Methods and devices for manipulation of magnetically collected material

Methods and devices are disclosed which are useful for collecting magnetic materials in either internally or externally generated magnetic gradient fields, followed by resuspension into solution with a simple manipulation of the magnetic device. The methods provide for removal of excess reagent, washing of magnetic material, and resuspension for analysis, among other uses. The methods are applicable to all types of biological material that are susceptible to magnetic labelling, including, for example, cells, viruses, proteins, hormones, and receptor-ligand complexes. Several devices are disclosed to take advantage of the method for cellular and immunoassay applications, including both internal and external devices. Both flow-through and static separators are disclosed.

FIELD OF THE INVENTION 
The present invention relates to a method for the magnetic collection of 
materials of interest with magnetic particles, and the subsequent 
controlled dispersion of the materials of interest. Several devices using 
this method of separation are disclosed. These devices include both 
internal and external magnetic gradients, as well as static and flow 
through-type separators. More specifically, biological substances such as 
cells, cell components, bacteria, viruses, toxins, nucleic acids, 
hormones, proteins, receptor-ligand complexes, other complex molecules or 
any combination thereof, can be first separated, and subsequently 
resuspended, while in the external magnetic field for further analysis, 
isolation or other use. 
BACKGROUND OF THE INVENTION 
A magnetic material or magnetic dipole will move in a magnetic field to the 
region of highest magnetic gradient. Magnetic gradients are broadly 
divided into two groups. Internal magnetic gradients are formed by 
inducing a magnetic field on some susceptible material placed in a 
magnetic field, giving rise to magnetic circuits which generate a 
gradient. Open field gradients are formed by magnetic circuits which exist 
around dipole magnets such as bar or horseshoe magnets or are formed by 
pole piece design, orientation or configuration. In the case of a simple 
rectangular bar magnet, field lines which form magnetic circuits 
conventionally move from North to South and are easily visualized with 
iron filings. From this familiar experiment in elementary physics it will 
be recalled that there is greater intensity of field lines nearest the 
poles. At the poles, the edges formed with the sides and faces of the bar 
will display an even greater density or gradient. Thus, a steel ball 
placed near a bar magnet is first attracted to the nearest pole and next 
moves to the region of highest gradient, typically the closest edge. For 
magnetic circuits any design which promotes increased or decreased density 
of field lines will generate a gradient. In opposing magnet designs, such 
as N-S-N-S quadrupole arrangements, opposing north poles or opposing south 
poles will have field lines which will not cross each other such that in 
the center of such an arrangement there will be zero field (no field lines 
crossing the center). From the circuits that result from North poles to 
each adjacent South pole such arrangements generate radial magnetic 
gradients. 
Internal high gradient magnetic devices have been employed for nearly 50 
years for removing weakly magnetic materials from slurries such as in the 
kaolin industry or for removing nanosized magnetic materials from 
solution. (See Kolm, Scientific American, Nov., 1975). Typically magnetic 
grade stainless steel wool is packed in a column which is then placed in a 
uniform magnetic field which induces gradients on the steel wool. See U.S. 
Pat. No. 3,676,337 to Kolm. Gradients as high as 200 kGauss/cm are easily 
achieved. The field gradient around a wire is inversely related to the 
wire diameter and the smaller the wire, the faster the gradient falls off. 
As will be described below, collection takes place on the wire surface 
when it is transverse to the external magnet field lines, but not when it 
is tangent to them. In using such a system, material to be separated is 
passed through the magnetic "filter" positioned in the external magnetic 
field. Next the material is washed and moved to a station outside the 
field where magnetic materials are removed, making the collector ready for 
reuse. Table I below indicates the strength of the magnetic gradient as a 
function of distance from ferromagnetic wires of diameters of 0.1 and 0.4 
mm as calculated from Maxwell's equation. These are typical wire 
thicknesses for the devices described above. Note that the thinner wire 
has a higher gradient at the wire surface, but that the gradient drops off 
much more quickly. 
TABLE I 
______________________________________ 
0.1 mm diameter wire 
Distance from 0.4 mm diameter wire 
rod surface 
Field Distance from rod 
Field 
(mm) Strength surface (mm) Strength 
______________________________________ 
0 mm 170 KG/cm 0 mm 42.5 KG/cm 
(rod surface) (rod surface) 
0.05 mm 21.1 KG/cm 0.2 mm 12.6 KG/cm 
0.10 mm 6.3 KG/cm 0.4 mm 5.3 KG/cm 
0.15 mm 2.7 KG/cm 0.6 mm 1.6 KG/cm 
0.20 mm 1.4 KG cm 0.8 mm 0.6 KG/cm 
______________________________________ 
Various attempts have been made to perform continuous (non-cycle) high 
gradient magnetic separation. Improvements include flowthrough devices 
with fluctuating fields to separate the magnetic material from the 
non-magnetic. See U.S. Pat. No. 3,902,994 to Maxwell. Removable screens of 
ferromagnetic material are also well known in the art. See U.S. Pat. No. 
4,209,394 to Kelland. In another device patented by Kelland, U.S. Pat. No. 
4,261,815, the movement of magnetic materials from the low gradient to 
high gradient sides (hereafter referred to as quadrants) of wires 
positioned transverse to an external magnetic field was utilized to 
perform continuous separation of magnetic materials from tailings. The 
device incorporates a vertical flow chamber having wires placed therein 
parallel to the direction of flow. It is placed in an external magnetic 
field such that the field is perpendicular to the flow and wires therein. 
As can be visualized, magnetic material in such an arrangement will move 
from the low gradient sides of wires to the high gradient sides. As flow 
proceeds, the tailings will be unaffected by the magnetic gradient. 
Therefore, the high gradient quadrants will contain the original 
concentration of tailing and two times the original concentrations of 
magnetic material. After sufficient travel down the wires when separation 
has taken place, baffles can be positioned in a quadrant fashion to 
prevent the magnetic and non-magnetic quadrants from mixing and to allow 
collection of the appropriate quadrants. In theory this will result in 
either magnetically enriched or depleted feed stock. By rerunning the 
magnetically enriched fractions, another doubling of enrichment is 
achieved. Repetition of this process will result in relatively pure 
magnetic materials. 
Another method of utilization of this same magnetic phenomenon was also 
devised by Kelland, et al, as described in U.S. Pat. No. 4,663,029. This 
patent teaches the use of a non-magnetic flow chamber adjacent at one side 
to a ferromagnetic rod or wire, which in the presence of a magnetic field 
will induce a magnetic gradient. Paramagnetic material will be attracted 
to the wire and diamagnetic material will be repelled from the wire, 
allowing collection of the magnetically unique material through ports 
either near or far from the wire. A similar gradation of materials by 
magnetic susceptibility was devised by Friedlaender, F. J., et al. and is 
the subject of U.S. Pat. No. 4,526,681. 
A method of separation useful for cells and other fragile particles was 
described by Graham et al. in U.S. Pat. No. 4,664,796. This apparatus 
contains a rectangular chamber within a cylinder. One pair of opposing 
sides of the chamber are made of non-magnetic material, while the other 
set of opposing sides are made of magnetic material. This flow chamber is 
packed with a magnetically responsive interstitial separation matrix such 
as steel wool. The material to be separated is run through this chamber 
which is located in a homogeneous magnetic field. In the collection mode, 
the chamber is aligned in the external magnetic field such that the 
magnetic sides of the chamber are parallel with the magnets, thus inducing 
a high gradient field on the interstitial matrix in the chamber. When the 
chamber is in this position, magnetically labeled cells are attracted to 
the matrix and held, while the non-magnetic components are eluted. Upon 
rotation of the chamber, its magnetic sides will be perpendicular to the 
magnets, which will "shunt" or "short-circuit" the magnetic field, 
lowering the gradient in the flow chamber, and allowing the particles of 
interest to be removed by the shear forces of the fluid flow. 
There are a variety of other internal magnetic devices whose gradient 
properties are used to achieve different applications. Commonly owned U.S. 
Pat. No. 5,200,084 teaches the use of thin ferromagnetic rods used to 
collect magnetically labeled cells from solution. Miltenyi (WO 90/07380) 
teaches the use of coated steel wool, or other magnetically susceptible 
material to separate cells. U.S. patent application Ser. No. 07/976,476, 
now abandoned by Liberti and Wang teaches an internal HGMS device useful 
for the immobilization of cells and subsequent sequential reactions of 
these cells. The teaching also allows for the observation of the 
immobilized cells. However, the resuspension and recovery of biological 
substances such as cells, which are either substantially undamaged or 
viable, remains a stumbling block that many recent patents have attempted 
to solve, but with only a limited degree of success. 
External gradient magnetic configurations can also be used to collect 
magnetically responsive particles, particularly more magnetic ones. These 
external devices are so-named because there is no other component of the 
magnetic collector except the magnets or the pole pieces. These devices 
rely solely on the gradients that are created via the magnetic circuits 
generated by the quantity and placement of magnets and in some cases by 
imperfections of field lines moving through space. In a standard bar 
magnet, gradients exist because the magnetic field lines follow non-linear 
paths and "fan out" or bulge as they move from North to South. These 
effects create gradients of about 0.1 to 1.5 kGauss/cm in high quality 
laboratory magnets. These relatively low gradients can be increased by 
manipulating the magnetic circuits so as to compress or expand field line 
density. For example, if the gradient at one pole of a bar magnet is of 
insufficient strength, moving a second bar magnet with an identical field 
in opposition to the first magnet would cause repulsion between the two 
magnets. The number of field lines would remain the same, but they would 
become compressed as the two magnets were forced closer together. Thus, an 
increased gradient would result. The addition of magnets of opposing field 
to this dipole configuration to form a quadrupole could further increase 
the size of the region of high gradients. Other configurations such as 
adjacent magnets of opposing fields would also create gradients higher 
than those seen in a bar magnet of equivalent strength. Yet another method 
of increasing gradients in external field devices is by adapting the pole 
piece design. For example, if the configuration of a standard dipole 
magnet were changed by making one of the magnets into a pointed magnet, 
all field lines would flow towards the point, dramatically increasing the 
gradient around that region. 
None of the effects described above are new. All have been described, used, 
and patented for use in various industries. For example, dipoles and 
quadrupoles have been used in the mining industry to separate clays and 
ores for decades. See U.S. Pat. No. 3,326,374 to Jones and U.S. Pat. No. 
3,608,718 to Aubrey. Dipoles have also reportedly been used to prevent 
scale and lime build up in water systems. See U.S. Pat. No. 3,228,878 to 
Moody and U.S. Pat. No. 4,946,590 to Herzog. Adjacent magnets of opposing 
polarity have been used in drum or rotor separators for the separation of 
ferrous and non-ferrous pieces, such as those generated in scrap yards, an 
improvement over the use of electromagnets. See U.S. Pat. No. 4,869,811 to 
Wolanski et al. and U.S. Pat. No. 4,069,145 to Sommer et al.. Other pole 
piece designs are well known in the literature. See Liberti & Feeley, Proc 
of J.Ugelstad Conference, 1991. 
External magnetic devices have also been used in the fields of cell 
separation and immunoassay. U.S. Pat. Nos. 3,970,518 and 4,018,886 to 
Giaever describe the use of small magnetic particles to separate cells 
using an actuating coil. Dynal Corp. (Oslo, Norway) exclusively uses 
simple external magnetic fields to separate the many particles which it 
markets for various types of cell separations. Commonly owned US patent 
applications Ser. Nos. 08/006,071, now U.S. Pat. No. 5,466,574, and 
08/228,818, now U.S. Pat. No. 5,541,072, disclose the use of external 
fields to separate cells, manipulating the magnetic particles and 
collection devices to form monolayers of cells or other biological 
components. However, resuspension and recovery of collected materials 
still requires removal of the collection vessel from the gradient field 
and some level of physical agitation to accomplish this. 
Turning now to the magnetic particles used in such collection devices, 
superparamagnetic materials have in the last 20 years become the backbone 
of magnetic separation technology in a variety of health care and 
bioprocessing applications. Such materials, regardless of their size (25 
nm to 100 microns,) have the property that they are only magnetic when 
placed in a magnetic field. Once the field is removed, they cease to be 
magnetic and can normally easily be dispersed into suspension. The basis 
for superparamagnetic behavior is that such materials contain magnetic 
material in size units below 20-25 nm, which is estimated to be below the 
size of a magnetic domain. A magnetic domain is the smallest volume for a 
permanent magnetic dipole to exist. Hence, these materials are formed from 
one or more or an assembly of units incapable of holding a permanent 
magnetic dipole. The magnetic material of choice is magnetite, although 
other transition element oxides and mixtures thereof can be used. 
Magnetic particles of the type described above have been used for various 
applications, particularly in health care, e.g. immunoassay, cell 
separation and molecular biology. Particles ranging from 2 to 5 microns 
are available from Dynal. These particles are composed of spherical 
polymeric materials into which has been deposited magnetic crystals. These 
materials, because of their magnetite content and size, are readily 
separated in relatively low fields (0.5 to 2 kGauss/cm) which can easily 
be generated with open field gradients. Another similar class of materials 
are those particles of Rhone Poulanc which typically are produced in the 
0.75 micron range. Because of their size, they separate more slowly than 
the Dynal beads in equivalent gradients. Another class of material is 
available from Advanced Magnetics. These particles are basically clusters 
of magnetite crystals, about 1 micron in size, which are coated with amino 
polymer silane to which bioreceptors can be coupled. These highly magnetic 
materials are easily separated in gradients as low as 0.5 kGauss/cm. Due 
to their size, both the Advanced Magnetics and Rhone Poulanc materials 
remain suspended for hours at a time. 
There is a class of magnetic material which has been applied to 
bioseparations and which has characteristics that places this type of 
material in a special category. These are nanosized colloids (see, for 
example, U.S. Pat. Nos. 4,452,773 to Molday, 4,795,698 to Owen et al, 
4,965,007 to Yudelson; and U.S. patent application Ser. No. 07/397,106 by 
Liberti, et al) . They are typically composed of single to multicrystal 
agglomerates of magnetite coated with polymeric material which render them 
aqueous compatible. Individual crystals range in size from 8 to 15 nm. The 
coatings of these materials have sufficient interaction with aqueous 
solvent to keep them permanently in the colloidal state. Typically, well 
coated materials below 150 nm will show no evidence of settling for as 
long as 6 months and even longer. These materials have substantially all 
the properties of ferrofluids which might be referred to as their 
non-aqueous compatible cousins. 
Because of their size and interaction with solvent water, substantial 
magnetic gradients are required to separate ferrofluids. It was customary 
in the literature to use steel wool column arrangements of the type 
described above which generate 100-200 kGauss/cm gradients. However, some 
years ago it was discovered that such materials must form "chains" in 
magnetic fields like beads on a string (markedly decreasing their Stokes' 
drag force) because separation can be achieved in gradient fields as low 
as 5 or 10 kGauss/cm. These discoveries lead to the development of devices 
using large gauge wires which generate relatively low gradients. Large 
gauge wires as well as other gradient surfaces can be used to cause 
ferrofluids to become deposited in a substantially uniform thickness upon 
collection. With the proper amounts of ferrofluid in a system, the 
thickness of the collected material is effectively the thickness of the 
magnetic colloid, meaning that a monolayer can be formed. Cells 
magnetically labeled can be made to easily form monolayers on macro wires 
or uniform gradient surfaces. See commonly owned U.S. Pat. No. 5,186,827 
and U.S. patent application Ser. No. 08/006,071, now U.S. Pat. No. 
5,466,574. 
Many techniques used in biotechnology require processes such as 
identification, separation, and/or manipulation of target entities, such 
as cells or microbes, within a fluid medium such as bodily fluids, culture 
fluids or samples from the environment. It is also often desirable to 
maintain the target entity intact and/or viable upon separation or 
manipulation in order to analyze, identify, or characterize the target 
entities. 
Identification techniques typically involve labeling the target entity with 
a reagent which can be detected according to a characteristic property. 
Entities which can be viewed optically such as cells or certain microbes, 
may be identified and/or characterized by using fluorescently labeled 
probes such as monoclonal antibodies or nucleic acids. Often the target of 
such probes is not accessible at the surface of the target entity or an 
excess of probe must be removed which requires washing steps and/or 
exposure to a variety of reagents facilitating the penetration of the 
probes. As the number of operations employed in such processes increases, 
a greater number of target entities are lost or no longer suitable for 
evaluation. Accurate microbial analyses employing such methodologies are 
difficult to achieve because of the small numbers of target entities 
involved, as well as the difficulty of washing away unbound labeling 
agent. 
For example, to measure the absolute and relative number of cells in a 
specific subset of leukocytes in blood, a blood sample is drawn and. 
incubated with a fluorescently labeled antibody specific for this subset. 
The sample is then diluted with a lysing buffer, optionally including a 
fixative solution, and the dilute sample is analyzed by flow cytometry. 
This procedure for analysis can be applied to many different antigens. 
However, the drawbacks to this procedure become apparent when large 
samples are required for relatively rare event analyses. In those 
situations, the time needed for the flow cytometer to analyze these 
samples becomes extremely long, making the analysis no longer feasible due 
to economic constraints. In addition, intra-cytoplasmic and/or 
intra-nuclear analysis of cell content is difficult since multiple 
incubations and washing steps require prohibitively long processing times. 
A particular nuisance that is often experienced when collection of magnetic 
materials is done with continuous flow-through devices is "piling-up" of 
the collected material on the inlet end of the device. This occurs because 
collection of magnetic material effectively extends the collection surface 
with collected material which in itself is magnetic. Thus collection 
distorts the surface and the concern over lack of uniformity of collection 
and of trapping of tailings become significant. 
Another problem with continuous separation is that once the collector 
surface has filled to capacity with magnetically labeled material, the 
separator must be physically removed from the field or somehow 
demagnetized so as to remove magnetic material for subsequent reuse of the 
device. 
SUMMARY OF THE INVENTION 
A method of magnetic collection followed by magnetic resuspension is 
provided which takes advantage of the gradients which are generated in 
high gradient magnetic separation (HGMS). In practicing this invention, 
differences in the direction and magnitude of the gradients are exploited 
by employing methods which involve collection of magnetic material in one 
part of the gradient, then reversing the gradient direction to 
repel/resuspend the magnetic material. By employing an apparatus which is 
designed to move a ferromagnetic element between selected positions within 
the gradient, magnetically labelled materials are exposed first to a field 
which leads to the collection of the material on a surface associated with 
the ferromagnetic element, then to a field which acts to repel the 
material off that surface. This invention is particularly adaptable to the 
immobilization and manipulation of microscopic entities, including 
biological entities, such as cells. Methods are provided which enable both 
the separation of cells from fluid medium, as well as for their subsequent 
release as intact entities into the same or different fluid medium. 
By operating in cycles which can involve filling of the collection chamber, 
magnetic collection, removal of tailings, washing, magnetic resuspension 
and harvesting of magnetic material, this invention obviates many of the 
above-noted problems encountered with traditional devices. Specifically, 
the present invention enables resuspension of collected, 
magnetically-labelled target substance in an efficient and reproducible 
manner so as to facilitate subjecting the target substances to detection 
or analysis systems, such as luminescence detectors, spectrophotometric 
analysis, fluorometers, flow cytometers, hematology analyzers, or other 
cell counting or analytical devices. This is achievable without the need 
for removing the magnetically-labelled target substance from the influence 
of the magnetic field, or for dislodging the collected target substance 
from its collection surface, followed by various forms of agitation such 
as stirring, shaking, vibration or mild sonication to resuspend the target 
substance. This cycling also allows the use of sequential reactions, which 
are important for the separation, labeling, and the manufacture of various 
substances, including the magnetic particles themselves. 
According to one aspect of this invention, a method is provided for 
separating magnetic particles from a non-magnetic carrier medium 
containing the particles and dispersing the separated magnetic particles 
in a suitable dispersion medium, the carrier 10 medium and the dispersion 
medium being the same or different. The method initially involves 
providing in proximity to a separation chamber a ferromagnetic element, 
having a collection surface for the magnetic particles associated 
therewith and introducing the carrier fluid medium into the separation 
chamber. 
A magnetic field is applied which intercepts the separation chamber, the 
magnetic field having a first region which exerts an attractive magnetic 
force urging magnetic particles in the carrier medium in a direction 
toward the ferromagnetic element and its associated collection surface and 
causing the magnetic particles to be collected on the surface, and a 
second region which exerts a repulsive force causing repulsion of the 
collected magnetic particles away from the ferromagnetic element and the 
collection surface, with the surface being movably positionable relative 
to said magnetic field. 
The aforesaid surface is then positioned relative to the magnetic field for 
exposure to the first region and collection of magnetic particles 
attracted to the ferromagnetic element on the collection surface, and 
thereafter repositioned relative to the magnetic field for exposure to the 
second region, thereby repelling magnetic particles from the ferromagnetic 
element and dispersing the repelled particles in the dispersion medium. 
According to another aspect of the present invention, there is provided an 
apparatus for separating magnetic particles from a non-magnetic fluid 
medium containing such particles. The apparatus comprises a housing, 
including an interior wall area, for receiving the fluid medium, and a 
ferromagnetic element, having a surface associated therewith, disposed in 
proximity to the housing. 
A magnetic field source provides a magnetic field that intercepts the 
housing. The magnetic field has a first region which exerts an attractive 
magnetic force urging magnetic particles in the fluid medium in a 
direction toward the ferromagnetic element and the aforementioned surface, 
and a second region which exerts a repulsive force urging the magnetic 
particles away from the ferromagnetic element and the surface. The surface 
associated with the ferromagnetic element is movably positionable relative 
to the magnetic field so as to serve as a collection zone for collecting 
magnetic particles attracted to the element in a first position and to 
serve as a dispersion zone for dispersing magnetic particles repelled from 
the element in a second position. 
The apparatus also includes means to move the surface selectively, such 
that in the first position the surface is in the first region and in the 
second position the surface is in the second region, and further includes 
barrier means in the housing, which comprises the surface associated with 
the ferromagnetic element and the interior wall area of the housing and 
which defines a chamber for confining the magnetic particles during 
movement of the surface from the first position to the second position. 
When used for cellular analysis, the instant invention also makes possible 
a significant reduction in volume for all types of samples, resulting in 
higher concentrations of the target entity, thus permitting shorter 
analysis time. The ease of resuspension eliminates the need for 
centrifugation, a significant factor contributing to cell loss. The 
immobilization of cells permits many types of reactions to be performed 
which involve these cells. 
When used for immunoassay, the instant invention provides a highly 
sensitive, but relatively small scale system for the collection of labeled 
analyte. The speed and reproducibility of collection and resuspension are 
important features for a relatively low-cost test for a multitude of 
different analytes. Further the resuspension principle is of significant 
benefit in constructing a reusable collection device making cleaning of 
the device an easier task as well.

DETAILED DESCRIPTION OF THE INVENTION 
In the case of internal gradient magnetic devices a simple system to 
visualize is the cross-section of a single, typically round ferromagnetic 
element placed in a homogeneous magnetic field with the wire positioned 
transverse (perpendicular) to the field lines of the external field, as 
depicted in FIG. 1. FIG. 1a shows set of two opposed magnets 11, which 
form a magnetic field 12 between them. A ferromagnetic wire or rod 13 will 
have induced on it a magnetic dipole 14 by the external field 12, with its 
North pole facing the South pole face of the external field and similarly 
its South pole facing the external field North. The circuit that this 
induced magnet forms will be from its North to South poles and, as shown 
in FIG. 1b, these vector field lines on the surface of that part of the 
wire confronting the pole faces have the same direction as the external 
field lines, which will tend to increase the magnetic field in this area. 
However, on the surface of that part of the wire perpendicular to the pole 
faces of the magnets, the vector field lines have a direction opposite the 
external field lines, which will tend to decrease the magnetic field in 
this area. The net result of this vector addition/subtraction is field 15, 
depicted in FIG. 1c, in which there is augmentation of field lines on 
those areas of the wire surface facing the poles with a diminution of 
field lines on the other two areas. 
FIG. 2 is a schematic representation of the force exerted on a particle in 
field 15 (not shown) near the ferromagnetic rod 13. A magnetically 
responsive particle 16 located in the region of canceled magnetic field 
will tend to move towards a region of higher magnetic field, which in this 
case is initially away from rod 13. Then the force on the particle will 
turn the particle back towards the rod to the region of reinforced 
magnetic field. The region of highest field gradient is at the surface of 
the rod 13 facing the external magnets 11 (not shown). The vector lines 
indicate that magnetic materials placed on the lower gradient surfaces of 
the wire will move along the field lines to the higher gradient side. One 
can view the circumference around a wire as being divided into quadrants. 
The two quadrants facing the external pole faces function as collection 
surfaces, the other two being non-collection surfaces. This distinction is 
easy to demonstrate by observing the collection patterns with magnetic 
colloids. In experiments on suspensions of magnetically labeled mammalian 
cells placed in a microtitre well with a single wire traversing the well 
horizontally and positioned transverse to an external magnetic field, 
cells can microscopically be observed collecting on the sides of the wire 
facing the poles of the external field. Additionally, cells adjacent to 
the wire on the "low" gradient sides can be observed to move away from the 
wire and then circle back onto the "high" gradient sides. Thus the path 
that these cells actually follow is the vector lines depicted in FIG. 2. 
Referring to FIGS. 3 and 4, the device shown is a ferromagnetic rod with 
four baffles or vanes, which will enable magnetic material to collect in 
chambers disposed on opposite sides of the rod. When the rod is turned 90 
degrees, magnetic force will act to repel the collected material from the 
rod back into solution. This apparatus may be used in either a batch-type 
mode to separate relatively small quantities of material or with certain 
materials in a continuous flow mode to decrease the volume of the sample 
significantly by collecting material from a large volume on a surface and 
resuspending it in a small volume. 
FIG. 3 shows the cross section of a ferromagnetic rod 13 of appropriate 
small diameter to generate a reasonably high gradient in a uniform 
magnetic field. The circumference of the wire or rod is sufficiently large 
that non-magnetic baffles 17 can be longitudinally attached to it, 
dividing the surface into four quadrants of approximately equal surface 
area. 
As illustrated in FIG. 4, the rod 13 and baffles or vanes 17 are inserted 
into a cylinder 18 such that four distinct zones are formed. In three 
dimensions these zones are actually chambers. When this apparatus is in a 
uniform magnetic field it will be appreciated, by comparison with FIG. 2, 
that magnetic materials will be attracted to the surface of the 
ferromagnetic rod in the quadrants labeled 19 and 19'. The force lines 
which exist in quadrants 20 and 20' are directed away from the wire 
surface towards the inner wall of cylinder 18. Given these forces, a 
simple cyclic separation/resuspension system can easily be implemented. 
Magnetic feedstock or sample to be separated is fed into one or both of 
the chambers 19 and 19' and separation is allowed to take place. Next, 
those chambers can be emptied to collect the "tailing" if that is the 
objective, or the collected magnetic material may be washed by the 
addition of fresh wash liquid. With the chamber now refilled, by turning 
the rod 13 with its vanes 17 90.degree., material collected on the rod 
will be repelled from the rod surface and into solution. If need be, the 
material can be recollected and resuspended at will merely by sequentially 
turning the collection surface 90.degree.. Optionally, the cylinder 18 can 
be moved as a single unit together with the rod and vanes. This apparatus 
should have inherently low non-specific binding to the collection device 
of material which is not magnetically labelled. Unlike most other internal 
gradient systems, there is little entrapment of particles due to the 
absence of steel wool mesh or other potentially entrapping arrangement of 
grids or wires. However, a coating on the wire may be necessary to reduce 
the oxidation of the apparatus over time. It should be apparent that the 
separation cycle could begin by filling the chamber in the non-collection 
mode. 
In another embodiment of this invention, a single collection chamber 53 may 
be provided in a housing 52 as illustrated in FIG. 5. When side 55 of the 
housing is positioned adjacent to magnet 56, magnetically responsive 
material in chamber 53 will collect on the surface of the rod 51. However, 
if the device or the magnet is repositioned such that the magnet is 
adjacent to side 54 of the housing, any magnetic material on the surface 
of rod 51 will be repelled from the rod surface and be resuspended into 
any fluid present in chamber 53. The entire apparatus may be alternated 
between the collection and resuspension modes and a single chamber used to 
manipulate magnetically labeled cells or other target substances. 
In yet another embodiment of the invention, one wall of the chamber need 
not be formed by a portion of the surface of the rod which generates the 
magnetic gradient. For example, a non-magnetic sleeve could be provided 
which surrounds and is substantially coextensive with the rod. The sleeve 
may be fabricated of a suitable biocompatible material for conducting 
biological separations, and provided with longitudinal vanes defining one 
or more separation chamber, as previously described. The sleeve may be 
affixed to, or rotatable relative to the ferromagnetic rod. In the latter 
embodiment, the chamber would be immediately adjacent to the rod, and 
either the rod or the chamber could be moved independently of one another. 
This would allow for removal of a disposable chamber, as may be necessary 
for some clinical applications. A removable rod might also be useful in 
some cases where variable field strengths (and thus variable wire 
diameters) might be desired. 
It will be understood from the foregoing description that although the 
collection surface for the magnetic particles is associated with the 
ferromagnetic element, it may or may not be integral therewith. When the 
exterior of the ferromagnetic element and the collection surface are one 
and the same, or when the aforementioned sleeve is affixed to the 
ferromagnetic element, such that the collection surface of the sleeve and 
the ferromagnetic element are manipulatable as a unit, the structure is an 
integral or unitary whole. 
It should be further understood that the rod or wire and associated 
collection chamber need not be placed in the homogeneous field generated 
between a pair of dipole magnets. Instead, the device could be placed 
adjacent to a magnetic field source which generates a sufficiently strong 
and homogeneous field to induce the appropriate gradient on the 
ferromagnetic element of such devices. A variety of such permanent or 
electromagnetic arrangements are known. Such an arrangement confers 
advantages not realized in a device sandwiched between two magnets. 
Namely, the field of collection need not be constrained to the side facing 
a magnet. In fact, with the proper materials engineering, a field of view 
can be established which leads to the complete exposure of the collected 
material to a viewing device. For example, in a very simple way, the 
device illustrated in FIG. 5 could be placed adjacent to a strong single 
magnet in a region where field lines are to the surface of the magnet 
facing the housing 52 and relatively homogeneous. If the device housing 52 
were made out of clear glass, clear plastic, or quartz, optical or 
spectrophotometric devices could be used to observe or analyze the 
materials immobilized on the collecting rod. This embodiment of the 
invention provides for the reading or imaging of luminescent, absorptive, 
fluorescent, and/or light scattered signals. 
Referring to FIGS. 6 and 7, an apparatus is shown which employs a 
quadrupole magnet arrangement with a collection vessel located in the 
field such that the magnetic material collects on one side of the vessel. 
When the vessel is moved to a different region within the field of the 
quadrupole, the magnetic force will repel the magnetic material, and 
eventually it will collect again on another side of the vessel. However, 
before this second collection occurs, the material can be removed. This 
apparatus can also be used in either batch or continuous modes of 
operation. 
The device of FIGS. 6 and 7 provides an alternative way of magnetically 
separating and resuspending material using the phenomenon of the magnetic 
force lines. This can be carried out by repositioning a collection chamber 
in magnetic gradients where gradient direction is reversed or altered. One 
way to achieve this objective is to employ the radial gradient generated 
in the quadrupole, or if less gradient is required, in a dipole 
arrangement. FIG. 6 also depicts the magnetic field lines 62 generated 
when magnets 61 are arrayed in a quadrupole arrangement. FIG. 7 depicts 
the manner in which separation may be effected by taking advantage of such 
force lines. A separation vessel 72 is placed in the radial field 62 (not 
shown) off center such that magnetic material is collected on the wall 
area 75 of vessel 72. By moving the vessel laterally to the region where 
wall area 75 experiences a magnetic gradient in the opposite direction as 
was previously the case, the magnetic material will resuspend. In time the 
magnetic material will recollect on the opposite wall of the tube. This 
apparatus can easily be used in either the batch or continuous mode. In 
batch mode operation, the collection vessel 72 could be a microtiter well 
for applications, such as immunoassay or a proportionally larger vessel 
and magnets, could be employed if needed for other applications. In 
continuous operation, the vessel would be provided with inlet/outlet ports 
at either end, which would be attached to tubing, through which the sample 
fluid could be flowed into the vessel. The non-magnetic components would 
be continuously removed through the opposite end. The vessel could be 
contained in a housing, which would control the placement of the vessel, 
allowing it to alternate between the two orientations within the field. It 
is also possible to simply place a piece of tubing through a quadrupole, 
securing it so that it remains fixed in place, and then moving the tubing 
or the magnets in relation to one another to resuspend the material. 
The present invention provides methods and apparatus for efficiently 
determining a broad range of target substances or analytes. In a preferred 
embodiment, the methods and devices of the invention are used for the 
determination of any constituent of a biological fluid or specimen that is 
capable of selective interaction with a specific binding substance. Thus, 
the term "target substance" as used herein, refers to a wide variety of 
substances of biological or medical interest which are measurable 
individually or as a group. Examples include cells, both eucaryotic (e.g., 
leukocytes, erythrocytes or fungi) and procaryotic (e.g., bacteria, 
protozoa or mycoplasma), viruses, cell components, molecules (e.g., 
proteins) and macromolecules (e.g., nucleic acids-RNA, DNA). These 
substances may be determined as discrete entities or in the form of 
complexes or aggregates. Such determinations are accomplished using 
certain methods of the invention which rely on the selective interaction 
of the specific binding substance with at least one characteristic 
determinant of the target substance or analyte of interest. 
The term "determinant" is used herein in its broad sense to denote an 
element that identifies or determines the nature of something. When used 
in reference to any of the foregoing target substances, "determinant" 
means that portion of the target substance involved in and responsible for 
selective binding to the specific binding substance, the presence of which 
is required for selective binding to occur. Cell-associated determinants 
include, for example, components of the cell membrane, cytoplasm or 
nucleus. Among such cell-associated structures are membrane-bound proteins 
or glycoproteins, including cell surface antigens of either host cell or 
viral origin, histocompatibility antigens, or membrane receptors. One 
class of specific binding substance that is used to selectively interact 
with the determinants is the class of antibodies capable of 
immunospecifically recognizing same. The term "antibody" as used herein 
includes immunoglobulins, monoclonal or polyclonal and immunoreactive 
immunoglobulin fragments. 
Further examples of characteristic determinants and their specific binding 
substances are: receptor-hormone, receptor-ligand, agonist-antagonist, RNA 
or DNA oligomers-complimentary sequences, Fc receptor of mouse IgG-protein 
A, avidin-biotin and virus-receptor. Still other determinant-specific 
binding pair combinations that may be determined using the methods of this 
invention will be apparent to those skilled in the art. 
The preferred magnetic particles for use in carrying out this invention are 
particles that behave as true colloids. Such particles are characterized 
by their sub-micron particle size, which is generally less than about 200 
nanometers (nm) (0.20 microns), and their stability to gravitational 
separation from solution for extended periods of time. Such small 
particles facilitate observation of the target entities via optical 
microscopy since the particles are significantly smaller than the 
wavelength range of light. Suitable materials are composed of a 
crystalline core of superparamagnetic material surrounded by molecules 
which may be physically absorbed or covalently attached to the magnetic 
core and which confer stability to the colloidal particles. The size of 
the colloidal particles is sufficiently small that they do not contain a 
complete magnetic domain, and their Brownian energy exceeds their magnetic 
moment. As a consequence, North Pole, South Pole alignment and subsequent 
mutual attraction/repulsion of these colloidal magnetic particles does not 
appear to occur even in moderately strong magnetic fields, contributing to 
their solution stability. Accordingly, colloidal magnetic particles are 
not readily separable from solution as such even with powerful 
electromagnets, but instead require a magnetic gradient to be generated 
within the test medium in which the particles are suspended in order to 
achieve separation of the discrete particles. 
Magnetic particles having the above-described properties can be prepared as 
described in U.S. Pat. No. 4,795,698, and U.S. patent application Ser. No. 
07/397,106, the entire disclosures of which are incorporated by reference 
in the present specification, as if set forth herein in full. 
For immobilization of cellular target entities, for example, the test 
medium typically comprises appropriately prepared fluids, for example, 
body fluids such as blood, urine, sputum or secretions. It is preferable 
to add the colloidal magnetic particles to the test medium in a buffer 
solution. A suitable buffer solution for this purpose comprises a mixture 
of 5% bovine serum albumin ("BSA") and 95% of a biocompatible phosphate 
salt solution, optionally including relatively minor amounts of dextrose, 
sodium chloride and potassium chloride. The buffer solution should be 
isotonic, with a pH about 7. The protein serves to decrease interactions 
which tend to interfere with the analysis. The target substance may be 
added to the test medium before, after or simultaneously with introduction 
of the magnetic particles. The practice of this invention takes advantage 
of the diffusion controlled solution kinetics of the colloidal magnetic 
particles, which may be further enhanced by the addition of heat to the 
test medium. The test medium is usually incubated to promote binding 
between the receptor and any ligand of interest present therein. 
Incubation is typically conducted at room temperature or at a temperature 
slightly above the freezing point of the test medium (i.e., 4.degree. C. 
in an aqueous medium). The period of incubation is normally of short 
duration (i.e., about 15 minutes). The test medium may be agitated or 
stirred during the incubation period to facilitate contact between 
receptors and ligands. 
The invention disclosed here can be utilized in conjunction with the 
invention disclosed in U.S. patent application Ser. No. 08/228,818, now 
U.S. Pat. No. 5,541,072 to achieve performance in relatively low gradient 
fields, as would be the case for relatively large diameter ferromagnetic 
collection rods. In the last-mentioned patent application, it is disclosed 
that a certain class of magnetic material, referred to as ferrofluids, 
form phases with non-ferrofluid solutions. For example when a dilute 
solution of aqueous soluble ferrofluid (see commonly owned US patent 
application Ser. No. 07/397,106) is layered onto the aqueous solvent of 
the ferrofluid, the ferrofluid solution will fall below the solvent and 
form two phases which will remain distinct. Further, these phases will be 
magnetically responsive. Thus if two phases, each 2 cm high, are formed in 
a 10.times.75 mm test tube, the ferrofluid phase being the bottom phase, 
and a bar magnet is brought to the side of the tube, the phases will 
reorient themselves vertically in the tube with the ferrofluid phase being 
closest to the magnet. Similarly, if the tube were placed in a radial 
gradient magnetic field, the ferrofluid would form an annular ring with an 
inner cylinder of solvent alone. This phasing phenomena can be used, as 
already disclosed, to position material which is to be separated 
substantially uniformly along a gradient surface and in closer proximity 
to the region of highest gradient than would be the case without phasing. 
This proximity of magnetic material to the region of highest gradient 
leads to more rapid and uniform separation. The ability to use the device 
and method described herein in conjunction with the ferrophasing 
phenomenon broadens the application possibilities of this invention. For 
example, to isolate infrequent cells, which are defined here as cells that 
occur at a frequency below one in 10.sup.2, Such as circulating malignant 
cells, fetal cells, progenitor cells, basophils, or eosinophils, this 
invention can be used in the flow through mode described above. Rare cells 
can be labeled with antibody and a magnetic ferrofluid capable of 
exhibiting the ferrophasing behavior. If the apparatus described above and 
illustrated in FIGS. 4 or 5 were filled with a non-magnetic buffer, then 
when the magnetically labeled cell sample is flowed into the apparatus, 
the ferrophasing phenomenon would cause submergence of the labelled sample 
through the non-magnetic buffer to the region adjacent to the rod along 
substantially the entire length thereof. This region is the region of the 
highest magnetic gradient, so that any magnetically labelled cells would 
be almost immediately captured. At the surface of the rod, the magnetic 
gradient would be sufficiently high to destroy the ferrophase, causing it 
to separate into magnetic and non-magnetic components. The non-magnetic 
material, including non-labeled cells would diffuse into the non-magnetic 
buffer, being carried out of the apparatus with the fluid flow. 
A further beneficial effect of the ferrophasing phenomenon is the resultant 
distribution of the magnetically collected cells along substantially the 
entire length of the rod, avoiding the effect the of "piling up" at the 
inlet, as seen in many HGMS systems. After the entire sample has been 
flowed into the magnetic collector, buffer could be used to remove the 
remaining non-magnetic material, and the apparatus could be rotated 
90.degree. with respect to the magnetic field. The magnetically labeled 
cells would then be resuspended and flowed out of the magnetic collector. 
It is important to note that this method of operation allows for a 
significant reduction in the volume of a sample. A relatively large volume 
of sample can be flowed through the system, with an extremely low 
incidence of rare cell events. Then after a short residence time in the 
apparatus, a significantly enriched cell population can be released in a 
small volume. 
The entire disclosure of the aforementioned U.S. patent application Ser. 
No. 08/228,818, now U.S. Pat. No. 5,541,072, is incorporated in the 
present specification by reference as if set forth herein in full. 
The advantages conferred by the instant invention over the state of the art 
are many. Devices such as those described above allow for relatively easy 
magnetic collection, resuspension, and if desired, the process can be 
repeated. In applications relating to cellular biology, the present 
invention obviates the need to perform multiple centrifugations for 
washing out unbound reagents. This is a tremendous advantage over the 
current art, eliminating the cell loss and clumping that often results 
from centrifugation. In applications relating to immunoassay, the ease of 
complete resuspension of labeled analyte without clumping is vital to 
reproducible analysis of analytes such as TSH, T4, hCG, etc. This 
advantage is also important in applications other than cellular biology or 
immunoassay. The ability to carry out resuspension in the separation 
chamber affords other advantages as labeled material can then be removed 
from the chamber for analysis elsewhere, such as to read a signal from the 
collected material, to measure the amount of collected material, to count 
the number of cells, etc. In addition, many types of reactions may be 
facilitated by collection of the magnetically labeled material, and 
washing out of the excess reactant. Examples include enzymatic reactions, 
labelling reactions, hybridization reactions, protein binding reactions, 
immunochemical reactions, which can be either single step or multi-step 
reactions. Such reactions can be performed with the target substance in 
the magnetically immobilized state where reactants can be removed by 
flowing them out of the chamber. This process not only provides for 
efficiency, but also for economy of reagents. The present invention also 
enables the performance of various analyses while the target substance is 
immobilized. 
Applications for this method of magnetic manipulation are many. In the 
field of diagnostic medicine, a blood sample could be incubated with 
fluorescently labeled antibodies identifying subsets of leukocytes and 
magnetically labeled monoclonal antibodies which recognize all leukocytes. 
The leukocytes may be magnetically removed from a blood sample and 
non-targeted cells (red blood cells and platelets) could be washed out of 
the system. The leukocytes, which may be collected as a monolayer, could 
be exposed to a variety of reagents, while immobilized on the collection 
surface. The ability to immobilize target cells permits the implementation 
of procedures such as in-situ hybridization (ISH), in-situ PCR, or 
functional assays. In the field of therapeutic medicine, genetically 
manipulated cells may be isolated from non-manipulated cells by the 
presence of a surface marker. In the field of food testing, pathogens 
present on carcasses or other surfaces could be analyzed for antibiotic 
susceptibility or pathogenicity. In the field of macromolecule separation 
such as immunoassays or molecular biology, the ability to resuspend 
magnetically collected materials could have many benefits, such as the 
ability to hybridize, do sequential reactions or protein/enzyme 
incubations in solution, while easily separating the desired material 
after the reactions in solution have occurred. Alternately, some reactions 
that have been practically impossible to do, become possible with such 
tight control over separation and resuspension. For example, it should be 
possible to do intracellular immunoassays, for example for HIV or 
carcinoembryonic antigen (CEA). In an intracellular immunoassay, the cell 
membrane could be permeabilized, then a reactant allowed to infuse the 
cell. Functional studies on cells could also be accomplished with the 
instant invention. A virus, bacteria, chemical agent, drug, or other 
biological molecule could be introduced to some cells immobilized in a 
collection chamber for a brief period, after which the buffer containing 
the pathogen or agent could be easily washed out. Then the effect of the 
pathogen or agent on the cell could be observed. Any effects would be 
solely due to the introduced material. For example, the cells could be 
exposed to foreign antigens or ligands and subsequently analyzed for 
intracellular phosphorylation of proteins involved in the activation 
pathways of the stimuli. This could lead to easier toxicity testing, 
disease resistance testing or cell function testing. 
Many different types of devices using this method of manipulation could be 
manufactured. The two internal rod devices and the external 
well/flow-though device that are illustrated in FIGS. 3-7 are some 
examples. However, this type of magnetic collection system need not stand 
alone in a clinical or laboratory situation. This type of device could be 
used as a front-end unit for cell analysis systems such as hematology 
analyzers or flow cytometers. For example, this device could be used with 
whole blood to collect leukocytes, obviating the need to gradient 
centrifuge whole blood to isolate mononuclear cells or erythrocyte lysed 
blood. 
Another application of the device of the instant invention involves use as 
an analysis chamber. A chamber could be constructed with a collection 
surface visible to some means of detection, such as fluorescent, 
chemiluminescent, spectrophotometric, or visual. The desired substance 
could be labeled with magnetic material and collected on the surface, 
preferably in a monolayer or approximately a monolayer. Optionally, the 
material could be subjected to any of the reactions or manipulations 
described previously. Then, the material could be observed, detected, 
measured, counted or otherwise quantified while the material remains 
immobilized in the device. 
The present invention also provides a device in which the housing moves 
independently of the rod. This type of housing could have two chambers, 
which have a different volume, and which are connected, but which also 
include a barrier that separates the two chambers. With the barrier 
closed, the larger chamber could be filled with material to be separated, 
which material is then collected on the rod. Then, with the barrier open, 
the housing could be rotated around the rod, not disturbing the cells 
immobilized on the rod such that the cells are encased within the smaller 
chamber. The barrier between the two chambers could then be closed. Since 
the second chamber has a volume smaller than the first chamber, this 
manipulation allows for resuspension in a smaller volume. 
Another aspect of the present invention involves the construction of a 
ferromagnetic probe which may be lowered into a vessel such as a 
microtiter well placed in a uniform magnetic field. In one orientation, 
the probe would collect magnetically responsive material. After a rotation 
of the probe 90.degree., the material collected would be dispersed. Thus, 
magnetic material could be removed from a first vessel and redispersed in 
a second vessel for washing or subsequent detection. One embodiment would 
resemble a small probe with a ferromagnetic segment having two opposing 
quadrants blocked to magnetic collection, covering approximately half of 
the ferromagnetic segment of the probe. The probe could be inserted into a 
microtiter well or other chamber with a sample containing an analyte of 
interest and placed in a uniform magnetic field. Magnetically labelled 
analyte would be collected upon the probe. The probe could optionally be 
washed, then turned 90.degree. for resuspension into an analysis solution, 
optionally containing substrate for chemiluminescent or other detection. 
Numerous other devices can be envisioned which exploit this method of 
magnetic separation, in addition to those described and exemplified 
herein. 
This method of magnetic separation and resuspension is especially adaptable 
to analysis of individual events, such as will be the case in the next 
several generations of particle analyzers. Devices for implementing the 
methods of the present invention are suitable for microfabrication, to 
separate very small samples of material, on the scale of microliters. The 
present invention may also be utilized as a front-end system of separation 
which could handle quantities of blood or other samples anywhere in the 
microliter to liter range. 
The following examples will serve to illustrate the principles of this 
invention; however, these examples should not be construed as limiting the 
scope of this invention. 
EXAMPLE 1 
Internal Collection Device for 
Resuspendable Magnetic Collection 
The fabrication of the embodiment of the invention illustrated in FIG. 5 
will now be described. 
A ferromagnetic element was manufactured (Jade, Huntingdon Valley, Pa.) out 
of soft metal iron rod with a radius of 1.0 mm and a length of 10 cm. A 
single collection chamber of a radius of 1.53 mm was formed in a solid 
acrylic body in the shape of a truncated cylinder which served as the 
housing around the rod. The collection chamber was parallel to the rod, 
and its circumference overlapped that of the rod, as shown in FIG. 5. 
Gradient field strength at the surface of the rod was approximately 5 
kGauss/cm, and the volume of the chamber was approximately 650 .mu.l. 
To demonstrate magnetic collection and resuspension, magnetically 
responsive particles coated with bovine serum albumin (BSA) were 
manufactured as disclosed in U.S. patent application Ser. No. 07/397,106. 
The particles were of a size approximately 100 nm diameter. The collection 
chamber was filled with this BSA ferrofluid at a concentration of 50 ug 
iron/ml in 20 mM sodium phosphate buffer at pH 7.2. The device was 
positioned horizontally into a yoked permanent magnet fixture having 
5.times.10 cm pole faces and a 1 cm gap near the midline of the pole 
faces. The yoke fixture positions two 5.times.10.times.2 cm rare earth 
magnets such that the pole face planes are vertical, the 10 cm dimension 
is horizontal and the gap opening is at the top and sides which allows 
easy insertion and viewing of the device. In the horizontal region near 
the center of the pole faces the field for this fixture was 7.5 kGauss and 
very homogeneous. By first inserting the ferrofluid filled 
collection/resuspension device into the magnetic field such that its 
orientation with respect to the field put the device in the "resuspension" 
mode, it could be determined visually that no collection occurred, nor was 
any other change visible. Next the device was rotated 90.degree. to the 
collection orientation, i.e., the position shown in FIG. 5, and collection 
of the ferrofluid on the ferromagnetic rod was observed. The device was 
then rotated back to its original orientation and the ferrofluid was 
observed to resuspend. This cycle of collection and resuspension was 
repeated several times. Additionally the device was removed from the 
magnetic field in the resuspension mode and the device emptied. The 
ferrofluid was observed to be fully resuspended and non aggregated. 
EXAMPLE 2 
The Effect of Ferrophasing 
To demonstrate the ferrophasing phenomenon two experiments were done. 
Holding the chamber in a vertical position it was half filled with 
ferrofluid at 50 ug iron/ml in phosphate buffer. Next buffer was layered 
onto the ferrofluid so that no bubbles remained in the chamber. The device 
in the same vertical position was carefully lowered into the magnetic 
field in the non-collection orientation until that portion containing the 
ferrofluid was completely in the field. At that point the device was 
rotated to the horizontal position and observed. The ferrofluid and buffer 
solution remained on their respective ends of the chamber. Next the device 
was rotated into the collection orientation whereupon the ferrofluid 
solution could be observed to immediately move under the buffer forming a 
uniform layer along the entire length of the collection rod. 
In a related experiment the device was filled with buffer placed in the 
magnetic field in the collection orientation and a small quantity of 
ferrofluid solution (approximately 20% of the chamber volume) was injected 
into the chamber. Immediately upon entry of the ferrofluid into the 
chamber it moved along the entire length of the collection rod forming a 
uniform phase which subsequently collected. 
While certain preferred embodiments of the present invention have been 
described and exemplified above, it is not intended to limit the invention 
to such embodiments, but various modifications may be made thereto, 
without departing from the scope and spirit of the present invention as 
set forth in the following claims.