US 6890426 B2
Apparatuses and methods for separating, immobilizing, and quantifying biological substances from within a fluid medium. Biological substances are observed by employing a vessel (6) having a chamber therein, the vessel comprising a transparent collection wall (5). A high internal gradient magnetic capture structure may be on the transparent collection wall (5), magnets (3) create an externally-applied force for transporting magnetically responsive material toward the transparent collection wall (5). The magnetic capture structure comprises a plurality of ferromagnetic members and has a uniform or non-uniform spacing between adjacent members. There may be electrical conductor means supported on the transparent collection wall (5) for enabling electrical manipulation of the biological substances. The chamber has one compartment or a plurality of compartments with differing heights. The chamber may include a porous wall. The invention is also useful in conducting quantitative analysis and sample preparation in conjunction with automated cell enumeration techniques.
1. An apparatus for observing magnetically responsive microscopic entities suspended in a fluid member, comprising:
a. a vessel having a transparent wall and a chamber formed therein for containing the fluid medium;
b. a ferromagnetic capture structure supported on the interior surface of the transparent wall;
c. magnetic means for inducing an internal magnetic gradient in the vicinity of the ferromagnetic capture structure, whereby the magnetically responsive entities are immobilized along the wall adjacent to the capture structure; and
d. electrical conductor means supported on the transparent wall for enabling electrical manipulation of the immobilized entities.
This is a divisional of Ser. No. 10/602,979, filed on Jun. 24, 2003, now allowed, which is a division of Ser. No. 09/856,672, filed on May 24, 2001, now U.S. Pat. No. 6,660,159, which is a 371 of PCT/US99/28231, filed on Nov. 30, 1999, which is a continuation-in-part of U.S. application Ser. No. 09/201,603, filed Nov. 30, 1998, now U.S. Pat. No. 6,136,182 which is a continuation-in-part of U.S. application Ser. No. 08/867,009, filed Jun. 2, 1997, now U.S. Pat. No. 5,985,853, which claims the benefit of U.S. Provisional Application No. 60/019,282, filed Jun. 7, 1996, and claims the benefit of U.S. Provisional Application No. 60/030,436, filed Nov. 5, 1996. Application Ser. No. 09/856,672, now allowed, U.S. Pat. No. 6,136,182 and U.S. Pat. No. 5,985,853 are all incorporated in full by reference herein.
The present invention relates to improved apparatus and methods for performing qualitative and quantitative analysis of microscopic biological specimens. In particular, the invention relates to such apparatus and methods for isolating, collecting, immobilizing, and/or analyzing microscopic biological specimens or substances which are susceptible to immunospecific or non-specific binding with magnetic-responsive particles having a binding agent for producing magnetically-labeled species within a fluid medium. As used herein, terms such as “target entity” shall refer to such biological specimens or substances of investigational interest which are susceptible to such magnetic labeling.
U.S. Pat. No. 5,985,853 describes an apparatus and method wherein an external magnetic gradient is employed to attract magnetically labeled target entities present in a collection chamber to one of its surfaces, and where an internal magnetic gradient is employed to obtain precise alignment of those entities on that surface. The movement of magnetically labeled biological entities to the collection surface is obtained by applying a vertical magnetic gradient to move the magnetically labeled biological entities to the collection surface. The collection surface is provided with a ferromagnetic collection structure, such as plurality of ferromagnetic lines supported on an optically transparent surface.
Once the magnetically labeled biological entities are pulled sufficiently close to the surface by the externally applied gradient, they come under the influence of an intense local gradient produced by the ferromagnetic collection structure and are immobilized at positions laterally adjacent thereto. The local gradient preferably exceeds adhesion forces which can hold the biological entities to the transparent surface after they collide with the surface. Alternatively, the adhesiveness of the surface must be sufficiently weak to allow the horizontal magnetic force to move the magnetically labeled biological entities towards the ferromagnetic structures. The smoothness and the hydrophobic or hydrophilic nature of the surface are factors that can influence the material chosen for the collection surface or the treatment of this surface to obtain a slippery surface.
In accordance with the present invention, there are described further alternative embodiments and improvements for the collection chamber, the interior geometry of the collection chamber, and further useful techniques that may be accomplished by use of a vertical magnetic gradient separator structure.
I. Vertical Gradient Collection and Observation of Target Entities
In a first embodiment of the invention, target entities such as cells are collected against a collection surface of a vessel without subsequent alignment adjacent to a ferromagnetic collection structure. The collection surface is oriented perpendicular to a magnetic field gradient produced by external magnets. In this embodiment, magnetic nanoparticles and magnetically labeled biological entities are collected in a substantially homogeneous distribution on an optically transparent surface while non-selected entities remain below in the fluid medium. This result can be accomplished by placing a chamber in a gap between two magnets arranged as shown in
The taper angle of the magnets 3 and the width of the gap between the two magnets determine the magnitude of the applied magnetic field gradient and the preferable position of the collection surface of the vessel. The field gradient produced by the magnets can be characterized as having a substantially uniform region, wherein the gradient field lines are substantially parallel, and fringing regions, wherein the gradient field lines diverge toward the magnets.
To illustrate the collection pattern of magnetic material on the collection surface area, a chamber with inner dimensions of 2.5 mm height (z), 3 mm width (x) and 30 mm length (y) was filled with 225 μl of a solution containing 150 nm diameter magnetic beads and placed in between the magnets as illustrated in FIG. 1A. The magnetic beads moved to the collection surface and were distributed evenly. When the vessel was elevated relative to the magnets, such that a significant portion of the top of the vessel was positioned in a fringing region, significant quantities of the magnetic particles parallel toward and accumulated at respective lateral areas of the collection surface positioned nearest the magnets.
In order to enhance uniformity of collection on the collection surface, the surface material can be selected or otherwise treated to have an adhesive attraction for the collected species. In such an adhesive arrangement, horizontal drifting of the collected species due to any deviations in positioning the chamber of deviations from the desired perpendicular magnetic gradients in the “substantially uniform” region can be eliminated.
An example of the use of the present embodiment discussed device is a blood cancer test. Tumor derived epithelial cells can be detected in the peripheral blood. Although present at low densities, 1-1000 cells per 10 ml of blood, the cells can be retrieved and quantitatively analyzed from a sample of peripheral blood using an anti-epithelial cell specific ferrofluid.
II. Ferromagnetic Collection Structures Producing Central Alignment of Cells
To provide for spatially patterned collection of target entities, a ferromagnetic collection structure can be provided on the collection surface of the vessel, in order to produce an intense local magnetic gradient for immobilizing the target entities laterally adjacent to the structures. The various ferromagnetic structures described below have been made by standard lithographic techniques using Nickel (Ni) or Permalloy (Ni—Fe alloy). The thickness of the evaporated metal layers was varied between 10 nm to 1700 nm. The 10 nm structures were partially transparent. The immobilizing force of these thin structures was, however, considerably less than those in the 200-700 nm thickness range. Although immobilization and alignment of magnetically labeled biological entities occurred sufficiently reliably, use of these moderately thicker structures was facilitated by a collection surface which had no or little adhesive force. Collection structures thicknesses between 200 and 1700 nm were effective in capturing the magnetically labeled biological entities and overcoming the surface adhesion.
Cells collected along the ferromagnetic collection structures can be detected by an automated optical tracking and detection system. The tracking and detection system, shown in
To evaluate the performance of the tracking and detection system and compare it to that of a flow cytometer, 6 μm polystyrene beads were prepared which were conjugated to ferrofluid as well as to four different amounts of the fluorochrome Cy5. The beads were used at a concentration of 105 ml−1 placed into a chamber with ferromagnetic collection structures of the type illustrated in FIG. 3C. The chamber was placed in the uniform gradient region between the two magnets and all beads aligned between the lines. The tracking and detection system was used to measure the fluorescence signals obtained while scanning along the ferromagnetic wires.
In applications where it is desired to simultaneously measure biological entities with significant differences in size, the collection structure can be configured to have a non-uniform geometry in order to centrally-align cells or other species of differing sizes. An example of such a structure is shown in
Many more collection structure patterns are possible within the scope of the invention for capturing and centrally aligning cells of varying sizes in a single sample. Four examples are illustrated in
An example of the utilization of custom designed ferromagnetic structure on the collection surface is a blood cancer test. Tumor derived epithelial cells can be detected in the peripheral blood and can be retrieved quantitatively from peripheral blood using anti-epithelial cell specific ferrofluids. The physical appearance of the tumor derived epithelial cells is extremely heterogeneous ranging from 2-5 μm size apoptotic cells to tumor cell clumps of 100 μm size or more. To accommodate this large range of sizes, triangular shaped ferromagnetic structures as schematically illustrated in
III. Addressable Ferromagnetic Collection Structures
In addition to using ferromagnetic structures to create high local magnetic gradients, they also can serve as electronic conductors to apply local electronic field charges. Furthermore, electronic conductors can be formed on the collection surface to allow electronic manipulation of the collected target entities. The ability to first move biological entities to a specific location followed by an optical analysis is schematically illustrated in FIG. 7A. Subsequent application of general or localized electronic charges, shown in
IV. Porous Chamber Surfaces for Excess Particle Removal
When large initial volumes of fluid samples are processed and reduced to smaller volumes by magnetic separation, the concentration of the nanometer sized (<200 nm) magnetic labeling particles increases proportionally. The collection surface in the chambers has a limited capacity for capturing unbound excess magnetic particles, and these particles may interfere with the positioning and observation of the magnetically labeled biological entities. An arrangement for separating unbound excess magnetic labeling particles form the magnetic labeled biological entities is illustrated in FIG. 9. The collection chamber comprises an outer compartment 1 and an inner compartment 2. The fluid sample containing unbound magnetic particles 3 and magnetically labeled and non-labeled biological entities 4 is placed in the inner compartment 2. At least one surface 5 of the inner chamber is porous, for example, a filter membrane having a pore size between 0.5 and 2 μm. Magnetic nanoparticles can pass through the pores, but the larger magnetically labeled cells cannot. The opposite surface of the inner chamber 6 consists of a transparent surface with or without ferromagnetic collection structures as described above.
After the inner chamber is filled with the fluid sample, the outer chamber is filled with a buffer. The vessel is then placed between the two magnets as shown in FIG. 9B. The chamber is positioned so that respective lateral portions of the vessel extend into the fringing magnetic gradient region. The unbound magnetic particles are transported by the magnetic gradient through the membrane (5) and toward respective lateral regions 8 of the outer chamber (1). This movement is consistent with the magnetic gradient field lines shown in FIG. 1B. The lateral accumulation of the particles is effectively aided by the horizontal movement of those nanoparticles which first hit the surface and then slide along the slippery surface (7).
Magnetically labeled biological entities such as cells also move according to the gradient lines (9) until they reach the membrane, whereas non magnetic biological entities settle to the bottom under the influence of gravity. After the separation of unbound particles is complete, the chamber is taken out of the magnetic separator and inverted (10). The chamber is repositioned in the uniform gradient region to optimize the homogeneity of the distribution of the cells at the collection surface, FIG. 9C. The magnetically labeled cells move towards the optically transparent surface (6) (indicated with 11 in
V. Longitudinal Variation of Chamber Height
The height of the chamber in concert with the concentration of the target entity determines the density of the distribution of target entities collected at the collection surface of a vessel such as described above. To increase the range of surface collection densities which are acceptable for accurate counting and analysis, one can vary the height of the chamber to eliminate the need to dilute or concentrate the sample, for analysis of samples where the concentration may vary widely. In
VI. Different Compartments in the Chamber
Different types of target entities present at different densities can be present in the sample. To permit simultaneous multiple analyses, chambers can be made with multiple compartments. An example of such a chamber is illustrated in FIG. 11A. The collection surface 1 and two separate compartments 2 and 3 in these chambers permit the usage of a different set of reagents. In case areas in the chamber are not separated by a wall, as illustrated with 4 in
The terms and expressions which have been employed are used as terms of description and not of limitation. There is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or any portions thereof. It is recognized, therefore, that various modifications are possible within the scope of the invention as claimed.