows a third type of sample processing method, in which a third type of flow connector connects the MAG outlet and the UFL entity fluid inlet.

[0119] FIG. 47 shows a fourth type of sample processing method including multiple UFLs, a fourth type of flow connector, and multiple MAGs.

[0120] FIG. 48 shows a fifth type of sample processing method including multiple UFLs, a fifth type of flow connector, and multiple MAGs.

[0121] FIG. 49 is a diagram showing a fifth type of sample processing method including multiple UFLs, a sixth type of flow connector, and multiple MAGs.

[0122] FIG. 50 is a diagram illustrating a seventh type of sample processing method including multiple MAGs, a fourth type of flow connector, and multiple UFLs.

[0123] FIG. 51 is a diagram illustrating an eighth type of sample processing method including multiple MAGs, a fifth type of flow connector, and multiple UFLs.

[0124] FIG. 52 is a diagram illustrating a ninth type of sample processing method including multiple MAGs, a sixth type of flow connector, and multiple UFLs.

[0125] FIG. 53 shows a tenth type of sample processing method that includes one or more of UFL and MAG, a fifth or sixth type of flow connector, and a different type of cell processing device.

[0126] Figure 54A shows an eleventh type of sample processing method that includes a multi-step MAG process.

[0127] Figure 54B shows a twelfth type of sample processing method that includes multiple cycle MAG steps.

[0128] Figure 54C shows a thirteenth type of sample processing method involving a multi-step UFL process.

[0129] FIG. 55A shows a first example of a closed, disposable fluid line for a third type of sample processing method.

[0130] FIG. 55B shows the fluid lines of FIG. 55A connected to or attached with various fluidic devices to realize a third type of sample processing method.

[0131] FIG. 56A shows a second example of a closed, disposable fluid line for a third type of sample processing method.

[0132] FIG. 56B shows the fluid lines of FIG. 56A connected to or attached with various fluidic devices to achieve a third type of sample processing method.

[0133] FIG. 57A shows an example of a closed, disposable fluid line for the first type of sample processing method.

[0134] FIG. 57B shows the fluid lines of FIG. 57A connected to or attached with various fluidic devices to implement a first type of sample processing method.

[0135] FIG. 58A shows an example of a closed, disposable fluid line for a second type of sample processing method.

[0136] FIG. 58B shows the fluid lines of FIG. 58A connected to or attached with various fluidic devices to achieve a second type of sample processing method.

[0137] FIG. 59A shows an example of a closed, disposable fluid line for sample processing through a single MAG.

[0138] FIG. 59B shows the fluid lines of FIG. 59A connected to or attached with various fluidic devices to achieve sample processing through a single MAG.

[0139] FIG. 60A shows an example of a closed, disposable fluid line for sample processing through a single UFL.

[0140] FIG. 60B shows the fluid lines of FIG. 60A connected to or attached with various fluidic devices to achieve sample processing through a single UFL.

[0141] FIG. 61A shows replacing the peristaltic pump of FIG. 56B by using a compression chamber on the input sample bag to transport fluid through the fluid line.

[0142] FIG. 61B shows replacing the peristaltic pump of FIG. 56B by using a vacuum chamber on the drain sample bag to transport fluid through the fluid line.

[0143] FIG. 62A shows replacing the peristaltic pump of FIG. 57B by using a compression chamber on the input sample bag to transport fluid through the fluid line.

[0144] FIG. 62B shows replacing the peristaltic pump of FIG. 57B by using a vacuum chamber on the drain sample bag to drive fluid through the fluid lines.

[0145] FIG. 63A shows replacing the peristaltic pump of FIG. 58B by using a compression chamber on the input sample bag to transport fluid through the fluid line.

[0146] FIG. 63B shows replacing the peristaltic pump of FIG. 58B by using a vacuum chamber on the drain sample bag to transport fluid through the fluid line.

[0147] FIG. 64A shows replacing the peristaltic pump of FIG. 59B by using a compression chamber on the input sample bag to transport fluid through the fluid line.

[0148] FIG. 64B shows replacing the peristaltic pump of FIG. 59B by using a vacuum chamber on the drain sample bag to transport fluid through the fluid line.

[0149] FIG. 65A shows replacing the peristaltic pump of FIG. 60B by using a compression chamber on the input sample bag to transport fluid through the fluid line.

[0150] FIG. 65B shows replacing the peristaltic pump of FIG. 60B by using a vacuum chamber on the drain sample bag to drive fluid through the fluid lines.

[0151] Figure 66 shows the first step flow for separating biological entities from peripheral blood using UFL and MAG.

[0152] Figure 67 shows a second process flow for separating biological entities from peripheral blood using MAG.

[0153] Figure 68 shows a third process flow for separating biological entities from peripheral blood using MAG.

[0154] Figure 69 shows a fourth process flow for separating biological entities from peripheral blood using MAG.

[0155] FIG. 70 shows a fifth process flow for separating biological entities from tissue samples using UFL and MAG.

[0156] FIG. 71 shows a sixth process flow for separating biological entities from tissue samples using MAG.

[0157] FIG. 72 shows a seventh step flow for separating biological entities from surface swab samples using UFL and MAG.

[0158] Figure 73 shows an eighth step flow for separating biological entities from surface swab samples using MAG.

[0159] Figure 74 shows a ninth step flow for separating biological entities from a solid sample using UFL and MAG.

[0160] Figure 75 shows a tenth process flow for separating biological entities from solid samples using MAG.

[0161] Figure 76A shows the addition of both magnetic and fluorescent labels to a fluid sample for specific binding to target cells or entities.

[0162] Figure 76B shows the simultaneous incubation of both magnetic and fluorescent labels to form specific binding to target cells or entities.

[0163] Figure 77A shows the removal of unbound free magnetic labels from the sample fluid by UFL prior to magnetic separation by MAG.

[0164] FIG. 77B shows the step of removing unbound free magnetic labels from the sample fluid by UFL after magnetic separation by MAG.

[0165] FIG. 78A shows the removal of unbound free magnetic and fluorescent labels from the sample fluid by UFL prior to magnetic separation by MAG.

[0166] FIG. 78B shows the step of removing unbound free magnetic and fluorescent labels from the sample fluid by UFL after magnetic separation by MAG.

[0167] Figure 79 shows the sequential processing of negative MAG samples through UFL and various cell processing devices and procedures.

[0168] FIG. 80 shows the sequential processing of a negative MAG sample through UFL and various particle or molecular processing devices.

[0169] FIG. 81 shows the identity analysis of negative MAG samples after MAG separation onto various analytical devices.

[0170] Figure 82 shows the sequential processing of a positive MAG sample through UFL and various cell processing devices and procedures.

[0171] FIG. 83 shows the sequential processing of a positive MAG sample through UFL and various particle or molecular processing devices.

[0172] Figure 84 shows the identity analysis of a positive MAG sample after MAG separation into various analytical devices.

[0173] FIG. 85A shows that immediately after negative MAG sample collection, a fluorescent label is added to specifically bind to a target entity within the negative MAG sample.

[0174] FIG. 85B shows that immediately after positive MAG sample collection, a fluorescent label is added to specifically bind to the target entity within the positive MAG sample.

[0175] FIG. 86 shows a cross-sectional view of a tenth embodiment of a MAG with a separation channel in a separation position.

[0176] FIG. 87A shows a cross-sectional view of an eleventh embodiment of the MAG.

[0177] FIG. 87B shows a cross-sectional view of a twelfth embodiment of the MAG.

[0178] FIG. 87C shows a cross-sectional view of a thirteenth embodiment of the MAG.

[0179] FIG. 88A shows a cross-sectional view of a fourteenth embodiment of the MAG.

[0180] FIG. 88B shows a cross-sectional view of a fifteenth embodiment of the MAG.

[0181] FIG. 88C shows a cross-sectional view of a sixteenth embodiment of the MAG.

[0182] Figure 89A shows the channel and its holder in an elevated position from the MAG.

[0183] FIG. 89B shows that mechanical vibration is applied to the channel through the second channel holder by a motor.

[0184] FIG. 89C shows that mechanical vibration is applied to the channel holder by a motor through a vibrator arm.

[0185] FIG. 90A shows a cross-sectional view of a portion of the UFL of FIG. 38A, including the solid fluid inlet, the buffer inlet, and a portion of the UFL.

[0186] FIG. 90B shows a cross-sectional view of the UFL of FIG. 38A with a PZT attached to the top cover of the flow channel.

[0187] FIG. 90C shows a top view of the UFL device of FIG. 38A with multiple PZTs attached to the same top cover of the same UFL device.

[0188] FIG. 91A shows a cross-sectional view of a UFL similar to FIG. 90B, but with flow channels having circular curved sidewalls.

[0189] FIG. 91B shows a cross-sectional view of a UFL similar to FIG. 91A, but in which a flow channel having a partially circular shape is formed within the UFL base.

[0190] FIG. 91C shows a cross-sectional view of a UFL similar to FIG. 91A, but in which flow channels having circular shapes are formed in both the UFL base and the UFL cover.

[0191] FIG. 92A shows a top view of a UFL device with two inlets and two outlets.

[0192] FIG. 92B shows a top view of one inlet and two outlets of the UFL device.

[0193] FIG. 93A shows the operation of the UFL device of FIG. 92A.

[0194] FIG. 93B illustrates the operation of the UFL device of FIG. 92B.

[0195] FIG. 94A shows an embodiment of the process flow between blood or bone marrow sample collection and UFL operation.

[0196] FIG. 94B shows another process flow embodiment between blood or bone marrow sample collection and UFL operation.

[0197] FIG. 95A shows an embodiment of the process flow between solid sample collection and UFL operation.

[0198] FIG. 95B shows an embodiment of the process flow between surface sample collection and UFL operation.

[0199] FIG. 96 shows an embodiment of the process flow after negative MAG sample collection including a UFL operation.

[0200] FIG. 97 shows another process flow embodiment after negative MAG sample collection including a UFL operation.

[0201] FIG. 98 shows an embodiment of the process flow after negative MAG sample collection including a UFL operation.

[0202] FIG. 99 shows another process flow embodiment after positive MAG sample collection including a UFL operation.

[0203] FIG. 100A shows an embodiment of a process flow involving two UFLs in serial operation.

[0204] FIG. 100B shows another process flow embodiment involving two UFLs in serial operation.

[0205] FIG. 101A shows another process flow embodiment involving two UFLs in serial operation.

[0206] FIG. 101B shows another process flow embodiment involving two UFLs in serial operation.

[0207] FIG. 102 shows an embodiment of a method for operating UFLs in a serial arrangement.

[0208] FIG. 103 shows an embodiment of another method for operating UFLs in a serial arrangement.

[0209] FIG. 104 shows another method embodiment for operating UFLs in a serial arrangement.

[0210] FIG. 105A shows an embodiment of another method for operating UFLs in a serial arrangement.

[0211] FIG. 105B shows another method embodiment for operating UFLs in a serial arrangement.

[0212] FIG. 106A shows an embodiment of the MAG in a modular configuration.

[0213] FIG. 106B shows an embodiment of the UFL in a modular arrangement.

[0214] FIG. 106C shows an embodiment of a system including a single module of MAG or a single module of UFL.

[0215] FIG. 106D shows an embodiment of a system including multiple modules of MAG and UFL.

[0216] FIG. 107 shows an embodiment of a system including multiple MAG and UFL modules where the liquid sample flows through the modules in series.

[0217] FIG. 108A shows the flexible channel of FIG. 33A attached to the output port of a peristaltic pump with an obstruction sensor near the flexible channel.

[0218] FIG. 108B shows the flexible channel of FIG. 108A expanding due to an obstruction in the fluid line and contacting the obstruction sensor.

[0219] FIG. 109A shows that a narrow inner diameter flow channel is used to replace the flow limiter of FIG. 108A to reduce flow rate pulsations.

[0220] FIG. 109B shows the flexible channel of FIG. 109A expanding due to an obstruction in the fluid line and contacting the obstruction sensor.

[0221] FIG. 110A shows an embodiment of a UFL with two inlets and optical detectors around the main channel of the UFL.

[0222] FIG. 110B shows an embodiment of a UFL with one inlet and optical detector around the main channel of the UFL.

[0223] FIG. 110C shows an embodiment of a UFL with optical detectors around the sample outlet channel of the UFL.

[0224] FIG. 110D shows another embodiment of a UFL with optical detectors around the enlarged channel.

[0225] FIG. 111A shows an embodiment of an optical detector in which an illuminator, a forward scatter sensor, and a backscatter sensor are used to detect biological entities.

[0226] FIG. 111B shows an embodiment of the optical detector of FIG. 111A embedded in the channel wall, with the backscatter sensor and illuminator centered along the same horizontal line.

[0227] FIG. 111C shows an embodiment of the optical detector of FIG. 111A embedded in the channel wall with the backscatter sensor above or below the illuminator.

[0228] FIG. 111D shows an embodiment of the optical detector of FIG. 111A embedded in the channel wall and covered within the channel by an optically transparent protective layer.

[0229] FIG. 111E shows an embodiment of the optical detector of FIG. 111A embedded in the channel wall and covered within the channel by an optically transparent protective layer, with a light absorbing layer at the bottom of the cover forming the upper wall of the channel.

[0230] FIG. 112A shows an embodiment of an optical detector in which an illuminator array and a forward scatter sensor array are used to detect small biological entities.

[0231] FIG. 112B shows an embodiment of an optical detector in which an illuminator array and a forward scatter sensor array are used to detect large biological entities.

[0232] FIG. 113A shows an embodiment of an optical detector in which one illuminator and a forward scatter sensor array are used to detect a biological entity at a first entity location.

[0233] FIG. 113B shows an optical detector embodiment in which one illuminator and a forward scatter sensor array are used to detect a biological entity at a second entity location.

[0234] FIG. 114A shows an embodiment of an optical detector in which an array of illuminators and one forward scatter sensor are used to activate a first illuminator and detect a biological entity at a first entity location.

[0235] FIG. 114B shows an embodiment of an optical detector in which an illuminator array and one forward scatter sensor are used to detect a biological entity at the first entity location, with a second illuminator operating after the first illuminator of FIG. 114A.

[0236] FIG. 114C shows an optical detector embodiment in which an illuminator array and one forward scatter sensor are used to activate a first illuminator and detect a biological entity at a second entity location.

[0237] FIG. 114D shows an embodiment of an optical detector in which an illuminator array and one forward scatter sensor are used to detect a biological entity at a second entity location, with a second illuminator operating after the first illuminator of FIG. 114C.

[0238] FIG. 115A shows examples of detector signal intensities at different sensor locations for the embodiment of FIGS. 113A and 113B.

[0239] FIG. 115B shows examples of detector signal intensities at different illuminator positions for the embodiment of FIGS. 114A-114D.

[0240] FIG. 116A shows an embodiment of an optical detector in which an illuminator array and a forward scatter sensor array are used to activate a first illuminator and detect the shape of a biological entity at a first entity location.

[0241] FIG. 116B shows an embodiment of an optical detector in which an illuminator array and a forward scatter sensor array are used to detect the shape of a biological entity at the location of the first entity, with a second illuminator operating after the first illuminator of FIG. 116A.

[0242] FIG. 117 shows examples of detector signal intensities at different sensor locations for the embodiment of FIGS. 116A and 116B in which elements of the illuminator array are activated individually.

[0243] FIG. 118A shows an embodiment of an optical detector in which the illuminator, forward scatter sensor, and backscatter sensor are embedded in the channel wall, but with optical components positioned between the channel wall and each of the illuminator and sensors.

[0244] FIG. 118B shows an embodiment of an optical detector in which an illuminator, a forward scatter sensor, and a backscatter sensor are positioned on the outer surface of the UFL facing the channel wall, with optical components positioned between the channel wall and each of the illuminator and sensors.

[0245] Figure 119A shows an embodiment of an optical detector in which an illuminator, forward scatter sensor, and backscatter sensor are located on the bottom surface of the UFL base, with optical components positioned in the leading optical path between the channel wall and each of the illuminator and sensor.

[0246] FIG. 119B shows an embodiment of an optical detector in which an illuminator, forward scatter sensor, and backscatter sensor are located on top of the UFL cover, with optical components positioned in the leading light path between the channel wall and each of the illuminator and sensor.

[0247] FIG. 120A shows an embodiment of an optical detector having an illuminator, a forward scatter sensor, and a backscatter sensor, with the optical detector controller embedded in the UFL base.

[0248] FIG. 120B shows an embodiment of an optical detector in which the illuminator, forward scatter sensor, and backscatter sensor are embedded in the UFL base, and the optical detector controller is outside the UFL.

[0249] FIG. 121A shows an embodiment of the controller of FIG. 120A of the optical detector in communication with an external computing device through an electrical connection.

[0250] FIG. 121B shows an embodiment of the controller of FIG. 120A of the optical detector that communicates with an external computing device through wireless means.

[0251] FIG. 122A shows an embodiment of an electrically controlled optical filter positioned between the UFL channel wall and the illuminator and scatter sensor of FIG. 118A.

[0252] FIG. 122B shows an embodiment of an electrically controlled optical lens positioned between the UFL channel wall and the illuminator and scatter sensor of FIG. 118A.

[0253] FIG. 122C illustrates an embodiment using an optical grating as an optical filter, optionally electrically positioned between the UFL channel wall and the illuminator and scatter sensor of FIG. 118A.

[0254] FIG. 123A shows the embedded illuminator, scattering sensor, and optical components of FIG. 118A formed on the UFL base after a first fabrication step during the UFL manufacturing process.

[0255] FIG. 123B shows a top layer deposited on the base surface of FIG. 123A covering the embedded illuminator, scattering sensor and optical components.

[0256] FIG. 123C shows the planarization of the top layer of FIG. 123B.

[0257] FIG. 123D shows a second etching step performed to form the main channel of the UFL in the upper layer of FIG. 123C.

[0258] FIG. 123E shows a protective layer deposited conformally over the top surface and etched channels of FIG. 123D.

[0259] FIG. 123F shows that a top cover is placed on the upper surface of FIG. 123E to form an enclosure for the main channel of the UFL.

[0260] FIG. 124A shows another embodiment of an optical detector in which an illuminator, a forward scatter sensor, and a backscatter sensor are used to detect biological entities.

[0261] FIG. 124B shows an embodiment of the optical detector of FIG. 124A in which the backscatter sensor and illuminator are embedded in the base of the UFL and the forward scatter sensor is embedded in the cover of the UFL.

[0262] FIG. 124C shows an embodiment of the optical detector of FIG. 124A in which the backscatter sensor and illuminator are embedded in the base of the UFL and the forward scatter sensor is attached to the top surface of the cover of the UFL.

[0263] FIG. 124D shows an embodiment of the optical detector of FIG. 124A in which the backscatter sensor and illuminator are embedded in the base of the UFL and the forward scatter sensor is positioned above the top surface of the cover of the UFL.

[0264] FIG. 124E shows an embodiment of the optical detector of FIG. 124A in which the backscatter sensor and illuminator are mounted on the bottom surface of the base of the UFL and the forward scatter sensor is mounted on the top surface of the cover of the UFL.

[0265] FIG. 125A shows an embodiment of the optical detector of FIG. 124A in which the illuminator is embedded in the cover of the UFL and the forward scatter sensor is embedded in the base of the UFL.

[0266] FIG. 125B shows an embodiment of the optical detector of FIG. 124A in which the illuminator is mounted on top of the cover of the UFL and the forward scatter sensor is embedded in the base of the UFL.

[0267] FIG. 125C shows an embodiment of the optical detector of FIG. 124A in which the illuminator is positioned below the bottom surface of the base of the UFL and the forward scatter sensor is positioned above the top surface of the cover of the UFL.

[0268] FIG. 125D shows an embodiment of the optical detector of FIG. 124A in which the illuminator is positioned above the top surface of the cover of the UFL and the forward scatter sensor is positioned below the bottom surface of the base of the UFL.

[0269] Figure 126A shows the embodiment of Figure 125C with an optical window formed in the cover to allow light to pass from the illuminator to the UFL channel and an optical window formed in the base to allow light to pass from the UFL channel to the forward scatter sensor.

[0270] Figure 126B shows the embodiment of Figure 125D with an optical window formed in the base to allow light to pass from the illuminator to the UFL channel and another optical window formed in the cover to allow light to pass from the UFL channel to the forward scatter sensor.

[0271] FIG. 127A shows an embodiment in which an illuminator is embedded in the cover or base of the UFL channel, and light from the illuminator passes through an optical grating that is embedded in the cover or base of the UFL channel.

[0272] FIG. 127B shows an embodiment in which a forward scatter sensor is embedded in the cover or base of the UFL channel, and light travels to the forward scatter sensor through an optical grating embedded in the cover or base of the UFL channel.

[0273] FIG. 127C shows an embodiment in which the illuminator is embedded in the cover or base of the UFL channel, and light from the illuminator passes through an optical phase plate that is embedded in the cover or base of the UFL channel.

[0274] FIG. 127D shows an embodiment in which a forward scatter sensor is embedded in the cover or base of the UFL channel, and light travels to the forward scatter sensor through an optical phase plate that is embedded in the cover or base of the UFL channel.

[0275] FIG. 128A shows an embodiment in which the illuminator is positioned outside the UFL channel and light from the illuminator passes through an optical grating embedded in the cover or base of the UFL channel.

[0276] FIG. 128B shows an embodiment in which the forward scatter sensor is placed outside the UFL channel and light travels to the forward scatter sensor through an optical grating embedded in the cover or base of the UFL channel.

[0277] FIG. 128C shows an embodiment in which the illuminator is positioned outside the UFL channel and light from the illuminator passes through an optical phase plate embedded in the cover or base of the UFL channel.

[0278] FIG. 128D shows an embodiment in which the forward scatter sensor is placed outside the UFL channel and light travels to the forward scatter sensor through an optical phase plate embedded in the cover or base of the UFL channel.

[0279] FIG. 129A shows an embodiment of multiple optical detectors, each with an illuminator, forward scatter sensor, or backscatter sensor positioned along the channel wall of a UFL channel to cause sequential optical detection of biological entities.

[0280] FIG. 129B shows an example of a fluorescent optical signal from the optical detector of FIG. 129A as a biological entity passes through the UFL channel.

[0281] FIG. 130A shows a method for aligning biological entities into linear flow in a channel for optical detection using fluid pressure nodes.

[0282] FIG. 130B shows a method for aligning biological entities into linear flow in a flow channel for optical detection using laminar flow.

[0283] FIG. 130C shows a method for aligning biological entities into linear flow in a flow channel for optical detection using laminar flow in combination with fluid pressure nodes.

[0284] FIG. 131A shows a method for using a spatially periodic illuminator or light path to detect biological entities.

[0285] FIG. 131B shows an example of a signal detected by the scattering sensor of FIG. 131A.

[0286] FIG. 131C shows enhanced detection of biological entities by using signal filtering based on spatial periodicity of FIG. 131A.

[0287] FIG. 132 shows a first embodiment of a biological entity sorting apparatus with the fluid pathway selector in a first sorting position.

[0288] FIG. 133 shows the sorting device of FIG. 132 in a second sorting position.

[0289] FIG. 134A shows the sorting device of FIG. 132 having four sorting positions and in the first sorting position.

[0290] FIG. 134B shows the sorting device of FIG. 134A in a second sorting position.

[0291] FIG. 134C shows the sorting device of FIG. 134A in a third sorting position.

[0292] FIG. 134D shows the sorting device of FIG. 134A in the fourth sorting position.

[0293] FIG. 135A shows the sorting device of FIG. 132 utilizing the coil line of the voice coil to connect to the device body.

[0294] FIG. 135B shows a view of the device of FIG. 135A along a first cross-sectional direction.

[0295] FIG. 135C shows a first example view of the device of FIG. 135A along a second cross-sectional direction.

[0296] FIG. 135D shows a view of a second example of the device of FIG. 135A along a second cross-sectional direction.

[0297] FIG. 135E shows a view of the device of FIG. 135A along a third cross-sectional direction.

[0298] FIG. 136A shows a diagram of the device of FIG. 135A with a first magnetic field application scheme.

[0299] FIG. 136B shows a diagram of the device of FIG. 135A with a second magnetic field application scheme.

[0300] FIG. 136C shows a diagram of the device of FIG. 135A with a third magnetic field application scheme.

[0301] FIG. 136D shows a diagram of the device of FIG. 135A with a fourth magnetic field application scheme.

[0302] Figure 137 shows a sorting device incorporating an actuator position decoder.

[0303] FIG. 138A shows a first current drive scheme for the actuator voice coil.

[0304] FIG. 138B shows a second current drive scheme for the actuator voice coil.

[0305] FIG. 139 shows a second embodiment of a biological entity sorting apparatus with the fluid pathway selector in the first sorting position.

[0306] FIG. 140 shows a third embodiment of the biological entity sorting device with the capacitive actuator in a first sorting position.

[0307] FIG. 141 shows a fourth embodiment of the biological entity sorting device, with the capacitive actuator in a first sorting position.

[0308] FIG. 142 shows a fifth embodiment of the biological entity sorting device, with the thermoelastic actuator in a first sorting position.

[0309] FIG. 143 shows a sixth embodiment of the biological entity sorting device, with the thermoelastic actuator in a first sorting position.

[0310] FIG. 144A shows an example of the device of FIG. 135A in which the flow channels in the base and the flow channels in the path selector are covered by separate top covers.

[0311] FIG. 144B shows a view of the device of FIG. 144A along a first cross-sectional direction.

[0312] FIG. 144C shows a view of the device of FIG. 144A along a second cross-sectional direction, where the actuator voice coil is not covered by the top cover.

[0313] FIG. 144D shows a view of the device of FIG. 144A along a second cross-sectional direction in which the actuator voice coil is covered with a top cover.

[0314] FIG. 144E shows a view of the device of FIG. 144A along a first cross-sectional direction, showing that an additional cover together with the device base forms an enclosure around the sorting arm and actuator.

[0315] Figure 145 shows an external controller drive for the sorting device.

[0316] FIG. 146 shows an example of using multiple devices of FIG. 134A in a cascade arrangement to increase sorting categories.

[0317] FIG. 147 shows a seventh embodiment of the biological entity sorting apparatus with the fluid pathway selector in the first sorting position.

[0318] FIG. 148 shows the sorting device of FIG. 147 in a second sorting position.

[0319] FIG. 149 shows an eighth embodiment of the biological entity sorting apparatus with the fluid pathway selector in the first sorting position.

[0320] FIG. 150 shows the sorting device of FIG. 149 in a second sorting position.

[0321] For purposes of clarity and conciseness, like elements and components have the same naming and numbering throughout the figures, but they are not necessarily drawn to scale. Detailed Description of the Invention

[0322] While the present invention may be embodied in many different forms, designs, or configurations, for the purposes of promoting an understanding of the principles of the invention, reference will be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation or restriction to the scope of the invention is intended thereby. Any modifications and further implementations of the principles of the invention as described herein would normally occur to one skilled in the art to which the invention pertains.

[0323] Biological entities referred to herein include cells, bacteria, viruses, molecules, particles containing RNA and DNA, cell clumps, bacterial clumps, molecular clumps, and particle clumps. Large and small entities refer to biological entities within the same fluid that have relatively larger and smaller physical sizes. In one embodiment, large entities include cells, bacteria, cell clumps, bacterial clumps, particle clumps, entities bound to magnetic labels, and entities bound to optical labels. In another embodiment, small entities include molecules, particles, viruses, cell debris, unbound free magnetic labels, and unbound free optical labels. In another embodiment, large entities have a physical size greater than 1 micrometer (μm), and small entities have a physical size less than 1 μm. In yet another embodiment, large entities have a physical size greater than 2 μm, and small entities have a physical size less than 500 nanometers (nm). In yet another embodiment, large entities have a physical size greater than 5 μm, and small entities have a physical size less than 2 μm. Biological samples include blood, body fluids, tissues extracted from any part of the body, bone marrow, hair, nails, bones, teeth, liquids and solids from bodily waste, or surface swabs from any part of the body. Substantial fluid or fluid samples or liquid samples or sample solutions include biological samples in their original liquid form, biological entities dissolved or dispersed in a buffer solution, or biological samples dispersed in a buffer solution after dissociation from the original non-liquid form of the biological sample. Biological entities and biological samples can be obtained from humans or animals. Biological entities can also be obtained from plants and the environment, including air, water, and soil. Substantial fluid or fluid samples or samples can contain various types of magnetic or optical labels or one or more chemical reagents that can be added during various steps within embodiments of the present invention. The sample flow rate is the volume of a fluid sample flowing through a cross section or fluid portion or flow path of a channel in a unit of time, where the volume may be in units of liters (L), milliliters (mL), microliters (μL), or nanoliters (nL), and the unit of time may be in units of minutes (min), seconds (s), milliseconds (ms), microseconds (μs), or nanoseconds (ns).Sample flow rate is the distance traveled by free molecules or entities within a liquid sample or fluid portion or flow path in a channel during a unit of time, and the distance can be measured in meters (m), centimeters (cm), millimeters (mm), or micrometers (μm). Separation efficiency is the percentage of target entities within a liquid sample that are successfully separated from the liquid sample by a method designed to separate the target entities. A buffer is a fluid medium that can dissolve or disperse biological entities without introducing additional biological entities.

[0324] FIG. 4 shows a cross-sectional view of a first embodiment of a magnetic separation device ("MAG") of the present invention. MAG 121 is composed of two magnetic field-generating poles, pole 102 and pole 103. Each of poles 102 and 103 is composed of a soft magnetic material, which may include one or more of the following elements: iron (Fe), cobalt (Co), nickel (Ni), iridium (Ir), manganese (Mn), neodymium (Nd), boron (B), samarium (Sm), and aluminum (Al). Pole 102 has a flux-collecting end 1023 and a tip portion 1021, and the shape of pole 102 converges from the flux-collecting end 1023 to the tip portion 1021. In FIG. 4, flux-collecting end 1023 is a flat surface that contacts or is adjacent to the north pole ("N") surface of permanent magnet 104. The permanent magnet 104 has a magnetization as shown by arrow 1041 in FIG. 4 , pointing from the south pole (“S”) face of the magnet 104 to the north face. The magnetization 1041 generates a magnetic field in free space, which can be described as magnetic flux lines 1046 emanating from the north face of the magnet 104 and returning to the south face. If the magnetic flux collecting end 1023 of the magnetic pole 102 is in contact with or close to the north face of the magnet 104 as shown in FIG. 4 because the magnetic pole 102 is a soft magnetic material, magnetic flux 1046 from the north face of the magnet 104 is collected by the magnetic pole 102 and passes through the magnetic flux collecting end 1023 into the body of the magnetic pole 102. Because of the converging shape of the magnetic pole 102, the collected magnetic flux is primarily guided within the soft magnetic material of the magnetic pole 102 and emanates from the tip 1021 of the magnetic pole 102. This proximity between the flux collecting end 1023 and the N-face of the magnet 104 can be such that the gap distance between the faces of 1023 is less than 1 mm. The tip 1021 can have a much smaller surface area than the flux collecting end 1023, which causes the fluid to exit the tip 1021 with a higher magnetic flux density, i.e., the magnetic flux 1045 is concentrated, and therefore a high local magnetic field and high magnetic field gradient around the tip 1021, than if the magnetic flux 1045 were emitted by the N-face of the magnet 104. The tip 1021 of the magnetic pole 102 is preferably as small as possible, e.g., a convergence point, to create the greatest magnetic flux concentration for achieving the maximum magnetic field.However, in practice, due to manufacturing processes, tip 1021 may have a curved or domed shape that does not affect the general concept of magnetic flux concentration by tip 1021. Magnetic pole 103 is similar to magnetic pole 102 in that magnetic pole 103 has a larger flux collecting end 1033 and a smaller tip 1031, with flux collecting end 1033 contacting or adjacent to the south face of permanent magnet 105. Magnetic pole 103 and magnet 105 are identical to magnetic pole 102 and magnet 104, but are preferably positioned to be mirror images of magnetic pole 102 and magnet 104 about centerline 1050. Magnetization 1051 of magnet 105 is opposite to magnetization 1041 of magnet 104. The magnetic flux 1047 collected by the magnetic flux collecting end 1033 from the south face of the magnetic pole 103 is opposite that of the magnetic pole 102, and the magnetic flux radiating from the tip 1031 of the magnetic pole 103 is opposite that of the tip 1021. Thus, between the gaps between the tips 1021 and 1031, the radiated magnetic flux forms a closed loop, further enhancing the magnetic field strength and magnetic field gradient around the tips 1021 and 1031. The dashed line 1045 is a schematic representation of the magnetic flux radiating from the tip 1021 and returning to the tip 1031. The magnetic flux lines 1045 closer to the tips 1021 and 1031 are denser, indicating a stronger magnetic field and a larger magnetic field gradient closer to the gap region. As shown in FIG. 4, the top of the magnetic pole 102 is tilted to the right, and the top of the magnetic pole 103 is tilted to the left. This sloped shape causes the magnetic flux in poles 102 and 103 to be diverted away from the bottom of the poles, placing tip 1021 of pole 102 and tip 1031 of pole 103 at the closest spacing between poles 102 and 103, helping to achieve a high magnetic field in the gap between tips 1021 and 1031 while minimizing magnetic flux leakage between the bottom half of pole 102 and the bottom half of pole 103. In Figure 4, the slope of the tops of poles 102 and 103 forms a triangular or convex top surface 1210 of MAG 121, which will hereinafter be referred to as the "MAG wedge" 1210 of MAG 121.The permanent magnets 104 and 105 may be composed of any of, but not limited to, Nd, Fe, B, Co, Sm, Al, Ni, Sr, Ba, O, NdFeB, AlNiCo, SmCo, strontium ferrite (SrFeO), barium ferrite (BaFeO), and cobalt ferrite (CoFeO).

[0325] The embodiment of Figure 4 includes a rigid, fixed-geometry channel 101. The channel 101 has channel walls that enclose a channel volume 1013, where a fluid sample can flow through the channel 101 in the channel volume 1013 along a length of the channel 101 that is perpendicular to the cross-sectional view of Figure 4. The channel 101 has a top surface 1012 and a bottom surface 1011. The bottom surface 1011 is formed with a shape that conforms to the MAG wedge surface 1210 such that when the channel 101 moves in a direction 1014 and contacts the magnetic poles 102 and 103 of the MAG 121, the bottom surface 1011 of the channel 101 contacts the MAG wedge surface 1210, with no or minimal gap between the bottom surface 1011 and the MAG wedge surface 1210. The top surface 1012 of the channel 101 is preferably conformal to the bottom surface 1011 to create a channel space 1013 shaped to maximize exposure of the fluid sample flowing through the channel 101 to the maximum magnetic field region of the MAG wedge gap magnetic field 1045.

[0326] In the embodiment of FIG. 4 , magnetic poles 102 and 103, magnets 104 and 105, and channel 101 extend in a direction perpendicular to the cross-sectional view of FIG. 4 , hereinafter referred to as the “lengthwise direction.” A fluid sample flows through channel 101 and is contained in channel space 1013 along the lengthwise direction. Channel 101 is a rigid, fixed-geometry channel, and the wall thickness of channel 101 at surface 1011 may be thinner than the wall thickness at surface 1012, such that the mechanical robustness of channel 101 is maintained by the thicker wall at surface 1012, the magnetic field effect on the fluid sample is enhanced by the thinner wall at surface 1011, and the fluid sample is allowed to be closer to MAG wedge 1210 and tips 1021 and 1031. Channel 101 may be attached to a non-magnetic channel holder 107 at top surface 1012. The channel holder 107 can adjust the position of the channel 101 relative to the MAG wedge 1210, move the channel 101 to a separation position in contact with the MAG 121, or lift the channel 101 away from the MAG 121 after magnetic separation. The channel holder 107 can be made of any non-magnetic material, including, but not limited to, metal, non-metallic elements, plastic, polymer, ceramic, rubber, silicon, and glass. In Figure 4, the flux collecting ends 1023 and 1033 of the soft magnetic poles 102 and 103 can be referred to as base ends 1023 and 1033.

[0327] Permanent magnets as described in different embodiments of the present invention, such as magnets 104 and 105 in FIG. 4, may each have an opposite magnetization direction to that described in each of the figures and embodiments without affecting the design, function, and process of the embodiment.

[0328] Figure 5 is a cross-sectional view of the first embodiment of the MAG of Figure 4, with channel 101 in a magnetic separation position. Channel 101 in Figure 4 moves along direction 1014, contacting the face of MAG wedge 1210 with bottom surface 1011. The MAG 121 gap formed by tips 1021 and 1031 contacts or is at a minimum distance from the walls of channel 101 and the fluid sample flowing through channel 101. The "C" shape of channel 101, matching the MAG wedge shape, facilitates a large cross-sectional area of ​​channel space 1013 to maintain a high flow rate while simultaneously confining cells 10 / 30 in the fluid sample flowing through channel 101 in the high field and high gradient region of the MAG 121 gap magnetic field, as indicated by magnetic field lines 1045. Compared to the prior art of FIGS. 3A and 3B, the first embodiment of the MAG 121 achieves comparable or better magnetic fields and field gradients on cells 10 / 30 flowing through the channel 101 while not introducing extraneous materials into the channel 101. Removing the magnetic poles 102 and 103 from the channel 101, along with the magnets 104 and 105, eliminates the magnetic field source and avoids the cell loss limitations associated with magnetic domains in the prior art. Compared to the prior art of FIG. 3C, the MAG wedge in FIG. 5, which is in contact with the wall of the channel 101, provides the maximum achievable magnetic field and field gradient for the fluid sample in the channel 101 for more efficient cell 10 / 30 separation. The shape of the channel 101, which conforms to the MAG wedge shape, allows the channel 101 to have a large cross-sectional sample flow area while avoiding the deficiency of the prior art in which cells 10 / 30 at the top of a circular channel experience a much lower magnetic field than at the bottom, ultimately limiting sample flow rate. Therefore, the sample flow rate in channel 101 can be higher than in the prior art while achieving better magnetic separation efficiency.

[0329] FIG. 6 is the same as FIG. 5 , except that biological entities, or for simplicity, cells 10 / 30, are included to illustrate magnetic separation by MAG 121 from a fluid sample 6. The fluid sample 6 carrying the cells 10 / 30 is flowed through the channel 101 along the length of the channel 101, perpendicular to the cross-sectional view of FIG. 6 . The MAG 121 gap magnetic field magnetizes SPL2 attached to the cells 10 / 30, and the magnetic field gradient pulls the cells 10 / 30 from the fluid 6 toward the MAG wedge, forming an aggregate layer against the bottom surface 1011 of the channel 101. Due to the design of MAG 121 and the shape of the channel 101, the magnetic field experienced by the cells 10 / 30 near the top surface 1012 is not significantly lower than that near the bottom surface 1011, and the distance traveled by the cells 10 / 30 from the top surface 1012 to the aggregate at the bottom surface 1011 is much shorter than in the prior art. These features enable MAG 121 to address the shortcomings of the prior art.

[0330] FIG. 7 is a side view of FIG. 6 along direction 61 of FIG. 6. A fluid sample 6 carrying cells 10 / 30 flows from left to right in channel 101, as indicated by arrow 1010. The MAG 121 gap magnetic field causes the cells 10 / 30 to separate from the fluid 6 and form aggregates on the channel wall at the bottom 1011. FIG. 7 shows that a majority of the cells 10 / 30 are separated from the fluid 6 earlier in the length of channel 101, as indicated by the dense population of cells 10 / 30. Because certain tail population cells 10 / 30 have relatively smaller SPL2 sizes or fewer SPL2 bound to their surfaces, it takes longer for such tail population cells to be attracted to the bottom 1011 than the nominal population while fluid 6 flows through channel 101. Thus, the population of separated cells 10 / 30 exhibits a decrease in density from the inlet to the outlet of channel 101.

[0331] FIG. 8A is a cross-sectional view of a second embodiment of a MAG of the present invention. MAG 122 of FIG. 8A is substantially similar to MAG 121, except that soft magnetic shield 106 is attached to the south face of magnet 104 and the north face of magnet 105. Magnetic flux from the south face of magnet 104 and the north face of magnet 105 forms a closed path within soft magnetic shield 106. Compared to MAG 121, MAG 122 has less magnetic flux leakage outside the MAG 122 structure, and the magnetic flux generated by magnets 104 and 105 is primarily confined within the soft magnetic material bodies of poles 102 and 103 and shield 106. MAG 122 is preferred in applications where it is desirable to minimize magnetic interference from MAG 122 to other surrounding equipment or devices.

[0332] Figure 8B is a cross-sectional view of a third embodiment of a MAG of the present invention. Compared to MAG 121, MAG 123 of Figure 8B incorporates only one permanent magnet 108 attached to both poles 102, 103, with the magnetic flux from the north face of magnet 108 and the magnetic flux from the south face of magnet 108 conducted by poles 102 and 103 to generate the MAG 123 gap field by tips 1021 and 1031. Compared to MAG 121, the magnetic flux generated by magnet 108 is primarily confined within the soft magnetic material bodies of poles 102 and 103, making MAG 123 relatively easy to assemble and with low magnetic flux leakage.

[0333] FIG. 9 shows a cross-sectional view of the first embodiment of the MAG 121 used for magnetic separation in combination with a flexible channel 201. FIG. 9 is similar to FIG. 4 except that the rigid channel 101 is replaced with the flexible channel 201. The flexible channel 201 can assume any shape, including a circular tubular form, in its undeformed state, but can be deformed into other shapes by an external force. The wall material of the channel 201 is deformable and can be composed of, but is not limited to, silicone, silicone rubber, rubber, PTFE, FEP, PFA, BPT, vinyl, polyimide, ADCF, PVC, HDPE, PEEK, LDPE, polypropylene, polymer, thin metal coated with a polymer layer, or fiber mesh. FIG. 9 also shows that the flexible channel 201 has a channel holder 107 attached to the back of the channel 201. The channel holder 107 can be composed of any non-magnetic material, including, but not limited to, metals, non-metallic elements, plastics, polymers, ceramics, rubber, silicon, and glass. Channel 201 may be attached to holder 107 through surface bonding, for example by adhesive or injection molding, or via mechanical attachment via element 1074 in FIG. 32. Holder 107 has a bottom surface 1070 that contacts the top surface of channel 201, surface 1070 preferably being substantially conformal to the wedge shape of MAG 121. In FIG. 9, holder 107 adjusts the position of attached flexible channel 201 relative to the MAG 121 wedge gap, moving channel 201 in direction 1014 toward the MAG wedge gap.

[0334] FIG. 10 shows flexible channel 201 being pressed against the MAG wedge of MAG 121 by channel holder 107. Pressure exerted by holder 107 on flexible channel 201 against the MAG wedge of MAG 121 causes channel 201 to deform in FIG. 10 such that bottom surface 2013 of channel 201 conforms and makes surface contact with MAG wedge surface 1210. Meanwhile, bottom surface 1070 of holder 107 may also be conformal to the MAG wedge shape, so that top surface 2012 of the channel may also be pressed to assume a substantially conformal shape against the MAG wedge. FIG. 10 shows the "separation position" of flexible channel 201 relative to MAG 121 during magnetic separation of cells 10 / 30 from sample fluid 6. The shape of flexible channel 201 is substantially similar to channel 101 of FIGS. 5 and 6 , except that such shape of channel 201 in the separation position is the result of self-alignment and self-adaptation of channel 201 to the MAG wedge, requiring no manufacturing process to obtain the shape of channel 101. Additionally, the flow space within channel 201 in the separation position can be adjusted to make the cross-sectional area of ​​the flow space of channel 201 larger or smaller so that the flow rate of fluid sample 6 through channel 201 and the efficiency of cell 10 / 30 magnetic separation can be optimized. Adjustment of the flow space can be achieved by varying the vertical distance 1071 from the top end of face 1070 of holder 107, which contacts upper surface 2012 of channel 201, to tips 1021 and 1031, or to an imaginary plane on which tips 1021 and 1031 reside. The longer the distance 1071, the less flexible channel 201 deforms, providing a larger flow space, thereby resulting in a slower flow rate for the same fluid flow rate. The shorter the distance 1071, the smaller the flow space of flexible channel 201, but also the closer the top end 2012 is to the MAG wedge gap and tips 1021 and 1031, which results in a stronger magnetic field and faster separation of cells 10 / 30. Therefore, optimization between flow rate and separation efficiency can be achieved by adjusting distance 1071 for certain combinations of MAG 121 design and flexible channel 201. In one embodiment, distance 1071 is greater than 0 mm and less than or equal to 1 mm.In another embodiment, distance 1071 is greater than 1 mm and less than or equal to 3 mm. In yet another embodiment, distance 1071 is greater than 3 mm and less than or equal to 5 mm. In yet another embodiment, distance 1071 is greater than 5 mm and less than or equal to 10 mm. In yet another embodiment, distance 1071 is greater than 2 times and less than or equal to 3 times the wall thickness of flexible channel 201. In yet another embodiment, distance 1071 is greater than 3 times and less than or equal to 5 times the wall thickness of flexible channel 201. In yet another embodiment, distance 1071 is greater than 5 times and less than or equal to 10 times the wall thickness of flexible channel 201. Flexible channel 201 in the separation position functions similarly to channel 101 in FIG. 6, and FIG. 10 also shows that during magnetic separation, cells 10 / 30 form aggregates along the channel 201 wall at the lower surface 2013 directly opposite MAG wedge surface 1210. The wall thickness of the channel 201 at the bottom surface 2013 may be thinner than the wall thickness of the channel 201 at the top surface 2012 .

[0335] FIG. 11 shows that after magnetic separation is completed in FIG. 10 , channel holder 107 moves in direction 1015 away from MAG 121, moving flexible channel 201 away from the MAG wedge of MAG 121 to a “raised position,” and flexible channel 201 may also return to its undeformed shape, e.g., a circular tube as shown in FIG. 11 . Magnetically separated cells 10 / 30 in FIG. 10 may retain aggregate morphology at the bottom of flexible channel 201 in the raised position. After flexible channel 201 reaches the raised position of FIG. 11 , a dissociation procedure may be performed on cells 10 / 30 in flexible channel 201 to disrupt aggregates, as described in FIGS. 22A-30B . The return of flexible channel 201 to its undeformed shape, e.g., a circular tube as shown in FIG. 11 , results in a larger cross-sectional area of ​​channel space 1013 as shown in FIG. 11 than in the separation position of FIG. 10 . Such a larger channel space 1013 may be preferable because it is easier to dissociate the cells 10 / 30 from the aggregate form. To assist the channel 201 in returning to its undeformed shape, additional buffer may be injected into the channel space 1013 of the channel 201 in the raised position.

[0336] MAG121 in FIGS. 9 to 11 can be replaced by MAG122 or MAG123 without being limited to the methods and steps described.

[0337] FIG. 12 shows a cross-sectional view of a fourth embodiment of MAG 124. MAG 124 has three soft magnetic poles 111, 112, and 113. Center pole 111 is attached to the N-face of permanent magnet 109 at its flux collecting end 1112, similar to the flux collecting end 1023 of pole 102 in FIG. 4. Magnetic flux 1048 from the N-face of magnet 109 is conducted by the soft magnetic body of pole 111 and then emanates from tip 1111, which has a much smaller area than flux collecting end 1112 of pole 111, and functions similarly to tip 1021 in FIG. 4 to concentrate the magnetic flux conducted from magnet 109, thereby creating a localized high magnetic field around tip 1111. Side poles 112 and 113 each have flux collecting ends 1122 and 1132, respectively, which are attached to the same top surface of soft magnetic bottom shield 114. Next, bottom shield 114 is attached to the south face of permanent magnet 109. Thus, magnetic flux 1049 from the south face of magnet 109 is conducted in the body of bottom shield 114, divided between magnetic poles 112 and 113, and further conducted to tips 1121 and 1131 of magnetic poles 112 and 113, respectively. Tip 1111 is formed in close proximity to tips 1121 and 1131. In one embodiment, tip 1111 may be recessed from an imaginary plane in which tips 1121 and 1131 exist toward magnet 109 by a distance of 0 mm to 1 mm. In another embodiment, tip 1111 may be recessed from an imaginary plane in which tips 1121 and 1131 exist toward magnet 109 by a distance of 1 mm to 5 mm. In yet another embodiment, tip 1111 may be recessed from an imaginary plane in which tips 1121 and 1131 reside toward magnet 109 by a distance of 5 mm to 10 mm. Preferably, tip 1111 is equally spaced from tips 1121 and 1131. The top of pole 112 is angled to the right, while the top of pole 113 is angled to the left, similar to poles 102 and 103 in FIG. 4. This angle is intended to maximize magnetic flux concentration around tips 1111, 1121, and 1131 and to increase the gap between the bodies of poles 112 and 113 relative to the body of pole 111 to reduce magnetic flux leakage.As magnetic flux emanates from tips 1111, 1121, and 1131, the magnetic flux 1048 conducted by central pole 111 opposes the magnetic flux 1049 conducted by side poles 112 and 113, causing the magnetic flux to form a ring between tips 1111 to 1112 and between tips 1111 to 1131. Thus, the magnetic flux generated by the north and south faces of magnet 109 is conducted within the body of poles 111, 112, 113 and shield 114, minimizing leakage outside the MAG 124 structure. The magnetic flux density is greatest around tip 1111, and tips 1121 and 1131 also produce high magnetic flux densities, all of which indicate high magnetic fields and field gradients around tips 1111, 1121, and 1131. Compared to MAGs 121, 122 and 123, MAG 124 has the advantage of less leakage and therefore a higher magnetic flux density around tip 1111, resulting in a more efficient magnetic flux confinement within the MAG 124 soft magnetic material which produces a higher magnetic field and magnetic field gradient in channel 301.

[0338] Channel 301 is a rigid channel similar to channel 101 in FIG. 4 and has a fixed shape similar to a rotated "D." Channel 301 is shown in a magnetic separation position in FIG. 12, with tips 1111, 1121, and 1131 all contacting the curved bottom surface 3011 of the "D" shape of channel 301, resulting in the maximum possible magnetic field and magnetic field gradient that MAG 124 can generate in the channel space through which the fluid sample flows in channel 301. In another embodiment, tip 1111 can contact surface 3011, and tips 1121 and 1131 are not in contact with surface 3011. In one embodiment, top surface 3012 of channel 301 can be on an imaginary plane on which tips 1121 and 1131 exist; in another embodiment, top surface 3012 can be 0 mm to 1 mm above the imaginary plane; and in yet another embodiment, top surface 3012 can be 1 mm to 5 mm above the imaginary plane. In one embodiment, the wall thickness of channel 301 at surface 3012 is thicker than the wall thickness at surface 3011. Channel 301 may be attached to non-magnetic channel holder 110 at top surface 3012. Channel holder 110 may adjust the position of channel 301 relative to the MAG gap of MAG 124 to move channel 301 to a separation position in contact with tips 1111 of MAG 124 poles 111, or lift channel 301 away from MAG 124 after magnetic separation.

[0339] FIG. 13 shows a cross-sectional view of the fourth embodiment of MAG 124 used for magnetic separation in combination with flexible channel 201, which is the same as FIG. 9 . Channel holder 110 may have a different shape than channel holder 107 of FIG. 9 . Prior to magnetic separation, channel holder 110 is attached to channel 201. Channel holder 110 adjusts the position of channel 201 relative to the MAG gap of MAG 124, which is composed of tip portions 1111, 1121, and 1131 as in FIG. 12 , and moves channel 201 in direction 1014 toward the MAG gap of MAG 124.

[0340] FIG. 14 shows flexible channel 201 in the separation position in MAG 124 of the fourth embodiment, with cells 10 / 30 separated and forming aggregates around the bottom and sidewalls of channel 201 near tips 1111, 1121, and 1131. In FIG. 14, flexible channel 201 is deformed similarly to FIG. 10 to conform to the MAG gap boundaries, primarily tips 1111, 1121, and 1131. Because flexible channel 201 conforms to the MAG gap boundaries under pressure from holder 110, the shape of flexible channel 201 may differ from channel 301 in the separation position. The shape of channel 201 in FIG. 14 may provide a higher liquid sample flow rate with higher separation efficiency than channel 301. The distance 1071 between the lower surface 1150 of holder 110 and tip 1111 may be adjusted to optimize the flow rate in channel 201. The range of the distance 1071 is the same as the range 1071 shown in FIG.

[0341] FIG. 15A shows a cross-sectional view of MAG 125 of the fifth embodiment. MAG 125 is the same as MAG 124 of FIG. 12, except that magnet 109 and bottom shield 114 of MAG 124 have been removed in MAG 125. As shown in FIG. 15A, permanent magnets 115 and 116 having opposite magnetizations 1151 and 1161 are positioned between poles 111 and 112 and between poles 111 and 113, respectively. Magnetizations 1151 and 1161 are horizontal in FIG. 15A, allowing center pole 111 to conduct north-face magnetic flux from both magnets 115 and 116, while side poles 112 and 113 each conduct south-face magnetic flux from magnets 115 and 116, respectively. Compared to MAG 124, MAG 125 may generate a higher magnetic field around tips 1111, 1121, and 1131 due to the use of two magnets 115 and 116. MAG 125 may also be easier to assemble than MAG 124.

[0342] 15B shows a cross-sectional view of the sixth embodiment of MAG 126. MAG 126 is the same as MAG 124, except that side poles 112 and 113 are attached to the south faces of permanent magnets 1092 and 1094, respectively, and magnetizations 1093 and 1095 are opposite to magnetization 1091 of magnet 109. Bottom shield 114 is attached to both the north faces of magnets 1092 and 1094 and the south face of magnet 109, thus forming an internal magnetic flux confinement in shield 114 between magnets 109, 1092, and 1094. Compared to MAG 124, MAG 126 can generate a higher magnetic field around tips 1111, 1121, and 1131 due to the use of three magnets 109, 1092, and 1094 in MAG 126.

[0343] Figure 15C shows a cross-sectional view of the seventh embodiment of MAG 127. MAG 127 is the same as MAG 126 of Figure 15B, except that bottom shield 114 has been removed.

[0344] FIG. 16 shows two of the third embodiment MAGs 123 used for magnetic separation on a pair of flexible channels 201. The pair of flexible channels 201 are fixed to the same channel holder 1020 in FIG. 16. The upper and lower MAGs 123 are substantially identical, with the upper MAG 123 upside down. The MAG wedges of the upper and lower MAGs 123 are substantially aligned with the centers of the upper and lower channels 201. The magnets 108 of both the upper and lower MAGs 123 may have the same magnetization direction as the arrows in the magnets 108 in FIG. 16, so that the magnetic fields generated by the upper and lower MAGs 123 in the upper and lower channels 201 during magnetic separation have horizontal magnetic field components in the same direction, thereby limiting magnetic flux leakage between the soft magnetic poles of the upper and lower MAGs 123.

[0345] FIG. 17 shows the two MAGs 123 of FIG. 16 being moved to a separation position relative to the two flexible channels 201, which is the same process as FIG. 10 . After reaching the separation position of FIG. 17 , the fluid sample carrying the cells 10 / 30 can flow through the channel 201 in a lengthwise direction perpendicular to the cross-sectional view to initiate magnetic separation of the cells 10 / 30 by the upper and lower MAGs 123. The distance 1071 between the surface of the holder 1021 that contacts the outer edge 2012 of the channel 201 and the tips 1021 and 1031 of the MAGs 123 or the imaginary plane on which the tips 1021 and 1031 exist can be adjusted to optimize the flow rate in each of the two channels 201. The adjustment range of the distance 1071 is the same as that of 1071 described in FIG. 10 .

[0346] 16 and 17 can be replaced by MAG121 or MAG122, and channel 201 can also be replaced by channel 101.

[0347] FIG. 18 shows that four of the fifth embodiment MAGs 125 are used for magnetic separation on four flexible channels 201. The four flexible channels 201 are fixed on the same channel holder 1040 as in FIG. 18. The four MAGs 125 are substantially identical. The MAG gaps of the four MAGs 125 are substantially aligned with the centers of the corresponding flexible channels 201. As shown in FIG. 18, the arrangement of the permanent magnets in each MAG 125 should be identical; for example, the central magnetic pole of each of the four MAGs 125 is attached to the north face of both magnets in each individual MAG 125, and the side magnetic pole of each of the four MAGs 125 is attached to the south face of the magnet in each MAG 125. Therefore, the adjacent MAGs 125 closest to the adjacent side magnetic poles have the same magnetic polarity, and side-to-side magnetic pole leakage between adjacent MAGs 125 can be minimized or avoided. Furthermore, the four MAGs used in four of the channels 201 of FIG. 18 are shown in FIG. 18 only as an example of multiple channel processing capabilities with a circular channel arrangement, with the channels located at the center of a circular array of MAGs 125. Fewer or more MAGs 125 used on a corresponding number of channels 201 can be achieved without limitation with the type of circular arrangement of FIG. 18. The circular arrangement of multiple channels of FIG. 18 with MAGs 125 is inherently more flexible than MAGs 123 as in FIG. 16 because, when the number of MAGs 123 is greater than two, the two-pole design of MAGs 123 in FIG. 16 can lead to magnetic flux leakage through the poles of adjacent MAGs 123.

[0348] FIG. 19 shows the four MAGs 125 of FIG. 18 being moved to a separation position relative to the four flexible channels 201, which is the same process as FIG. 14 . After reaching the separation position of FIG. 19 , a fluid sample carrying cells 10 / 30 can flow through the channels 201 in a lengthwise direction perpendicular to the view of FIG. 19 to begin magnetic separation of the cells 10 / 30 by the four MAGs 125. As in FIG. 17 , the distance 1071 between the surface of the holder 1040 that contacts the outer edge 2012 of the channels 201 and the tip 1111 of the central magnetic pole 111 of the MAG 125 for each channel 201 and MAG 125 pair can be adjusted to optimize the flow rate in each of the four channels 201. The adjustment range of the distance 1071 is the same as that of 1071 described in FIG. 10 .

[0349] 18 and 19 may be replaced by MAG 124, MAG 126 or MAG 127, and channel 201 may be replaced by channel 301.

[0350] FIG. 20A shows a sixth embodiment of MAG 128 having a rotated "D"-shaped rigid channel 320 in the separation position. MAG 128 is similar to MAG 123, except that the MAG wedge of MAG 123 has been changed from a triangular shape to a flat surface. The pole 1022 of MAG 128 is similar to pole 102 of MAG 123, but has a flat surface 1042 on pole 1022 instead of a tip on pole 102. A similar flat surface 1052 is present on pole 1032, which is similar to pole 103 of MAG 123. Due to the flat surface of the MAG wedge in MAG 128, the rigid channel 320 can have a flat bottom surface 1062 that mates with and contacts the MAG wedge flat surface in the separation position to obtain the maximum magnetic field and field gradient region from MAG 128. The channel 320 can be attached to a non-magnetic channel holder 1102 at its top surface. The channel holder 1102 can adjust the position of the channel 320 relative to the MAG wedges of the MAG 128, move the channel 320 to a separation position in contact with the tips of the MAG 128 poles 1022 and 1032, or lift the channel 320 away from the MAG 128 after magnetic separation.

[0351] 20B shows the sixth embodiment MAG 128 in use on a flexible channel 201, which is attached to a channel holder 1102. The channel holder 1102 moves the channel 201 along direction 1014 toward the MAG wedge of the MAG 128.

[0352] FIG. 20C shows the sixth embodiment of MAG 128 in which flexible channel 201 of FIG. 20B is moved to the separation position, causing cells 10 / 30 to separate from the liquid sample and form aggregates at the bottom of channel 201 against the upper flat surface of the MAG wedge of MAG 128. Holder 1102, which presses channel 201 against the flat surface of the MAG wedge of MAG 128, pushes channel 201 to form a rotated "D"-shaped channel, and the shape of channel 201 in the separation position resembles channel 320 of FIG. 20A . Distance 1071 between bottom surface 1062 of holder 1102, which contacts channel 201 upper end 2012, and magnetic pole faces 1042 and 1052 of MAG 128 can be adjusted to optimize the flow rate in channel 201. The adjustment range of distance 1071 is the same as that of 1071 described in FIG. 10 .

[0353] Magnet 108 of MAG 128 may be replaced by an arrangement of magnets 104 and 105 as in MAG 121 and by an arrangement of magnets 104 and 105 and bottom shield 106 as in MAG 122.

[0354] 21A shows a seventh embodiment of MAG 129 having a "V" shaped rigid channel 330 in a separation position. MAG 129 differs from MAG 123 in the shape of its poles, with poles 1024 and 1034 of MAG 129 having flux concentrating tips 3301 and 3302 that form a "V" shaped concave surface, instead of the triangular wedge shape of MAG 123. The V-shaped MAG concave surface of MAG 129 also makes rigid channel 330 V-shaped, with bottom ends 3303 and 3304 in direct contact with the surfaces of tips 3301 and 3302. Additionally, channel 330 may preferably have a V-shaped notch in the channel at top end 3305 following the V-shape of the edges of 3303 and 3304, which confines the fluid sample in V-shaped channel space 3306 and helps it flow closer to the magnetic pole faces 3303 and 3004, which provide the high magnetic field and magnetic field gradient. Channel 330 may be attached to a non-magnetic channel holder 1103 at top surface 3305. Channel holder 1103 may adjust the position of channel 330 relative to the concave MAG surface of MAG 129, move channel 323 into a separation position in contact with the faces of the tips of magnetic poles 1024 and 1034, or lift channel 330 away from MAG 129 after magnetic separation.

[0355] 21B shows a seventh embodiment of MAG 129 used with a flexible channel 201, where the channel 201 is attached to a channel holder 1103 at the top end of the channel 201. The channel holder 1103 moves the channel 201 toward the recess of MAG 129 along direction 1014. The channel holder 1103 has a triangular shape, with the convergence point of the triangle meeting the top end of the channel 201.

[0356] 21C shows the seventh embodiment of MAG 129 in which flexible channel 201 of FIG. 21B is moved to the separation position, and cells 10 / 30 are separated from the liquid sample and form aggregates at the bottom of channel 201 against the upper surfaces of the MAG concave surfaces of tips 3301 and 3302 of MAG 129. Channel 201 is pressed into a "V"-shaped channel by holder 1103. In FIG. 21C, holder 1103 presses channel 201 against the MAG concave surfaces of MAG 129 with a downward convergence point, deforming the upper wall of channel 201 downward and moving closer to tips 3301 and 3302, while the same force also forces the lower wall of channel 201 to conform to the MAG concave surfaces of MAG 129 and into contact with upper surfaces 3303 and 3304 of tips 3301 and 3302. 21C in the separation position exhibits a V-shape similar to that of channel 330 in FIG. 21A, forcing cells 10 / 30 in channel space 3306 closer to high magnetic field and high gradient tips 3301 and 3302 and tip surfaces 3303 and 3304. Vertical distance 1071 between the lower convergence point of holder 1103, which contacts upper end 2012 of channel 201, and tips 3301 and 3302 of MAG 129 or the imaginary plane on which tips 3301 and 3302 exist, can be adjusted to optimize the flow rate in channel 201. The adjustment range of distance 1071 is the same as that of 1071 described in FIG. 10.

[0357] Magnet 108 of MAG 129 may be replaced by an arrangement of magnets 104 and 105 as in MAG 121, and by an arrangement of magnets 104 and 105 and bottom shield 106 as in MAG 122.

[0358] 22A-27D describe various methods for demagnetizing or dissociating cells 10 / 30 magnetically separated from aggregates in a MAG channel. For ease of explanation, a flexible channel 201 is used. However, the channels in FIGS. 22A-27D may be labeled "201 / 101," indicating that, when used for explanation, the flexible channel 201 may be replaced with a rigid channel 101 without affecting the function and results of the described method. Also, for ease of explanation, MAG 123 is used in FIGS. 22A-27D, while any other MAG embodiment may be used without limitation under the same concept with the corresponding channel as described in the preceding figures.

[0359] FIG. 22A is substantially similar to FIG. 10, with channel 201 in a separation position and cells 10 / 30 separated by a magnetic field from a MAG. In FIG. 22A, MAG 123 is used instead of MAG 121 of FIG. 10. Channel holder 1081 may differ from channel holder 107 of FIG. 10 by having a notch on its top surface that allows a demagnetizing or dissociating magnetic structure ("DMAG"), which in FIG. 22A is permanent magnet 120, to approach channel 201 / 101 to provide a magnetic field sufficient to demagnetize or dissociate cells 10 / 30 from aggregates in channel 201 / 101. Such a notch is preferred, but not required. DMAG magnet 120 is positioned away from MAG 123 in FIG. 22A without affecting the magnetic separation of cells 10 / 30 by MAG 123. The magnetization of DMAG magnet 120 is shown as vertical 1201, but could also be horizontal without any functional difference. The position of channel 201 / 101 relative to MAG 123 and DMAG 120 in Figure 22A is "Position 1."

[0360] 22B is similar to FIG. 11, except that channel holder 1081 moves channel 201 / 101 away from MAG 123, bringing it into contact with or close to DMAG magnet 120 on the top surface of holder 1081, which can fit into a notch in holder 1081 to provide maximum magnetic field on the cell 10 / 20 aggregate in channel 201 / 101. Cells 10 / 30 form aggregates after magnetic separation by MAG and do not automatically fall out of the aggregate due to SPL2 on cells 10 / 30 not self-demagnetizing when part of the aggregate. By gradually removing the cells 10 / 30 using the magnetic field gradient from magnet 120, for example, using the higher magnetic moment SPL2, which responds faster to the weaker magnetic field from DMAG 120, the aggregate can reach a critical volume, where the remaining cells 10 / 30 in the aggregate do not encounter sufficient static magnetic field from other cells 10 / 30 and self-demagnetize into individual cells 10 / 30 due to the recovery of the superparamagnetic properties of SPL2. Therefore, removing a certain amount of cells 10 / 30 to dissociate them from the aggregate, or dividing the aggregate from a continuous large fragment into multiple smaller fragments, can help achieve self-demagnetization of the cells 10 / 30. The position of channel 201 / 101 relative to MAG 123 and DMAG 120 in Figure 22B is "Position 2." Compared to channel 101, channel 201 may have advantages during dissociation of cells 10 / 30 with DMAG magnet 120 because channel 201 provides a larger channel space that allows further separation between free cells 10 / 30 and from aggregates or between divided aggregate fragments, which helps to reduce magnetostatic coupling and increases the self-demagnetization rate of SPL2 on cells 10 / 30. For flexible channel 201, it is preferable to fill channel 201 with additional buffer before or at position 2 to return channel 201 to a circular shape for the larger channel space.

[0361] FIG. 22C shows cell 10 / 30 in channel 201 / 101 of FIG. 22B being dissociated from the aggregate by DMAG magnet 120 at position 2.

[0362] Figure 22D shows channel holder 1081 moving channel 201 / 101 from DMAG position 2 in Figure 22C to a position called "position 3" between MAG 123 and DMAG magnet 120. At position 3, the combined magnetic field on cells 10 / 30 in channel 201 / 101 may be minimal, which may help SPL2 self-degauss. Channel 201 / 101 may be maintained at position 3 for an extended period of time to allow SPL2 and cells 10 / 30 to completely self-degauss and dissociate aggregates.

[0363] For effective disintegration of the aggregates, mechanical agitation can be applied to the aggregates by the magnetic forces applied by the MAG and DMAG magnets. For example, the channel holder 1081 can be swapped with the channel 201 / 101 between positions 1 and 2, or between positions 2 and 3, or between positions 1, 2, and 3. The swapping of the magnetic forces by the MAG and DMAG can then move all or part of the aggregates in the channel space, thus breaking the aggregates into smaller fragments or helping enough cells 10 / 30 break free from the aggregates so that the aggregates can self-dissociate. After the aggregates are sufficiently dissociated, the free cells 10 / 30 can be flushed out of the channel 201 / 101 at positions 3 or 2.

[0364] 23A shows that when channel 201 / 101 is in position 2 or position 3 in FIGS. 22B and 22C, mechanical vibrations can be applied to channel holder 1081 by motor 130. Such vibrations can be transmitted from holder 1081 through the walls of channel 201 / 101 to the fluid within channel 201 / 101, creating localized turbulence at various locations within channel 201 / 101, which can serve to mechanically break up aggregates and aid in dissociation of the aggregates.

[0365] Figure 23B shows that ultrasonic vibrations by a piezoelectric transducer ("PZT") 131 can be applied to the channel holder 1081. Similar to Figure 23A, the ultrasonic vibrations can be transmitted to the fluid within the channel 201 / 101 to create localized high frequency turbulence within the channel 201 / 101, which can serve to mechanically break up the aggregates and aid in dissociation of the aggregates.

[0366] FIG. 23C shows that the mechanical vibration of FIG. 23A can be applied directly to the walls of the channel 201 / 101 by a motor 130.

[0367] FIG. 23D shows that the ultrasonic vibrations of FIG. 23B can be applied directly to the walls of the channel 201 / 101 by a PZT 131.

[0368] Figure 23E is a side view of channel 201 / 101 along direction 61, as in Figure 22D. Arrow 1030 indicates that pulsed fluid flows in alternating directions may be applied to the channel liquid sample to create turbulent flow in the liquid within channel 201 / 101, which may also create localized turbulence with the fluid in channel 201 / 101 to help mechanically break aggregates into smaller pieces and assist in self-dissociation of the aggregates. The alternating pulsed flows of Figure 23E may be applied to channel 201 / 101 at positions 2 or 3 in Figures 22B-22D in combination with the vibration method of Figures 23A-23D.

[0369] If the aggregates in the channel 201 / 101 are large in size, multiple rounds of cell 10 / 30 dissociation and flushing of the cells 10 / 30 from the channel 201 / 101 by the method of Figures 22B-23E may be used. During each flush, a certain portion of the cells 10 / 30 may be washed out of the channel, making it easier to dissociate the remaining cells 10 / 30 in the aggregates in the channel 201 / 101 in the next round.

[0370] FIG. 24A is similar to FIG. 22B, in which channel holder 1081 contacts or is in close proximity to DMAG magnet 120 after cells 10 / 30 have been magnetically separated by MAG 123. Unlike FIG. 22B, DMAG magnet 120 in FIG. 24A is positioned to the side of and away from MAG 123, and holder 1081 has also been rotated compared to FIG. 22B to align the cuts in its upper surface with magnet 120. The position of magnet 120 in FIG. 24A may reduce magnetic field interference between MAG 123 and DMAG magnet 120. The position of channel 201 / 101 relative to MAG 123 and DMAG 120 in FIG. 24A is "position 12."

[0371] Figure 24B shows that after cell 10 / 30 is dissociated with positron 12 of Figure 24A, channel 201 / 101 along with channel holder 1081 of Figure 24A rotates away from magnet 120 of Figure 24A to a position between MAG 123 and DMAG magnet 120, where the combined magnetic fields from MAG 123 and DMAG magnet 120 on channel 201 / 101 and cell 10 / 30 therein are lowest, which is similar to position 3 of Figure 22D. The position of channel 201 / 101 relative to MAG 123 and DMAG 120 in Figure 24B is "position 13."

[0372] FIG. 25A shows a DMAG structure that is the same as FIG. 22B, except that the DMAG structure includes only a permanent magnet 120 having a magnetization 1201.

[0373] 25B shows a DMAG structure including a permanent magnet 120 and a soft magnetic pole 1202 with a converging shape toward channel 201 / 101. The converging shape of soft magnetic pole 1202 helps to concentrate the magnetic flux from magnet 120 to create a high magnetic field and high magnetic field gradient at cells 10 / 30 in channel 201 / 101 at position 2, more effectively demagnetizing and dissociating the clusters of cells 10 / 30.

[0374] 25C shows a DMAG structure including a permanent magnet 120 and a pair of soft magnetic poles 1203 and 1204. The magnetization 1201 of magnet 120 is horizontal, and each of poles 1203 and 1204 has a converging shape toward channel 201 / 101. The converging ends of poles 1203 and 1204 form a DMAG gap located at or near a notch in the top surface of channel holder 1081. Magnetic flux from magnet 120 is conducted by poles 1203 and 1204 and concentrated in the DMAG gap to generate a high magnetic field and a high magnetic field gradient on cells 10 / 30 in channel 201 / 101 at position 2, thereby more effectively demagnetizing and dissociating the cell 10 / 30 aggregates.

[0375] FIG. 25D shows a DMAG structure including an electromagnet including a soft magnetic core 1205 and a coil 1206, where a current flowing through the coil 1206 can generate magnetization in the core 1205 in a direction 1207, and the core 1205 functions like a magnet 120 to generate a magnetic field at the cells 10 / 30 in the channel 201 / 101 at position 2, demagnetizing or dissociating the cell 10 / 30 cluster. By varying the current amplitude and direction in the coil 1206, the magnetic field from the core 1205 on the cells 10 / 30 can be varied in strength and direction. In one embodiment, a DC current is applied to the coil 1206. In another embodiment, an AC current with alternating polarities is applied to the coil 1206. In yet another embodiment, the current applied to the coil 1206 is programmed to vary the amplitude, direction, or frequency or rate of increase or decrease of the amplitude to more effectively demagnetize and dissociate the cell 10 / 30 cluster.

[0376] FIG. 25E shows that a motor 130 such as that shown in FIG. 23A can generate mechanical vibrations on the DMAG structure of FIG. 25C, and such vibrations can be transmitted from the DMAG structure to holder 1081 through contact from the DMAG structure to holder 1081, and finally to the fluid in channel 201 / 101, and the DMAG structure can be modified to any of the DMAG structures described in FIGS. 25A-25D.

[0377] FIG. 25F shows that PAT131 as shown in FIG. 23B can generate ultrasonic vibrations on the DMAG structure of FIG. 25C, and such vibrations can be transmitted from the DMAG structure to holder 1081 through contact from the DMAG structure to holder 1081, and finally to the fluid in channel 201 / 101, and the DMAG structure can be modified to any of the DMAG structures described in FIGS. 25A-25D.

[0378] To achieve demagnetization and dissociation of cells 10 / 30 from aggregates in channels 201 / 101, alternative methods such as those described in Figures 26A-26D can be used without using a DMAG structure, with the function of the DMAG structure being achieved with the same MAG.

[0379] Figure 26A is the same as Figure 22A, except that channel holder 1082 does not have to have a notch on the top surface like holder 1081, and channel 201 / 101 is in the separation position, with cells 10 / 30 being separated in channel 201 / 101 by the magnetic field of MAG 123. The position of channel 201 / 101 relative to MAG 123 in Figure 26A is "position 21."

[0380] 26B shows that the channel 201 / 101 of FIG. 26A is lifted from the MAG 123 to a lower magnetic field position, "position 22." At position 22, the channel 201 / 101 may rotate, preferably 180 degrees, about its center as indicated by arrow 210. Such rotation may require that the channel 201 / 101 not be permanently fixed to the holder 1082.

[0381] FIG. 26C shows channel 201 / 101 of FIG. 26B after being rotated 180 degrees at position 22, where the clusters of cells 10 / 30 that form on the inner walls of channel 201 / 101 rotate with the channel walls to come to the upper end of channel 201 / 101 relative to MAG123.

[0382] FIG. 26D shows that channel 201 / 101 is moved from position 22, closer to MAG123, to position 23, between positions 21 and 22, where the magnetic field from MAG123 on cells 12 / 30 is stronger than at position 22 but weaker than at position 21. Next, cells 10 / 30 in the aggregate at the top end of channel 201 / 101 may be pulled away from the aggregate by the MAG123 magnetic field, and demagnetization and dissociation of the aggregate may begin. The steps of FIGS. 26B-26D may be repeated multiple times, with channel 201 / 101 making another rotation from position 23 back to position 22 and then back to position 23, until cells 10 / 30 are sufficiently dissociated in channel 201 / 101. Once demagnetization is complete, cells 10 / 30 may be flushed out of channel 201 / 101, preferably at position 22. Mechanical vibrations and flow disturbances as described in Figures 23C-23E can be applied to channel 201 / 101 at positions 22 and 23.

[0383] Figure 27A is the same as Figure 26A, with channel 201 / 101 in the separation position and cells 10 / 30 being separated by the magnetic field of MAG 123. The position of channel 201 / 101 relative to MAG 123 is "position 21." Channel 201 / 101 is attached to holder 1082 in Figure 27A.

[0384] FIG. 27B shows that the channel 201 / 101 of FIG. 27A is lifted by a holder 1082 from the MAG 123 to a lower magnetic field position 22.

[0385] FIG. 27C shows that at position 22, dissociation of cells 10 / 30 in channel 201 / 101 can be achieved solely through mechanical vibrations exerted by motor 130. FIG. 27C shows motor 130 applying mechanical vibrations to holder 1082; such vibrations can be transmitted from holder 1082 through the walls of channel 201 / 101 to the fluid within channel 201 / 101, creating localized turbulence at various locations within channel 201 / 101, which can help mechanically break up the aggregates and aid in the self-dissociation of cell 10 / 30 aggregates. Instead of through holder 1082, motor 130 can also exert vibrations directly in channel 201 / 101, as shown in FIG. 23C. Simultaneously with the application of motor 130 vibrations, alternating-direction pulsed fluid flow, as described in FIG. 23E, can be applied to the channel liquid sample to create turbulent flow in the liquid within channel 201 / 101.

[0386] FIG. 27D shows that at position 22, dissociation of cells 10 / 30 in channel 201 / 101 can be achieved primarily through ultrasonic vibrations exerted by PZT 131. FIG. 27D shows PZT 131 applying ultrasonic vibrations to holder 1082, which can transmit the ultrasonic vibrations to the fluid in channel 201 / 101, creating localized high-frequency turbulence within channel 201 / 101, which can help mechanically break up the aggregates and aid in the self-dissociation of cell 10 / 30 aggregates. As shown in FIG. 23D, PZT 131 can also apply ultrasonic vibrations directly to channel 201 / 101. Simultaneously with the application of ultrasonic vibrations by PZT 131, an alternating-direction pulsed fluid flow, as described in FIG. 23E, can be applied to the channel liquid sample to create turbulent flow in the liquid within channel 201 / 101.

[0387] Figures 28A-30B describe embodiments of methods for assisting dissociation of cell 10 / 30 aggregates by mechanical agitation, which may be added to channel 201 / 101 at position 22 as in Figure 27B, and may be added to channel 201 / 101 as in Figures 22B-22D, 23A-23D, 24A-25F, 26B-26D, and 27B-27D.

[0388] FIG. 28A shows a side view of the channel 201 / 101 and holder 1082 along direction 61 in FIG. 27B, in which cells 10 / 30 are magnetically separated by the magnetic field of the MAG and form aggregates on the underside of the channel 201 / 101 wall. A channel mount 1073 can be used to attach the channel 201 / 101 to the channel holder 1082. The channel mount 1073 can secure the channel 201 / 101 at the portion attached to the mount 1073 as an anchor against deformation, compression, or stretching of the channel 201 / 101 during the mechanical agitation process. The channel mount 1073 can also function as a valve to close fluid flow into and out of the portion of the flexible channel 201 between the two channel mounts 1073 before the mechanical agitation process in FIG. 28A, thereby allowing fluid trapped in the channel 201 to more efficiently create localized turbulence within the channel 201. 28A shows that an externally applied force 300 can stretch or deform the channel 201 / 101 in a direction away from the holder 1082, e.g., perpendicular to the length of the channel 201 / 101. This deformation or stretching of the channel 201 / 101 causes elastic energy to be stored in the channel 201 / 101 wall material.

[0389] Figure 28B shows that force 300 in Figure 28A is released, and the elastic energy stored in the walls of channel 201 / 101 acts to push channel 201 / 101 back toward its original, undeformed, and unstretched position. Depending on the wall material properties of channel 201 / 101, such rebounding may result in transient turbulence at various locations within channel 201 / 101, which may serve to mechanically break the cell 10 / 30 aggregate into smaller pieces and assist in the self-dissociation of the cell 10 / 30 aggregate. After release of force 300 and rebounding of channel 201 / 101, alternating flow 1030 may be applied, similar to Figure 23E, to assist in the dissociation process of the cell 10 / 30 aggregate, where valve 1073 may be deactivated to allow fluid flow within channel 201 / 101.

[0390] The process of deforming / stretching and releasing the channel 201 / 101 of Figures 28A and 28B can be repeated as many times as necessary until the cell 10 / 30 aggregates are sufficiently dissociated, which can then be flushed out of the channel 201 / 101 by a buffer solution.

[0391] Figure 29A shows an alternative method of mechanical agitation from Figure 28A. All aspects are the same as Figure 28A, except that a compressive force 302 can be applied to compress channel 201 in a direction perpendicular to the length of channel 201, for example, to compress channel 201 against channel holder 1082 as shown in Figure 29A. Because the liquid in channel 201 has limited compressibility, force 302 can cause the walls of channel 201 / 101 to expand in a direction perpendicular to the view of Figure 29A, i.e., perpendicular to both the length of the channel and the direction of force 302. This expansion of the walls of channel 201 / 101 also stores elastic energy in the wall material of channel 201.

[0392] Figure 29B is the same in all respects as Figure 28B, except that after the compressive force 302 of Figure 29A has been released, the elastic energy stored in the walls of channel 201 acts to cause channel 201 to rebound to its original, uncompressed shape. Such rebounding may result in strong transient turbulence at various locations within channel 201, which may serve to mechanically break the cell 10 / 30 aggregates into smaller pieces and assist in the self-dissociation of the cell 10 / 30 aggregates. After the release of force 302 and the rebounding of channel 201 shape, an alternating flow 1030 may be applied, similar to Figure 23E, to assist in the dissociation process of the cell 10 / 30 aggregates, at which point the valve function of 1073 may cease.

[0393] The process of compressing and releasing the channel 201 of FIGS. 29A and 29B can be repeated as many times as necessary until the cell 10 / 30 aggregates are sufficiently dissociated and can then be flushed out of the channel 201.

[0394] Figure 30A illustrates another alternative method of mechanical agitation. As shown in Figure 30A, all aspects are the same as Figure 28A, except that a rotational torsional force 303 or 304 can be applied to channel 201 to twist channel 201 along its length. In one embodiment, only one of rotational forces 303 or 304 is applied to one end of channel 201. In another embodiment, both rotational force 303 and force 304 are applied to different ends of channel 201 in opposite rotational directions such that channel 201 is twisted along its length. This torsional deformation of channel 201 re-stores elastic energy in the wall material of channel 201.

[0395] Figure 30B is the same in all respects as Figure 28B, except that Figure 30B is after rotational forces 303 and 304 of Figure 30A have been released, and the elastic energy stored in the walls of channel 201 acts to bounce channel 201 back to its original, untwisted shape. Such bouncing may result in strong transient turbulence at various locations within channel 201, which may serve to mechanically break the cell 10 / 30 aggregates into smaller pieces and assist in the self-dissociation of the cell 10 / 30 aggregates. After the release of forces 303 and 304 and the rebounding of channel 201 shape, alternating flow 1030 may be applied, similar to Figure 23E, to assist in the dissociation process of the cell 10 / 30 aggregates, at which point the valve function of 1073 may cease.

[0396] The process of twisting and releasing channel 201 of Figures 30A and 30B can be repeated as many times as necessary until the cell 10 / 30 aggregates are sufficiently dissociated, which can then be flushed out of channel 201 with a buffer solution.

[0397] Mechanical forces 300, 302, 303 and 304 can be applied by a mechanical structure that is motorized and capable of repeatedly applying such forces to channel 201, examples can include a flap to provide force 300, a compressor to provide force 302 and a twister to provide forces 303 and 304.

[0398] Figure 31 is a schematic diagram showing a method for using MAG to separate biological entities, e.g., cells 10 / 30, complexed with magnetic labels, from a fluid solution. The MAG channel in Figure 31 can be any of channels 101, 201, 301, 320, or 330 described in any of the figures herein, and the MAG in Figure 31 can be any of MAGs 121, 122, 123, 124, 125, 126, 127, 128, or 129, as described with the corresponding channel in any of the figures. The method of Figure 31 can include, in order, the following steps: In step 400, the MAG channel is placed in a separation position, i.e., position 1 or position 21, as shown in Figures 5, 10, 12, 14, 17, 19, 20A, 20C, 21A, 21C, 22A, 26A, and 27A, with its outer wall in contact with the MAG wedge surface or the tip of the magnetic pole. In step 401, a fluid sample is flowed through the MAG channel in the separation position. Next, in step 402, positive entities with attached magnetic labels SPL2, e.g., cells 10 / 30, and free magnetic labels SPL2 within the fluid sample, are attracted by the magnetic field of the MAG and aggregate at the MAG channel wall against the MAG wedge or MAG pole tip. Meanwhile, in step 4020, negative entities without attached magnetic labels SPL2 pass through the MAG channel without being attracted. The negative entities can then be processed directly in a subsequent step, as indicated by path 427, which may include entity analysis 407, such as steps included in Figures 79-81, or the negative entities can be passed to a subsequent step 408, such as through a UFL device, as shown in Figures 46A-46C, 50-52, or through repetition of the MAG step, as in Figures 54A and 54B. After step 402, in step 403, the sample can be depleted in the input of the MAG channel, completing the magnetic separation of the positive entities. In optional step 404, with the MAG channel still in the separation position, buffer can be flowed through the MAG channel to wash away negative entities that are free of magnetic label SPL2, but may be present along with the aggregates of positive entities due to non-specific binding.Then, in step 405, the MAG channel can be removed from the MAG to a dissociation position, including positions 2 and 22 in Figures 11, 22B, 22D, 24B, 26B, 26D, and 27B, and magnetic dissociation 451 as shown in Figures 22A-26D or mechanical dissociation 452 as shown in Figures 27C-30B or magnetic dissociation with mechanical dissociation 453 can be applied to the positive entities in the MAG channel. In step 406, a buffer solution can be flowed through the MAG channel to flush out the dissociated positive entities. If the positive entities are not completely dissociated, 465 indicates that repeated dissociation steps 405 can be applied to the positive entities remaining in the MAG channel after the preceding flushing step until the positive entities are sufficiently dissociated and flushed out of the MAG channel. If the volume of the fluid sample is large, the fluid sample may be separated into multiple smaller volumes, and after the smaller volume steps from step 400 to step 406, the next smaller volume may be introduced into the MAG channel starting from step 400 for continued steps as indicated by 461 until the large volume fluid sample is completed. After the positive entities are recovered after step 406, they may be processed in subsequent steps as indicated by path 428, which may include entity analysis 407 or continued steps 408.

[0399] FIG. 32 illustrates a method for aligning the channel 201 / 101 relative to the MAG gap of the MAG 123 device. As described in embodiments of the present invention, it is important to precisely align the channel 201 / 101 relative to the MAG wedge or MAG pole tip. In FIG. 32 , side fixtures 1074 can be used to align and position the channel 201 / 101 relative to a designated location on the channel holder 1081 or 1082, and the fixtures 1074 can align with predetermined slots, notches, clips, or other physical features on the side of the channel holder 1081 / 1082. In one embodiment, the channel 201 / 101 can be slightly stretched along its length, so that the channel 201 / 101 may have a narrower width 2011 between the fixtures 1074; however, this stretching helps ensure that the channel is straight, which can then be aligned to match the straight MAG wedge of the MAG 123. After channel 201 / 101 is attached to holder 1081 / 1082 by fixture 1074, holder 1081 / 1082 can then move channel 201 / 101 to a separation position, where holder 1081 / 1082 can have a predetermined physical orientation, e.g., a hinge, relative to MAG 123, which precisely adjusts the position of channel 201 / 101 relative to the MAG wedge or MAG pole tip of MAG 123. Fixture 1074 can be the same as 1073 in Figures 28A-30B.

[0400] 33A-37 illustrate methods for utilizing peristaltic pumps in embodiments of the present invention.

[0401] FIG. 33A shows a typical peristaltic pump 500 including a rotor 501, a driver 502 attached to the rotor 501, and pump tubing 504 / 505, where tubing 504 is a fluid inlet portion and tubing 505 is a fluid outlet portion of the same pump tubing. When rotor 501 rotates in direction 503, driver 502 compresses the pump tubing, forcing fluid to move from inlet portion 504 to outlet portion 505 in directions 5041 and 5051, respectively. When rotor 501 rotates in the opposite direction to direction 503, fluid moves from outlet portion 505 to inlet portion 504 of the pump tubing. Connectors 506 and 507 may be optional connections to inlet and outlet fluid lines 508, respectively. An advantage of peristaltic pumps is that tubing 504 / 505 may be included as a continuous portion of a closed fluid line, may be disposable and single-use, and may be sterile for clinical purposes, as shown in FIGS. 55A-60B. However, due to the spacing of the drivers 502 around the circumference of the rotor 501, the flow rate of the fluid discharge from section 505 has a pulsating behavior, with the flow rate increasing and decreasing with the movement of each driver 502. Such pulsation is undesirable for MAG and UFL fluid actuation. FIG. 33A shows that the discharge portion 505 discharges fluid through a connector into a channel 508. The channel 508 is preferably a flexible tube. The channel 508 can also be part of channel 201. The flow restrictor portions 509 and 510 function together to effectively clamp against the channel 508 to reduce the fluid flow rate through the restrictor. As the flow rate through the restrictor decreases, the continuous discharge of fluid from portion 505 of the pump 500 into the channel 508 increases the fluid pressure within the channel 508. Due to the flexibility of the channel 508, the channel 508 can expand in width perpendicular to the channel length, creating a fluid accumulation within the channel 508 due to elastic stresses built up in the channel walls.During a pulse of output flow from pump 500, as flow rate 5051 increases, channel 508 widens, increasing stress on the 508 channel walls and pressure within channel 508, and the volume of channel 508 increases to absorb most of the instantaneous inflow flow while exhibiting a smaller increase in flow rate 520 through restrictor 509 / 510 into channel 501. As flow rate 5051 decreases, the accumulated elastic stress on the channel 508 walls and fluid pressure in channel 508 continue to push fluid through restrictor 509 / 510, exhibiting a smaller decrease in flow rate 520.

[0402] FIG. 33B shows a top view of the internal structure of a first type of flow restrictor 509 along direction 63. FIG. 33B shows that flow restrictor 509 has a shaped trench 5011 that allows fluid to flow through channel 508 when restrictors 509 and 510 are pressed onto channel 508 as in FIG. 33A . Trench 5011 has an inlet width 511 to the incoming fluid and an outlet width 512 to channel 201, where width 511 may be wider than width 512. As the width of trench 5011 narrows from 511 to 512, the flow rate through restrictor 509 / 510 decreases. Flow restrictor 510 may have the same top view and structure as restrictor 509 when viewed in the direction opposite 63.

[0403] Figure 33C shows a second type of flow restrictor in the same view as Figure 33A, where after flow restrictors 509 and 510 are pressed onto channel 508, flow restrictors 509 / 510 form an effective opening at 514 towards channel 508 and an opening at 513 towards channel 201. Opening 513 can be smaller than opening 514, which reduces the flow rate through restrictors 509 / 510.

[0404] Figure 34A is the same as Figure 33A, except that flow restrictor 509 / 510 is removed from flexible channel 508, so that flow from pump 500 through channel 508 and channel 201 is continuous without restrictor 509 / 510 and no elastic stress builds up in the channel 508 wall.

[0405] Figure 34B is a schematic diagram of fluid flow rate 520 as in the situation of Figure 34A, showing large pulsations in flow rate 520. Figure 34B shows example 520 flow rate values ​​versus run time of pump 500 from start of pumping to end of pumping. Value 521 indicates a high flow rate, and value 522 indicates a low flow rate pulsating behavior.

[0406] FIG. 35A is the same as FIG. 33A, in which flow restrictor 509 / 510 is pressed onto flow channel 508, reducing the flow rate through flow restrictor 509 / 510, widening the channel width of channel 508, and causing elastic stress to build up in the walls of channel 508.

[0407] Figure 35B is a schematic diagram of fluid flow rate 520 as in the situation of Figure 35A, showing a reduction in pulsations in flow rate 520 compared to Figure 34B. Value 523 corresponds to value 521 in Figure 34B, and value 524 corresponds to value 522 in Figure 34B. Figure 35B shows that restrictors 509 / 510 effectively reduce 520 flow rate pulsations. Because channel 508 liquid pressure increases at the start of pumping and channel 508 liquid pressure dissipates at the end of pumping, a flow rate increase gradient 5221 after pumping starts and a flow rate decrease gradient 5222 after pumping ends may exist in Figure 35B while restrictors 509 and 510 are engaged.

[0408] 36A and 36B show a method for using flow restrictors 509 / 510 to generate short, instantaneous pulses of high flow rate through channel 201 to flush out magnetically separated entities, such as dissociated cells 10 / 30.

[0409] Figure 36A shows the situation in Figures 33A and 35A, where pump 500 pumps fluid into channel 508 while flow restrictors 509 and 510 press onto flexible channel 508, causing pressure to build up in flexible channel 508 and elastic stress to build up in the walls of channel 508. Line 525 represents the continuous channel 201 from after restrictors 509 / 510 to channel 201 on the MAG structure. Flow rate 5201 represents the average flow rate of flow rates 523 and 524 in Figure 35B when flow restrictors 509 and 510 are fitted.

[0410] Figure 36B shows that flow restrictors 509 and 510 are removed from flexible channel 508, similar to the situation in Figure 34A, while pump 500 is still pumping fluid through channel 508, or immediately after pump 500 stops pumping and before the pressure in channel 508 dissipates. Upon release of restrictors 509 and 510, the liquid pressure in channel 508 and elastic stresses in the walls of channel 508 can create an instantaneous high-velocity fluid pulse flow 5202 into channel 201, flushing magnetically separated entities out of channel 201. Such high-velocity, short-pulse flow 5202 can help achieve complete flushing of cells 10 / 30 with the small volume of fluid originally contained in channel 508 of Figure 36A. FIG. 36B also shows that a rigid cladding structure 1075 may be in contact with the channel 201 to reduce deformation of the flexible channel 201 during the flushing of the cells 10 / 30 and help maintain the flow rate through the channel 201.

[0411] FIG. 37 is a schematic diagram of the fluid flow pulses produced by operation of the flow restrictors of FIGS. 36A-36B, where 5201 is the fluid flow rate in channel 201 before restrictors 509 and 510 are released, and 5202 is the peak value of the flow rate after restrictors 509 and 510 are released.

[0412] 33A-36B, channel 508 is a flexible channel, while channel 201 may be replaced by rigid channel 101, 301, 320 or 330. In FIG.

[0413] 38A-43 describe various embodiments of microfluidic chips ("UFLs") and methods of use.

[0414] 38A shows a top view of a first UFL embodiment, UFL600, in which the microfluidic channels are formed as trenches in a base material 611. The UFL contains an inlet 602 for a substantial fluid 6020, an inlet 604 for a buffer solution 6040, a main channel 601, an outlet 607 for a large entity 6070, and an outlet 609 for a small entity 6090. Two side channels 603 connect the inlet 602 to the main channel 601 from two sides of the main channel 601. The inlet 604 is directly connected to the main channel 601 at its center. The main channel 601 connects to the outlet 607 at the center of the main channel 601 and connects to the outlet 609 from two sides of the main channel 601 through two side channels 608. The substantial fluid 6020 contains both the large entity 6070 and the small entity 6090. Buffer solution 6040 is a fluid for providing UFL function but without biological entities. The large entity 6070 fluid from outlet 607 contains mainly large entities 6070 and buffer solution 6040. The small entity 6090 fluid from outlet 609 contains mainly small entities 6090 and substance fluid 6020, and may contain a certain amount of buffer solution 6040. During UFL 600 operation, buffer solution 6040 and substance fluid 6020 are simultaneously pumped into outlets 604 and 602, respectively, with buffer solution 6040 flowing along the centerline of main channel 601 and substance fluid flowing near the two sides of the main channel in a laminar flow. Buffer solution 6040 carries large entities 6070 to outlet 607, and substance fluid carries the remaining small entities 6090 to outlet 609. The channel 601 is substantially straight and linear along its length from the inlet 604 to the outlet 607 .

[0415] FIG. 38B is a cross-sectional view of a portion of UFL 600 of FIG. 38A along direction 64, including solid fluid inlet 602, buffer inlet 604, and a portion of UFL main channel 601. FIG. 38B shows that UFL 600 is comprised of two components: base 611 and cover 610. Inlets 602 and 604, outlets 607 and 609, and channels 601, 603, and 608 are formed in base 611 as trenches of the same depth 627, preferably formed in a single step. In one embodiment, depth 627 is between 100 nm and 500 nm. In another embodiment, depth 627 is between 500 nm and 1 μm. In yet another embodiment, depth 627 is between 1 μm and 10 μm. In yet another embodiment, depth 627 is between 10 μm and 100 μm. In yet another embodiment, depth 627 is between 100 μm and 1 mm. Cover 610 contains external access ports to the inlets and outlets of UFL 600, allowing entity fluid 6020 and buffer 6040 to enter through inlets 602 and 604, and large entity 6070 and small entity 6090 fluids to exit through outlets 607 and 609. Inlets 602 and 604 and outlets 607 and 609 are shown as circular in FIG. 38A but may be any other shape, including oval, square, rectangular, triangular, or polygonal, as appropriate for the application. The access ports in cover 610 are gaps, or holes, through cover 610 that directly cover inlets and outlets 602, 604, 607, and 609. FIG. 38B shows example gaps for access ports 621 and 641 that match the locations of inlets 602 and 604. After fabrication of UFL 600 base 611 with inlet, outlet and channel trenches and cover 610 with access ports, cover 610 is placed over base 611 to form closed channels 601, 603 and 608, and cover 610 may be bonded to base 611 through any of the following: (1) face-to-face van der Waals forces; (2) adhesion; or (3) ultrasonic thermal fusion if one or both of base 611 and cover 610 are made of plastic or polymeric material.The access port gaps in cover 610, e.g., 621 and 641 to inlets and outlets 602, 604, 607, and 609, are preferably smaller in size than the corresponding inlets and outlets, allowing for positioning errors during alignment of cover 610 to base 611 without causing loss of UFL functionality due to misalignment. Next, injectors 6021 and 6041 illustrate examples of possible external fluid injection into the inlets of UFL 611 through the access port gaps in cover 610, where injectors 6021 and 6041 may have larger nozzle sizes than matching access ports 621 and 641 to manage positioning errors between the injectors and the access ports. Figure 38B shows that a substance fluid 6020 containing large entities 612 and small entities 613, which can be injected by injector 6021, passes through access port 621, enters inlet 602 and enters main channel 601 as a lateral laminar flow, while a buffer solution 6040 can be injected by injector 6041, passes through access port 641, enters inlet 604 and enters main channel 601 as a central laminar flow.

[0416] The base 601 can be made of glass, silicone, aluminum-titanium-carbon (AlTiC), plastic, polymer, ceramic, or metal, and the metal can be made of any one of iron, nickel, chromium, platinum, tungsten, and rhenium, or any alloy thereof. In one embodiment, forming the inlets, outlets, and channels in the base 611 includes the following steps: (1) providing the base 611 with a substantially flat surface; (2) forming an etching mask on the upper surface of the flat surface; and (3) etching the base by a first etching method, including wet etching using a fluid chemical, dry etching using a chemical gas, plasma dry etching, sputter etching using ion plasma, and ion beam etching (IBE). In forming the etching mask in step (2), the etching mask can be made of photoresist (PR), which can include depositing or spin-coating PR on the flat surface; exposing it to light or ion / electron beams in the pattern of the inlets, outlets, and channels; and developing the PR after the exposure, where the remaining PR with the pattern becomes the etching mask. The etching mask can also be made from a hard mask material that has a lower etching rate than the base material under a first etching method, and step (2) can include the following steps: depositing a hard mask layer on the planar surface; depositing or spin-coating a PR layer on the hard mask layer; exposing the PR to light or ion / electron radiation in a pattern of inlets, outlets, and channels, and developing the PR after the light exposure (wherein the remaining PR with the pattern serves as an etching mask for the hard mask); etching the hard mask using a second etching method, including wet etching using a fluid chemical, dry etching using a chemical gas, plasma dry etching, or sputter etching using an ion beam; and removing the remaining PR layer. The second etching method and the first etching method can be different in type or chemicals.

[0417] In another embodiment, the inlets, outlets, and channels in the base 611 may be formed by heat pressing, which involves using a heated stencil having the physical pattern of the inlets, outlets, and channels to melt and deform a portion of the base 611 to create the inlets, outlets, and channels, then cooling the base 611 and removing the stencil. In heat pressing, the base material is preferably a plastic or polymer. In yet another embodiment, the inlets, outlets, and channels in the base 611 may be formed by stamping, which involves using a stencil having the physical pattern of the inlets, outlets, and channels to stamp into a partially or fully melted base 611, then cooling the base 611, and finally removing the stencil, with the cooled base retaining the pattern transferred from the stencil of the inlets, outlets, and channels. In stamping, the base material is preferably a plastic or polymer. In another embodiment, the inlets, outlets, and channels are formed in the base 611 by injection molding, where molten base 611 material is poured into a mold cavity and the body of the base 611 with the inlets, outlets, and channels engraved therein is defined by the mold cavity. The cover 610 can be constructed similarly to the material of the base 611, and the access ports of the cover 610 can be formed in the cover 610 similar to the inlets, outlets, and channels formed in the base 611 as described above.

[0418] FIG. 38C is a schematic diagram illustrating a single fluid pressure node 615 created between two sidewalls of the UFL 600 channel 601 of FIG. 38A by ultrasonic vibrations generated by the PZT 614. FIG. 38C is a cross-sectional view along direction 65 of FIG. 38A of a portion of the UFL 600, including the main channel 601, base 611, cover 610, and PZT transducer 614 attached to the bottom of the base 611. FIG. 38C shows that after injection of the substantive fluid 6020 and buffer solution 6040, the substantive fluid 6020, containing the large entities 612 and the small entities 613, flows primarily laminarly along the edges of the channel 601. An AC voltage is applied to the PZT 614, with the frequency (Fp) of the AC voltage preferably matching the PZT resonant frequency (Fr). The PZT 614 generates ultrasonic vibrations at frequency Fp relative to the base 611. The ultrasonic vibrations are transmitted to the fluid contained in channel 601. Channel 601 has a channel width 625, defined as the vertical distance between two sidewalls of channel 601. In one embodiment, width 625 is between 100 nm and 1 μm. In another embodiment, width 625 is between 1 μm and 10 μm. In yet another embodiment, width 625 is between 10 μm and 100 μm. In yet another embodiment, width 625 is between 100 μm and 500 μm. In yet another embodiment, width 625 is between 500 μm and 5 mm. If channel width 625 is a half wavelength or an integer multiple of a half wavelength of an ultrasonic mode in the fluid within channel 601 at frequency Fp, a standing wave may exist between the two sidewalls of channel 601, as indicated by dashed line 626. Figure 38C shows that when channel width 625 is one-half wavelength of the fluid ultrasonic mode at frequency Fp, a single fluid pressure node 615 is formed along the centerline of channel 601 in the direction of the channel length perpendicular to the view of Figure 38C. In another embodiment, channel width 625 is an integer multiple of one-half wavelengths of the fluid ultrasonic mode at frequency Fp, where the integer is greater than one, and this integer number of fluid pressure nodes can be formed across width 635, each node being a line along the direction of the channel length.The presence of standing waves 626 and pressure nodes 615 exert an acoustic radiation force, shown in FIG. 38D as arrows 628, on entities in the laminar flow of the entity fluid along the sidewall of channel 601, causing large entities 6070 to move closer to central node 615 as they flow through channel 601. This acoustic radiation force 628 has the following characteristics: (1) maximum amplitude near the sidewall of channel 601, with the force direction pointing from the sidewall to node 615; (2) minimum or near zero force around node 615; (3) linearly proportional to entity size; and (4) is a function of the density and compressibility of both buffer solution 6040 and the entity. Due to these characteristics, by appropriately optimizing the buffer composition, the laminar flow velocity of the buffer solution 6040, and the laminar flow velocity of the entity fluid 6020, larger acoustic radiation forces act on the entity 612, concentrated around the central nodal point 615, and the entity 612 can be optimized to preferably break through the laminar flow barrier and enter the buffer laminar flow.

[0419] Figure 38D is a schematic diagram showing the hydroacoustic waves of Figure 38C moving larger entities 612 into the laminar buffer flow around the center of channel 601. When the fluid in channel 601 exits the channel to outlets 607 and 609, the central subchannel width 651 of channel 601 in Figure 38A to outlet 607 can be much narrower than the width 625 of channel 601, thus only allowing large entities 612 in the central flow within channel 601 to exit outlet 607 as large entity 6070 fluid. Smaller entities 613, primarily near the sidewall laminar flow, exit channel 601 through side channels 308 and exit outlet 609 as small entity 6090 fluid.

[0420] In one embodiment, the frequency of vibration Fp of PZT 614 is 100 kHz to 500 Hz, in another embodiment, 500 kHz to 1 MHz, in yet another embodiment, 1 MHz to 3 MHz, in yet another embodiment, 3 MHz to 10 MHz, and in yet another embodiment, 10 MHz to 100 MHz. In Figures 38C and 38D, PZT 614 can also be attached to the top of cover 610 in Figures 38C and 38D, and ultrasonic vibrations from PZT 614 are transmitted from PZT 614 through cover 610 to the fluid in channel 601, or through cover 601 to base 611, and then to the fluid in channel 601.

[0421] FIG. 39 is a schematic diagram illustrating a method for separating biological entities of different sizes using a UFL, which may be UFL 600 of FIG. 38A or 40A, or UFLs 620, 630, and 640 of FIG. 41A-43. As shown in FIG. 41B and 42B, the sequential steps of 701-705 and 706 are substantially similar to those described in FIG. 38A, 38B, 38C, and 38D, except that steps 703 and 704 refer to the possibility of multiple pressure nodes. The step 707 entity analysis may be performed on both large entities 6070 and small entities 6090 and may include steps 903, 904, 905, 906, 5824, 5825, and 5826, as described in FIG. 53, 79, 80, 82, and 83, in the corresponding UFL discharge sample. For example, step 708 continues through a MAG device as shown in Figures 44A-45C, 47-49, or through a cascaded UFL process as in Figure 54C.

[0422] FIG. 40A is a cross-sectional view of a portion of UFL 650 similar to FIG. 38B. UFL 650 is identical to UFL 600 from a top-down view, as in FIG. 38A, except that a uniform soft magnetic layer (“SML”) 616 is deposited on a base 611 of UFL 650 and patterned with base 611 to form inlets 602 and 604, outlets 607 and 609, and channels 601, 603, and 608. SML 616 can be composed of at least one element from iron (Fe), cobalt (Co), and nickel (Ni). Thickness 6164 of SML 616 is 10 nm to 100 nm in one embodiment, 100 nm to 1 μm in another embodiment, 1 μm to 10 μm in yet another embodiment, 10 μm to 100 μm in yet another embodiment, 100 μm to 1 mm in yet another embodiment, and 1 mm to 3 mm in yet another embodiment. Deposition of SML layer 616 on base 611 can be by electroplating, vacuum plating, plasma vapor deposition (PVD), atomic layer deposition (ALD), or chemical vapor deposition (CVD). Etching of layer 616 together with base 611 to form inlets 602 and 604, outlets 607 and 609, and channels 601, 603, and 608 can be by dry etching, plasma dry etching, ion plasma etching, and IBE. Layer 616 can be a continuous layer along the length of channel 601 and formed as part of the sidewalls of channel 601.

[0423] FIG. 40B is similar to FIG. 38D and shows a schematic diagram illustrating that if large entities 612 are concentrated in the center of channel 601 around pressure node 615 by acoustic radiation force 628, small entities 613 will remain primarily around the sidewalls of channel 601. Additionally, a magnetic field 617 is applied to the surface, inducing magnetization 6162 in SML layer 616. For SML layer 616, which is disposed as part of the sidewall of channel 601, magnetization 6162 generates a local magnetic field 6163, which has the strongest field strength and field gradient near the sidewalls of channel 601. Magnetic field 6163 may help keep small magnetic entities, such as free magnetic label SPL2 that is part of entity fluid 6020 in a positive sample after MAG separation as shown in FIGS. 82 and 83, in the laminar flow near the sidewalls of channel 601 and be expelled from outlet 609 in FIG. 38A.

[0424] 40C shows that after etching the SML layer 616 together with the base 611 and before attaching the cover 610 to the base 611, the exposed surfaces of the SML layer 616 and base 611 can be coated to deposit a passivation layer 6172, which can help isolate fluid reactions with the material of the SML layer 616. Layer 6172 can be deposited on the etched surfaces of the SML 616 and base 611, preferably conformally, by vacuum plating, electroplating, PVD, ALD, CVD, molecular beam deposition (MBE), and diamond-like carbon (DLC) deposition. Layer 6172 can be an oxide, nitride, or carbide of any one or more of the elements Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr, Zn, Mg, Nb, Mo, Ni, Co, Fe, Ir, Mn, Ru, Pd, and C. Layer 6273 may be composed of at least one of Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr, Zn, Mg, Nb, Mo, Ni, Co, Fe, Ir, Mn, Ru, Pd, and C. Layer 6273 may be a DLC layer. The thickness of layer 6273 may be 1 nm to 10 nm in one embodiment, 10 nm to 100 nm in another embodiment, 100 nm to 10 μm in another embodiment, and 10 μm to 100 μm in another embodiment.

[0425] FIG. 41A shows a top view of a second UFL embodiment, UFL620, which is the same as FIG. 38A except that it includes a wider main channel portion 6012 connecting between the inlet 604 and the narrower channel portion 601 of FIG. 38A. A slope 6016 corresponds to the transition 6016 from the wider portion 6012 to the narrower portion 601. The channel portions 6012 and 601 are substantially straight and linear along the length of the channel. The transition portion 6016 can be part of the main channel, which includes the channel portion 6012 that connects to the channel portion 601 through the transition portion 6016. The transition portion 6016 functions to converge the fluid flow from the wider portion 6012 to the narrower portion 601. The channel wall of the transition portion 6016 can intersect with the straight wall of the wider portion 6012 at the beginning of the transition. The channel walls of the transition portion 6016 may intersect with the straight walls of the narrower portion 601 at the transition stop point. In one embodiment, the channel shape of the transition portion 6016 between the transition start point and the transition stop point may be a straight slope, as shown in FIG. 41A. In another embodiment, the channel shape of the transition portion 6016 between the transition start point and the transition stop point may be curved, but the curvature may be tangential to one or both of the channel walls of the wider portion 6012 and the channel walls of the narrower portion 601.

[0426] Figure 41B is a cross-sectional view of UFL 620 along direction 66 in Figure 41A across wider portion 6012. Wider portion 6012 has a channel width 6252, which is a full wavelength of the ultrasonic mode in the liquid in channel portion 6012 at the operating frequency Fp of PZT 614, as shown in Figure 38C, effectively doubling the channel width 625 of channel 601 as in Figures 38C and 41C. Because channel width 6252 is equal to a full wavelength of the ultrasonic mode at Fp, two pressure nodes may exist in channel portion 6012, and acoustic radiation forces from the ultrasonic mode may move and focus large entity 612 at each of the two nodes from the channel wall entity laminar flow.

[0427] Figure 41C is a cross-sectional view of UFL 620 along direction 65 of Figure 41A, across narrower portion 601, and is identical to Figure 38D. After the fluid in channel portion 6012 flows through transition 6016 into channel portion 601, a single pressure node in channel portion 601 forces large entities 612 to concentrate at the center of channel portion 601, the same as in Figure 41C. Wider portion 6012 causes a first stage of separation of large entities 612 from smaller entities 613. After transition 6016, due to the narrowing of the channel width from 6252 to 625, the flow rates of the central buffer laminar flow and the laminar flow of entities at the channel sidewalls increase by approximately two times the velocity of the same flow in portion 6012. Channel portion 601, together with the increased flow rate in channel portion 601, may provide a second stage of separation of large entities from smaller entities, improving the purity of large entities 612 in the 6070 fluid discharge from outlet 607, as well as the purity of small entities 613 in the 6090 fluid discharge from outlet 609, compared to UFL 600 of FIG. 38A.

[0428] Figure 42A shows a top view of a third UFL embodiment, UFL630, which is a further improvement over UFL620 of Figure 41A. Compared to UFL620 of Figure 41A, all aspects of Figure 42A are the same as Figure 41A, except that UFL630 of Figure 43A includes an additional side channel 6013 that connects around transition section 6016 to side channel 608, or in another embodiment, directly to outlet 609, to divert the sidewall laminar flow of small entities 613 from the wider section, referred to as first-stage section 6012, or directly to outlet 6090 without entering the narrower section as shown in Figure 42B, or referred to as second-stage section 601. Channel sections 6012 and 601 are substantially straight and linear along the length of the channel. In one embodiment, the side channel 6013 connects from the first stage portion 6012 before the transition start point of portion 6012 intersects with portion 6016. In another embodiment, the side channel 6013 connects from the transition start point of portion 6012 where it intersects with portion 6016. In yet another embodiment, the side channel 6013 connects from within the transition 6016, between the transition start point of portion 6012 where it intersects with portion 6016 and the transition stop point of portion 6012 where it intersects with portion 601. In yet another embodiment, the side channel 6013 connects from the transition stop point of portion 6012 where it intersects with portion 601. In yet another embodiment, the side channel 6013 connects from the second stage portion 601 after the transition stop point of portion 6012 where it intersects with portion 601.

[0429] Figure 42B is a cross-sectional view of UFL 630 along direction 66 in Figure 42A across wider portion 6012. Figure 42B is identical to Figure 41B.

[0430] Figure 42C is a cross-sectional view of UFL 630 along direction 65 in Figure 42A across narrower portion 601 and side channel 6013. In comparison to Figure 41C, side channel 6013 connecting from around transition section 6016 in Figure 42A contains primarily or purely small entities 613. Channel 601 in Figure 42C shows the separation of larger entities 612 and concentration toward a pressure node in the center of channel 601, similar to Figure 41C, but the small entities 613 around the channel walls of portion 601 are at a reduced density when compared to Figure 41C. Due to the diversion of small entities in pre-channel section 601 by side channel 6013, UFL 630 can be even more enriched in large entities 612 in the 6070 fluid discharge from outlet 607 and more enriched in small entities 613 in the 6090 fluid discharge from outlet 609.

[0431] FIG. 43 is a top view of a fourth UFL embodiment, UFL 640, having a multi-stage UFL channel with a continuously narrowing channel width along the channel flow path. FIG. 43 illustrates a further enhancement of large-entity purity at 6070 and an increase in small-entity purity at 6090. FIG. 43 illustrates that an additional, wider-width section 6014 is added between inlet 604 and channel section 6012. The channel width of 6014 may be three half-wavelengths of the ultrasonic mode of the liquid flowing through the UFL 640 channel at the PZT frequency Fp, which is one-half wavelength wider than the channel width 6252 of section 6012. The channel width of 6014 may also be wider than the channel width 6252 of the next stage channel section 6012 by an integer number of half-wavelengths, where the integer is greater than one. Channel section 6014 transitions into narrowing channel section 6012 through transition section 6017. Side channel 6015 connects around transition 6017 to side channels 6013 or 608, or directly to outlet 609, to divert small entities 613 from the channel sidewall laminar flow in portion 6014 away from inlet portion 6012, thereby improving the purity of the large entity concentration in portion 6012. Channel portions 6014, 6012, and 601 are substantially straight and linear along the length of the channel.

[0432] As a further extension from FIG. 43 , the multi-stage UFL 640 can have multiple channel sections along the UFL 640 channel flow path, with each initial section of the UFL channel along the channel flow path having a channel width that is wider than the channel width of the immediately succeeding section by an integer multiple of half wavelengths of the ultrasonic mode of the fluid flow at the PZT frequency Fp, where the integer is equal to or greater than 1. The final channel section before the flow exits the UFL 640 outlet preferably has a channel width equal to said half wavelength in one embodiment, but can also have a channel width equal to an integer multiple of said half wavelength in another embodiment, where the integer is equal to or greater than 1. Side channels connecting each transition region between adjacent channel sections divert small entities from the earlier channels in the entity laminar flow closer to the earlier channel wall toward the outlet 609, reducing the number of small entities entering the immediately succeeding channel section.

[0433]

[00130] Figures 44A through 65B illustrate various embodiments of methods for utilizing MAG and UFL devices to separate biological entities from a biological fluid. For simplicity, the UFL 600 and MAG 123 with channel 201 of Figure 38A are used in the figures for illustration. However, UFL 600 can be replaced with UFLs 650, 630, and 640 of Figures 40A, 41A, 42A, and 43, while MAG 123 can be replaced with MAGs 121, 122, 124, 125, 126, 127, 128, and 129 and the corresponding channel types described in the preceding figures, without limitation and without sacrificing performance.

[0434] Figure 44A shows a first type of sample processing method in which a biological sample first passes through the UFL 600, then the large-entity effluent 6070 of the UFL 600 passes through the channel 201 adapted for the MAG123, and a first type of flow connector 801 connects the UFL 600 large-entity outlet 607 and the MAG inlet flow, as in step 401 of Figure 31 or step 708 of Figure 39. For sequential operation of the UFL 600 and MAG123 devices, the optimal flow rates for the UFL channel 601 and the MAG channel 201 may be different. The optimal flow rate for acoustic radiation force separation of large and small entities in the UFL channel 601 is determined by laminar flow conditions and the separation efficiency between large and small entities. The optimal flow rate for MAG123 separation is determined by the length of the channel 201 and the magnetic field force on the magnetic labels attached to the entities. Direct fluid coupling from UFL600 outlet 607 to MAG channel 201 inlet would force the flow rates through the UFL600 channel and MAG channel 201 to be the same, which could adversely affect the separation efficiency for either or both of the UFL600 and MAG123. It is necessary to decouple the fluid flow through the UFL600 and MAG123 channels 201. Flow connector 801 serves to decouple the flow rates of UFL600 and MAG123. The discharge fluid 6070 is first injected into connector 801 through inlet 8011, and the fluid in connector 801 flows as in stages 401 / 708 to the inlet of MAG123 channel 201 and is discharged through outlet 8012. Both UFL600 and MAG123 channels 201 can operate at their individual optimal flow rates. In one embodiment where the optimal flow rate of MAG123 channel 201 is greater than the optimal flow rate of UFL 600, MAG123 extracts fluid 401 / 708 from connector 801 faster than UFL 600 injects fluid 6070 into connector 801. A fluid level sensor 100 may be attached to connector 801 to sense the fluid level remaining in connector 801.If the fluid level drops below a low threshold, as in the intake of stage 401 / 708, sensor 100 may signal MAG 123 to pause flow and wait for the liquid level inside connector 801 to rise to another, higher level before MAG 123 can resume extracting liquid from connector 801 as in stage 401 / 708. In another embodiment where the optimal flow rate of MAG 123 channel 201 is less than the optimal flow rate of UFL 600, MAG 123 extracts fluid from connector 801 as in stage 401 / 708 more slowly than UFL 600 injects fluid 6070 into connector 801. If the fluid level increases above a low threshold, sensor 100 may signal UFL 600 to pause discharging flow 6070 and wait for the liquid level inside connector 801 to drop to another, lower level before UFL 600 can resume discharging fluid 6070 into connector 801. Flow connector 801 may be designed as shown in FIG. 44A , where inlet 8011 is at a higher vertical position than outlet 8012, and flow 6070 enters connector 801 and accumulates at outlet 8012 inside 801 by gravity. Alternatively, the liquid sample may first be processed completely through UFL 600 and stored in connector 801. MAG 123 then extracts fluid from connector 801 as input to MAG 123 channel 201, completing processing of the entire liquid sample from connector 801. The connector 801 can be made as part of a closed fluid line, where the fluid sample is not exposed to air and is sterile during passage of flow 6070 from the outlet 607 of the UFL 600 to the inlet 8011 of the connector 801 to flow to the inlet of the channel 201 as in step 401 / 708, to the outlet 8012.

[0435] FIG. 44B illustrates the first type of sample processing method of FIG. 44A by using a second type of flow connector 802 connecting the UFL600 large-volume outlet 607 and the MAG123 channel 201 inlet. The connector 802 as shown in FIG. 44B takes a form similar to a vial. Stream 6070 enters the connector 802 through the short inlet tube 8021 of the connector 802 and drips to the bottom of the connector 802 due to gravity. Streams like those in steps 401 / 708 are extracted from the fluid at the bottom of the connector 802 by the long outlet tube 8022 to the input of channel 201. A fluid level sensor 100 can be attached to the connector 802 to detect the fluid level in the connector 802. Both the UFL600 and the MAG123 can operate at their respective optimal flow rates, and the fluid level sensor 100 can function to pause UFL600 operation or MAG123 operation in the same manner as described in FIG. 44A. Alternatively, the liquid sample can be processed completely through UFL 600 and stored in connector 802. MAG 123 then extracts the fluid from connector 801 as input to MAG 123 channel 201, completing processing of the entire liquid sample from connector 802. Connector 802 can be made as part of a closed fluid line similar to connector 801.

[0436] FIG. 44C illustrates the first type of sample processing method of FIG. 44A by using a third type of flow connector 803 connecting the UFL600 large-volume outlet 607 and the MAG123 channel 201 inlet. The connector 803 as shown in FIG. 44C takes a form similar to a fluid bag or blood bag. Flow 6070 enters the connector 803 through the bottom inlet 8031 ​​and fills the connector 803 from the bottom of the connector 803 by gravity. Flow, like steps 401 / 708, is extracted from the fluid at the bottom of the connector 803 through outlet 8032 to the input of channel 201. A fluid level sensor 100 can be attached to the connector 803 to detect the fluid level in the connector 803. Both the UFL600 and the MAG123 can operate at their respective optimal flow rates, and the fluid level sensor 100 can function to pause UFL600 operation or MAG123 operation in the same manner as described in FIG. 44A. Alternatively, the liquid sample can be processed completely through UFL 600 and stored at connector 803. MAG 123 then extracts fluid from connector 801 as input to MAG 123 channel 201, completing processing of the entire liquid sample from connector 803. Connector 803 can be made as part of a closed fluid line similar to connector 801.

[0437] Figure 45A shows a second type of sample processing method in which the biological sample first passes through UFL 600, then the small entity output 6090 of UFL 600 passes through MAG 123, and a first type of flow connector 801 connects the UFL small entity 6090 outlet 609 and the MAG 123 channel 201 inlet. Figure 45A is identical in all aspects to Figure 44A except that the small entity flow 6090 from outlet 609 is injected into the inlet 8011 of connector 801.

[0438] Figure 45B shows a second type of sample processing method in which the biological sample first passes through UFL 600, then the small entity output 6090 of UFL 600 passes through MAG 123, and a second type of flow connector 802 connects the UFL small entity 6090 outlet 609 and the MAG 123 channel 201 inlet. Figure 45B is identical to Figure 44B in all aspects, except that the small entity flow 6090 from outlet 609 is injected into the inlet 8021 of connector 802.

[0439] Figure 45C shows a second type of sample processing method in which the biological sample first passes through UFL 600, then the small entity output 6090 of UFL 600 passes through MAG 123, and a third type of flow connector 803 connects the UFL small entity 6090 outlet 609 and the MAG 123 channel 201 inlet. Figure 45C is identical to Figure 44C in all aspects, except that the small entity flow 6090 from outlet 609 is injected into the inlet 8031 ​​of connector 803.

[0440] 46A shows a third type of sample processing method in which a biological sample first passes through MAG123 channel 201, and then, after step 427 or 428 of FIG. 31 , the output of MAG123 channel 201 passes through UFL 600 as entity fluid 6020 to inlet 602, as in step 408 of FIG. 31 , with a first type of flow connector 801 connecting the outlet of MAG123 channel 201 and the inlet 602 for entity fluid 6020 of UFL 600. In FIG. 46A , the output from MAG123 can be either a negative entity without SPL2 attached, or a positive entity that is separated by the MAG123 magnetic field and subsequently dissociated and flushed out of channel 201, as described in FIG. 31 . As in FIG. 44A , MAG123 and UFL 600 can each be operated at their individual optimal flow rates. A fluid level sensor 100 may be attached to connector 801 to detect the fluid level remaining in connector 801. Fluid level sensor 100 may operate similarly to FIG. 44A to detect fluid in connector 801 and, depending on the flow rate difference between MAG 123 and UFL 600, pause the flow of MAG 123 or UFL 600 to maintain the fluid level in connector 801 above a low level or below a high level. Alternatively, the liquid sample may first be processed completely through MAG 123 and stored in connector 801. UFL 600 then extracts fluid from connector 801 as input to inlet 602, completing processing of the entire liquid sample from connector 801. Connector 801 may be made part of a closed fluid line similar to FIG. 44A.

[0441] FIG. 46B is the same in all respects as FIG. 46A except that connector 801 is replaced by connector 802, and the operation of connector 802 and attached sensor 100 is the same as that described in FIG. 44B.

[0442] FIG. 46C is the same in all respects as FIG. 46A except that connector 801 is replaced by connector 803, and the operation of connector 803 and attached sensor 100 is the same as that described in FIG. 44C.

[0443] Figure 47 shows a fourth type of sample processing method in which a biological sample is first passed through multiple UFLs 600, and then the output streams from the UFLs 600, which can be either large entities 6070 or small entities 6090, are fed into the inlet 8011 of a fourth type flow connector 8010 and out the connector 8010 outlet 8012 to the inlets of the channels 201 of the multiple MAGs 123. Figure 47 is functionally similar to Figures 44A and 45A. Connector 8010 is also functionally identical to connector 801, except that the inlet 8011 of connector 8010 accepts multiple fluid outputs from multiple UFLs 600 and the outlet 8012 of connector 8010 outputs to the inputs of the multiple channels 201 of the multiple MAGs 123.

[0444] Figure 48 illustrates a fifth type of sample processing method in which a biological sample first passes through multiple UFLs 600, and then the output streams from the UFLs 600, which can be either large entities 6070 or small entities 6090, are fed into an inlet 8021 of a fifth type flow connector 8020 and out an outlet 8022 of the connector 8020 to the inlets of channels 201 of multiple MAGs 123. Figure 48 is functionally similar to Figures 44B and 45B. Connector 8020 is also functionally identical to connector 802, except that the inlet 8021 of connector 8020 accepts multiple fluid outputs from multiple UFLs 600 and the outlet 8022 of connector 8020 outputs to the inputs of multiple channels 201 of multiple MAGs 123.

[0445] Figure 49 illustrates a sixth type of sample processing method in which a biological sample is first passed through multiple UFLs 600, and then the output streams from the UFLs 600, which can be either large entities 6070 or small entities 6090, are fed into an inlet 8031 ​​of a sixth type flow connector 8030 and out an outlet 8032 of the connector 8030 to the inlets of channels 201 of multiple MAGs 123. Figure 49 is functionally similar to Figures 44C and 45C. Connector 8030 is also functionally identical to connector 803, except that the inlet 8031 ​​of connector 8030 accepts multiple fluid outputs from multiple UFLs 600 and the outlet 8032 of connector 8030 outputs to the inputs of multiple channels 201 of multiple MAGs 123.

[0446] In each of Figures 47, 48, and 49, in one embodiment, the same biological sample is split and processed simultaneously through multiple UFLs 600. In another embodiment, each UFL 600 processes a different biological sample. The output from each UFL 600, either a large entity 6070 fluid from outlet 607 or a small entity fluid from outlet 609, shown as dashed lines in Figures 47, 48, and 49, can be individually fed into inlet 8011 of connector 8010 in Figure 47, or into inlet 8021 of connector 8020 in Figure 48, or into inlet 8031 ​​of connector 8030 in Figure 49, as shown by solid lines 6070 / 6090 in each of Figures 47, 48, and 49. From outlets 8012, 8022, 8032 in Figures 47, 48, and 49, respectively, after step 401 or 708, each of the MAGs 123 in Figures 47, 48, or 49 can extract a fluid sample from corresponding connectors 8010, 8020, and 8030 into its corresponding channel 201. Each UFL 600 and each MAG 123 in Figures 47, 48, or 49 can operate at its own individual optimal sample flow rate, which can vary between different UFLs 600 and between different MAGs 123 within the same figure. Because of the presence of connectors 8010, 8020, and 8030, flow rate interference between different UFLs 600 and MAGs 123 in Figures 47, 48, and 49, respectively, is minimized or eliminated. Fluid level sensors 100 may be attached to buffers 8010, 8020, and 8030 to sense the fluid level remaining in each of flow connectors 8010, 8020, and 8030. Fluid level sensors 100 operate similarly to FIGS. 44A-44C in sensing fluid in flow connectors 8010, 8020, and 8030, and may deactivate one or more MAGs 123 or deactivate one or more UFLs 600 in each figure depending on the flow rate differential between the MAGs 123 and UFLs 600 in each figure to maintain the fluid level in the corresponding connector 8010, 8020, or 8030 above a low level threshold or below a high level threshold.Alternatively, the liquid sample may first be processed completely through the entire UFL 600 and stored in the corresponding connector 8010, 8020, or 8030 in each of Figures 47, 48, and 49. MAG 123 then extracts the fluid from the corresponding connector 8010, 8020, or 8030 in each figure, completing processing of the entire liquid sample from each corresponding connector 8010, 8020, or 8030. Similar to those depicted in Figures 44A-44C, connectors 8010, 8020, and 8030 may be made as part of a series of closed fluid lines, which may include connections from UFL 600, channel 201, and UFL 600 to each connector 8010, 8020, 8030, and from each connector 8010, 8020, and 8030 to channel 201, respectively.

[0447] Figure 50 shows a seventh type of sample processing method in which the biological sample first passes through multiple MAG123s, and then the output flow from the MAG123 channels 201 is fed into the inlet 8011 of the flow connector 8010 of Figure 47 and from the flow connector 8010 outlet 8012 into the solid fluid inlets 602 of multiple UFLs 600.

[0448] Figure 51 shows an eighth type of sample processing method in which the biological sample first passes through multiple MAG123s, and then the output flow from the MAG123 channels 201 is fed into the inlet 8021 of the flow connector 8020 of Figure 48 and from the flow connector 8020 outlet 8022 into the solid fluid inlets 602 of multiple UFLs 600.

[0449] Figure 52 shows a ninth type of sample processing method in which the biological sample first passes through multiple MAG123s, and then the output flow from the MAG123 channels 201 is fed into the inlet 8031 ​​of the flow connector 8030 of Figure 49 and from the flow connector 8030 outlet 8032 into the solid fluid inlets 602 of multiple UFLs 600.

[0450] In each of Figures 50, 51, and 52, in one embodiment, the same biological sample is divided and processed simultaneously through multiple MAGs 123. In another embodiment, each of the MAGs 123 processes a different biological sample. The output from each MAG 123, either a negative entity after step 427 or a positive entity after step 428, can be individually fed into inlet 8011 of connector 8010 in Figure 50, or into inlet 8021 of connector 8020 in Figure 51, or into inlet 8031 ​​of connector 8030 in Figure 52, as indicated by solid lines 427 / 428 in each of Figures 50, 51, and 52. From outlets 8012, 8022, 8032 in Figures 50, 51, and 52, respectively, after step 408, each of the UFLs 600 in Figures 50, 51, or 52 can extract a fluid sample as solid fluid 6020 from corresponding connectors 8010, 8020, and 8030 to its corresponding solid inlet 602, and each UFL 600 and each MAG 123 in Figures 50, 51, or 52 can operate at its own individual optimal sample flow rate, which can vary between different UFLs 600 within the same figure and between different MAGs 123. Because of the presence of connectors 8010, 8020, and 8030, flow rate interference between different UFLs 600 and MAGs 123 in Figures 50, 51, and 52, respectively, is minimized or eliminated. Fluid level sensors 100 may be attached to flow connectors 8010, 8020, and 8030 to sense the fluid level remaining in each of the flow connectors. Fluid level sensor 100 operates similarly to FIGS. 46A-47C in sensing fluid in flow connectors 8010, 8020, and 8030, and may pause operation of one or more MAGs 123 or pause operation of one or more UFLs 600 in each figure, depending on the flow rate differential between MAGs 123 and UFLs 600 in each figure, to maintain the fluid level in the corresponding connector 8010, 8020, and 8030 above a low level threshold or below a high level threshold. Alternatively, the liquid sample may first be processed completely through all MAGs 123 and stored in the corresponding connector 8010, 8020, or 8030 in each of FIGS. 50, 51, and 52.UFL 600 then extracts fluid from the corresponding connector 8010, 8020, or 8030 in each figure, completing processing of the entire liquid sample from each corresponding connector 8010, 8020, or 8030. As shown in Figures 46A-46C, flow connectors 8010, 8020, and 8030 may each be made part of a series of closed fluid lines that may include connections from UFL 600, channel 201, and channel 201 to each connector 8010, 8020, 8030, and from each connector 8010, 8020, and 8030 to UFL 600.

[0451] Figure 53 shows a tenth type of sample processing method in which the biological sample after passing through one or more of UFL600 or MAG123, and the output fluids from UFL600 and MAG123, are pumped into the inlet of flow connector 8020 or flow connector 8030, and from the outlets of flow connectors 8020 and 8030 to different types of cell processing devices. Similar to Figures 51 and 52, Figure 53 shows that an example of the liquid sample output from MAG123 channel 201, including the negative entity after step 427 and the positive entity after step 428, can be injected into inlet 8021 of connector 8020 or inlet 8031 ​​of connector 8030. Alternatively, large entity output 6070 of UFL 600 from outlet 607 or small entity output 6090 from outlet 609 can also be injected into inlet 8021 of connector 8020 or inlet 8031 ​​of connector 8030, as in Figures 48 and 49. After the sample fluid has been completely processed through UFL 600 or MAG123 and injected into and stored in connector 8020 or connector 8030, entity analysis can be performed, such as step 407 in Figure 31 and step 707 in Figure 39, by pumping the entity-containing sample fluid from connector 8020 or connector 8030 to either a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, and a DNA or RNA sequencer 906. Further entities may be fed to a DNA or RNA sequencer 906 after the cell counter 903 as at 936, or after the cell imager 904 as at 946, or after the flow cytometer or sorter 905 as at 956. To feed sample fluid from the outlet 8022 of the connector 8020 or from the outlet 8032 of the connector 8030, a pressurized chamber 800 may be used to contain the connector 8020 or connector 8030 and to force the sample fluid out of the connector 8020 or connector 8030 in a steady and continuous flow. The chamber 800 may be a chamber filled with pressurized air therein. The vial-type connector 8020 may have an additional air port 8023 opening to the internal pressurized air of the chamber 800 to help force the sample fluid out of the connector 8020.Connector 8030 may be in the form of a flexible blood bag that automatically contracts under the pressurized air in chamber 800 to push sample fluid out through outlet 8032. To avoid backflow into UFL600 or MAG123 channel 201, shut-off valves 805 may be implemented on the exhaust lines from MAG123 channel 201 and UFL600 to connector 8020 or connector 8030.

[0452] Figure 54A shows an eleventh type of sample processing method in which, as in step 408 of the continuous process, the biological sample after passing through the first MAG123 channel 201 during magnetic separation can discharge a negative entity fluid after step 427 or a positive entity fluid after step 428 into the inlet of the second MAG123 channel 201 input. Figure 54A shows a multi-step MAG process.

[0453] Figure 54B shows a twelfth type of sample processing method in which, after a biological sample has passed through MAG123 for magnetic separation, the outlet fluid from MAG123 channel 201, containing either negative or positive entities, can be diverted to stream 913 through T-connector 912. Stream 913 can then be re-injected into the input of channel 201 of MAG123 for another round of magnetic separation through T-connector 911. T-connector 911 allows for the initial fluid sample input, as in step 401, and the input of recirculation stream 913 into channel 201. T-connector 912 allows for the output from channel 201 to recirculation stream 913 or output from MAG123, as in steps 427 and 428. In one embodiment, recirculation stream 913 contains negative entities, and the repetition of magnetic separation in Figure 54B helps achieve complete depletion of all magnetic entities in the negative entity stream before output into steps 427 / 428. In another embodiment, the recirculation stream 913 contains the positive entities after dissociation, and a repeated process such as that shown in Figure 54B serves to increase the purity in the positive magnetic entities and allow for washing away non-magnetic entities that may be in the aggregate due to non-specific binding. Figure 54B shows the use of the same MAG123 as in the multi-cycle MAG process.

[0454] Figure 54C shows a thirteenth type of sample processing method in which the biological sample after passing through the first UFL 600, the discharge fluid from the first UFL 600, such as a large entity 6070 fluid from outlet 607 or a small entity 6090 fluid from outlet 609, can be passed to the entity fluid inlet 602 of one or more subsequent UFLs 600 as a multi-stage UFL process.

[0455] FIG. 55A shows a first embodiment of a closed, disposable fluid line for the third type of sample processing method as shown in FIG. 46A, in which connector 801 can be replaced with, without limitation, connector 802 or connector 803. Input line 923 can connect to a sample liquid container. Input line 924 can connect to a MAG buffer container. Input line 923 and input line 924 are connected through a T-connector 921 to the inlet of first pump tube 504 / 505, which can be mounted on a peristaltic pump. The outlet of first pump tube 504 / 505 connects to channel 201, which can be used as part of MAG 123. The output of channel 201 connects to T-connector 922, which connects to output line 925 and output line 926. Output line 925 can connect to a MAG Out sample container, and output line 926 connects to the inlet of connector 801. In one embodiment, the outlet line 925 can discharge negative entities into the MAG Out sample container, and the outlet line 926 can discharge positive entities into the connector 801. In another embodiment, the outlet line 925 can discharge positive entities into the MAG Out sample container, and the outlet line 926 can discharge negative entities into the connector 801. The outlet of the connector 801 connects to the input line 9271 of the second pump tube 504 / 505. The outlet of the second pump tube 504 / 505 then connects to the UFL600 sample input line 6020. The input line 9272 can connect to a UFL buffer container and connects to the inlet of the third pump tube 504 / 505. The outlet of the third pump tube 504 / 505 then connects to the UFL600 buffer input line 6040. The output line of the UFL600 large entity 6070 can connect to a large entity sample container. The UFL600 small entity 6090 outlet line may connect to a small entity sample container. Figure 55A shows that in addition to input and output lines 923, 924, 925, 9272, 6070, and 6090 connecting to external containers, the entire flow path from the sample liquid input to line 923, all pumps, MAG123, and other fluid line components to the sample being discharged into lines 925, 6070, 6090 are externally attached to the lines of Figure 55A. Thus, the lines of Figure 55A are internally closed and suitable for single-use disposable and sterile applications.

[0456] Figure 55B shows the fluid lines of Figure 55A connected to or attached with various fluidic components. Input line 923 connects to a liquid sample container 928 in the form of a blood bag. Input line 924 connects to a buffer container 929. Valves 935 and 936 are attached to lines 923 and 924 to control the flow of either sample liquid from bag 928 or buffer from container 929 through T-connector 921 and into first pump tube 504 / 505. First, second, and third pump tubes 504 / 505 each lead to peristaltic pump 500. The three pumps 500 operate to pump either sample fluid or buffer to MAG123 and UFL600. To reduce flow pulsations from pumps 500, flow restrictors 509 / 510 may be attached to the outlet lines from each pump 500, including lines 201, 6020, and 6040. Channel line 201 is attached to MAG123. Outlet line 925 connects to MAG Out sample container 934. Valve 940 is attached to line 925 and valve 937 is attached to line 926, which controls the negative or positive entities from MAG123 entering either container 934 or connector 801 through T-connector 922. Valves 940 and 937 can both block flow in lines 925 and 926 during the demagnetization / dissociation step of MAG123. Input line 9272 can connect to UFL buffer container 931. UFL outlet line 6070 connects to large entity container 932 and outlet line 6090 connects to small entity 933. Adjustable valves 939 and 938 may be attached to lines 6070 and 6090 to adjust the flow rate in each line, 6070 and 6090, which in turn controls the laminar flow rate in the UFL channel relative to the buffer flow in the center of the channel and the solid sample flow at the edges of the channel.

[0457] Figure 56A shows a second embodiment of a closed, disposable fluid line for the third type of sample processing method as shown in Figure 46A. Figure 56A is identical to Figure 55A except that outlet line 925 is connected to MAG sample container 934, UFL outlet line 6070 is connected to large solid container 932, and UFL outlet line 6090 is attached to small solid container 933. Figure 56A shows that containers 934, 932, 933 are in the form of blood bags. Bags 932, 933, 934 as part of the boxed lines in Figure 56A are disposable and can be sterilized and separated from the line after the separation steps of steps 407 and 408 in Figure 31 or steps 707 and 708 in Figure 39.

[0458] Figure 56B illustrates the same process as Figure 55B for connecting sample container 928, buffer container 929, and buffer container 931 to lines 923, 924, and 9272, respectively. Containers 928, 929, and 931 are in the form of blood bags. Also similar to Figure 55B, three pump tubes 504 / 505 are installed in three peristaltic pumps 500, valves 935, 936, 940, 937, 939, and 938 are attached to the corresponding lines, and flow restrictors 509 / 510 can be attached to the outlet line of each pump 500, as in Figure 55B.

[0459] Figure 57A shows an embodiment of a closed, disposable fluid line for the first type of sample processing method as shown in Figure 44A, in which connector 801 can be replaced with, without limitation, connector 802 or connector 803. Input line 9271 can connect to a UFL sample liquid container and also to the inlet of first pump tube 504 / 505, which further connects to UFL600 substance input line 6020. Input line 9272 can connect to a UFL buffer container and also to the inlet of second pump tube 504 / 505, which further connects to UFL600 buffer input line 6040. UFL600 large substance output line 6070 connects to the inlet of connector 801. UFL600 small substance output line 6090 can connect to a small substance container. The outlet of connector 801 connects to MAG sample input line 923. MAG buffer input line 924 can connect to MAG buffer solution. Input lines 923 and 924 are connected to the inlet of third pump tube 504 / 505 through T-connector 921. The outlet of third pump tube 504 / 505 connects to channel 201, which may be used as part of MAG 123. The output of channel 201 connects to T-connector 922, which connects to output line 925 and output line 926. Output lines 925 and 926 may each connect to a MAG Out sample container. In one embodiment, output line 925 may discharge negative entities into a first MAG Out sample container, and output line 926 may discharge positive entities into a second MAG Out sample container. Figure 57A shows the entire flow path from UFL sample and UFL buffer input lines 9271 and 9272 to sample output lines 6090, 925 and 926, as well as input and output lines 9271, 9272, 924, 6090, 925 and 926 that connect to external containers, with all pumps, MAG123 and other fluid line components attached externally to the lines in Figure 57A. Thus, the lines in Figure 57A are internally closed and suitable for single use disposable and sterile applications.

[0460] Figure 57B shows the fluid lines of Figure 57A connected to or attached to various fluidic components. First, second, and third pump tubes 504 / 505 each lead to a peristaltic pump 500. The three pumps 500 operate to pump either sample fluid or buffer solution into MAG 123 and UFL 600. To reduce flow pulsations from the pumps 500, flow restrictors 509 / 510 may be attached to the outlet lines from each pump 500, including lines 201, 6020, and 6040. Input line 9271 connects to a liquid sample container 928 in the form of a blood bag. Input line 9272 connects to a UFL buffer container 931, also in the form of a blood bag. UFL outlet line 6090 connects to a small solid container 933 in the form of a blood bag. Adjustable valves 939 and 938 are attached to lines 6070 and 6090 to adjust the flow rates in each of lines 6070 and 6090, which in turn controls the laminar flow rate in the UFL600 channel for buffer flow in the center of the channel and bulk sample flow at the edges of the channel. Input line 924 connects to MAG buffer reservoir 929. Valves 935 and 936 are attached to lines 923 and 924 to control the flow of either sample liquid from connector 801 or buffer fluid from reservoir 929 through T-connector 921 to third pump tube 504 / 505. Channel line 201 is attached to MAG123. Output line 925 connects to first MAG Out sample reservoir 934. Output line 926 connects to second MAG Out sample reservoir 9342. Valve 940 is attached to line 925 and valve 937 is attached to line 926 to control the negative and positive entities from MAG 123 entering either vessel 934 or vessel 9342 through T-connector 922. Both valves 940 and 937 can block flow in lines 925 and 926 during the demagnetization / dissociation step of MAG 123.

[0461] Figure 58A shows an embodiment of a closed, disposable fluid line for the second type of sample processing method as shown in Figure 45A. Figure 58A is identical in all respects to Figure 57A, except that a UFL 600 small entity discharge line 6090 connects to the inlet of connector 801 instead of discharge line 6070 as in Figure 57A. The large entity discharge line 6070 in Figure 58A can connect to a large entity vessel.

[0462] Figure 58B shows the fluid lines of Figure 58A connected to or attached with various fluidic components. Figure 58B is identical in all respects to Figure 57B except that the UFL 600 small entity outlet line 6090 connects to the inlet of connector 801 instead of outlet line 6070 as in Figure 57B. The large entity outlet line 6070 in Figure 58B connects to a large entity container 932 in the form of a blood bag.

[0463] Figure 59A shows an embodiment of a closed, disposable fluid line for sample processing through a single MAG. Input line 923 may connect to a sample liquid container. Input line 924 may connect to a MAG buffer container. Input line 923 and input line 924 are connected through T-connector 921 to the inlets of pump tubes 504 / 505, which may be mounted on a peristaltic pump. The outlet of pump tube 504 / 505 connects to channel 201, which may be used as part of MAG 123. The output of channel 201 connects to T-connector 922, which connects to output lines 925 and 926. Output lines 925 and 926 may each connect to a MAG Out sample container.

[0464] Figure 59B shows the fluid lines of Figure 59A connected to or attached to various fluidic components. Input line 923 connects to liquid sample container 928. Input line 924 connects to buffer container 929. Valves 935 and 936 are attached to lines 923 and 924 to control the flow of either sample liquid from bag 928 or buffer from container 929 through T-connector 921 and into first tube 504 / 505. Pump tube 504 / 505 is attached to peristaltic pump 500. Pump 500 operates to pump either sample fluid or buffer into MAG 123. A flow restrictor 509 / 510 can be attached to outlet line 201 from pump 500 to reduce pulsations in the flow rate from pump 500. Channel line 201 is attached to MAG 123. Outlet line 925 connects to MAG Out sample container 934. Exhaust line 926 connects to MAG Out sample container 9342. Valve 940 is attached to line 925 and valve 937 is attached to line 926, which control the negative and positive entities from MAG 123 entering either container 934 or container 9342. Both valves 940 and 937 can block flow in lines 925 and 926 during the demagnetization / dissociation step of MAG 123. Figure 59B shows that containers 928, 929, 934 and 9342 can be in the form of blood bags, but can also be other physical forms such as vials or bottles.

[0465] Figure 60A shows an embodiment of a closed, disposable fluid line for sample processing through a single UFL 600. Input line 9271 may connect to a UFL sample liquid container and also connects to the inlet of a first pump tube 504 / 505, which further connects to a UFL 600 substance input line 6020. Input line 9272 may connect to a UFL buffer container and also connects to the inlet of a second pump tube 504 / 505, which further connects to the UFL 600 buffer input line 6040. UFL 600 large substance output line 6070 may connect to a large substance container. UFL 600 small substance output line 6090 may connect to a small substance container.

[0466] Figure 60B shows the fluid lines of Figure 60A connected to or attached with various fluidic components. First and second pump tubes 504 / 505 are each attached to a peristaltic pump 500. The two pumps 500 operate to pump sample fluid and buffer into the UFL 600. To reduce pulsations in the flow rate from the pumps 500, flow restrictors 509 / 510 may be attached to the output lines from each pump 500, including lines 6020 and 6040. Input line 9271 connects to liquid sample container 928. Input line 9272 connects to UFL buffer container 931. UFL output line 6070 connects to large solid container 932. UFL output line 6090 connects to small solid container 933. Adjustable valves 939 and 938 can be attached to lines 6070 and 6090 to adjust the flow rate in each line 6070 and 6090, which in turn controls the laminar flow rate in the UFL 600 channel for the buffer flow in the center of the channel and the solid sample flow at the edges of the channel. Figure 60B shows that containers 928, 931, 932 and 933 can be in the form of blood bags, but can also be other physical forms such as, without limitation, vials or bottles.

[0467] FIG. 61A shows replacing the peristaltic pump of FIG. 56B by using a compression chamber 800 on an input sample bag to transport fluid through a fluid line. In FIG. 61A, pump 500, pump tubing 504 / 505, and flow restrictor 509 / 510 of FIG. 56B are eliminated. Channel 201 is connected directly to T-connector 921. Connector 801 is replaced with a connector 803 bag. UFL entity liquid line 6020 is connected to connector 803. Sample liquid bag 928, MAG buffer bag 929, connector 803 bag, and UFL buffer bag 931 are each enclosed within pressure chamber 800. Pressure chamber 800 can operate by increasing the pressure of a chamber medium, such as air or other fluid, such that the bag enclosed in the chamber is submerged in the chamber medium. As the chamber medium pressure increases, liquid contained in the bag can be forced out of the bag and into the fluid line. The operation of FIG. 61A may require separate operation of MAG123 and UFL600. In a first step, valve 941 attached to line 6020 is closed. Pressure in chamber 800 enclosing bags 803 and 931 is released. Pressure is increased in chamber 800 enclosing bags 928 and 929 to force sample fluid or buffer into channel 201 and initiate MAG123 separation. After MAG123 separation and depletion of sample fluid in bag 928, bag 934 and connector 803 are filled with the effluent sample from MAG123 after MAG separation, respectively. Then, in a second step, valve 937 is closed and valve 941 is opened. Pressure is increased in chamber 800 around connector 803 and bag 931 to force the sample in connector 803 and the buffer in 931 out and into UFL600, initiating UFL separation. After the sample in connector 803 is depleted and separation of UFL 600 is complete, bags 932 and 933 contain the large and small entities from the UFL discharge. Connector 803 can be replaced by connector 8020 of FIG. 52, which has an air port 8023.

[0468] FIG. 61B illustrates replacing the peristaltic pump of FIG. 56B by using a vacuum chamber 806 on the exhaust sample bag to transport fluid through the fluid lines. FIG. 61B is the same as FIG. 61A except that the pressure chamber 800 has been removed. Bags 934, 932, 933, and connector 803 are each enclosed in a vacuum chamber 806. The vacuum chambers 806 may be operated by increasing the vacuum level within each chamber 806, and fluid from the fluid lines connected to the bags is forced into the enclosed bags because the fluid line pressure is greater than the vacuum pressure. The operation of FIG. 61B may also require the operation of the separate MAG 123 and UFL 600. In a first step, valve 941 attached to line 6020 closes. The vacuum in chamber 806 enclosing bags 932 and 933 is released. The vacuum in chamber 806 enclosing bags 934 and 933 is increased to force sample fluid or buffer into channel 201, initiating MAG123 separation. After MAG123 separation and depletion of sample fluid in bag 928, bag 934 and connector 803 are each filled with the sample outlet from MAG123 after MAG separation. Then, in a second stage, valve 937 is closed and valve 941 is opened. The vacuum in chamber 806 around connector 803 is released. The vacuum in chamber 806 enclosing bags 932 and 933 is increased to force the sample and buffer in connector 803 in 931 into UFL600, initiating UFL separation. After the sample in connector 803 is depleted and UFL600 separation is complete, bags 932 and 933 contain large and small entities from the UFL outlet. Connector 803 may be replaced by connector 8020 of FIG. 52 having air port 8023 .

[0469] FIG. 62A shows replacing the peristaltic pump of FIG. 57B by using a compression chamber 800 on the input sample bag to transport fluid through the fluid line. In FIG. 62A, pump 500, pump tubing 504 / 505, and flow restrictor 509 / 510 of FIG. 57B are removed. Channel 201 is connected directly to T-connector 921. Connector 801 is replaced with Connector 803 bag. MAG sample line 923 is connected to Connector 803. Sample fluid bag 928, MAG buffer bag 929, Connector 803 bag, and UFL buffer bag 931 are each enclosed in pressure chamber 800. FIG. 62A can separate the operation of UFL 600 and MAG 123. In a first stage, valve 935 attached to line 923 closes. Pressure in chamber 800 enclosing bag 803 is released. Pressure is increased in chamber 800 surrounding bags 928 and 931, forcing sample fluid and UFL buffer into the UFL600 inlet and initiating the UFL600 separation. After the UFL600 separation and the sample fluid in bag 928 are depleted, bag 933 contains the small entity fluid and connector 803 contains the large entity fluid from the UFL600 separation. Then, in a second stage, valve 939 is closed and valve 935 is opened. Pressure is increased in chamber 800 around connector 803 and bag 929, forcing the large entity fluid sample in connector 803 or the MAG buffer in 929 into channel 201 of MAG123 and initiating the MAG123 separation. After the sample in connector 803 is depleted and the MAG123 separation is completed, bags 934 and 9342 contain the positive and negative samples from the MAG123 channel 201 outlet. Connector 803 can be replaced by connector 8020 of FIG.

[0470] Figure 62B shows replacing the peristaltic pump of Figure 57B by using a vacuum chamber 806 on the exhaust sample bag to transport fluid through the fluid lines. Figure 62B is the same as Figure 62A, except that pressure chamber 800 has been removed. Bags 934, 9342, 933, and connector 803 are each enclosed in vacuum chamber 806. Operation of Figure 62B can separate the operation of MAG123 and UFL600. In the first stage, valve 935 attached to line 923 closes. The vacuum in chamber 806, which encloses bags 933 and 803, is increased to force sample fluid and UFL buffer into the inlet of UFL600, initiating UFL600 separation. After UFL600 separation and depletion of sample fluid in bag 928, bag 933 contains small entity fluid, and connector 803 contains large entity fluid from UFL600 separation. Next, in a second stage, valves 938 and 939 are closed and valve 923 is opened. The vacuum in chamber 806 surrounding connector 803 is released. The vacuum in chamber 806 enclosing bags 934 and 9342 is increased to force the connector 803 bulk sample or MAG buffer in 929 into MAG123 channel 201, initiating the MAG123 separation. After the sample in connector 803 is depleted and the MAG123 separation is complete, bags 934 and 9342 contain the positive and negative samples from the MAG123 channel 201 outlet. Connector 803 can be replaced by connector 8020 of FIG. 52.

[0471] Figure 63A shows replacing the peristaltic pump of Figure 58B by using a compression chamber 800 on the input sample bag to transport fluid through the fluid lines. Figure 63A is identical to Figure 62A in fluid line layout and operation of UFL 600 with chamber 800 and MAG 123, except that UFL large substance output 6070 connects to a large substance container 932 in the form of a blood bag, and small substance output 6090 connects to connector 803.

[0472] Figure 63B shows replacing the peristaltic pump of Figure 58B by using a vacuum chamber 806 on the discharge sample bag to transport fluid through the fluid lines. Figure 63A is identical to Figure 62B in fluid line layout and operation of UFL 600 with chamber 806 and MAG 123, except that UFL large substance discharge 6070 connects to a large substance container 932 in the form of a blood bag, a small substance container 932 enclosed in vacuum chamber 806 replaces container 933 of Figure 62B, and small substance discharge 6090 connects to connector 803.

[0473] FIG. 64A shows replacing the peristaltic pump of FIG. 59B by using a compression chamber 800 on input sample bags 928 and 929 to drive fluid through channel 201 of MAG 123. In FIG. 64A, pump 500, pump tubing 504 / 505, and flow restrictor 509 / 510 of FIG. 59B are eliminated. Channel 201 is directly connected to T-connector 921. Sample fluid bag 928 and MAG buffer bag 929 are each enclosed in a pressure chamber 800. Increasing pressure in chamber 800 enclosing bags 928 and 929 forces sample fluid or buffer into channel 201, initiating separation of MAG 123. After MAG 123 separation and depletion of sample fluid in bag 928, bag 934 and bag 9342 are filled with either negative or positive entities from MAG 123 after MAG separation, respectively.

[0474] Figure 64B shows replacing the peristaltic pump of Figure 59B by using a vacuum chamber 806 on the exhaust sample bags 934 and 9342 to drive fluid through channel 201 of MAG123. Figure 64B is the same as Figure 64A except that pressure chamber 800 has been removed. Exhaust sample bags 934 and 9342 are each enclosed in vacuum chamber 806. Increasing the vacuum in chamber 806 enclosing bags 934 and 9342 forces solid sample from bag 928 or MAG buffer from bag 929 into channel 201 of MAG123, initiating the MAG123 separation. After the sample in bag 928 is depleted and the MAG123 separation is complete, bags 934 and 9342 contain the positive and negative samples from the MAG123 channel 201 output.

[0475] FIG. 65A shows replacing the peristaltic pump of FIG. 60B by using a compression chamber 800 on sample liquid bag 928 and UFL buffer bag 931 to transport fluid through UFL 600. In FIG. 65A, pump 500, pump tubing 504 / 505, and flow restrictors 509 / 510 of FIG. 60B are removed. Sample liquid bag 928 and UFL buffer bag 931 are each enclosed in a pressure chamber 800. Increasing pressure in chamber 800 enclosing bags 928 and 931 forces sample fluid and UFL buffer into the inlet of UFL 600, initiating UFL 600 separation. After UFL 600 separation, the sample fluid in bag 928 is depleted, bag 932 contains the large entity fluid, and bag 933 contains the small entity fluid.

[0476] Figure 65B shows replacing the peristaltic pump of Figure 60B by using a vacuum chamber 906 on exhaust sample bags 932 and 933 to transport fluid through UFL 600. Figure 65B is the same as Figure 65A except that pressure chamber 800 has been removed. Exhaust sample bags 932 and 933 are each enclosed in vacuum chamber 806. Increasing the vacuum in chamber 806 enclosing bags 932 and 933 forces sample fluid from bag 928 and UFL buffer from bag 931 through UFL 600, initiating UFL separation. After the sample fluid in bag 928 is depleted and UFL 600 separation is complete, bag 932 contains the large entity fluid and bag 933 contains the small entity fluid.

[0477] The structures, components and methods described in Figures 55A-65B for a closed fluid line including one UFL 600 and one MAG 123 can be applied to Figures 47-52 without limitation, and a closed fluid line including multiple MAG 123 and multiple UFL 600 can be achieved by replicating the components in one UFL 600 and one MAG 123 from Figures 55A-65B in each of the UFL 600 and MAG 123 of Figures 47-52.

[0478] Figures 66-88 illustrate process flow embodiments for utilizing MAG and UFL devices to separate biological entities from various biological samples. For simplicity, the terms UFL and MAG are used in these figures for descriptive purposes. However, a UFL can be any of UFLs 600, 650, 620, 630, and 640 in Figures 40A, 41A, 42A, and 43, while a MAG can be any of MAGs 121, 122, 123, 124, 125, 126, 127, 128, and 129 with corresponding channel types as described in the preceding figures, without limitation and without sacrificing performance. When a component or structure in Figures 66-88 shares the same name as a component or structure in a preceding figure, it refers to the same component or structure as the preceding figure.

[0479] 66 shows a first process flow embodiment for separating biological entities from peripheral blood using UFL and MAG. In step 5801, a peripheral blood sample is collected from a patient or person under test; in step 5802, the peripheral blood sample may be subjected to red blood cell lysis, and in other embodiments, step 5802 may be omitted; in step 5803, the blood sample from step 5802 or directly from step 5801 is injected into UFL entity fluid inlet 602, while UFL buffer is injected into outlet 604; in step 5804, a P unit attached to the UFL is inserted to generate standing waves and pressure nodes in the UFL fluid. setting the frequency and vibration intensity of the ZT; in step 5805, allowing the UFL outlet 607 to discharge the target sample containing the large entities or cells; in step 5806, adding to the target sample from step 5805 a magnetic label that is hybridized with an antibody or ligand that specifically binds to a surface antigen or receptor on the target cells or entities; in step 5807, incubating the target sample from step 5806 to form a magnetic label that binds to the target cells or entities; in step 5808, magnetic separation During step 5808, the target sample from step 5807 is flowed through the MAG channel in a positive direction, and a negative MAG sample may be transferred, as in step 5815, to be collected in step 5813; in step 5809, target cells or entities bound to the magnetic labels are separated by MAG in the MAG channel; in step 5810, after step 5809, a buffer solution may be flowed through the MAG channel to wash away remaining non-target entities without magnetic labels, and the washed-out fluid may be returned to step 5816, as in step 5817. 3, and step 5810 may be omitted in another embodiment; in step 5811, after step 5810 or immediately after step 5809, the separated entity aggregates in the MAG channel may be dissociated into isolated cells or entities; in step 5812, buffer is flowed through the MAG channel to wash away the dissociated cells and entities in the MAG channel, which may be collected as a positive MAG sample in step 5814, as indicated by 5817.

[0480] The peripheral blood sample in Figure 66 can also be other bodily fluids including, but not limited to, saliva, tears, mucus, urine, and secretions from various organs of the body.

[0481] Figure 67 shows a second process flow embodiment for separating biological entities from peripheral blood using MAG. All other aspects of Figure 67 are the same as Figure 66, except that steps 5803, 5804, and 5805 of Figure 66 are omitted between steps 5802 and 5806 of Figure 67. Meanwhile, in Figure 67, the blood sample from step 5802 or directly from step 5801 is centrifuged in step 6201 to extract a target sample containing white blood cells. The target sample form step 6201 is then sent to step 5806, and from step 5806, the flow of Figure 67 is the same as Figure 66.

[0482] Figure 68 shows a third process flow embodiment for separating biological entities from peripheral blood using MAG. All other aspects of Figure 68 are the same as Figure 66, except that steps 5803, 5804, and 5805 of Figure 66 are removed between steps 5802 and 5806 of Figure 68. Meanwhile, in Figure 68, a peripheral blood sample collected from a patient or person under test, such as step 6301, which is the same as step 5801 of Figure 66, is considered the target sample. The target sample from step 5802, either after red blood cell lysis in step 6301 or directly from step 6301, is then sent to step 5806. From step 5806, the flow of Figure 68 is the same as Figure 66.

[0483] Figure 69 shows a fourth process flow embodiment for separating biological entities from peripheral blood using MAG. All other aspects of Figure 69 are the same as Figure 66, except that steps 5801, 5802, 5803, 5804, and 5805 of Figure 66 are removed prior to step 5806 of Figure 69. Meanwhile, in Figure 69, a target sample is collected following apheresis of a peripheral blood sample collected from a patient or person under test. The target sample form step 6401 is then sent to step 5806. From step 5806, the flow of Figure 69 is the same as Figure 66.

[0484] FIG. 70 shows an embodiment of a fifth process flow for separating biological entities from a tissue sample using UFL and MAG. All other aspects of FIG. 70 are the same as FIG. 66, except that steps 5801, 5802, and 5803 are omitted before step 5804 in FIG. 70. In FIG. 70, a tissue sample is collected in step 6501. In step 6502, the tissue sample from step 6501 is dissociated in a fluid base. In step 6503, the dissociated tissue fluid from step 6502 is injected into the UFL channel through inlet 602, and UFL buffer is injected through inlet 604. From step 5804, the flow of FIG. 70 is the same as FIG. 66. The tissue sample in FIG. 70 can include any of human tissue aspirates, human organ tissue aspirates, bone marrow, animal body, or organ tissue aspirates. The target cells or entities in FIG. 70 can be rare disease cells, e.g., cancer cells, or microorganisms, e.g., bacteria.

[0485] Figure 71 shows a sixth process flow embodiment for separating biological entities from a tissue sample using MAG. All other aspects of Figure 71 are the same as Figure 70, except that steps 6503, 5804, and 5805 are removed prior to step 5806 in Figure 71. In Figure 71, the tissue sample from step 6501 is dissociated in a fluid base in step 6502 to form a target sample, and the process continues in step 5806. From step 5806, the flow in Figure 71 is the same as Figure 70.

[0486] FIG. 72 shows an embodiment of a seventh process flow for separating biological entities from a surface swab sample using UFL and MAG. All other aspects of FIG. 72 are the same as FIG. 66, except that steps 5801, 5802, and 5803 are removed before step 5804 in FIG. 72. In FIG. 72, surface entities are collected by a swab in step 6701. In step 6702, the surface entities collected on the swab are dissolved in a fluid base. In step 6703, the fluid base with dissolved surface entities from step 6702 is injected into the UFL channel through inlet 602, and UFL buffer is injected through inlet 604. From step 5804, the flow of FIG. 72 is the same as FIG. 66. The surface entities in FIG. 72 can be collected by swabbing from objects including any of the human body, saliva, bodily fluids, human waste, animals, plants, soil, air, water, and commercial products. The target cells or entities in Figure 72 may include cells from the human or animal body or plants, or may include microorganisms such as bacteria, fungi or spores.

[0487] Figure 73 shows an embodiment of an eighth process flow for separating biological entities from a surface swab sample using MAG. All other aspects of Figure 73 are the same as Figure 72, except that steps 6703, 5804, and 5805 are eliminated prior to step 5806 in Figure 73. In Figure 73, surface entities recovered on the swab in step 6701 are dissolved in a fluid base in step 6702 to form a target sample, and the process continues in step 5806. From step 5806, the flow of Figure 73 is the same as Figure 72.

[0488] Figure 74 shows an embodiment of a ninth process flow for separating biological entities from a solid sample using UFL and MAG. All other aspects of Figure 74 are the same as Figure 66, except that steps 5801, 5802, and 5803 are removed before step 5804 in Figure 74. In Figure 74, a solid sample is collected in step 6901. In step 6902, the solid sample from step 6901 is dissociated in a fluid base. In step 6903, the dissociated solid sample fluid from step 6902 is injected into the UFL channel through inlet 602, and UFL buffer is injected through inlet 604. From step 5804, the flow in Figure 74 is the same as Figure 66. The tissue sample in Figure 70 can include any solid biological product or waste material, powder, and soil produced by humans, animals, or plants. The target cells or entities in Figure 74 may include cells from the human or animal body or plants, or may include microorganisms such as bacteria, molds or spores.

[0489] Figure 75 shows an embodiment of a tenth process flow for separating biological entities from a solid sample using MAG. All other aspects of Figure 75 are the same as Figure 74, except that steps 6903, 5804, and 5805 are removed prior to step 5806 in Figure 75. In Figure 75, the solid sample from step 6901 is dissociated in a fluid base to form a target sample in step 6902, and the target sample continues to be processed in step 5806. From step 5806, the flow of Figure 75 is the same as Figure 74.

[0490] Figure 76A shows the addition of both magnetic and fluorescent labels to a fluid sample for specific binding to target cells or entities. Figure 76A shows that step 5806 of Figures 66-75 can be modified to step 58061, where in addition to the magnetic label, a fluorescent label that hybridizes to an antibody or ligand that specifically binds to a surface antigen or receptor on the target cell or entity can also be added in the target sample from step 5805.

[0491] Next, Figure 76B shows that incubation step 5807 of Figures 66-75 can also be modified to step 58071, which includes simultaneous incubation of both magnetic and fluorescent labels to form specific binding to target cells or entities. The binding sites of the magnetic and fluorescent labels on the same target cell or entity can be different.

[0492] Steps 5806 and 58061 may be implemented in a flow connector including any one of 801, 802, 803, 8010, 8020, 8030 of the preceding figures, where the flow connector may contain pre-loaded hybridization magnetic and fluorescent labels in the form of a liquid solution or a dry powder. Steps 5807 and 58071 may also be performed in said flow connector, where said flow connector may also be placed in a temperature-controlled chamber to control the rate and quality of incubation. In another embodiment, a temperature control circuit may be attached to or embedded in said flow connector to control incubation in the flow connector.

[0493] Figure 77A shows the step of removing unbound free magnetic labels from the sample fluid by UFL before magnetic separation by MAG. Figure 77A shows that steps 5818 and 5819 can be added between steps 5807 and 5808 for each of Figures 66-75. After incubating the target sample in step 5807, in step 5818, the target sample can be injected into the second UFL through inlet 602, and a buffer can be injected into the second UFL through inlet 604. In step 5819, the second UFL ejects the target sample containing large entities from outlet 607, and unbound free magnetic labels are ejected from second UFL outlet 609. Next, in step 5808, the target sample containing large entities from second UFL outlet 607 is passed through the MAG channel for magnetic separation. The target sample in step 5819 can contain cells or entities bound to magnetic labels. Step 5819 envisions a second UFL fitted with a PZT operating at a particular ultrasonic vibration amplitude and frequency to generate standing waves in the second UFL channel fluid.

[0494] Figure 77B shows the step of removing unbound free magnetic labels from the sample fluid by UFL after magnetic separation by MAG. Figure 77B shows that, for each of Figures 66-75, instead of passage 5817, steps 5820 and 5821 can be added between steps 5812 and 5814. After the magnetic aggregates in the MAG channel are dissociated and the positive MAG sample entities are flushed from the MAG channel as in step 5812, the flushed positive MAG sample can be injected into a third UFL through inlet 602, and buffer can be injected into the third UFL through inlet 604. In step 5821, the third UFL ejects the positive MAG sample containing the large entities from outlet 607, and the unbound free magnetic labels are ejected from third UFL outlet 609. Next, in step 5814, the positive MAG sample with reduced or depleted free magnetic labels can be collected. In step 5821, a third UFL is envisioned that is fitted with a PZT that operates at a particular ultrasonic vibration amplitude and frequency to generate standing waves in the third UFL channel fluid.

[0495] Figure 78A shows the removal of unbound free magnetic and fluorescent labels from a sample fluid by UFL prior to magnetic separation by MAG. Figure 78A is similar to Figure 77A, replacing step 5807 of Figure 77A with step 58071 of Figure 76B and step 5819 with step 58191. After adding magnetic and fluorescent labels to the target sample as in step 58061 of Figure 76A, the target sample is incubated in step 58071 as in Figure 76B to form magnetic and fluorescent labels that bind to target cells or entities. In step 5818, the target sample can be injected into a second UFL through inlet 602, and a buffer can be injected into the second UFL through inlet 604. In step 58191, the second UFL discharges the target sample containing large entities from outlet 607, and unbound free magnetic and fluorescent labels are discharged from second UFL outlet 609. Next, in step 5808, the target sample containing large entities from the second UFL outlet 607 is flowed through the MAG channel for magnetic separation. The target sample in step 58191 may contain cells 30 or entities bound with magnetic and fluorescent labels. In step 58191, a second UFL is envisioned that is fitted with a PZT operating at a specific ultrasonic vibration amplitude and frequency to generate standing waves in the second UFL channel fluid.

[0496] Figure 78B shows the removal of unbound free magnetic and fluorescent labels from the sample fluid by UFL after magnetic separation by MAG. Figure 78B is similar to Figure 77B, replacing step 5821 of Figure 77A with step 58211. The separated entities in steps 5812 and 5820 of Figure 78B may contain cells 30 or entities bound to magnetic and fluorescent labels, unbound free magnetic labels, and small amounts of unbound free fluorescent labels due to nonspecific binding to aggregates in the MAG channel during magnetic separation. In step 58212, the third UFL discharges the positive MAG sample containing the large entities from outlet 607, and the unbound free magnetic and free optical labels are discharged from third UFL outlet 609. Next, in step 5814, the positive MAG sample, reduced or depleted of free magnetic and free fluorescent labels, can be collected. Step 58212 contemplates a third UFL equipped with a PZT operating at a particular ultrasonic vibration amplitude and frequency to generate standing waves in the third UFL channel fluid.

[0497] Figure 79 shows the sequential processing of the negative MAG sample after MAG separation, such as step 408 in Figure 31, through a UFL to remove small entities and transfer the large entities to various cell processing devices and procedures. Step 5813 is the same as Figures 66-75, in which the negative MAG sample is collected during MAG separation of the target sample. In step 5822, the negative MAG sample of step 5813 is injected into fourth UFL inlet 602, and UFL buffer is injected into fourth UFL inlet 604. In step 5823, the fourth UFL discharges the negative MAG sample containing the large entities from outlet 607, and the small entities are removed from the large entities and discharged from fourth UFL outlet 609. A PZT attached to the fourth UFL and operating at a specific ultrasonic vibration amplitude and frequency to generate a standing wave in the fourth UFL is envisioned. Finally, the negative MAG sample containing large entities from the outlet 607 of the fourth UFL can be sent for analysis by either a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, or a DNA / RNA sequencer 906. Alternatively, the output from the cell counter 903 or the output from the cell imaging device 904 or the output from the flow cytometer or sorter 905 can be further sent for processing by a DNA / RNA sequencer 906, as shown by paths 936, 946, and 956, respectively. In step 5823, the negative MAG sample containing large entities from the outlet 607 of the fourth UFL can also be sent to the process of cytogenetic modification and cell proliferation 5824. Prior to DNA / RNA sequencing in DNA / RNA sequencer 906, a polymerase chain reaction (PCR) procedure may be performed on the DNA / RNA sample obtained from cell lysis of large entities from outlet 607 of the fourth UFL from step 5823, where the PCR may target one or more target DNA / RNA sequences and amplify the number of target DNA / RNA sequences in the DNA / RNA sample.

[0498] FIG. 80 illustrates the sequential processing of a negative MAG sample after MAG separation, such as step 408 in FIG. 31, through a UFL to recover small entities and transfer the small entities to various molecular or small entity processing devices. After step 5813 in FIGS. 66-75, in which the negative MAG sample is recovered during MAG separation of the target sample, in step 5822, the negative MAG sample of step 5813 is injected into fourth UFL inlet 602 and UFL buffer is injected into fourth UFL inlet 604. In step 5825, the fourth UFL ejects the negative MAG sample containing large entities from outlet 607, and small entities including DNA, RNA, molecules, and other small particles are ejected from fourth UFL outlet 609. A PZT attached to the fourth UFL is envisioned, operating at a specific ultrasonic vibration amplitude and frequency to generate a standing wave in the fourth UFL. Finally, the small entities from outlet 609 of the fourth UFL may be sent for analysis by either particle counter 5835, particle imager 5836, flow cytometer or sorter 905, or DNA / RNA sequencer 906. Alternatively, the output from particle counter 5835 or the output from particle imager 5836 or the output from flow cytometer or sorter 905 may be further sent for processing by DNA / RNA sequencer 906, as shown by paths 5827, 5828, and 956, respectively. DNA / RNA sequencer 906 may contain a PCR step on the small entities from outlet 609 of the fourth UFL from step 5825 prior to DNA / RNA sequencing, where PCR may target and amplify in large quantities one or more specific DNA / RNA sequences.

[0499] Figure 81 illustrates the substantive analysis of negative MAG samples after MAG separation, such as step 407 in Figure 31, into various analytical devices. After step 5813 in Figures 66-75, in which negative MAG samples are collected during MAG separation of the target sample, the collected negative MAG samples can be sent for analysis by any of a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, a particle counter 5835, a particle imaging device 5836, or a DNA / RNA sequencer 906. Alternatively, the output from the cell counter 903 or the output from the cell imaging device 904 or the output from the flow cytometer or sorter 905 or the output from the particle counter 5835 or the output from the particle imaging device 5836 can be further sent for processing by the DNA / RNA sequencer 906, as shown by paths 936, 946, 956, 5827, and 5828, respectively. The negative MAG sample may also be sent to the step of cytogenetic modification and cell proliferation 5824. The DNA / RNA sequencer 906 may contain (1) DNA / RNA obtained after cell lysis of the cells contained in the negative MAG sample; and (2) a PCR step on the DNA / RNA / molecules contained in the negative MAG sample. Prior to DNA / RNA sequencing, PCR may target one or more specific DNA / RNA sequences for mass amplification.

[0500] Figure 82 shows the sequential processing of a positive MAG sample after MAG separation, such as step 408 in Figure 31, through a UFL to remove small entities and transfer the large entities to various cell processing devices and procedures. Step 5814 is the same as Figures 66-75, in which the positive MAG sample is collected after MAG separation of the target sample. In step 5829, the positive MAG sample of step 5814 is injected into the fifth UFL inlet 602, and UFL buffer is injected into the fifth UFL inlet 604. In step 5830, the fifth UFL ejects the positive MAG sample containing the large entities from outlet 607, and the small entities are removed from the large entities and ejected from the fifth UFL outlet 609. A PZT attached to the fourth UFL is envisioned, operating at a specific ultrasonic vibration amplitude and frequency to generate a standing wave in the fifth UFL. Finally, the positive MAG sample containing large entities from the outlet 607 of the fifth UFL can be sent for analysis by either a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, or a DNA / RNA sequencer 906. Alternatively, the output from the cell counter 903 or the output from the cell imaging device 904 or the output from the flow cytometer or sorter 905 can be further sent for processing by the DNA / RNA sequencer 906, as shown by paths 936, 946, and 956, respectively. The positive MAG sample containing large entities from the outlet 607 of the fifth UFL in step 5830 can also be sent to the process of cytogenetic modification and cell proliferation 5824. Prior to DNA / RNA sequencing, the DNA / RNA sequencer 906 may contain a PCR step on the DNA / RNA obtained after cell lysis of large entities from the outlet 607 of the fifth UFL from step 5830, in which the PCR may target and amplify in large quantities one or more specific DNA / RNA sequences.

[0501] Figure 83 shows the sequential processing of a positive MAG sample after MAG separation, such as step 408 of Figure 31, through a UFL to recover small entities and transfer the small entities to various molecular or small entity processing devices. After step 5814 of Figures 66-75, in which a positive MAG sample is recovered after MAG separation of the target sample, in step 5829, the positive MAG sample of step 5814 is injected into the fifth UFL inlet 602 and UFL buffer is injected into the fifth UFL inlet 604. In step 5831, the fifth UFL ejects the positive MAG sample containing large entities from outlet 607, and small entities, including DNA, RNA, molecules, and other small particles to which magnetic labels are attached, are ejected from the fifth UFL outlet 609. A PZT attached to the fifth UFL and operating at a specific ultrasonic vibration amplitude and frequency to generate a standing wave in the fifth UFL is envisioned. Finally, the small entities from outlet 609 of the fifth UFL may be sent to either particle counter 5835, particle imager 5836, flow cytometer or sorter 905, or DNA / RNA sequencer 906. Alternatively, the output from particle counter 5835 or the output from particle imager 5836 or the output from flow cytometer or sorter 905 may be further sent for processing by DNA / RNA sequencer 906, as shown by paths 5827, 5828, and 956, respectively. DNA / RNA sequencer 906 may contain a PCR step on the small entities from outlet 609 of the fifth UFL from step 5831 prior to DNA / RNA sequencing, where PCR may target and amplify in large quantities one or more specific DNA / RNA sequences.

[0502] Figure 84 illustrates the substantive analysis of a positive MAG sample after MAG separation, such as step 407 in Figure 31, into various analytical devices. After step 5814 in Figures 66-75, in which a positive MAG sample is collected after MAG separation of a target sample, the collected positive MAG sample can be sent for analysis by any of a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, a particle counter 5835, a particle imaging device 5836, or a DNA / RNA sequencer 906. Alternatively, the output from the cell counter 903 or the output from the cell imaging device 904 or the output from the flow cytometer or sorter 905 or the output from the particle counter 5835 or the output from the particle imaging device 5836 can be further sent for processing by the DNA / RNA sequencer 906, as shown by paths 936, 946, 956, 5827, and 5828, respectively. The positive MAG sample may also be sent to the step of cytogenetic modification and cell proliferation 5824. Prior to DNA / RNA sequencing, the DNA / RNA sequencer 906 may contain: (1) DNA / RNA obtained after cell lysis of the cells contained in the positive MAG sample; and (2) a PCR step on the DNA / RNA / molecules contained in the positive MAG sample, where the PCR may target and amplify in large amounts one or more specific DNA / RNA sequences.

[0503] Figure 85A shows the addition of a fluorescent label to specifically bind to target entities within the negative MAG sample immediately after negative MAG sample collection. Figure 85A shows that immediately after step 5813, when the negative MAG sample is collected during MAG separation, in step 58131, a fluorescent label that hybridizes with an antibody or ligand and specifically binds to a surface antigen or receptor on the target cell or entity is added to the negative MAG sample, and the negative MAG sample is then incubated to form a fluorescent label that binds to the target cell or entity. Step 58131 can be inserted in Figures 79 and 80 between step 5813 and step 5822, or in Figure 81 immediately after step 5813 and before the device or steps 903, 904, 905, 906, 5824, 5825, and 5826.

[0504] Figure 85B shows the addition of a fluorescent label to specifically bind to target entities within the positive MAG sample immediately after positive MAG sample collection. Figure 85B shows that immediately after step 5814, when the positive MAG sample is collected after MAG separation, in step 58141, a fluorescent label that hybridizes with an antibody or ligand and specifically binds to a surface antigen or receptor on the target cell or entity is added to the positive MAG sample, and the positive MAG sample is then incubated to form a fluorescent label that binds to the target cell or entity. Step 58141 can be inserted between steps 5814 and 5829 in Figure 82 and Figure 82, or can be inserted immediately after step 5814 and before the device or steps 903, 904, 905, 906, 5824, 5825, and 5826 in Figure 84.

[0505] FIG. 86 shows a cross-sectional view of a tenth embodiment of MAG 1241. MAG 1241 in FIG. 86 has the same design as MAG 124 in FIG. 12 but differs from MAG 123 in FIG. 12. MAG 1231 in FIG. 86 differs from MAG 123 in FIG. 12 in that its flux-collecting ends 11211 and 11311 are flat and function primarily to collect magnetic flux emanating from the tip 1111 of the central pole 111, forming a magnetic flux closure within the main pole 111, side poles 1120 and 1130, and bottom shield 114. When the flux-collecting ends 11211 and 11311 are flat, the maximum magnetic field and maximum magnetic field gradient are near the tip 1111 of the main pole 111, thus facilitating the movement of biological entities 10 / 30 in the channel 301 toward the tip 1111. In the embodiment of FIG. 86 , the magnetic flux collecting ends 11211 and 11311 can also be described as soft magnetic shields for the tip 1111 of the main pole 111, and the soft shields 11211 and 11311 can help confine the magnetic flux within the gap between the tip 1111 and the shields 11211 and 11311 and can also increase the effective magnetic force exerted on the biological entity 10 / 30 in the channel 301 toward the tip 1111. In FIG. 86 , the flat ends of the soft shields 11211 and 11311 are parallel to each other and form a physical space with the tip 1111, i.e., the MAG gap of the MAG 1241, while the channel 301 is aligned with the soft shields 11211 and 11311 to contact the tip 1111 at the bottom of the channel 301. In FIG. 86, the magnetic flux generated by the north and south faces of magnet 109 is conducted within the body of poles 111, 1121, 1131 and shield 114, minimizing leakage outside the MAG1241 structure. The magnetic flux density is greatest around tip 1111, and soft shields 11211 and 11311 create lower magnetic flux densities, allowing for high magnetic fields and field gradients around tip 1111. Compared to MAGs 121, 122, 123, and 124, MAG1241 has the advantage of minimal flux leakage and therefore a higher magnetic flux density around tip 1111, and more efficient flux containment within the MAG1241 soft magnetic body, creating a higher magnetic force in channel 301 and in the biological entity 10 / 30.

[0506] Channel 301 in FIG. 86 is a rigid channel similar to channel 301 in FIG. 12. Channel 301 may be attached to a non-magnetic channel holder 110 at its upper surface 3012. Channel holder 110 may adjust the position of channel 301 relative to the MAG gap of MAG 1241, move channel 301 to a separation position in contact with tip 1111 of MAG 1241 pole 111, or lift channel 301 away from MAG 1241 after magnetic separation. Channel 301 may be substituted for soft channel 201 in FIGS. 13 and 14 and operated by MAG 1241 in FIG. 86 in the same manner as MAG 124 in FIGS. 13 and 14.

[0507] FIG. 87A shows a cross-sectional view of MAG 1251 of the eleventh embodiment. MAG 1251 is the same as MAG 1241 of FIG. 86 except that magnet 109 and bottom shield 114 of MAG 1241 of FIG. 86 have been removed in MAG 1251. Permanent magnets 115 and 116, having opposite magnetizations 1151 and 1161, respectively, are positioned between poles 111 and 1121 and between poles 111 and 1131, as shown in FIG. 87A. Magnetizations 1151 and 1161 are horizontal in FIG. 87A, allowing center pole 111 to conduct north-face magnetic flux from both magnets 115 and 116, while side poles 1120 and 1130 conduct south-face magnetic flux from magnets 115 and 116, respectively. Compared to MAG 1241, MAG 1251 can generate a higher magnetic field around tip 1111 because two magnets 115 and 116 are used.

[0508] FIG. 87B shows a cross-sectional view of a twelfth embodiment of MAG 1261. MAG 1261 is the same as MAG 1241 of FIG. 86, except that side poles 1120 and 1130 are attached to the south faces of permanent magnets 1092 and 1094, respectively, and magnetizations 1093 and 1095 are opposite to magnetization 1091 of magnet 109. Bottom shield 114 is attached to both the north faces of magnets 1092 and 1094 and the south face of magnet 109, thus forming internal magnetic flux containment in shield 114 between magnets 109, 1092, and 1094. Compared to MAG 1241, MAG 1261 can generate a higher magnetic field around tip 1111 due to the use of three magnets 109, 1092, and 1094 in MAG 1261.

[0509] Figure 87C shows a cross-sectional view of a thirteenth embodiment of MAG 1271. MAG 1271 is the same as MAG 1261 of Figure 87B, except that bottom shield 114 is removed.

[0510] FIG. 88A shows a cross-sectional view of a fourteenth embodiment of MAG 1242. MAG 1242 is the same as MAG 1241 of FIG. 86 except that side shielding surfaces 11212 and 11312 are not parallel to one another, but rather side shielding surfaces 11212 are substantially parallel to main pole 111 tip bevel 11112, side shielding surfaces 11213 are substantially parallel to main pole 111 tip bevel 11113, and tip bevels 11112 and 11113 meet to form tip 1111. The MAG gap formed by tip end 1111, side shielding surfaces 11212, and 11312 of FIG. 88A may result in a higher magnetic flux concentration at tip 1111 in channel 301 / 201 and a higher effective magnetic force on biological entity 10 / 30 when channel 301 / 201 may contact tip 1111. In FIG. 88A, the channel 301 / 201 may contact the main pole tip 1111 of the MAG 1242, or may be close to but not contact the main pole tip 1111.

[0511] Figure 88B shows a cross-sectional view of a fifteenth embodiment, MAG 1243. MAG 1243 is the same as MAG 1242 of Figure 88A, except that tip 1111 of Figure 88A is replaced with flat tip 11114 of Figure 88B. When channel 301 / 201 can contact tip 11114, to maximize the high magnetic field effective area within channel 301 / 201, the MAG gap formed by flat tip 11114, side shield surfaces 11212 and 11312 of Figure 88B can avoid magnetic flux saturation of flat tip 11114 of main pole 111, thus increasing the effective magnetic force exerted on biological entity 10 / 30 in channel 301 / 201. The flat tip of Figure 88B can also be used to replace the tip 1111 of the main pole 111 of the MAG embodiments of Figures 12-15C, 18, 19, and 86-87C. In Figure 88B, the channel 301 / 201 may be in contact with the main pole tip 11114 of the MAG 1243, or may be close to but not in contact with the main pole tip 11114.

[0512] Figure 88C shows a cross-sectional view of MAG 1244 of the sixteenth embodiment. MAG 1244 is the same as MAG 1242 of Figure 88A, except that the side shielding surfaces 11214 and 11314 of Figure 88C are positioned above the main pole tip 1111, and each of the side shielding surfaces 11214 and 11314 slopes substantially toward the main pole tip 1111, forming a funnel-shaped slope with a smaller opening between the surfaces 11214 and 11314 when closer to the main pole tip 1111. The tips of the side shielding surfaces 11214 and 11314 can be above the main pole tip 1111. The tips of the side shielding surfaces 11214 and 11314 and the main pole tip 1111 can also be positioned in a horizontal plane. The positioning of the side shielding surfaces 11214 and 11314 in Figure 88C can help to produce a high magnetic field and high magnetic field gradient in the channel 301 / 201 and therefore a higher effective magnetic field on the biological entity 10 / 30 in the channel 301 / 201 when the channel 301 / 201 is in contact with the tips of the surfaces 11214 and 11314 and may be in contact with or close to the tip 1111 of the MAG 1244. The positioning of the side shielding surfaces 11214 and 11314 in Figure 88C can also help to adjust the position of the main pole tip 1111 and the channel 301 / 201 during positioning of the channel 301 / 201 towards the main pole tip 1111. In FIG. 88C, the channel 301 / 201 may be in contact with the main pole tip 1111 of the MAG 1244, or may be close to but not in contact with the main pole tip 1111.

[0513] 86-88C are cross-sectional views of different embodiments of MAG designs, while the MAG designs extend in a direction into or out of the cross-section. The tenth through fifteenth embodiments of MAGs in FIGS. 86-88C are similar to the first through ninth embodiments of MAGs as shown in the previous figures, while the soft shields 11211, 11311, 11212, 11312, 11213, 11313, tip 1111, upper tip 11114, and channels 101 / 201 / 301 that may contact or be adjacent to tip 1111 or upper tip 11114 extend in the direction 62 as shown in FIG. 32 and are parallel to one another. The attachment of permanent magnets 115, 116, 1092, 109, 1094 to main pole 111, side poles 1120 and 1130 as in Figure 87A, 87B or 87C may be applied to Figures 88A, 88B and 88C, respectively.

[0514] Figure 89A shows a cross-sectional view of channel 301 / 201 / 101 similar to Figure 27B, in which after magnetic separation of biological entities 10 / 30 in channel 301 / 201 / 101, channel 301 / 201 / 101 is lifted by holder 1082 from the MAG gap of the MAG embodiment of Figures 4 to 21C and Figures 86 to 88B to a lower magnetic field position 22.

[0515] Figure 89B shows that at position 22 in Figure 89A, dissociation of cells 10 / 30 in channel 301 / 201 / 101 can be achieved by using a second channel holder 1301 in contact with channel 301 / 201 / 101, where a motor 130 is mechanically coupled to channel holder 1301 to generate mechanical vibrations to channel holder 1301, and the mechanical vibrations from motor 130 can then be transmitted through channel holder 1301 to channel 301 / 201 / 101 in contact with channel holder 1301, causing localized turbulence at various positions within channel 301 / 201 / 101, which can encourage mechanical disruption of the cell 10 / 30 aggregates into small fragments to assist in self-dissociation of the aggregates. In one embodiment, the channel holder 1301 can push the channel 301 / 201 / 101 away from the channel holder 1082 during dissociation of the cells 10 / 30. The direction of vibration exerted on the channel 301 / 201 / 101 through the channel holder 1301 can be direction 61001, or direction 61002, or alternating between directions 61001 and 61002. The channel holder 1301 can be shaped with a thinner handle that connects to the motor 130 and a wider holder arm 1302 that contacts the channel 301 / 201 / 101 for effective vibration transmission from the motor 130 to the channel 301 / 201 / 101. The length of the channel holder 1301 in the direction into and out of the cross-section of FIG. 89B can be much shorter than the channel holder 1082. The channel holder 1301 can contact the channels 301 / 201 / 101 through existing clearances in the channel holder 1082 without physical contact with the channel holder 1082.

[0516] Figure 89C shows that at position 22 in Figure 89A, dissociation of cells 10 / 30 in channel 301 / 201 / 101 can be achieved by using transducer arm 1303 to contact channel holder 1082. A motor 130 is mechanically coupled to transducer arm 1303 to generate mechanical vibrations to transducer arm 1303. The mechanical vibrations from motor 130 can then be transmitted through transducer arm 1303 to channel holder 1082 and then to channel 301 / 201 / 101 in contact with channel holder 1082, creating localized turbulence at various locations within channel 301 / 201 / 101, which can help mechanically break the aggregates into small pieces and aid in the self-dissociation of cell 10 / 30 aggregates. The direction of vibration exerted on the channel 301 / 201 / 101 through the channel holder 1082 can be direction 61001, or direction 61002, or alternating between directions 61001 and 61002. The transducer arm 1303 can be bifurcated, with a thinner handle that connects to the motor 130 and a wider transducer end 1304 that contacts the channel holder 1082 for effective vibration transmission from the motor 130 to the channel holder 1082. The transducer end 1304 can have a locking mechanism that mechanically secures it onto the channel holder 1082 to generate effective vibration transmission.

[0517] The channels 101, 201, 301 in Figures 4 to 7, 9 to 14, 16 to 30B, 32 to 36B, 44A to 65B, and 86 to 89C may have channel wall thicknesses in any of the following ranges: 0.01 mm to 0.02 mm, 0.02 mm to 0.05 mm, 0.05 mm to 0.1 mm, 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, 0.4 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, and 2 mm to 5 mm, respectively.

[0518]

[0519] FIG. 90A is a cross-sectional view of a portion of UFL 600 of FIG. 38A along direction 64, including solid fluid inlet 602, buffer inlet 604, and a portion of UFL main channel 601. FIG. 90A shows that UFL 600 is composed of two parts, base 611 and cover 610, with channels 601, 603, and 608 formed in base 611 as grooves of equal depth 627, preferably formed in one step from the first surface of base 611. In one embodiment, depth 627 is between 100 nm and 500 nm. In another embodiment, depth 627 is between 500 nm and 1 μm. In yet another embodiment, depth 627 is between 1 μm and 10 μm. In yet another embodiment, depth 627 is between 10 μm and 100 μm. In yet another embodiment, depth 627 is between 100 μm and 1 mm. Unlike the embodiment of Figure 38B, the access ports for injecting fluid into inlets 602 and 604 and for expelling fluid from outlets 607 and 608 are formed in base 611 as a single clearance, like inlets 602, 604 and outlets 607 and 608, allowing fluid to be injected or expelled from a second face of base 611 opposite the first face on which channels 601, 603, and 608 are formed. Figure 90A shows an example in which access port 621 and inlet 602 are formed as a single clearance connecting from the second face at the bottom of base 611 to channel 603 formed from the first face at the top of base 611, while access port 641 and inlet 604 are formed as a single clearance connecting from the second face at the bottom of base 611 to main channel 601 formed from the first face at the top of base 611. In Figure 90A, unlike Figure 38B, cover 610 is a uniform cover with no clearance features. Access ports in base 611, which are also the inlet and outlet of UFL 600, allow entity fluid 6020 and buffer 6040 to enter inlets 602 and 604, and allow large entity 6070 and small entity 6090 fluids to exit through outlets 607 and 609. In the embodiment of Figure 90A, alignment of the access ports to the inlet and outlet as in Figure 38B is avoided.During fabrication of UFL 600, after base 611 is patterned on the top first surface with grooves 601, 603, 608, clearances can be formed at the locations of inlets 602, 604 and outlets 607, 609 through base 611 to connect from the grooves to the bottom second surface. Cover 610 can then be placed as a uniform piece on the top first surface of base 611 to form enclosed channels 601, 603, and 608, and cover 610 can be placed on the top first surface of base 611 to form enclosed channels 601, 603, and 608. It can be bonded to base 611 through either (1) surface-to-surface van der Waals forces; (2) adhesion; or (3) ultrasonic thermal fusion if one or both of base 611 and cover 610 are made of a plastic or polymer material. Next, syringes 6021 and 6041 show examples of possible external fluid injection into the inlet of UFL 600 through the access port clearance of base 611, and syringes 6021 and 6041 can have larger nozzle sizes than matching access ports 621 and 641 to accommodate positioning errors between the syringe and the access port. Figure 90A shows that a substance fluid 6020 containing large entities 612 and small entities 613, which can be injected by injector 6021, passes through access ports 621 / 602 and enters main channel 601 as a lateral laminar flow, while a buffer solution 6040, which can be injected by injector 6041, passes through access ports 641 / 604 and enters main channel 601 as a central laminar flow.

[0520] 90A may each be composed of glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon oxide, alumina, plastic, PDMS, polymer, ceramic, or metal, which may be composed of any one of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver, or any alloy thereof. Cover 610 may be composed of a different material than base 611.

[0521] In one embodiment, forming the access ports, inlets, outlets, and channels in the base 611 includes the following steps: (1) providing a base 611 having two substantially flat surfaces; (2) forming an etching mask for the channels on the first flat surface; (3) etching the base by a first etching method, including wet etching with a fluid chemical, dry etching with a chemical gas, plasma dry etching, sputter etching with an ion plasma, and ion beam etching (IBE), to form the channels in the base; (4) forming an inlet and outlet etching mask on a second flat surface of the base opposite the first flat surface; and (5) etching the base by the first etching method to form inlets and outlets in the base that connect from the second flat surface through the base to the channels formed in step (3). In forming the etching mask in steps (2) and (4), the etching mask may be made of photoresist (PR), which may include depositing or spin-coating PR on the flat surface, then exposing it to light or ion / electron radiation in a channel pattern, and developing the PR after the exposure, so that the remaining PR with the pattern serves as the etching mask. The etching mask may also be made from a hard mask material that has a lower etching rate than the base material under a first etching method, and steps (2) and (4) may include depositing a hard mask layer on the flat surface, depositing or spin-coating a PR layer on the hard mask layer, then exposing the PR to light or ion / electron radiation in a channel pattern, and developing the PR after the light exposure, so that the remaining PR with the pattern serves as the etching mask for the hard mask; etching the hard mask using a second etching method, which may include wet etching using a liquid chemical, dry etching using a chemical gas, plasma dry etching, or sputter etching using an ion beam; and removing the remaining PR layer. The second etching method and the first etching method may be of different types or different chemistries.

[0522] In another embodiment, the inlets, outlets, and channels in the base 611 may be formed by heat pressing, which involves using a heated stencil having the physical pattern of the inlets, outlets, and channels to melt and deform a portion of the base 611 to create the inlets, outlets, and channels, then cooling the base 611 and removing the stencil. In heat pressing, the base material is preferably a plastic or polymer. In yet another embodiment, the inlets, outlets, and channels in the base 611 may be formed by stamping, which involves using a stencil having the physical pattern of the inlets, outlets, and channels to stamp into a partially or fully melted base 611, then cooling the base 611, and finally removing the stencil, with the cooled base retaining the pattern transferred from the stencil of the inlets, outlets, and channels. In stamping, the base material is preferably a plastic or polymer. In another embodiment, the inlets, outlets and channels are formed in the base 611 by injection molding, where molten base 611 material is poured into a mold cavity and the base 611 body with the inscribed inlets, outlets and channels is defined by the mold cavity.

[0523] FIG. 90B is a cross-sectional view of a portion of UFL 600, including main channel 601, base 611, and cover 610, taken along direction 65 in FIG. 38A. The embodiment of FIG. 90B functions similarly to FIG. 38C, except that PZT 614 is attached to cover 610, and ultrasonic vibrations from PZT 614 are transmitted to ULF 600 channel 601 through cover 610. In FIG. 90B, thickness 6101 of cover 601 is preferably equal to or less than thickness 6111 of base 611. In one embodiment, thickness 6101 is less than thickness 6111 minus depth 627 of channel 601. Thickness 6101 of cover 610 can be any one of 1 mm to 2 mm, 0.5 mm to 1 mm, 0.2 mm to 0.5 mm, or 0.1 mm to 0.2 mm.

[0524] FIG. 90C shows a top view of the UFL 600 of FIG. 38A with multiple PZTs attached to the same UFL 600 device. Two or more of the PZTs 6141, 6142, 6143, and 6144 are connected to and cover the UFL 600 at different locations along the main channel 601. The PZT 6144 covering at least one outlet or at least one inlet of the UFL 600 should be attached to the base 611 of the UFL 600 in FIG. 38B, while it should be attached to the cover 601 in FIG. 90A, opposite the outlet or inlet opening in both embodiments. The PZTs 6141, 6142, and 6143 can be attached to the cover 601 or the base 611, respectively. In one embodiment, two or more of the PZTs 6141, 6142, 6143, and 6144 are attached to the cover 601 in FIG. 90A. In another embodiment, two or more of PZTs 6141, 6142, 6143, 6144 are attached to base 611 of Figure 38B. In yet another embodiment, two or more of PZTs 6141, 6142, 6143 are attached to cover 601 of Figure 38B. In yet another embodiment, two or more of PZTs 6141, 6142, 6143 are attached to base 611 of Figure 90A. In one embodiment, at least two PZTs selected from any of 6141, 6142, 6143, 6144 attached to UFL 600 operate at the same frequency. In another embodiment, at least two PZTs selected from any of 6141, 6142, 6143, and 6144 attached to the UFL 600 operate at different frequencies, with each different PZT having a different frequency, thereby generating different standing wave modes in the channel 601 directly covered by each PZT, and simultaneously, the channel 601 may have a varying channel width between the inlet and outlet of the UFL 600. In one embodiment, each PZT has a length 61411 along the channel 601 direction and a width 61412 perpendicular to the length 61411 direction, and each PZT attached to the UFL 600 has the length 61411 longer than the width 61412. In another embodiment, each PZT attached to the UFL 600 has the length 61411 shorter than the width 61412.In yet another embodiment, each PZT selected from any of 6141, 6142, 6143, and 6144 attached to the UFL 600 is identical, each PZT is attached to the same cover 601 surface or the same base surface 611 of the UFL 600, and the same AC voltage is applied simultaneously to drive each PZT attached to the UFL 600 at the same frequency.

[0525] FIG. 91A shows a cross-sectional view of a UFL similar to FIG. 90B but with a flow channel having spherically curved sidewalls. The channel 601 in FIG. 91A is in the shape of a truncated circle, with the sidewalls 60102 and 60103 being portions of the same circle, while the diameter of that circle is a half wavelength, or an integer multiple of a half wavelength, of the ultrasonic mode in the fluid within the channel 601 at the resonant frequency, or drive frequency, Fp, of the PZT 614. A standing ultrasonic wave can exist between the two sidewalls 60102 and 60103 of the channel 601, as indicated by dashed line 626. The center of the circle of which the sidewalls 60102 and 60103 are a part is preferably at the center of the channel, as indicated by point 62601, so that the bottom end wall 60101 and the top end wall 60104 are substantially parallel to each other and have the same width. The top end wall 60104 is formed by the top cover 610, which covers the base 611. The truncated circle shape of channel 601 in FIG. 91A may be formed by etching base 611 by isotropic, or partially isotropic and partially anisotropic, etching methods, including wet and dry etching, that etch sidewalls 60101 and 60103 into a substantially circular curvature, while the lower channel wall 60101 may be kept flat during this etching by having a slower etching, or etch stop, layer at the location of lower channel wall 60101 that does not etch as easily as walls 60102 and 60103 of base 611. Base 611 may be a multi-layer structure having an etch stop layer 60111 to form lower wall 60101 during etching of channel 601, and an etchable layer 60112 over etch stop layer 60111 to enable etching of channel 611 into the truncated circle shape of the channel in FIG. 91A.

[0526] FIG. 91B shows a cross-sectional view of a UFL similar to FIG. 91A , but in which a channel having a partially circular shape is formed in UFL base 611. Channel 601 in FIG. 91B has a circular shape truncated only at the top, and sidewall 60105 is close to a perfect circle, while the diameter of the circle is a half wavelength, or an integer multiple of a half wavelength, of an ultrasonic mode in the fluid in channel 601 at the resonant frequency, or driving frequency, or Fp, of PZT 614, so that standing ultrasonic waves can exist within the circle in channel wall 60105, as indicated by dashed line 626. Top end wall 60104 is formed by top cover 610 covering base 611. The truncated circular shape of channel 601 in FIG. 91B can be formed by etching base 611 with an isotropic, or partially isotropic and partially anisotropic, etching method, including wet etching and dry etching, to etch sidewall 60105 into a substantially circular shape within base 611.

[0527] Figure 91C shows a cross-sectional view of a UFL similar to Figure 91A, but in which a flow path having a circular shape is formed in both the UFL base and the UFL cover. Channel 601 in Figure 91C is circular in shape, with a substantially semicircular lower channel wall 60105 formed in base 611 and a substantially similar semicircular upper channel wall 60104 formed in top cover 610, having the same diameter as the circle of 60105. The diameter of the circle formed by 60104 and 60105 is a half wavelength, or an integer multiple of a half wavelength, of an ultrasonic mode in the fluid in channel 601 at the resonant frequency, or drive frequency, Fp, of PZT 614, so that a standing ultrasonic wave can exist within the circle of channel walls 60104 and 60105, as indicated by dashed line 626. The semicircular shape 60105 of the channel 601 in the base 611 and the semicircular shape 60104 of the cover 610 in Figure 91C may be formed by etching the base 611 and walls by isotropic, or partially isotropic and partially anisotropic, etching methods, including wet and dry etching, that etch 60104 and 60105 into substantially circular shapes in the cover 610 and base 611, respectively. An alignment step is then performed to align the channel walls 60104 and 60105 to the surrounding channel 601 to form the channel shape of Figure 91C.

[0528] In Figures 91A-91C, the base 611, cover 601 and etch stop layer 60111 may be glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon oxide, alumina, plastic, PDMS, polymer, ceramic or metal, which may be composed of any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver.

[0529] In the case of channels 601, 603, 608, inlets 602, 603, and outlets 607, 609 in Figures 38A, 38B, and 91A, the channels, inlets, and outlets after being patterned into their shapes may be coated with one or more layers of any of silicon oxide, SiN, SiC, alumina, aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, and silver by PVD, CVD, PE-CVD, oxidation, ALD, or PE-ALD processes, and such coated layers come into contact with the liquid sample flowing through UFL 600 during operation.

[0530] Figure 92A shows a top view of a UFL device 600 similar to UFL 600 of Figure 38A, with two inlets 602 and 604 and two outlets 607 and 609. Input subchannel 6042, leading from inlet 604 to main channel 601, has channel width 6511. The main channel has channel width 6510. Input side channel 603, leading from inlet 602 to main channel 601, has channel width 6514. Output subchannel 6072, leading from main channel 601 to outlet 607, has channel width 6512. Output side channel 608, leading from main channel 601 to outlet 609, has channel width 6513. In one embodiment, channel width 6512 is smaller than channel width 6511. Channel width 6512 can be a percentage of channel width 6511, where the percentage is within any of the following ranges: 10% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, 80% to 100%, 100% to 150%, 150% to 200%, 200% to 500%, or 500% to 1000%. In another embodiment, channel width 6512 is smaller than channel width 6513. Channel width 6512 can be a percentage of channel width 6513, where the percentage is within any of the following ranges: 10% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, 80% to 100%, 100% to 150%, 150% to 200%, 200% to 500%, or 500% to 1000%. The channel width 6512 and the side channel width 6513 of output subchannel 6072 can be adjusted to allow the output fluid flow rates through 607 and 609 to be different. For example, if 6512 is less than or equal to 6513, the output flow rate from outlet 607 is less than outlet 609. The ratio of the output fluid flow rate from outlet 607 to the output fluid flow rate from outlet 609 can be estimated as (channel width 6512) divided by (2 times channel width 6513). In one embodiment, channel width 6511 is smaller than channel width 6510, channel width 6512 is smaller than channel width 6511, channel width 6512 is smaller than channel width 6513, channel width 6513 is larger than channel width 6514, and channel width 6514 is smaller than channel width 6510.In one embodiment, the ratio of channel width 6512 to channel width 6513 is less than the ratio of channel width 6511 to channel width 6514. In another embodiment, the ratio of channel width 6512 to channel width 6513 is greater than the ratio of channel width 6511 to channel width 6514. In yet another embodiment, the ratio of channel width 6512 to channel width 6513 is the same as the ratio of channel width 6511 to channel width 6514. An ultrasonic generator 614, e.g., a PZT, can be attached to UFL 600 similar to FIG. 38C or FIGS. 90B-91C.

[0531] Figure 92B shows a top view of UFL device 6000, which is identical to Figure 92A in all other respects except that it has only one inlet 6022 and two outlets 607 and 609. Main channel 601 connects directly from inlet 6022 to output side channel 608 and output sub-channel 6072. In one embodiment, channel width 6512 is smaller than channel width 6513, and channel width 6514 is smaller than channel width 6510. An ultrasonic generator 614, e.g., a PZT, can be attached to UFL 6000 similar to Figure 38C or Figures 90B-91C.

[0532] Figure 93A shows the operation of the UFL600 device of Figure 92A. Substantial fluid 6020 of Figure 38A, containing large entities 10 / 20 / 30 / 612 and smaller entities 613, is injected into the UFL600 channel through inlet 602 and then passes through input side channel 603 into main channel 601 at an effective volumetric flow rate 6031. Buffer fluid 6040 of Figure 38A is injected into the UFL600 channel through inlet 604 and then passes through input subchannel 6042 of Figure 92A into main channel 601 at an effective volumetric flow rate 6041. Buffer fluid 6040 and substantive fluid 6020 meet in main channel 601, forming a laminar flow, where buffer fluid 6040 flows down the center of main channel 601 and substantive fluid 6020 flows alongside buffer fluid 6040 through channel 601 and along the sidewalls of channel 601. Due to the laminar flow, buffer fluid 6040 and substantive fluid 6020 do not mix while passing through channel 601. To achieve said laminar flow, buffer fluid 6040 may have a different fluid density than substantive fluid 6020, or buffer fluid 6040 may have a different viscosity than substantive fluid 6020, or buffer fluid 6040 may have a different compressibility than substantive fluid 6020. In one embodiment, buffer fluid 6040 is denser than the substantive fluid. In one embodiment, buffer fluid 6040 is more viscous than the substantive fluid. In one embodiment, buffer fluid 6040 is more compressible than the substantive fluid. In main channel 601, substantive fluid 6020 may have a linear flow velocity 6033 along the edges of channel 601, and buffer fluid 6040 may have a linear flow velocity 6043 at the center of channel 601. Linear velocities 6033 and 6043 may be different. In one embodiment, velocity 6033 may be lower than velocity 6043; in another embodiment, velocity 6033 may be higher than velocity 6043; and in yet another embodiment, velocity 6033 may be substantially the same as velocity 6043.

[0533] In Figure 93A, as shown in Figures 38C and 38D, ultrasonic standing wave modes generated in main channel 601 caused by ultrasonic generator 614 cause larger entities 10 / 20 / 30 / 612 to move from a substance flow 6020 flowing at the wall of channel 601 at velocity 6033 to a buffer flow 6040 flowing through the center of channel 601 at velocity 6043, while all or most of the smaller entities 613 are retained in substance flow 6020. Small entities 613 may be retained in substance flow 6020 by virtue of their smaller size, greater density, or less compressibility than the larger entities 10 / 20 / 30 / 612. When the laminar flow in channel 601 containing 6020 and 6040 reaches the end of channel 601, a portion of central buffer fluid 6040 containing larger sized entities 10 / 20 / 30 / 612 flows through output sub-channel 6072 of FIG. 92A and exits through outlet 607 at effective volumetric flow rate 6071. Entity flow 6020 containing smaller entities 613 at the sidewall of channel 601 passes through output side channel 608 at effective volumetric flow rate 6081 and exits through outlet 609. In one embodiment, flow rate 6071 is less than flow rate 6081 and channel width 6512 is less than channel width 6513 of FIG. 92A. In another embodiment, flow rate 6071 is greater than flow rate 6081 and channel width 6512 is greater than channel width 6513 of FIG. 92A. In yet another embodiment, flow rate 6071 is substantially similar to flow rate 6081 and channel width 6512 is similar to 6513 in Figure 92A.

[0534] Figure 93B shows the operation of the UFL6000 apparatus of Figure 92B. The entity fluid 6020 of Figure 38A containing large entities 10 / 20 / 30 / 612 and smaller entities 613 is injected into the UFL6000 channel through inlet 6022, with an effective volumetric flow rate 6021 entering the main channel 601. An ultrasonic standing wave mode, also called an acoustic standing wave mode, generated by an ultrasonic or acoustic generator 614 in the main channel 601 similar to that shown in Figures 38C and 38D causes the large entities 10 / 20 / 30 / 612 to flow along the end walls of the channel 601 and move toward the center of the channel 601 at a velocity 6043, i.e., concentration of the large entities 10 / 20 / 30 / 612 at the channel 601 centerline, while all or most of the smaller entities 613 remain less concentrated in the entity flow 6020. Small entities 613 may be maintained in entity stream 6020 without concentration by being smaller, or having a greater density, or less compressibility than larger entities 10 / 20 / 30 / 612. When the entity stream in channel 601 reaches the terminal end of channel 601, the central portion of entity stream 6020, containing most or all of the large entities 10 / 20 / 30 / 612, and a small percentage of the smaller entities 613, flows through outlet subchannel 6072 in FIG. 92B and exits through outlet 607 at an effective volumetric flow rate 6071. The side portions of entity stream 6020, containing most or all of the smaller entities 613, pass through output side channel 608 at an effective volumetric flow rate 6081 and exit through outlet 609. In one embodiment, flow rate 6071 is less than flow rate 6081, and channel width 6512 is less than channel width 6513 in FIG. 92B. In another embodiment, flow rate 6071 is greater than flow rate 6081 and channel width 6512 is greater than 6513 in FIG. 92B. In yet another embodiment, flow rate 6071 is substantially similar to flow rate 6081 and channel width 6512 is similar to 6513 in FIG. 92B.

[0535] In Figure 93B, when both large entities 10 / 20 / 30 / 612 and smaller entities 613 are present in the incoming entity fluid 6020, the UFL 6000 of Figure 93B functions to discharge 6070 fluid through outlet 607, resulting in a higher and more concentrated percentage of larger entity 10 / 20 / 30 / 612 content and a lower percentage of smaller entities 613 in 6070 than in the original incoming entity fluid 6020, while the fluid 6090 discharge from outlet 609 is depleted of the large entity 10 / 20 / 30 / 612 population. If the substantive fluid 6020 contains only large entities 10 / 20 / 30 / 612, the UFL 6000 of FIG. 93B will function to primarily eject the 6070 fluid, resulting in a reduced fluid volume compared to the substantive fluid 6020 through outlet 607, with a higher concentration of the larger entities 10 / 20 / 30 / 612 in fluid 6070 than in the substantive fluid 6020. The UFL 6000 function of FIG. 93B can be effectively achieved with the UFL 600 of FIG. 93A by injecting the substantive stream 6020 using inlet 602 and not injecting the buffer stream 6040 through inlet 604, or by injecting the substantive stream 6020 using inlet 604 and not injecting the buffer stream 6040 through inlet 602 of FIG. 93A.

[0536] 93A and 93B, flow rates 6071 and 6081 are intrinsic output flow rate values ​​for UFL 600 or UFL 6000, which means that when outlets 607 and 609 are not connected to any external conduits and fluids 6070 and 6090 in FIG. 38A flow freely out of outlets 607 and 609. If outlets 607 and 609 are connected to external conduits to direct fluids 6070 and 6090 away from outlets 607 and 609, these conduits can be used to create additional fluid resistance in either 6070 or 6090, resulting in an exogenous modification to flow rates 6071 and 6081. In one embodiment, the flow resistance of fluid 6070 through the conduit connected to outlet 607 is greater than the flow resistance of fluid 6090 through the conduit connected to outlet 609, and therefore 6071 is less than the effective volumetric flow rate of fluid 6090 through outlet 609, which is twice the value of 6081 in Figures 93A and 93B. In another embodiment, the flow resistance of fluid 6070 through the conduit connected to outlet 607 is less than the flow resistance of fluid 6090 through the conduit connected to outlet 609, and therefore 6071 is greater than the effective volumetric flow rate of fluid 6090 through outlet 609 in Figures 93A and 93B.

[0537] Figure 94A shows an embodiment of a process flow between blood or bone marrow sample collection and UFL operation. In step 5801, a peripheral blood sample or bone marrow sample may be collected from a patient or individual under test, and in step 5802, red blood cell lysis may be performed on the sample from step 5801; in step 5803, the sample from step 5802 after lysis is injected at UFL entity fluid inlet 602, while UFL buffer fluid is injected at outlet 604; and in step 5804, the UFL is operated similarly to step 5804 in Figure 66. After step 5804, steps after step 5804 may be performed, such as in Figures 66, 70, 72, and 74.

[0538] Figure 94B shows another embodiment of the process flow between blood or bone marrow sample collection and UFL operation. In step 5801, a peripheral blood sample or bone marrow sample is collected from a patient or individual under test; in step 5802, red blood cell lysis is performed on the sample of step 5801; in step 5806, the target sample from step 5802 is affixed with a magnetic label and / or a fluorescent molecule that hybridizes with an antibody or ligand that specifically binds to a surface antigen or receptor on the target cell or entity; in step 5807, the target sample from step 5806 is incubated to form antibody-antigen or ligand-receptor binding to the target cell or entity; in step 5803, the target sample from step 5807 is injected at UFL entity fluid inlet 602, while UFL buffer fluid is injected at outlet 604; in step 5804, the UFL is operated similarly to step 5804 of Figure 66. After step 5804, steps after step 5804 such as those in Figures 66, 70, 72, and 74 may be performed.

[0539] Figure 95A shows an embodiment of the process flow between solid sample collection and UFL operation. In step 6901, a solid tissue sample is collected; in step 6902, the solid tissue sample from step 6901 is subjected to fluid-based dissociation; in step 5806, the target sample from step 6902 is affixed with a magnetic label and / or a fluorescent molecule that hybridizes with an antibody or ligand that specifically binds to a surface antigen or receptor on the target cell or entity; in step 5807, the target sample from step 5806 is incubated to form an antibody-antigen or ligand-receptor bond to the target cell or entity; in step 5803, the target sample from step 5807 is injected at UFL entity fluid inlet 602, while UFL buffer fluid is injected at outlet 604; in step 5804, the UFL is operated similarly to step 5804 of Figure 66. After step 5804, steps after step 5804, such as those in Figures 66, 70, 72, and 74, can be performed.

[0540] Figure 95B shows an embodiment of the process flow between surface sample collection and UFL operation. In step 6701, surface entities are collected by swabbing; in step 6702, the surface entities collected in the swab are dissolved in a fluid base; in step 5806, the target sample from step 6702 is affixed with a magnetic label and / or a fluorescent molecule that hybridizes with an antibody or ligand that specifically binds to a surface antigen or receptor on the target cell or entity; in step 5807, the target sample from step 5806 is incubated to form antibody-antigen or ligand-receptor bonds to the target cell or entity; in step 5803, the target sample from step 5807 is injected at UFL entity fluid inlet 602, while UFL buffer fluid is injected at outlet 604; in step 5804, the UFL is operated similarly to step 5804 in Figure 66. After step 5804, steps after step 5804, such as those in Figures 66, 70, 72, and 74, can be performed.

[0541] Figure 96 shows an embodiment of a process flow after negative MAG sample collection, including a UFL operation. Step 5813 is the same as in Figures 66-75, and involves collecting a negative MAG sample during MAG separation of a target sample. After step 5813, the negative MAG sample of step 5813 is concentrated by UFL without the use of UFL buffer, including either (a) in step 58222, injecting the negative MAG sample of step 5813 into inlet 602 or 604 of the fourth UFL 600 of Figure 92A without injecting UFL buffer, or (b) in step 58223, injecting the negative MAG sample of step 5813 into inlet 6022 of the fourth UFL 600 of Figure 92B. After step 58222 or step 58223, in step 58231, the fourth UFL outlet 607 outputs a sample enriched in large entities, with fewer small entities than the negative MAG sample of step 5813. Finally, the negative MAG sample containing large entities from the fourth UFL outlet 607 can be sent for analysis by either a cell counter 903, a cell imaging device 904, a flow cytometer or sorter 905, or a DNA / RNA sequencer 906. Alternatively, the output from the cell counter 903 or the output from the cell imaging device 904 or the output from the flow cytometer or sorter 905 can be further sent for processing by the DNA / RNA sequencer 906, as shown by paths 936, 946, and 956, respectively. The negative MAG sample containing large entities from the fourth UFL outlet 607 in step 5823 can also be sent to the process of cytogenetic modification and cell proliferation 5824. Prior to DNA / RNA sequencing in DNA / RNA sequencer 906, a polymerase chain reaction (PCR) procedure may be performed on the DNA / RNA sample obtained from cell lysis of large entities from outlet 607 of the fourth UFL from step 5823, where the PCR may target one or more target DNA / RNA sequences and amplify the number of target DNA / RNA sequences in the DNA / RNA sample.

[0542] Figure 97 shows another process flow embodiment after negative MAG sample collection, including a UFL operation. Step 5813 is the same as in Figures 66-75, and involves collecting a negative MAG sample during MAG separation of a target sample. After step 5813, the negative MAG sample of step 5813 is concentrated by UFL without the use of UFL buffer, including either (a) in step 58222, injecting the negative MAG sample of step 5813 into inlet 602 or 604 of the fourth UFL 600 of Figure 92A without injecting UFL buffer, or (b) in step 58223, injecting the negative MAG sample of step 5813 into inlet 6022 of the fourth UFL 600 of Figure 92B. After step 58222 or step 58223, in step 58251, fourth UFL outlet 609 outputs a sample having small sized entities including DNA / RNA / molecules / small particles, depleted of the larger entities originally contained in the negative MAG sample of step 5813. Finally, the small entities from fourth UFL outlet 609 can be sent for analysis by either particle counter 5835, particle imaging device 5836, flow cytometer or sorter 905, or DNA / RNA sequencer 906. Alternatively, the output from particle counter 5835 or particle imaging device 5836 or flow cytometer or sorter 905 can be further sent for processing by DNA / RNA sequencer 906, as shown by paths 5827, 5828, and 956, respectively. The DNA / RNA sequencer 906 may contain a PCR step on the small entities from the outlet 609 of the fourth UFL from step 5825 prior to DNA / RNA sequencing, where the PCR may target and amplify in large quantities one or more specific DNA / RNA sequences.

[0543] Figure 98 illustrates an embodiment of a process flow following positive MAG sample collection, including a UFL operation. Step 5814 is the same as in Figures 66-75, in which a positive MAG sample is collected after MAG separation of the target sample. After step 5814, the positive MAG sample of step 5814 is concentrated by UFL without UFL buffer, including either (a) in step 58291, injecting the positive MAG sample of step 5814 into inlet 602 or 604 of the fifth UFL 600 of Figure 92A without injecting UFL buffer, or (b) in step 58292, injecting the positive MAG sample of step 5814 into inlet 6022 of the fifth UFL 600 of Figure 92B. After step 58291 or step 58292, in step 58301, fifth UFL outlet 607 outputs a sample enriched in large entities, which has fewer small entities than the positive MAG sample of step 5814. Finally, the positive MAG sample containing large entities from fifth UFL outlet 607 can be sent for analysis by any of cell counter 903, cell imaging device 904, flow cytometer or sorter 905, and DNA / RNA sequencer 906. Alternatively, the output from cell counter 903 or cell imaging device 904 or flow cytometer or sorter 905 can be further sent for processing by DNA / RNA sequencer 906, as shown by paths 936, 946, and 956, respectively. The positive MAG sample containing large entities from fifth UFL outlet 607 in step 5823 can also be sent to a process of cytogenetic modification and / or cell expansion 5824. Prior to DNA / RNA sequencing in DNA / RNA sequencer 906, a polymerase chain reaction (PCR) procedure may be performed on the DNA / RNA sample obtained from cell lysis of large entities from outlet 607 of the fourth UFL from step 5823, where the PCR may target one or more target DNA / RNA sequences and amplify the number of target DNA / RNA sequences in the DNA / RNA sample.

[0544] Figure 99 illustrates another process flow embodiment after positive MAG sample collection, including a UFL operation. Step 5814 is the same as in Figures 66-75, where a positive MAG sample is collected after MAG separation of the target sample. After step 5814, the positive MAG sample of step 5814 is concentrated by UFL without UFL buffer, including either (a) in step 58291, injecting the positive MAG sample of step 5814 into inlet 602 or 604 of the fifth UFL 600 of Figure 92A without injecting UFL buffer, or (b) in step 58292, injecting the positive MAG sample of step 5814 into inlet 6022 of the fifth UFL 6000 of Figure 92B. After step 58291 or step 58292, in step 58311, fifth UFL outlet 609 outputs a sample with small entities including DNA / RNA / molecules / small particles, depleted of the large entities originally contained in the positive MAG sample of step 5814. Finally, the small entities from fifth UFL outlet 609 can be sent for analysis by either particle counter 5835, particle imaging device 5836, flow cytometer or sorter 905, or DNA / RNA sequencer 906. Alternatively, the output from particle counter 5835 or particle imaging device 5836 or flow cytometer or sorter 905 can be further sent for processing by DNA / RNA sequencer 906, as shown by paths 5827, 5828, and 956, respectively. The DNA / RNA sequencer 906 may contain a PCR step on the small entities from the outlet 609 of the fourth UFL from step 5825 prior to DNA / RNA sequencing, where the PCR may target and amplify in large quantities one or more specific DNA / RNA sequences.

[0545] FIG. 100A shows an embodiment of a process flow including two UFLs operating in series. In step 58051, an input sample is injected into inlet 602 or 604 of UFL 600 in FIG. 92A or inlet 6022 of UFL 6000 in FIG. 92B, and then UFL outlet 607 outputs a target sample containing large cells or entities from the original input sample. After step 58051, the target sample from step 58051 is concentrated by UFL without UFL buffer, including either (a) in step 58222, injecting the target sample from step 58051 into inlet 602 or 604 of a fourth UFL 600 in FIG. 92A, and no UFL buffer is injected, or (b) in step 58223, injecting the target sample from step 58051 into inlet 6022 of a fourth UFL 6000 in FIG. 92B. After step 58222 or step 58223, in step 58231, the fourth UFL outlet 607 outputs a sample enriched in large entities that has fewer small entities than in the target sample from step 58051. After step 58231 in Figure 100A, steps may similarly be performed as described in Figure 96 utilizing steps 903, 904, 905, 906, 5824 after step 58231.

[0546] Figure 100B shows another process flow embodiment involving two UFLs in sequential operation. Figure 100B is the same as Figure 100A, except that after step 58222 or step 58223, in step 58251, the fourth UFL outlet 609 outputs a sample with small entities including DNA / RNA / molecules / small particles that are depleted of the larger entities originally contained in the target sample from step 58051. After step 58251 in Figure 100B, steps such as those described in Figure 97 utilizing steps 5835, 5836, 905, and 906 can similarly be performed after step 58251.

[0547] Figure 101A shows another process flow embodiment involving two UFLs in continuous operation. In step 58052, an input sample is injected into inlet 602 or 604 of UFL 600 in Figure 92A or inlet 6022 of UFL 6000 in Figure 92B, and then UFL outlet 609 outputs a target sample containing smaller cells or entities from the original input sample. After step 58052, the target sample from step 58052 is concentrated by UFL without UFL buffer, including either (a) in step 58222, injecting the target sample from step 58052 into inlet 602 or 604 of a fourth UFL 600 in Figure 92A and not injecting UFL buffer, or (b) in step 58223, injecting the target sample from step 58052 into inlet 6022 of a fourth UFL 6000 in Figure 92B. After step 58222 or step 58223, in step 58226, fourth UFL outlet 607 outputs a first sample enriched in large entities with fewer small entities than in the target sample from step 58052, and fourth UFL outlet 609 outputs a second sample with small entities containing DNA / RNA / molecules / small particles that are depleted of the large entities originally contained in the target sample from step 58052. After step 58226 of Figure 101A, steps such as those described in Figure 96 utilizing steps 903, 904, 905, 906, and 5824 after step 58231 can be similarly performed on the first sample from step 58226; after step 58251, steps such as those described in Figure 97 utilizing steps 5835, 5836, 905, and 906 can be similarly performed on the second sample from step 58226.

[0548] Figure 101B shows another process flow embodiment involving two UFLs in continuous operation. In step 58052, an input sample is injected into inlet 602 or 604 of UFL 600 in Figure 92A or inlet 6022 of UFL 6000 in Figure 92B, and UFL outlet 609 then outputs a target sample containing smaller cells or entities from the original input sample. After step 58052, in step 58227, the target sample from step 58052 is injected into inlet 602 and buffer fluid is injected into inlet 604 of the sixth UFL 600 in Figure 92A. After step 58227, in step 58226, sixth UFL outlet 607 outputs a first sample having primarily large entities depleted of smaller entities originally contained in the target sample from step 58052; outlet 609 outputs a second sample having primarily smaller entities including DNA / RNA / molecules / small particles depleted of larger entities originally contained in the target sample from step 58052. After step 58228 of Figure 101B, steps such as those described in Figure 96 utilizing steps 903, 904, 905, 906, 5824 after step 58231 can similarly be performed on the first sample from step 58228; after step 58251, steps such as those described in Figure 97 utilizing steps 5835, 5836, 905, 906 can similarly be performed on the second sample from step 58228.

[0549] Figure 102 shows an embodiment of a method for operating multiple UFLs in a series or cascade arrangement. Figure 102 shows that a biological sample passes through multiple first-stage UFLs 600, and the output fluid from the UFLs 600, which can be either large entities 6070 or small entities 6090, is then pumped into an inlet 8011 of a fourth type flow connector 8010 and from the connector 8010 outlet 8012 to (a) the inlet 602 or inlet 604 of one or more second-stage UFLs 600; or (b) the inlet 6022 of one or more second-stage UFLs 6000.

[0550] Figure 103 shows another method embodiment for operating multiple UFLs in a series or cascade arrangement. Figure 102 shows that a biological sample passes through multiple first-stage UFLs 600, and then the output stream from the UFLs 600, which can be either large entities 6070 or small entities 6090, is fed into an inlet 8021 of a fifth type flow connector 8020, and from the connector 8020 outlet 8022, to (a) the inlet 602 or inlet 604 of one or more second-stage UFLs 600; or (b) the inlet 6022 of one or more second-stage UFLs 600.

[0551] Figure 104 shows another method embodiment for operating multiple UFLs in a series or cascade arrangement. Figure 104 shows that a biological sample passes through multiple first-stage UFLs 600, and then the output stream from the UFLs 600, which can be either large entities 6070 or small entities 6090, is fed to an inlet 8031 ​​of a sixth type flow connector 8030, and from the connector 8030 outlet 8032, to (a) the inlet 602 or inlet 604 of one or more second-stage UFLs 600; or (b) the inlet 6022 of one or more second-stage UFLs 6000.

[0552] To achieve the function of a multi-stage UFL, the series or cascade structures of Figures 102, 103 and 104 may be used sequentially, for example, large entities 6070 or small entities 6090 from the outlets 607 and 609 of the second stage UFL 600 or UFL 6000 of Figures 102, 103 and 104, respectively, may be similarly injected into the third stage UFL 600 or UFL 6000 of Figures 102, 103 and 104, respectively, through another intermediate 8010 / 8020 / 8030 connector in any combination.

[0553] Figure 105A shows another method embodiment for operating multiple UFLs in a series or cascade arrangement. Figure 105A shows that the biological sample first passes through the first UFL 600, and the output target sample from UFL 600 can be a large entity 6070 from outlet 607, as in step 58051 in Figure 100A or Figure 100B, or a small entity 6090 from outlet 609, as in step 58052 in Figure 101A or Figure 101B. UFL 6001 and UFL 6002 are similar in design or operation to UFL 600.

[0554] In the first embodiment of Figure 105A, a target sample of large entities 6070 from UFL600 can then be injected into the inlet 602 of UFL6001, and the large entities 6070 from UFL600 can then be further separated by UFL6001 into a first sample containing larger population entities of 6070 from UFL600 and an effluent from outlet 607 of UFL6001, or into a second sample containing smaller population entities of 6070 from UFL600 and an effluent from outlet 609 of UFL6001. In the first embodiment of Figure 105A, the ultrasonic vibration generator 6145 attached to the UFL 600 may operate at a higher vibration intensity, a higher driving voltage, a higher resonant frequency, or have a larger area size than the ultrasonic vibration generator 6146 attached to the UFL 6001; the main channel 601 of the UFL 600 may have a narrower channel width or a deeper channel depth than the main channel 601 of the UFL 6001; and the buffer fluid 6040 entering the inlet 604 of the UFL 600 may have a lower density, lower viscosity, higher compressibility, or slower flow rate than the buffer fluid 6040 entering the inlet 604 of the UFL 6001.

[0555] In the second embodiment of Figure 105A, a target sample of smaller entities 6090 from UFL600 can then be injected into the inlet 602 of UFL6002, and the smaller entities 6090 from UFL600 can then be further separated by UFL6001 into a third sample containing larger population entities of 6090 from UFL600 and a drain from outlet 607 of UFL6002, or a fourth sample containing smaller population entities of 6090 from UFL600 and a drain from outlet 609 of UFL6002. In the second embodiment of FIG. 105A, the ultrasonic vibration generator 6148 attached to the UFL 6002 may operate at either a higher vibration intensity, a higher driving voltage, a higher resonant fre...

Claims

1. 1. An apparatus for separating biological entities in a fluid sample, comprising: an input channel having an outlet; a first exhaust channel having a first inlet fluidly connected to the outlet; a second exhaust channel having a second inlet fluidly connected to the outlet; an optical detector disposed around the input channel; an actuator having a selector gate and a voice coil; a magnetic field exhibiting opposite magnetic polarity applied across the voice coil; Including; the input channel, the first output channel, and the second output channel are contained within a base; the fluid sample is passed through the input channel and the optical detector detects the first entity from the fluid sample before the first entity passes through the outlet; an apparatus wherein a current is applied to the voice coil and activated to move the actuator to a first sorting position, the selector gate blocks the second inlet, and the first entity passes through the outlet and the first inlet to the first exhaust channel.

2. 2. The apparatus of claim 1, wherein the actuator is attached to a route selector disposed above a hinge; the selector gate is attached to the route selector; movement of the actuator causes rotation of the route selector about the hinge; the rotation causes the selector gate to block the first entrance; and the hinge is part of the base.

3. The voice coil a conductive wire disposed on top of the actuator; a conductive wire disposed within the actuator; and Metal wire containing either copper, silver or gold The device of claim 1 , wherein

4. The device of claim 1 , wherein the input channel, first output channel, and second output channel are fabricated in the base during the same manufacturing stage.

5. The device of claim 1 , wherein the base is constructed from one of glass and silicon.

6. 2. The device of claim 1, wherein the base is constructed from any one of quartz, aluminum-titanium-carbon, silicon carbide, silicon nitride, silicon oxide, alumina, a polymer, a ceramic, or a metal; and the metal is constructed from any one of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, and silver, or any alloy thereof.

7. 2. The device of claim 1, wherein the optical detector detects the second entity from the fluid sample before the second entity passes through the outlet; the current applied to the voice coil changes polarity and is activated to move the actuator to a second sorting position, causing the selector gate to block the first inlet; and the second entity passes through the outlet and the second inlet to the second exhaust channel.

8. The current DC current; DC current with decreasing amplitude; AC current; and AC Current with Bias Change The device according to claim 1 ,

9. The apparatus of claim 1 , wherein the base includes a position sensor that detects the position of the actuator and confirms that the actuator reaches the first sort position.

10. The base includes a controller that communicates with the optical detector, the voice coil, and the position sensor through electrical connections, the controller comprising: (i) controlling the optical detector to detect the first entity; (ii) applying the current to the voice coil; (iii) controlling the position sensor to detect when the actuator reaches the first sorting position; 10. The apparatus of claim 9, operative for:

11. The apparatus of claim 1 , wherein the optical detector comprises at least one optical illuminator and at least one optical sensor.

12. the input channel is formed by three surfaces within the base and a top surface from the top cover; the three surfaces include two opposing side surfaces and a bottom surface; the top surface faces the bottom surface; the at least one optical illuminator and at least one optical sensor are disposed on opposing surfaces of the input channel; The opposing surfaces are the top surface and the bottom surface; said two opposing sides; The device of claim 11 , comprising:

13. The device described in claim 1, wherein at least one spring connects the actuator to the base.

14. The device of claim 1 , wherein the optical detector detects the first entity by sensing light emitted from fluorescent molecules contained in the first entity.

15. 10. The device of claim 1, wherein an acoustic generating component is attached to the device to create an acoustic standing wave within the input channel to align the entity with a detection region of the optical detector within the input channel.

16. The magnetic field is Permanent magnets; soft magnetic pole pieces that connect to the permanent magnet; electromagnet The device of claim 1 , wherein the voltage is applied by either

17. 1. An apparatus for separating biological entities in a fluid sample, comprising: an input channel having an outlet; a path selector having a first path and a second path; a first exhaust channel having a first inlet; a second exhaust channel having a second inlet; an optical detector disposed around the input channel; an actuator having a voice coil and connected to the path selector; a magnetic field exhibiting opposite magnetic polarity applied across the voice coil; Including, the input channel, the first output channel, and the second output channel are contained within a base; the fluid sample is passed through the input channel and the optical detector detects the first entity from the fluid sample before the first entity passes through the outlet; an apparatus wherein a current is applied to the voice coil and activated to move the actuator to a first sorting position, and the first entity passes sequentially through the outlet, the first path, the first inlet, and into the first discharge channel.

18. 18. The apparatus of claim 17, wherein the optical detector detects the second entity from the fluid sample before the second entity passes through the outlet; and the current applied to the voice coil changes polarity and operates to move the actuator to a second sorting position, causing the second entity to pass sequentially through the outlet, the second path, the second inlet, and into the second discharge channel.

Images