Patent Application
20140248679
None
The present invention relates to apparatus and methods for rare cell separation and biological analyses using biological samples, and more specifically, apparatus and methods to enhance effectiveness of rare cell separation using magnetic fields and magnetic nano-particles (MNPs).
Rare cell separation is of utmost importance in experimental biology and medicine. The rapid development and progress in cell separation methods have been driven by both academic interest and practical clinical needs, catalyzed by ever increasing demands for sensitivity and selectivity with this process.
Some diseases are related to specific type of cells (or target cells) which can be used as a biomarker for diagnosing and monitoring the progression of a particular disease. For example, circulating tumor cells (CTCs) can be present in blood stream of carcinoma (or cancer) patients. Detection of CTCs is of profound clinical and scientific interest. Metastasis, the process of carcinoma tumor cells spreading and growth from a primary carcinoma tumor site to a distant secondary site, is a significant problem in carcinoma research and treatment. Recently, it has been found that CTCs are present in patients with various metastatic carcinomas. It is also beneficial for evaluation of a patient's response to therapy, especially for those patients with metastatic carcinoma. The number of CTCs in the blood has been shown to correspond to the clinical course of the disease and is a predictor of patient's overall survival rate. Some clinical studies have demonstrated correlation between CTCs counts and progression of disease for certain types of carcinoma, such as metastatic breast cancer, colorectal cancer, and prostate cancer. Better understanding of CTCs properties is expected to revolutionize carcinoma treatment. There is therefore a strong need to develop technologies to enable biological studies with rare cells such as CTCs.
However, understanding of CTCs properties is limited in both clinical and medical research since CTCs are rare cells which are difficult to be separated from their host samples. Cell separation devices are helpful and, in many cases, even mandatory for advance in related areas, including, but not limited to, life science, pharmaceutical research, and medical practice. Separated cells, after enrichment, isolation, and purification, can be used in various subsequent downstream tests and measurements, including, but not limited to, cell counts, analysis of DNA mutation and RNA/protein expression even on single cell level, study of tumor formation mechanism and metastatic processes, detection and monitoring of various diseases, performing pathology analyses, authenticating a person's identity, and classifying animal species.
Tremendous efforts have been made in developing methods to capture and separate rare biological cells in a complex suspension of cells. One of the most commonly used methods is magnetic cell separation, also known as immunomagnetic cell enrichment, where magnetic nano-particles (MNPs), also referred to as magnetic beads, are used to separate target cells from various specimen including blood, tumor tissues, biopsies, or bone marrow, etc. Such methods rely on separation of target cells by specific antibodies conjugated to MNPs which are mixed with specimen. Target cells are captured in the presence of an applied magnetic field. Captured cells are then cleaned by washing them in certain liquid solution to remove non-specific cells and unwanted materials. Finally, target cells are released or separated in order to be collected for further downstream analysis as aforementioned.
An important advantage of magnetic cell separation over other affinity-based separation methods is its capability to collect magnetically-tagged target cells and to control their movement within fluidic systems. This capability provides tremendous flexibility in downstream sub-cellular level analysis and utilization of cells for specific applications.
Magnetic nanoparticles (MNPs) are a valuable class of nano-materials with unique properties which are distinct from those of their bulk counterparts. In general, when MNPs size or diameter descends from micron range to tens of nm range, depending on specific material composition, the magnetic state migrates from multi-domain state, through single-domain state, and eventually to the super-paramagnetism regime.
One class of MNPs is of particular interest. The total magnetic energy barrier is proportional to the product of the volume of MNPs and the magnetic anisotropy (or stiffness). When the magnetic energy barrier of MNPs is on the same order of magnitude as the thermal energy, MNPs become super-paramagnetic. An extremely important and useful characteristic of super-paramagnetic MNPs is that they exhibit maximum magnetic moment in the presence of a small applied magnetic field. Upon removal of the field, they tend to exhibit zero net magnetic moment over time since their magnetic moment can be constantly disturbed and randomly flipped due to thermal energy. In this condition, MNPs exhibit low coercivity, that is, low resistance to magnetic moment reversal. This unique property makes MNPs of small size (diameter in tens of nanometers) highly manipulatable with an applied magnetic field. Zero net magnetic moment over time in the absence of external magnetic field also offers the advantage of reduced risk of particle aggregation [1].
In the case that size of MNPs is above the limit of super-paramagnetism regime, MNPs can exhibit either single-domain or multi-domain state in the absence of applied magnetic fields, depending on the particle size and magnetic property of the material. To be specific, when the particle size is larger than the characteristic exchange length of the material, MNPs exhibit multi-domain state, in which overall magneto-static energy is minimized. In another word, different portions of MNPs carry magnetic moment in different directions. On the other hand, when the particle size is smaller than the characteristic exchange length of the material, MNPs exhibit single-domain state, that is, all portions of MNPs carry magnetic moment in the same direction.
In the presence of applied magnetic field, MNPs originally in the multi-domain state can transform into the single-domain state with magnetic moment aligned with the magnetic field direction, and MNPs originally in the single-domain state reorient their magnetic moment along the field direction. In either case, the magnetic field strength is required to be large enough to overcome energy barrier associated with either demagnetization field or intrinsic magnetic stiffness of the material. Therefore, sufficient magnetic field strength is required for successful magnetic cell separation.
The magnetic force exerted on MNPs directly contributes to effectiveness of magnetic cell separation and is governed by the following equation,
F∝V*M*∇H (1)
where V is the active volume of MNPs (excluding non-magnetic materials enclosing MNPs), and M is the magnetization of the magnetic materials, or net magnetic moment per unit volume. Therefore, V*M actually denotes total magnetic moment of MNPs. ∇H is the gradient of the magnetic field, that is, the rate of magnetic field strength change versus linear spacing from the source of the magnetic field. It should be noted that once M is saturated with sufficient magnetic field strength H, further increasing H no longer contributes to increase of magnetic force on MNPs. Instead, field gradient ∇H becomes the dominant term to enhance magnetic force.
A well-designed magnetic cell separation apparatus should be flexible to work with MNPs of various sizes and material properties in order to accommodate a wide range of applications. Depending on MNPs size and material properties, apparatus to generate magnetic field for magnetic cell separation should have either sufficient magnetic field gradient to maximize magnetic force in the case of MNPs exhibiting super-magnetism or single-domain behaviors, or optimized magnetic field strength to ensure complete saturation of MNPs otherwise. It should be emphasized that, for MNPs exhibiting super-magnetism or single-domain behaviors, field gradient plays a more critical role than magnetic field strength itself and should be maximized to enhance effectiveness of magnetic cell separation, as indicated by Equation (1).
One main advantage of small size MNPs is their large effective surface areas for easy bonding to target cells and low sedimentation rates with enhanced stability [2]. Another advantage of small MNPs is that the magnetic dipole-dipole interaction between MNPs is significantly reduced since it scales as inverse of r̂6, where r is the radius of MNPs [3].
MNPs can have size ranging from nanometers up to microns, such that their dimension is smaller than or comparable to those of biological entities of interest, such as cells (10-100 micron), virus (20±500 nm), proteins (5-50 nm), or even genes (2 nm wide and 10±100 nm long). In the specific case of cells, such as CTCs, multiple MNPs can be bonded to the target cell surface. Total magnetic force exerted on multiple MNPs, and therefore total attraction force on targets cells for effective separation, can be maximized. In addition, smaller MNPs size also minimizes disturbance or even damage to target cells and simplifies downstream analyses including cell identification.
Tremendous efforts have been made in developing methods to capture and separate rare biological cells. As one of the most commonly used methods for cell separation and purification, magnetic cell separation relies on the tagging, or labeling, of a specific cell population in a heterogeneous mixture with magnetic nano-particles (MNPs). These magnetic tags can be tailored to target specific antigens, enabling magnetic capture of these entities with an applied magnetic field. This technique can be used to separate and enrich target cells from various samples, including blood, tumor tissues, biopsies, or bone marrow etc., for further downstream analysis.
As one specific example, CTCs are extremely difficult to detect using conventional blood analysis methods and have been difficult to separate until recently. Using advanced commercial magnetic cell separation equipment such as CellSearch system (by Johnson & Johnson), it was shown that CTCs are consistently present in the blood stream of carcinoma patients. Scientists further demonstrated that CTCs have significant clinical values as prognostic markers using the CellSearch system [4]. However, detection of CTCs still has not been adopted in routine clinical practice by American Society of Clinical Oncology (ASCO) guideline since greater sensitivity is needed to detect CTCs and more downstream analysis of CTCs are required to better understand their properties.
In addition to CellSearch system, there are additional magnetic enrichment technologies being developed. For example, techniques described in U.S. Pat. No. 3,970,518, by Giaever et al, entitled “Magnetic Separation of Biological Particles”, U.S. Pat. No. 5,200,084, by Liberti et al, entitled “Apparatus and Methods for Magnetic Separation”, U.S. Pat. No. 5,837,144, by Bienhaus et al, entitled “Method of Magnetically Separating Liquid Components”, U.S. Pat. No. 8,071,395, by Davis, entitled “Method and Apparatus for Magnetic Separation of Cells”, and techniques utilized by various commercial products including AutoMACS separation (Miltenyi Biotec), Ariol (Microsystems), RoboSep (StemCell), and MagSweeper [5]. Using these technologies and products, EpCAM or Cytokeratin positive cells can be enriched and thereafter detected.
Despite progress in method and apparatus for rare cell separation, many problems remain unsolved. Firstly, all aforementioned methods employ permanent magnet to generate magnetic field. Due to the nature of permanent magnets, the magnetic field generated is fixed. One problem with permanent magnets as magnetic field source is, therefore, that the magnetic field is not adjustable without physically changing the spacing between magnets and specimen in study. In the specific case of MagSweeper system, it is required to alternatively attach and detach the capture probe cap from the permanent magnet probe, in order to change the magnetic field strength generated by the magnetic probe for capturing and releasing target cells tagged with MNPs. This requirement complicates cell separation operation, severely limits the effectiveness of target cell collection, and compromises the sensitivity of cell separation devices. As a result, this greatly hinders wide-spread adoption and further development of advanced cell separation techniques.
Some approaches attempt to improve sensitivity by repeating capturing steps without releasing captured target cells. However, capturing efficiency is only marginally improved by this approach, and there is also concern with target cell damage. Other approaches attempt to improve cell capture sensitivity by replacing the capture probe cap enclosing the magnetic probe after each round of capture, wash, and release cycle. However, such procedure is cumbersome and time consuming with compromised operation efficiency.
In addition, it should also be pointed out that the saturation magnetization of the permanent magnet, which determines the maximal achievable magnetic field and field gradient, is usually compromised due to stringent requirements for coercivity, a measure of resistance to magnetization reversal. Since extremely high coercivity is required for permanent magnets, doping of non-magnetic materials is required, overall magnetization is therefore diluted.
In order to conveniently control magnetic field strength and direction without physically changing spacing and position between magnets and specimen in study, electromagnets can be used in lieu of permanent magnets. In this case, magnetic field is generated with driving coils winding around magnetic cores, which are in general made of soft magnetic materials, that is, magnetic moment of which can be easily reversed by applied magnetic field. Magnetic field strength and direction can be conveniently controlled by electrical current magnitude and direction. It is also flexible to control magnetic field by adjusting electrical current over time.
To precisely control magnetic field profile, Carpino et al. reported a Quadrupole electromagnet structure which consists of four separate electromagnets with spherically-shaped pole tips and was used for field-flow fractionation (FFF) as an analytical separation and characterization technique for macromolecules and particles [6]. The Quadrupole electromagnet generates a unique field profile with radially symmetric magnetic field which linearly increases with radial distance from the center axis, while the field gradient is constant within the aperture between pole tips. The channel for specimen flow occupies a thin annular space within the aperture, thereby avoiding the center axial region where magnetic field strength is small.
Another benefit of the electromagnet is that the magnetic field can be conveniently controlled over time with user-defined ramp-up and ramp-down rate to facilitate customized characterization. This feature could not be conveniently realized with permanent magnets with fixed magnetic field.
However, there is limitation with aforementioned electromagnet system. First of all, four pieces of electromagnets are required. This makes magnetic cell separation system bulky with increased power consumption and cost. In addition, while the system provides benefit of fixed gradient, the magnetic field strength in the center axis region is low and increases linearly with radius. Since sufficient magnetic field is required to make MNPs magnetically saturated, this limits the usable space for cell separation operation between magnetic pole pieces.
According to Equation (1), the magnetic force is proportional to both M and ∇H, where M is the magnetization of MNPs and increases with applied magnetic field. Until magnetization reaches saturation, higher magnetic field strength is needed for increased magnetic attraction force and effective separation. However, once MNPs are magnetically saturated, M no longer increases with increasing magnetic field, and magnetic attraction force is only dependent on the field gradient ∇H. Therefore, an effective magnetic separation apparatus requires sufficient magnetic field strength for saturation of MNPs magnetization, and more importantly, maximum field gradient after magnetic saturation of MNPs. In the specific case of small MNPs approaching super-paramagnetism regime, the field gradient requirement is dominant while the magnetic field strength requirement can be relaxed due to aforementioned reasons. In the case of MNPs with size above super-paramagnetism limit, the magnetic field strength must be at least sufficient to magnetically saturate MNPs. Once meeting minimal magnetic field strength requirement, field gradient then needs to be maximized.
Therefore, what is needed is apparatus and method to effectively manipulate and enhance magnetic field strength as well as magnetic field gradient. Furthermore, the apparatus and method must work effectively with a wide range of MNPs types with different sizes and magnetic properties. To be specific, the applicable range of the apparatus and method must encompass MNPs sizes in super-magnetism regime and beyond without compromising effectiveness of magnetic cell separation.
To overcome the limitations in the prior art described above, and to overcome other limitations which will become apparent upon reading and understanding the present specification, the present invention discloses methods and corresponding magnet designs to enhance magnetic field gradient for effective magnetic cell separation. Various embodiments of the present invention are described herein.
One preferred embodiment in accordance with the present invention is in the form of an electromagnet with one magnetic core with an open gap formed between pole tip surfaces and two separate driving coils. Electrical current magnitude and direction through two coils can be independently controlled. In the specific case when two magnetic pole tips emanate magnetic fields, in opposite direction and preferably with different magnitude, the field gradient, and therefore the magnetic force exerted on MNPs, is advantageously enhanced. The magnetic core can further comprise a removable section such that the electromagnet is reconfigurable.
In another embodiment, an electromagnet can be combined with a permanent magnet. The pole tip of the electromagnet and the pole tip of the permanent magnet form an open gap. The pole tip of the electromagnet and the permanent magnet emanates magnetic fields, in opposite direction and preferably with different magnitude, resulting in advantageously enhanced magnetic field gradient.
In yet another embodiment, the magnetic field can be generated by two separate pieces of permanent magnets. The pole tip of the first permanent magnet and that of the second permanent magnet form an open gap. The pole tip of the first permanent magnet and the pole tip of the second permanent magnet both emanate magnetic field, in opposite direction and preferably with different magnitude, resulting in advantageously enhanced magnetic field gradient.
These and other features and advantages of the invention will be apparent upon reading of the following detailed description of preferred embodiments taken in conjunction with the Figures in which like reference numerals indicate like elements throughout.
In the following description of the embodiments, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration of the specific embodiments in which the invention may be practiced. It should be noted that the figures discussed herein are not drawn to scale and thicknesses of lines are not indicative of actual sizes. It is to be understood that other embodiments may be utilized since structural changes may be made without departing from the scope of the present invention.
Furthermore, in the following detailed description of embodiments of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be recognized by one of ordinary skill in the art that embodiments of the present invention may be practiced without these specific details. In other instances, well known methods, procedures, and components have not been described in detail as not to unnecessarily obscure aspects of the present invention.
The discussion will begin with description and overview of magnetic nano-particles (MNPs). Magnetic nanoparticles (MNPs) possess unique properties which are distinct from those of their bulk counterparts. When MNPs size or diameter descends from the micron range to the tens of nm range, depending upon specific material properties, the magnetic state of MNPs migrates from multi-domain state, through single-domain regime, and eventually to the super-paramagnetic state.
A typical M-H loop of MNPs, that is, magnetization vs. applied magnetic field curve, is shown in
In the case that size of MNPs is larger than the limit of super-paramagnetism regime, MNPs can exhibit either single-domain or multi-domain state in the absence of applied magnetic field, depending on the size of MNPs and the magnetic properties. To be specific, when the particle size is larger than characteristic exchange length of the material, MNPs exhibit multi-domain state 101, in which overall magnetic energy is minimized. In another word, different portion of MNPs can carry magnetic moment in different directions. When the particle size is smaller than characteristic exchange length of the material, MNPs exhibit single-domain state. In the presence of applied magnetic field, MNPs originally in multi-domain states transform into single-domain states with magnetic moment aligned with the field direction, while MNPs originally in single-domain states reorient their magnetic moment along the applied field direction. In either case, the magnetic field strength is required to be large enough to overcome energy barrier associated with either demagnetization field or intrinsic magnetic stiffness. In general, the higher the magnetic moment, the higher the applied magnetic field is required. For example, if MNPs saturation magnetization Ms is 500 emu/cc, or 6,300 Guass (4π*500) in SI units, the field required to saturate MNPs is on the order of 6,300 Oe (Orested).
According to Equation (1), the magnetic attraction force on MNPs is proportional to both magnetization M of MNPs and magnetic field gradient ∇H. One important implication of
However, there is limitation with aforementioned electromagnet apparatus. First of all, four pieces of electromagnets are required and make magnetic cell separation system bulky with increased power consumption and cost. In addition, while the system provides benefit of fixed gradient, the magnetic field in the center axis area is low. Since certain magnetic field strength, depending on MNPs size and magnetic properties, is required to magnetically saturate MNPs, this confines the usable space between magnetic pole pieces.
Before embodiments in accordance with the present invention are described, and for the purpose of comparison, a reference electromagnet structure, as shown in
The magnetic field profile along center axis 207 is calculated and plotted in
The shape of the magnetic core 340 along the magnetization direction 361 and 362 is preferably C-shaped for Embodiment #1. The cross-section shape of the magnetic core 320 is preferably square, rectangular, or circular.
Specific material properties of the magnetic core 320 are required for practical electromagnet applications. Such requirements include high permeability μ and high saturation magnetization Ms. Permeability (mu) is a measure of easiness for magnetic materials to be magnetized in an applied magnetic field and is defined as the ratio between magnetization of the magnetic core vs. the applied magnetic field. Materials with high permeability are usually referred to as soft magnetic materials. High permeability is necessary to reduce electrical current needed to induce magnetization in the magnetic core and therefore effectively generate large magnetic field for a given current. Before pole tips reach magnetic saturation, the magnetic field strength H along center axis of pole tips 361 and 362 can be expressed as
H=μ*(N1*I1+N2*I2)/L (2)
where N1 and N2 are the number of turns of coils 311 and 312, respectively. I1 and I2 are the magnitude of the electrical current 351 and 352 following through coils 311 and 312, respectively. L is the length of the open gap 340 along the direction of induced magnetization 361/362, or equivalently the distance between surfaces of pole tips 331/332. It should be noted that once pole tips of the magnetic core reach saturation, the maximum magnetic field no longer increases with further increased driving current and is limited by saturation magnetization Ms of pole tip materials. Therefore, higher saturation magnetization Ms is required for the magnetic core 320, and especially pole tips 361/362.
For aforementioned reasons, both high permeability and high saturation magnetization are required. Specifically, for effective practice of embodiment #1, the magnetic core 320 should have permeability greater than 500 and the pole tips 361/362 should have saturation magnetization Ms greater than 0.5 Tesla. Examples of soft magnetic materials which meet these requirements include, but are not limited to, Permalloy, Permendur, etc. For the purpose of reference, if Ms of the pole tip material is 0.5 Tesla, maximal achievable magnetic field strength at pole tip surfaces is 5,000 Oe with two pole tips forming an open gap.
Magnetic field strength and field gradient generated by the electromagnet Embodiment #1 in the operation mode (Mode 1) as illustrated in
The solid line in
While the operation mode (Mode 1) illustrated in
H=μ*(N1*I1−N2*I2)/L (3)
where N1 and N2 are the number of turns of coils 311 and 312, respectively. I1 and I2 are the magnitude of the electrical current 351′ and 352′ following through coils 311 and 312, respectively. L is the length of the open gap 340 along the direction of induced magnetization 361′/362′, or equivalently the distance between surfaces of pole tips 331/332. It should be noted that, compared to Equation (2), plus ‘+’ in parenthesis is replaced with minus ‘−’ due to opposing magnetic fields generated by said first and second electrical current.
It is apparent that the operation mode (Mode 2) shown in
In summary, with the same Embodiment #1 as shown in
To quantify the benefit with the present invention,
As stated earlier, the magnetic field is substantially perpendicular to the surface of pole tips 331 and 332 in the center region of the pole tip surfaces, while its direction starts to deviate from perpendicular at the edge of the pole tips. This fringing effect can be controlled by varying the ratio of the length of the open gap 340 versus the width of the pole tip surface 331/332. A smaller ratio leads to an increased usable center region with the field substantially perpendicular to pole tip surfaces and is desirable for magnetic cell separation, as long as there is sufficient physical space between pole tips for specimen in study.
In another embodiment (Embodiment #2), an electromagnet can be combined with a permanent magnet, using the hybrid configuration as illustrated in
In yet another embodiment (Embodiment #3), the magnetic field can be generated by two separate pieces of permanent magnets, as illustrated in
The operation mode of
The operation mode of
Magnetic field and field gradient can be further improved in addition to aforementioned embodiments. Since the magnetic field emanates from pole tips, the most effective approach for further improvement is to optimize configuration of pole tips and the open gap.
To avoid magnetic saturation of pole tips, the tapered sections 21′/22′ further consist of magnetic materials which preferably have higher saturation magnetization than that of the main magnetic cores 21/22. This allows main sections of magnetic cores 21/22 and tapered sections 21′/22′ to be optimized independently. Specifically, the tapered sections 21′/22′ require higher saturation magnetization to avoid magnetic saturation and degradation of magnetic field gradient, while the main magnetic cores 21/22 require higher permeability for efficient generation of magnetic flux. For example, Permendur has higher saturation magnetization, therefore higher flux carrying capability than that of Permalloy, and is preferred for pole tips.
With the advantage of enhanced magnetic field as well as field gradient, there are numerous methods to utilize preferred embodiments for magnetic cell separation. Several examples are described herein.
It should be noted that, while
Again, it should be noted that, while
It should be pointed out that the magnetic field profile generated by miniaturized embodiments is scale invariant due to the nature of magneto-static field. In another word, preferred embodiments with scaled-down dimension inherit the same field profile versus spacing from pole tip surface when normalized to dimension of pole tips. Miniaturized embodiments can be manufactured using nano-fabrication techniques and thin film processes which are widely adopted in semiconductor industry and hard disk drive (HDD) industry.
Although the present invention has been described in terms of specific embodiments, it is anticipated that alterations and modifications thereof will no doubt become apparent to those more skilled in the art. It is therefore intended that the following claims be interpreted as covering all such alterations and modification as fall within the true spirit and scope of the invention.
The present application is related to U.S. provisional patent application Ser. No. 61/771,802, filed on Mar. 2, 2013, by ZHANG et al., which is included by reference herein and for which benefit of the priority date is hereby claimed.
| Number | Date | Country | |
|---|---|---|---|
| 61771802 | Mar 2013 | US |
