JP4593672B2 - Magnetic separator and method thereof - Google Patents

Description

(Cross-reference to related applications)
This document claims priority to US Provisional Application No. 60 / 019,282 filed June 7, 1996 and US Provisional Application No. 60 / 030,436 filed November 5, 1996. No. 5,985,853, a continuation-in-part of US patent application Ser. No. 08 / 867,009 filed Jun. 2, 1997, Nov. 30, 1998. Is a continuation-in-part application of US application Ser. Priority is also claimed herein for US Provisional Application No. 60,110,280, filed November 30, 1998. Each of the above applications and patents are incorporated herein by reference.

  The present invention relates to an improved apparatus and method for performing qualitative and quantitative analysis of microscopic biological samples. The present invention more particularly relates to microscopic susceptibility to immunospecific or non-specific binding with magnetically reactive particles having a binding agent to generate magnetically labeled species in a fluid medium. The present invention relates to an apparatus and method for performing segregation, collection, restraint and / or analysis of a biological sample or substance. As used herein, terms such as “target entity” refer to biological samples or materials for research purposes that are susceptible to such magnetic labeling.

  US Pat. No. 5,985,853 utilizes an external magnetic gradient to attract a magnetically labeled target entity present in the collection chamber to one of its surfaces, An apparatus and method is described that utilizes gradients to accurately align these entities on the surface. To move a magnetically labeled biological entity to the collection surface, a perpendicular magnetic gradient is applied to move the magnetically labeled biological entity to the collection surface. The collection surface comprises a ferromagnetic collection structure such as a plurality of ferromagnetic lines supported on an optically transparent surface.

  This magnetically labeled biological entity is placed under the influence of a strong local gradient generated by the ferromagnetic collection structure once attracted sufficiently close to the surface by an externally applied gradient. On the other hand, it is restrained at a position adjacent in the lateral direction. This local gradient is desirably above the adhesive force that allows the biological entity to be retained on the surface after it has collided with the transparent surface.

In the alternative, the adhesion of this surface must be weak enough for the magnetically labeled biological entity to move toward the ferromagnetic structure by horizontal magnetic force. The smoothness of the surface as well as the hydrophobicity or hydrophilicity are factors that can affect the material chosen for the collection surface or the treatment to impart slipperiness to the surface.
In accordance with the present invention, further alternative embodiments and improvements to the collection chamber, the internal shape of the collection chamber, and further more useful techniques that can be accomplished by using a perpendicular magnetic gradient separation structure are described.

US Pat. No. 5,985,853

  The present invention relates to an improved apparatus and method for performing qualitative and quantitative analysis of microscopic biological samples.

  The isolation of microscopic biological samples or substances that are susceptible to immunospecific or non-specific binding to magnetically reactive particles with a binding agent to generate magnetically labeled species in a fluid medium; Apparatus and methods for performing collection, restraint and / or analysis are provided.

FIG. 1A is a schematic diagram of a magnetic separator. FIG. 1B is a diagram showing a magnetic field in the magnetic separator of FIG. 1A. It is a microscope picture of the sample collected in the magnetic separator. FIG. 5 is a plan view of an alternative ferromagnetic collection structure used in a magnetic separator. 1 is a schematic diagram of an optical tracking and detection mechanism for analyzing species collected in a magnetic separator. Figure 5 is a histogram of fluorescence signals obtained from the magnetic separator (5A) and flow cytometer (5B) used to quantify species in the same fluid sample. It is a microscope picture of the material collected in the magnetic separator. 2 is a continuous schematic showing a charge enhanced collection method in a magnetic separator. FIG. 2 is a cross-sectional view and a plan view, respectively, of a ferromagnetic and conductive composite collection structure for a magnetic separator. 2 is a continuous schematic diagram illustrating a method for separating particles within a magnetic separator. 2 is a continuous schematic diagram illustrating a method for measuring particle density in a fluid having an unknown particle density. FIG. 3 is a cross-sectional view of a separation vessel configured to perform multiple simultaneous analyzes of fluids that include multiple target species having different concentrations.

1. Vertical Gradient Collection and Target Entity Observation In a first embodiment of the invention, a target entity, such as a cell, is collected without subsequent alignment to the collection surface of the container adjacent to the ferromagnetic collection structure. The collection surface is oriented to be orthogonal to the magnetic gradient generated by the external magnet. In this embodiment, nanoparticles and magnetically labeled biological entities are collected in a substantially uniform distribution on an optically transparent surface, while unselected entities remain down in the fluid medium. .

  The result is that the chamber is placed in the gap between two magnets arranged as shown in FIG. 1A, so that the transparent collection surface of the chamber is effective against the perpendicular magnetic gradient generated by the external magnet 3. This is achieved by making them orthogonal. The magnet 3 has a thickness of 3 mm and is tapered toward a gap of 3 mm. The magnet 3 is held in a yoke 1 at the top of the housing 2.

  The container support 4 holds the container 6 in a region between magnets that are oriented so that the magnetic field lines are substantially perpendicular to the collection surface 5 of the container 6. The collecting surface of the container is preferably a polycarbonate member having a thickness of 0.1 mm. The collecting surface is parallel to the upper surface of the external magnet 3 and is 2 mm below this. The space between the inner edge of the magnet and the top surface edge is 3 mm.

  The taper angle of the magnet 3 and the gap width between the two magnets determine the strength of the applied magnetic field gradient and the desired location of the collection surface on the container. The magnetic field gradient generated by the magnet may be characterized by having a substantially uniform region in which the gradient magnetic field lines are substantially parallel and a peripheral region in which the gradient magnetic field lines diverge toward the magnet.

  FIG. 1B shows a mathematically approximated magnetic field gradient for such a magnet arrangement. The magnetic field lines (not shown) are overwhelmingly parallel to the chamber surface, while the gradient lines are overwhelmingly perpendicular to it. In order to collect a uniformly distributed layer of target entities, the container is positioned so that the transverse magnetic gradient element substantially places the chamber in a uniform region, thereby magnetically labeled biology The target entity is transferred laterally to the collection surface.

  To illustrate the collection pattern of magnetic material on the collection surface area, a chamber with internal dimensions of 2.5 mm height (z), 3 mm width (x) and 30 mm length (y) is 150 nm. Filled with 225 μl of solution containing diameter magnetic beads and placed between magnets as shown in FIG. 1A. The magnetic beads were distributed evenly by moving towards the collection surface. When the container is raised with respect to the magnet so that a significant portion of the top of the container is located in the peripheral region, a significant amount of magnetic particles will be in each lateral region of the collection surface closest to the magnet. Moved in parallel and accumulated there.

  In order to increase the uniformity of collection on the collection surface, the surface material may be selected or otherwise treated to adhesively attract the collected species. This adhesion consideration can eliminate the horizontal drift of collected species due to displacement of the positioning of the chamber or displacement from the desired orthogonal magnetic gradient in the “substantially uniform” region. is there.

  In the example of the device studied using this embodiment, a blood cancer test is used. Tumor-induced epithelialized cells are detectable in peripheral blood. Although it is low density, 1 to 1000 cells in 10 ml of blood can be detected and quantitatively analyzed from a peripheral blood sample using a ferrofluid for each anti-epithelialized cell.

  FIG. 3 shows an example in which peripheral blood-selected epithelialization-induced tumor cells are localized, distinguished, and enumerated using chambers and magnets that do not have ferromagnetic structures on the collection surface. In this example, 5 ml of blood was cultured for 15 minutes using 35 μg of epitheliogenic ferrofluid (EPCAM-FF, Imunicon, Huntingdon Valley, Pa.). The sample was placed in a quadrupole magnetic separator (Immunicon QMS17) for 10 minutes and the blood was discarded.

  The container is removed from the separator, and the collected cells on the wall of the separation container are resuspended in 3 ml of detergent-containing buffer to permeabilize the cells (Immunicon's Imuno Palm) for 10 minutes. Returned inside. The detergent-containing buffer was discarded, the container was removed from the separator, and the cells collected on the wall were labeled with the UV excitable nucleic acid dye DAPI (Molecular Probes) and the fluorescent dye Cy3. Resuspended in 200 μl of buffer containing cytokeratin monoclonal antibody (to identify epithelial cells).

  These cells were incubated for 15 minutes, after which the container was placed in a separator. After 5 minutes, the uncollected portion containing excess reagent was discarded, the container was removed from the separator, and the collected cells were resuspended in 200 μl of isotonic buffer. This solution was placed in a collection chamber and placed in the magnetic separator shown in FIG. 1A.

Cells labeled with ferrofluid and free ferrofluid particles immediately migrated to the collection surface and were evenly distributed along the surface as shown in FIG. 2A. The figure shows a representative area on the collection surface using transmitted light and a 20x objective lens. In FIG. 2B, the same magnetic field is shown, but in this case a filter cube is used for excitation and emission of Cy3. Two objects are identified by the numbers 1 and 2.

FIG. 2C shows the same magnetic field, but here the filter cube has been switched to one with an excitation / emission filter cube for DAPI. The objects at positions 1 and 2 are both colored with DAPI at positions 3 and 5 so that they are identified as epithelial forming cells. Additional non-epithelializing cells and other cellular element cells are distinguished by the DAPI colorant; an example is shown at number 4.

II. Ferromagnetic collection structure centered on cells To collect target entities in a spatial pattern, a ferromagnetic collection structure is equipped on the collection surface of the container to generate a strong local magnetic gradient , Accordingly, the target entity may be constrained by being adjacent to the structure in the lateral direction.

The various ferromagnetic structures described below can be manufactured by standard lithographic techniques using nickel (NI) or permalloy (nickel-iron alloy). The thickness of the deposited metal layer varies in the range of 10 to 1700 nm. The 10 nm structure is partially transparent. However, the restraining force of these thin structures is considerably inferior to that of structures in the thickness range of 200-700 nm.

  Although restraints and alignment of magnetically labeled biological entities occurred reliably enough, these medium thickness structures are easier to use with a collection surface that has little or no adhesion. It was. A collection structure with a thickness in the range of 200-1700 nm was effective in capturing magnetically labeled biological entities and surpassing surface adhesion.

3A-3I show various magnets for the ferromagnetic collection structure.
In FIG. 3B, the ferromagnetic collection structure comprises a nickel wire with a spacing comparable to the cell diameter (10 microns nominal). As the spacing between the wires shown in FIG. 3C decreases , the position of the cells relative to the wire edge becomes much more uniform. Almost all cells appear to be aligned at the center. However, some parts of each cell overlap and are obscure due to the nickel wire.

  Cells collected along the ferromagnetic collection structure can be detected by an automated optical tracking and detection system. This tracking / detection system shown in FIG. 4 uses a computer-controlled power stage that moves the magnet and chamber in the X and Y directions under a laser beam having an elliptical spot of 2-15 μm. The maximum speed of the table is 2 cm / second in the Y direction and 1 mm / second in the X direction. Using two cylindrical lenses (1) and (2) from a Sony compact disc player and a position-adjustable objective lens (3), a 635 nm laser diode (see plug 5) In 4), a 2 × 15 μm elliptical spot on the sample was made.

  The light beam reflected from the sample is projected onto the photomultiplier tube through the dichroic mirror (7), the spherical lens (8), the diaphragm (9), and the band pass filter (10). Quadrant photodiode reflected from wire through mirror (12), dichroic mirror (7), quarter wave plate (13), polarization beam splitter (14), cylindrical lens (15) and spherical lens (16) (11) Using the measurement of the difference in polarization direction of the light beam projected onto it, the position of the laser spot on the sample is determined, and the signal is fed back to the objective lens to allow any displacement of the ancestor value ( (See plug-in 17). A photodiode (18) was used at right angles to the sample to measure the light scattered from the irradiated event.

  The feedback mechanism of this tracking system was optimized to keep the laser beam in the same X and Z position relative to the line while scanning in the Y direction at a maximum speed of 1 cm / sec. At the end of the 2 cm long line, the objective lens position was polarized to the next line, which was repeated until all of the chamber lines were scanned.

  In order to evaluate the performance of the tracking and detection system and compare it with the performance of the flow cytometer, 6 μm polystyrene beads were created for four different amounts of fluorochrome Cy5 different from the ferrofluid. And joined. These beads were used at a concentration of 105 ml-1 in a chamber with a ferromagnetic collection structure of the type shown in FIG. 3C. This chamber was placed in a uniform gradient region between the two magnets and all beads were aligned between the lines. A tracking and detection system was used to measure the fluorescence signal obtained while scanning along the ferromagnetic wire.

  FIG. 5A shows a histogram of the fluorescence signal of the bead mixture. Four clearly resolved peaks distinguish the Cy5 absent, implicit, intermediate and explicitly labeled beads. A mixture consisting of the same beads was made and measured with a flow cytometer equipped with a 635 nm laser diode (FACScaibur, BDIS, San Jose, Calif.).

  A histogram of this fluorescence signal is shown in FIG. 5B, which shows that the four different populations can be distinguished, but the resolution is inferior to that obtained when the sample was measured with the magnetic flux cytometer of the present invention. It is clear. These results indicate that the matching by the system described in this document provides better sensitivity and accuracy in measuring fluorescent beads than with a flow cytometer.

In applications where it is desirable to measure biological entities that are significantly different in size at the same time, the collection structure can be configured to have a non-uniform shape to focus on cells of different sizes and other species. What is necessary is just to match.

  An example of such a structure is shown in FIG. 3D. One collection structure pattern was formed from one collection surface area with wires having a period of 10 μm and a spacing of 7 μm and another area having wires with a period of 25 μm and a spacing of 7 μm. This was used to collect both small platelets and large white blood cells from whole blood. Prior to collection, blood was incubated with ferrofluids specific to platelets and leukocytes, ie, ferrofluids labeled with monoclonal CD41 and ferrofluid labeled with monoclonal antibody CD45, respectively.

  Platelets and leukocytes align along the wire in their respective regions on the collection surface, as shown in FIG. 3D. Platelet measurement can be performed in a region where the space between the wires is small, and white blood cell measurement can be performed in a region where the space between the wires is large. By changing the width of the gap that occurs along the extension of the ferromagnetic structure, the collected cells of different sizes are aligned linearly along a common centerline.

  Many more collection structure patterns are possible within the scope of the present invention to capture cells of various sizes and center them in one sample. Four examples are shown in FIGS. 3E, 3F, 3G, 3H and 3I. FIG. 3E shows a wire spacing similar to that shown in FIG. 3C, but the wires have lateral protrusions formed along their entire length. In the case of the shape of FIG. 3E, two positions have been selected by the cell, ie right or left side of the protrusion as shown. In such a design, cells are positioned periodically on both axes on the collection plane.

  As shown in FIG. 3F, adding the “prong” edge shape of an asymmetric triangle instead of “bar” removes the minor (left-right) asymmetry observed in FIG. 3E. Adding a large asymmetric triangular “prong” edge shape as shown in FIG. 3G is also effective for cells of various sizes. A more acute triangle style is shown in FIG. 3H.

  FIG. 3I shows an array of isolated triangles spaced along one axis to fit the cell size. It is larger than the size of the spacing cells along the other axis, so that the cells are free to move towards a position between more closely spaced sides of the triangle.

  An example of utilizing a custom designed ferromagnetic structure on the collection side is a blood cancer test. Tumor-induced epithelial cells can be detected in peripheral blood, and can be quantitatively retrieved from peripheral blood using ferrofluids by anti-epithelializing cells. The physical appearance of tumor-derived epithelial forming cells ranges from a very uniform range of apoptotic cells with a size of 2-5 μm to tumor cell aggregation with a size of 100 μm or more. To accommodate this large range of sizes, a triangular ferromagnetic structure such as that shown schematically in FIG. 3G or 3H may be used.

  An example of positioning peripheral blood derived cancer cells is shown in FIG. In this example, 5 ml of blood was cultured in a ferrofluid (Immunicon, EPCAM-FF) for each epitheliogenic cell and treated in the same manner as described above. The final cell suspension was placed in a magnetic separator. The ferrofluid labeled cells and ground ferrofluid immediately migrated to the collection surface.

  FIG. 6A shows a collection surface using transmitted light and a 20 × objective lens. The ferromagnetic collection structure is 1, the open wide collection space is 2, the narrow collection space is 3, and the large object is 4. FIG. 6B shows the same region using ultraviolet excitation only here. This large object is actually a large cell, as confirmed by coloring with the nucleic acid dye indicator 5, and is well aligned. The tracking system shown in FIG. 4 was used to successfully scan along the ferromagnetic structure shown in FIGS. 3H and 6A.

III. Addressable ferromagnetic collection structure A ferromagnetic structure can be used to create a local high magnetic gradient using a ferromagnetic structure, in addition to acting as an electronic conductor and to generate a local electron field charge. Can also be applied. In addition, electronic conductors can be formed on the collection surface to manipulate the collected target entity electronically. The function of first moving a biological entity to a specific location and then optically analyzing is schematically illustrated in FIG. 7A. A general or local electronic charge can then be applied as shown in FIG. 7B, adding another dimension to the utility of the described system.

  In applications involving cells, RNA, proteins and DNA, it is known to apply local electronic charges usefully. A schematic diagram of one design of such a collection surface is shown in FIG.

For optimal control of the electronic charge, a specific pattern / layer of aluminum 1 is first deposited on an optically transparent substrate 4 so that the electrons for 5 of the individual ferromagnetic structures shown in FIG. 8B. It becomes a circuit. Nickel or by depositing the following layers of other ferromagnetic materials on the substrate 2 in FIG. 8A, to create a separate ferromagnetic structure shown in FIG. 8B. The insulating layer 3 can be obtained by vapor-depositing SiO 2 or other hypoviscous material.

  A magnetically labeled biological entity 7 is localized between the ferromagnetic structures. Next, by applying an electronic charge to improve the specificity of the immunospecific binding and changing the orientation of the captured biological entity according to its electronic polarity, or by applying an electronic charge to the conductor It is possible to modify the properties of an entity (eg “explode it”). Biological entities can be studied before and / or after application of electronic charge.

IV. Porous chamber surface to remove excess particles When a large initial volume of fluid sample is processed by magnetic separation and its volume is reduced, the concentration of magnetically labeled particles of nanometer size (<200 nm) increases proportionally To do. The collection surface in the chamber has a limited capacity to capture extra unbound magnetic particles, and these particles are an obstacle to the positioning and observation of magnetically labeled biological entities. It is.

  An arrangement for separating unbound extra magnetically labeled particles from magnetically labeled biological entities is shown in FIG. The collection chamber comprises an outer compartment 1 and an inner compartment 2. A fluid sample containing unbound magnetic particles 3 and magnetically labeled and unlabeled biological entities 4 is placed in the inner compartment 2.

  At least one surface of the inner chamber is porous, for example a filter membrane, whose pore size is in the range 0.5-2 μm. Nano-sized magnetic particles can pass through this pore, but larger magnetically labeled cells cannot. The opposite surface of the inner chamber 6 comprises a transparent surface with or without a ferromagnetic collection structure as described above.

  Once the inner chamber is filled with the fluid sample, the outer chamber is filled with buffer. Next, the container is placed between two magnets as shown in FIG. 9B. The chamber is positioned such that each lateral portion of the container extends into the peripheral magnetic gradient region.

  Unbound magnetic particles travel through the membrane (5) to the respective lateral region 8 of the outer chamber (1) due to the magnetic gradient. This motion is consistent with the magnetic gradient field line shown in FIG. 1B. The accumulation in the lateral direction of the particles is effectively assisted by the horizontal movement of these nanoparticles which first strike the surface and then slide along the slippery surface (7 ').

  Magnetically labeled biological entities such as cells also move according to the gradient line (9) until they reach the membrane, while non-magnetic biological entities settle to the bottom under the influence of gravity. . When separation of unbound particles is complete, the chamber is removed from the magnetic separator and inverted (10). The chamber is repositioned in a uniform gradient region, thereby optimizing the homogeneity of cell distribution at the collection surface, as shown in FIG. 9C.

  Magnetically labeled cells move towards the optically transparent surface (6) (indicated by number 11 in FIGS. 9B and 9C), while non-magnetic biological entities are membranes under the influence of gravity. Precipitate in (5). Free magnetic nanoparticles move in a vertical direction towards the surface 6. Free magnetic nanoparticles no longer exist on the observation path, so that magnetically labeled biological entities can be examined. The system described above is particularly suitable for applications where the target cell count is low so as to prevent membrane clogging.

V. Longitudinal variation in chamber height The height of the chamber in harmony with the concentration of the target entity determines the distribution of the target entity collected on the collection surface of the container as described above. To increase the range of surface collection densities that are acceptable for accurate counting and analysis, the chamber height can be varied to dilute or enrich the sample for analysis of samples where the concentration can vary significantly. It is sufficient to eliminate the need to

  FIG. 10A shows a cross-sectional view of the chamber in which the collection surface 1 and the six compartments have different heights. Target cells are randomly placed in the chamber. FIG. 10B shows the same cross section, but here the cells have moved to the collection surface due to the influence of the magnetic gradient. In the highest area of the chamber, the density of the cells is too high to be measured accurately, while in the deepest part of the chamber it is too few to accurately count the cells.

  To further illustrate this principle, a histogram of cell density shown along the collection surface is shown in FIG. 11C. Note that the number of cells in the highest density region is underestimated. The method described here increases the concentration range that can be measured accurately as compared with the cell number measurement conventionally used in hematology analyzers and flow cytometers.

VI. Different compartments in the chamber There can be different types of target entities with different concentrations in the sample. To allow multiple analyzes at the same time, the chamber may be created with multiple compartments. An example of such a chamber is shown in FIG. 11A. Due to the presence of the collection surface 1 and the two separate compartments 2 and 3 in such a chamber, different reagent collections can be used.

  If the region in the chamber is not separated by a wall as illustrated by number 4 in FIG. 11B, the reagents used will move all magnetically labeled cell types toward the top. As an example, for example, a ferrofluid specific to leukocytes and a ferrofluid specific to platelets may be used simultaneously.

  Since the density of platelets is much greater than the density of white blood cells, as in the arrangement shown in FIG. 3D, platelet measurements are performed in the shallow part of the chamber (which has a relatively small line spacing on the collection surface). The leukocyte measurement is performed in a deep region of the chamber, which has a relatively large line spacing on the collection surface.

  The terms and expressions used as explanation terms are not restrictive. The use of such terms and expressions is not intended to exclude the features illustrated and described or any equivalents thereof. Accordingly, it will be appreciated that various modifications are possible within the scope of the claimed invention.

Claims (6)

In a method of optically analyzing a microbiological sample suspended in a fluid medium, the method comprises:
Magnetically labeling the microbiological sample;
Including the fluid medium in a container having a chamber therein for receiving the fluid medium and having a transparent top member;
Positioning the container in a magnetic field having a substantially uniform region of a vertical magnetic gradient, thereby allowing a chamber to be placed in the uniform region;
Collecting a non-uniformly distributed layer of the magnetically labeled microbiological sample on the interior surface of the chamber bound by the transparent member;
Optically analyzing the microbiological sample while retaining the sample collected on the internal surface of the chamber coupled with the transparent member;
Only including,
Here, the step of positioning the container, the rear exit of steps including how to position the container in the gap between the pair of magnets each having a surface tapered facing the gap. Before SL optical analysis step includes the step of observing with a microscope the samples along the observation path extending vertically in said chamber and the magnet with each other between in the gap;
The method of claim 1.   The method of claim 1, comprising gluing the inner surface of the chamber coupled with the transparent member and the sample to prevent horizontal movement of the collected sample. In a method of collecting and observing a microbiological sample in a fluid medium, the method includes:
A step of attaching magnetically labeling the sample by contacting a plurality of magnetically labeled particles to the sample;
Placing the fluid medium in a container having a chamber with a transparent surface and a porous wall;
Applying a magnetic field gradient to the chamber to remove excess of the magnetically labeled particles through the porous wall of the chamber while holding the labeled sample;
Attracting the labeled sample towards the transparent wall and observing after removal of the excess particles;
Only including,
Here, the container, including the method to be subjected positioned in the gap between the pair of magnets each having a surface tapered facing the gap, infra. In a method of optically analyzing a microbiological sample suspended in a fluid medium, the method comprises:
Magnetically labeling the microbiological sample;
Including the fluid medium in a container having a chamber for receiving the fluid medium, a transparent top member, and a chamber having two collection regions of different heights;
Placing the container in a magnetic field having a substantially uniform region with a vertical magnetic gradient, thereby allowing the chamber to be placed in the uniform region;
Collecting a magnetically labeled microbiological sample on a respective region of the inner surface of the transparent top member corresponding to the collection region of the chamber;
Only including,
Wherein the container is positioned in a gap between a pair of magnets each having a tapered surface facing the later gap .   The method of claim 5, comprising providing a barrier between the collection areas of the chamber.

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