U.S. patent application number 16/156487 was filed with the patent office on 2019-02-07 for method and device for the manipulation of microcarriers for an identification purpose.
This patent application is currently assigned to MYCARTIS NV. The applicant listed for this patent is MYCARTIS NV. Invention is credited to Kevin BRAECKMANS, Stefaan Cornelis DE SMEDT, Joseph DEMEESTER, Emmanuel Marie Paul Ernest GUSTIN, Marc Jan Rene LEBLANS, Christiaan Hubert Simon ROELANT.
Application Number | 20190041304 16/156487 |
Document ID | / |
Family ID | 8172155 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190041304 |
Kind Code |
A1 |
LEBLANS; Marc Jan Rene ; et
al. |
February 7, 2019 |
METHOD AND DEVICE FOR THE MANIPULATION OF MICROCARRIERS FOR AN
IDENTIFICATION PURPOSE
Abstract
A method and apparatus for the manipulation for an
identification purpose of a microcarrier. The method comprising the
steps of: (a) an identification purpose step of the microcarrier;
and (b) a positioning and orientation step prior to or during the
identification purpose step. The apparatus comprising means for
identification purposes such as a microscope or labelling means
such as a high spatial resolution light source, and means for the
positioning and orientation of the microcarriers.
Inventors: |
LEBLANS; Marc Jan Rene;
(Kontich, BE) ; GUSTIN; Emmanuel Marie Paul Ernest;
(Vosselaar, BE) ; ROELANT; Christiaan Hubert Simon;
(Leuven, BE) ; DE SMEDT; Stefaan Cornelis;
(Mariakerke, BE) ; DEMEESTER; Joseph; (Gent,
BE) ; BRAECKMANS; Kevin; (Destelbergen, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MYCARTIS NV |
Zwijnaarde |
|
BE |
|
|
Assignee: |
MYCARTIS NV
ZWIJNAARDE
BE
|
Family ID: |
8172155 |
Appl. No.: |
16/156487 |
Filed: |
October 10, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14279867 |
May 16, 2014 |
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16156487 |
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12888187 |
Sep 22, 2010 |
8735172 |
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14279867 |
|
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10399921 |
Apr 16, 2003 |
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PCT/EP01/12194 |
Oct 19, 2001 |
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12888187 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/0054 20130101;
G01N 21/6428 20130101; B01J 19/0046 20130101; G01R 15/20 20130101;
G09F 3/00 20130101; G01R 33/1269 20130101; B01J 2219/00596
20130101; B01J 2219/00547 20130101; B01J 2219/00542 20130101; G01N
33/58 20130101; C40B 70/00 20130101; B01J 2219/005 20130101; B01J
2219/00587 20130101; G01N 2001/002 20130101; G01N 33/53 20130101;
G01N 1/38 20130101; Y10T 436/25 20150115; B01J 2219/00702 20130101;
G01N 27/745 20130101 |
International
Class: |
G01N 1/38 20060101
G01N001/38; B01J 19/00 20060101 B01J019/00; G01N 33/53 20060101
G01N033/53; G01N 33/58 20060101 G01N033/58; G09F 3/00 20060101
G09F003/00; G01N 21/64 20060101 G01N021/64; G01R 15/20 20060101
G01R015/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2000 |
EP |
00203627.5 |
Claims
1. A method for manipulation of a microcarrier for the purpose of
identifying the microcarrier, said method comprising the steps of:
a detection step for detecting an encoded microcarrier, wherein the
encoded microcarrier includes a code written thereon; and a
positioning and orientation step prior to or during the detection
step, wherein said positioning and orientation step comprises:
distributing a plurality of microcarriers, that includes said
encoded microcarrier, in a one-layer system which results in a
plane configuration having two dimensions (X, Y), and restricting
rotational movement of the plurality of microcarriers, wherein the
plurality of microcarriers have an ellipsoidal or cylindrical shape
and wherein the positioning and orientation step results from the
ellipsoidal or cylindrical shape of the plurality of
microcarriers.
2. The method according to claim 1, wherein the step of
distributing the plurality of microcarriers in the one-layer system
results in a line configuration.
3. The method according to claim 1, wherein said encoded
microcarrier is encoded by a code written thereon by exposure to a
high spatial resolution light source.
4. The method according to claim 1, wherein the method further
comprises: (i) encoding said encoded microcarrier by writing a code
thereon, and (ii) allowing a target-analyte reaction on or in said
encoded microcarrier.
5. The method according to claim 4, wherein step (ii) precedes step
(i).
6. The method according to claim 4, wherein the step of
distributing the plurality of microcarriers in the one-layer system
results in a line configuration.
7. The method according to claim 4, wherein said encoded
microcarrier is encoded by a code written thereon by exposure to a
high spatial resolution light source.
8. The method according to claim 4, wherein said encoded
microcarrier is encoded by a process selected from the group
comprising: photochroming, chemical etching, material deposition,
photobleaching, or exposing said encoded microcarrier to a high
spatial resolution light source.
9. The method according to claim 1, wherein the encoded
microcarrier includes one or more ligands bound to a surface
thereof.
10. The method according to claim 1, wherein the detection step is
performed using an optical identification means.
11. The method according to claim 10, wherein said optical
identification mean comprises a laser beam, a transmission
microscope, a confocal microscope, or a fluorescence
microscope.
12. The method according to claim 1, wherein said method for
manipulation is for performing a target analyte assay.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 14/279,867, filed May 16, 2014, which is a divisional of
U.S. application Ser. No. 12/888,187, filed Sep. 22, 2010 (now U.S.
Pat. No. 8,735,172, issued May 27, 2014), which is which is a
continuation of U.S. Ser. No. 10/399,921 filed Apr. 16, 2003 (now
abandoned), which is a U.S. National Stage Application of
International Application No. PCT/EP2001/012194, filed Oct. 19,
2001, which claims priority from European Patent Application No.
00203627.5, filed Oct. 19, 2000, said patent applications hereby
fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the manipulation of microcarriers
for an identification purpose, and more specifically but not
limited to the manipulation of microcarriers having codes written
on them. An example of these microcarriers is described in the
prior filed, and at the time of the priority not yet published
patent application no. PCT/EP00/03280. Said application is hereby
enclosed by reference. Any reference in this disclosure to codes
written "on" the microcarriers includes codes written on the
surface of the microcarriers as well as codes written at an
internal depth of the microcarriers. Identification purposes are
for example the reading or detection and the labeling or encoding
of the microcarrier.
BACKGROUND OF THE INVENTION
[0003] Drug discovery and drug screening in the chemical and
biological arts commonly involve performing assays on very large
numbers of compounds or molecules. These assays typically include
screening chemical libraries for compounds of interest, screening
for particular target molecules in test samples, and testing
generally for chemical and biological interactions of interest
between molecules. The assays described above often require
carrying out thousands of individual chemical or biological
reactions. For example, a drug discovery assay may involve testing
thousands of compounds against a specific target analyte. Any
compounds that are, observed to react, bind, or otherwise interact
with the target analyte may hold promise for any number of
utilities where the observed interaction is believed to be of
significance.
[0004] A number of practical problems exist in the handling of the
large number of individual interactions required in the assays
described above. Perhaps the most significant problem is the
necessity to label and track each reaction. For example, if a
reaction of interest is observed in only one in a group of
thousands of reactions, the researcher must be able to determine
which one of the thousands of initial compounds or molecules
produced that reaction.
[0005] One conventional method of tracking the identity of the
reactions is by physically separating each reaction into an
individual reaction vessel within a high-density array and
maintaining a record of the identity of the individual reactants
were used in each vessel. Thus, for example, when a reaction of
interest is observed in a vessel labeled as number 5 of 1000, the
researcher can refer to the record of reactants used in the vessels
and will learn from the record of vessel 5 what specific reactants
were present to lead to the reaction of interest. Examples of the
high-density arrays referred to above are 384-, 864-, 1,536-,
3,456-, and 9,600-well microtiter plate containers, where each well
of a microtiter plate constitutes a miniature reaction vessel.
Miniaturized reaction wells are used because they conserve space,
allow to increase speed and reduce the cost of reagents used in the
assays.
[0006] The use of microtiter plate containers in chemical and
biological assays, however, carries a number of disadvantages. For
example, the use of the plates requires carefully separating a very
large number of discrete reaction vessels, rather than allowing for
all reactions to take place freely, and often more conveniently, in
one reaction vessel. Furthermore, the requirement that the reaction
volumes be spatially separated carries with it a physical
limitation to the size of microtiter plate used, and thus to the
number of different reactions that may be carried out on the
plate.
[0007] In light of the limitations described above in the use of
microtiter plates, some attempts have been made to develop other
means of tracking individual reactions in high-throughput assays.
These methods have abandoned the concept of spatially separating
the reactions, and instead track the individual reactions by other
means. For example, methods have been developed to carry out
high-throughput assays and reactions on microcarriers as supports.
Each microcarrier may contain one particular ligand bound to its
surface to act as a reactant, and the microcarrier can additionally
contain a "code" that identifies the microcarrier and therefore
identifies the particular ligand bound to its surface. These
methods described above allow for "random processing", which means
that thousands of uniquely coded microcarriers, each having a
ligand bound to their surface, may all be mixed and subjected to an
assay simultaneously. Those microcarriers that show a favorable
reaction of interest between the attached ligand and target analyte
may then have their code read, thereby leading to the identity of
the ligand that produced the favorable reaction.
[0008] A main problem in the prior art is the random position of
microcarriers for identification purposes and therefore lacking
efficiency in the encoding and in the identification. Merely
positioning a encoded microcarrier on a support is not sufficient
for allowing an efficient encoding and identification. Several
documents disclose a positioning on a solid support. The practice
of random processing described above requires accurate encoding of
each of the microcarriers separately, and requires accurate,
reliable, and consistent identification of the codes. Because
assays using random processing rely heavily on the coding of the
microcarriers for their results, the quality of the assays depends
largely on the reliability, readability, unique code, number of
codes, precise dimension and readability of the codes on the
microcarriers. Attempts to code microcarriers are still limited to
differential coloring (Dye-Trak microspheres), fluorescent labeling
(Fluorospheres; Nu-flow), so-called remotely programmable matrices
with memories (IRORI; U.S. Pat. No. 5,751,629), detachable tags
such as oligonucleotides and small peptides (U.S. Pat. No.
5,565,324; U.S. Pat. No. 5,721,099; U.S. Pat. No. 5,789,172), and
solid phase particles that carry transponders (U.S. Pat. No.
5,736,332). WO 98/40726 describes a solid support being an optical
fiber bundle sensor in which separate microspheres carrying
different chemical functionalities may be optically coupled to
discrete fibers or groups of fibers within the bundle. The
functionalities are encoded on the separate microspheres using
fluorescent dyes and then affixed to wells etched in the end of the
bundle. The disclosures of the patents cited above are incorporated
by reference herein.
SUMMARY OF THE INVENTION
[0009] The invention provides in a first aspect a method for the
manipulation of microcarriers wherein an improved position and
orientation is obtained. In its broadest scope, the invention
provides a method for the manipulation for an identification
purpose of a microcarrier comprising the steps of:
[0010] (a) an identification purpose step of the microcarrier;
and
[0011] (b) a positioning and orientation step prior to or during
the identification purpose step.
Although this method requires both a positioning and an orientation
step prior or during the identification purpose step, the invention
surprisingly results in a better, more efficient and more reliable
identification purpose step. A main reason is the lack of
randomness in the degree of freedom of the position of the
microcarrier.
[0012] The present invention is especially suitable for enabling
the reading or writing of a code on a microcarrier, whereby the
code is generated by a spatial modulation created inside the
microcarrier or on its outer surface. This spatial modulation may
be defined as a known arrangement of a finite number of distinct
volume elements located inside or on the surface of the
microcarrier. The known arrangement of distinct volume elements can
be generated by (i) changing one or more properties of the material
in an individual volume element, or (ii) by removing material from
an individual volume element, or (iii) by depositing material on an
individual volume element, or (iv) by leaving an individual volume
element unchanged, or a combination of the above possibilities.
This known arrangement for example, may be such that these volume
elements lie on one or more dimensions such as on a line
arrangement or in a plane. The main object of the invention is then
to position and orient the microcarrier in reference to the writing
instrument and the reading instrument, such that knowledge on the
position and orientation of the microcarrier allows the writing
instrument to generate the code by creating a known arrangement of
a finite number of distinct volume elements, which code can
subsequently be reliably resolved by the reading instrument using
said knowledge on the position and orientation of the microcarrier
on which the code is written. Resolving the code is performed by
measuring the properties of those volume elements that together
constitute the code which is located within the microcarrier or on
the surface of the microcarrier. The orientation may be done with
reference to one, two, or all three axes, depending on the symmetry
of the arrangement of the volume elements. If this known
arrangement is designed to be symmetric around one or more axes,
the microcarrier does not need to be oriented with reference to
rotation around these axes.
[0013] The present invention provides a method for the manipulation
for an identification purpose of a microcarrier comprising the
steps of (a) an identification purpose step of the microcarrier;
and (b) a positioning and orientation step prior to or during the
identification purpose step. According to an embodiment, the
identification purpose step is a detection step for the detection
of an identifiable or encoded microcarrier. According to another
embodiment, the identification purpose step is a labeling step
resulting in an identifiable or encoded microcarrier.
[0014] In another embodiment, the present invention provides a
method for the manipulation for an identification purpose of a
microcarrier, wherein said microcarrier is an encoded microcarrier
encoded by a code written on the microcarrier. According to yet
another embodiment, said microcarrier is encoded by a code written
on the microcarrier by exposing the microcarrier to a high spatial
resolution light source.
[0015] An embodiment of the method according to the invention is a
method for the manipulation for identification purposes of a
population of microcarriers, whereby the positioning and
orientation step further comprises:
[0016] (b. 1) the distribution of the population of microcarriers
in a one-layer system; and
[0017] (b. 2) restricting the rotational movement of the
microcarriers.
[0018] Another embodiment according to the invention is a method,
whereby the distribution of step b. 1 results in a plane
configuration having two dimensions (X, Y).
[0019] Another embodiment according to the invention is a method,
wherein the distribution of step b. 1 results in a line
configuration. A one dimensional configuration results in a faster
detection.
[0020] Another embodiment according to the invention is a method,
wherein the distribution step is caused by transportation of the
microcarriers preferably according to a laminar flow pattern in a
liquid, gaseous or semi-solid environment. Transport of the
microcarrier results in the possibility that the detection means
can have a fixed position, thereby further improving the detection
speed and dismissing any calibration of the detection means.
[0021] Another embodiment according to the invention is a method,
wherein the laminar flow pattern in a liquid environment is
provided in a capillary tube. Besides the laminar flow pattern,
other flow patterns are possible.
[0022] Another embodiment according to the invention is a method,
wherein the distribution step is caused by the positioning of the
microcarriers in a semi-liquid or a liquid support, wherein said
semi-liquid or liquid support may have a differential viscosity or
density or can be composed of two or more semi-liquid or liquid
layer with different viscosity or density. The microcarrier may
then float or be positioned on or in the support at the interface
of a viscosity or a density change. The position may vary according
to the microcarrier density. The absence of a flow in said
distribution of the microcarrier results in the possibility that
the detection means could be mobile.
[0023] Another embodiment according to the invention is a method,
whereby the positioning and orientation step results from a
physical, mechanical, chemical or biological interaction on or near
the microcarrier. As an example, chemical interaction can be any
kind of interaction such as covalent or Vanderwaals interactions. A
biological interaction can be obtained via a direct or indirect
coupling of the microcarrier to a support or to a carrier realized
via e.g. avidin/biotin, antibody/antigen, antibody/hapten,
receptor/ligand, sugar/lectin, complementary nucleic acid (RNA or
DNA, or combination thereof), enzyme/substrate, enzyme/cofactor,
enzyme/inhibitor and/or immunoglobulin/Staphylococcal protein A
interaction.
[0024] Another embodiment according to the invention is a method,
whereby the positioning and orientation step restricts the
rotational movement of the microcarrier as a result of a magnetic
field imposed on the microcarrier.
[0025] Another embodiment according to the invention is a method,
whereby the positioning and orientation step restricts the
rotational movement of the microcarrier as a result of an
electrical field imposed on the microcarrier.
[0026] Another embodiment according to the invention is a method,
whereby the positioning and orientation step results from the
non-spherical configuration of the microcarrier, and more in
particular by the ellipsoidal or cylindrical configuration of the
microcarrier.
[0027] In a second aspect the invention relates to an apparatus for
the manipulation for identification purposes of a microcarrier
comprising means for reading or detection, or identification
purposes such as optical means, electronic means, physical means,
chemical means and magnetic means, or labeling means such as a high
spatial resolution light source, and means for the positioning and
orientation of the microcarriers.
[0028] In an embodiment, the invention relates to an apparatus for
the manipulation for identification purposes of a microcarrier
comprising means for identification purposes such as a microscope
or labeling means such as a high spatial resolution light source,
and means for the positioning and orientation of the
microcarriers.
[0029] An embodiment according to the invention is an apparatus,
whereby the means for positioning and orientation of the
microcarriers comprises a solid support comprising a number of
wells each suitable for housing at least one microcarrier and
rotation restriction means.
[0030] An embodiment according to the invention is an apparatus,
whereby the means for positioning and orientation of the
microcarriers comprises a semi-liquid or a liquid support and
rotation restriction means. According to another embodiment, said
semiliquid or liquid support may have a differential viscosity or
density or can be composed of two or more semi-liquid or liquid
layers with different viscosity or density. The microcarrier may
then float or be positioned and oriented on or in the support at
the interface of a viscosity or a density change. The position and
orientation may vary according to the microcarrier density.
[0031] Another embodiment according to the invention is an
apparatus, whereby the rotation restriction means are provided via
a magnetic and/or electrical field.
[0032] Another embodiment according to the invention is an
apparatus further comprising a reservoir suitable for containing a
population of microcarriers, which reservoir is connectable to a
capillary tube and pressure differential means for providing a
laminar flow pattern in the capillary tube.
[0033] Another embodiment according to the invention is an
apparatus, whereby further a magnetic and/or electrical field is
provided for the restriction of the rotation of the
microcarriers.
[0034] In a third aspect of the invention, a microcarrier is
provided useful in the method of the first aspect which
microcarrier is encoded by a code written on the microcarrier.
[0035] An embodiment according to the invention is a microcarrier,
whereby the encoded microcarrier is characterized in that the code
has been written by exposing the microcarrier to a high spatial
resolution light source.
[0036] An embodiment according to the invention is a microcarrier,
whereby the encoded microcarrier is characterized in that the code
has been written by deposition of material on the surface or at the
internal depth of said microcarrier.
[0037] Another embodiment according to the invention is a
microcarrier further comprising a net electrical charge, an
electrical dipole moment or a magnetic dipole moment. The
microcarrier may also be ferro-, ferri- or paramagnetic as such, or
has an anisotropy in its shape, an anisotropy in its mass
distribution or any combination of these features.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In FIG. 1 a spherical microcarrier is shown with a magnetic
dipole moment coming from magnetic material inside. The magnetic
field caused by the coils holds the microcarrier into place and
orients it at the same time. When the magnetic material is placed
outside the center of the microcarrier as is illustrated, a
complete 3D orientation is obtained because of the gravitation and
the magnetic attraction.
[0039] In FIG. 2 spherical microcarriers are shown with a magnetic
dipole moment transported by a fluid flowing through a capillary
with velocity v. Two coils are provided that can induce a magnetic
field parallel to the capillary. Outside the magnetic field, the
carriers will rotate because of the friction of the fluid. Inside
the coils, the magnetic field will try to align the dipole moment
antiparallel to itself, thus eliminating the rotation in the
direction of the movement of the particle.
[0040] In FIG. 3 a schematic representation is shown of the
capillary system used to examine the positioning of microcarriers
transported by a laminar flow inside a capillary using a confocal
microscope.
[0041] FIG. 4 shows a confocal image with one particle flowing
inside the capillary. The arrow at the right indicates the inside
dimension of the capillary: 80 .mu.m. The capillary and the water
are completely dark since they do not emit fluorescent light. The
field of view is 0.92 mm.times.0.10 mm One particle is seen as a
set of three separate lines rather than an actual disk because of
the velocity of the particles and the particular way a confocal
image is taken.
[0042] Since FIG. 4 is one of the pictures of a complete time
series, the particle of FIG. 4 can indeed be found in FIG. 5 at the
same position since it is the addition of all the pictures from the
time series into one picture. From FIG. 5 it becomes clear that the
particles indeed have a certain position when being transported by
a laminar flow through the capillary (Pressure about 0.05 atm,
wherein about as cited herein refers to plus or minus 15%). The
particles follow one straight line at a constant distance from the
capillary wall (the line seems to be tilted but that's because the
capillary itself was positioned that way in the field of view).
[0043] FIG. 5 shows a composite picture of all the individual
pictures of one time series, wherein all the particles that have
passed in that time interval (pressure about 0.05 atm) are shown.
It is clear that the particles all move along one straight line at
a constant distance from the wall (but not in the center) of the
capillary.
[0044] FIGS. 6 and 7 also show a composite picture from two
different time series. FIG. 6 shows a composite picture with the
same positioning at a higher pressure (about 0.1 atm). FIG. 7 shows
a composite picture with the same positioning at a higher pressure
(about 0.15 atm). The only difference between FIGS. 5, 6 and 7 is
the applied pressure (about 0.05, 0.1 and 0.15 atm respectively),
and thus the fluid velocity. The positioning is therefore valid at
higher pressures as well.
[0045] FIG. 8 shows a confocal image of the top of a
green-fluorescent 40 .mu.m microsphere coated with ferromagnetic
CrO2 particles.
[0046] FIG. 9 shows a confocal image of the central plane of 40
.mu.m green fluorescent ferromagnetic-coated particles.
[0047] FIG. 10 shows a confocal image of a simple pattern that was
bleached at the central plane of a ferromagnetic-coated
particle.
[0048] FIG. 11 shows an image of a 28 .mu.m photochromic
microsphere (before UV illumination) with red light in transmission
light mode. The microscope was focused at the central plane.
[0049] FIG. 12 shows a transmission image of the microsphere after
photochroming of a 3 .mu.m square in said microsphere.
[0050] FIG. 13 shows a transmission image of a completely colored
and transparent microsphere.
[0051] FIG. 14 shows a confocal image of three `dotcodes`
microsphere (left) and a normalized intensity profile measured
through the middle code (right). Each division along the image axes
is 2 .mu.m.
[0052] FIG. 15 shows a confocal image of a photobleached
microsphere in DMSO (left). The second image on the right was taken
three hours later.
[0053] FIG. 16 shows a schematic representation of ferro-magnetic
microcarriers in a support consisting of two liquids or semi-liquid
of different density. Two coils are provided that can induce a
magnetic field. Inside the coils, the magnetic field will try to
align the dipole moment antiparallel to itself, thus positioning
and orienting the microcarrier in a specific manner.
[0054] FIG. 17 shows a schematic representation of device
comprising a Confocal Laser Scanning Microscope (CLSM) coupled to a
powerful laser combined with a fast optical switch. The light
source used is a Spectra Physics Stabilite 2017 Ar ion laser, tuned
at a single wavelength, e.g. 488 nm. The AOM causes the laser light
to be diffracted into multiple beams. The first order beam is then
coupled to an optical fiber. The AOM is controlled by a PC and
dedicated software to switch the intensity of the first order beam
between two levels: a weak imaging beam and a strong bleaching
beam. The fiber end is coupled into a `dual fiber coupling` so that
the light coming out of the fiber can be combined with the light
from another laser (but is not used in the bleaching experiments).
Finally the light enters the confocal scanning laser microscope
(CSLM) and is focused on the sample. A bleaching pattern can be
designed in dedicated software. While taking an image, which is
done by scanning the laser light in a raster pattern, dedicated
software controls the optical switch in such a way that low and
high power laser light reaches the sample according to the designed
pattern.
[0055] FIG. 18 shows a confocal image of a bleached barcode, using
three widths and two intensity levels, in the central plane of a 45
micron polystyrene fluorescent microsphere.
[0056] FIG. 19 shows a confocal image of a bleached barcode, using
8 different intensity levels, in the central plane of a polystyrene
fluorescent microsphere (right), and a normalized intensity profile
measured through the middle code (right).
[0057] FIG. 20 shows two confocal images of microspheres wherein
bar codes of different geometry e.g. letters or numbers, are
bleached.
[0058] FIG. 21 shows images of 40 micron ferromagnetic fluorescent
beads flowing in a flow cell.
[0059] FIG. 22 represents a cylindrically symmetric bead wherein
the codes are written in a circle around the Z' axis, with a
control pattern which indicates the beginning of the code.
[0060] FIG. 23 represents a spherical bead wherein code bits are
written along the symmetry axis of said bead.
[0061] FIG. 24 shows magnetic beads flowing through a capillary,
and passing through the focus of a laser beam. Coils, carrying an
electric current, create a magnetic field and orient the beads
along the direction of motion.
[0062] FIG. 25 shows an example of a coding scheme using 4
different intensities, each intensity represented by a color and a
number from 0 to 3. This coding scheme has 28 characters,
symbolically represented by the 26 letters of the Roman alphabet
and two extra punctuation marks. Each character consists of 4
coding elements (i.e. 4 possible intensities (or colors)) with the
extra condition that no two identical elements may follow each
other, not even when two characters are placed next to each
other.
[0063] FIG. 26 represents a capillary surrounded by a coil
generating a variable magnetic field B and a bead containing a
closed conductor with induced magnetic field B', which is parallel
when the magnetic field B is increasing.
[0064] FIG. 27 represents a bead containing a closed conductor
flowing in a capillary that is placed between two magnetic plates
and submitted to a magnetic field perpendicular to the flow
direction.
[0065] FIG. 28 represents a schematic drawing of an experimental
set-up wherein a reservoir containing ferromagnetic green
fluorescent microsphere suspension was placed on a Bio-Rad MRC1024
confocal microscope attached to an inverted microscope so that it
was possible to use a Nikon 60.times. water immersion objective
lens to look at the beads through the bottom microscope slide. The
microspheres were illuminated by a 488 nm laser beam. The
microspheres were oriented by an external magnetic field B induced
by a strong permanent magnet positioned 20 cm from the
reservoir.
[0066] FIG. 29 represents images of ferromagnetic microspheres
wherein an arrow was bleached at the central plane said
microsphere. In image (a), the microsphere was oriented in an
external magnetic field of a magnet. In images (b-i), the
microsphere was oriented in a second moving external magnetic
field. In images (j-1), the microsphere returned to the original
orientation after taking away the second magnet.
[0067] FIG. 30 represents images of ferromagnetic microsphere
wherein an arrow was bleached at the central plane said
microsphere. In image (a), the microsphere was oriented in an
external magnetic field of a magnet. In images (bj), the same
magnet was used to rotate the microsphere by moving 360 around the
reservoir and placing it back in its exact original position. Image
j shows that the microsphere did not return to its original
orientation due to a relatively strong polymer-glass interaction.
In images (k-1), the microsphere was loosened by quickly moving a
second magnet near the reservoir and was observed to return
immediately to its original orientation.
[0068] FIG. 31: Drawings (a, b, i, j) represent schematic field of
views of a microcarrier flowing in front of a microscope objective.
Drawings (a, i) show the field of view before the microcarrier
arrives into the focused laser beam for reading/writing the
code.
[0069] Drawings (bj) show the field of view with a microcarrier at
the focus position.
[0070] Drawings (c, d, e, f, g, h) represent side view of the
microscope objective placed in front of a flow cell. Drawing (c)
shows the case where the focus of the reading/writing laser beam
scans along the symmetry axis of the microcarrier. Drawing (f)
shows the case where the focus of the reading/writing the laser
beam scans below the symmetry axis of the microcarrier. Drawing (d,
g) represents the case where an auxiliary laser beam illuminates a
microcarrier and produces a shadowing effect on the other side of
said microcarrier.
DETAILED DESCRIPTION OF THE INVENTION
[0071] Prior to discussing embodiments on how a microcarrier can be
positioned and oriented in a certain way, it is necessary to
describe the third aspect of the invention, i.e. the different
types of microcarriers that can be used.
[0072] As used herein a "microcarrier" also termed "microsphere",
"bead" or "microparticle" relates to a reaction volume or a support
which may be made from, for example, any materials that are
routinely employed in high-throughput screening technology and
diagnostics. For example, the microcarriers may be made from a
solid, a semi-solid, or a combination of a solid and a semi-solid,
and can be supports such as chemical and biological assays and
syntheses. Non-limiting examples of these materials include
cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, agar,
pore-glass, silica gel, polystyrene, brominated polystyrene,
polyacrylic acid, polyacrylonitrile, polyamide, polyacrolein,
polybutadiene, polycaprolactone, polyester, polyethylene,
polyethylene terephthalate, polydimethylsiloxane, polyisoprene,
polyurethane, polyvinylacetate, polyvinylchloride,
polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene,
polyvinylidene chloride, polydivinylbenzene,
polymethylmethacrylate, polylactide, polyglycolide, poly
(lactide-co-glycolide), polyanhydride, polyorthoester,
polyphosphazene, polyphosophaze, polysulfone, grafted copolymer
such as polyethylene glycol/polystyrene, cross-linked dextrans,
methylstyrene, polypropylene, acrylic polymer, paramagnetic,
carbon, graphite, polycarbonate, polypeptide, hydrogels, liposomes,
proteinaceous polymer, titanium dioxide, latex, resin, lipid,
ceramic, charcoal, metal, bentonite, kaolinite, rubber,
polyacrylamide, latex, silicone, e.g., polydimethyldiphenyl
siloxane, dimethylacrylamide, and the like or combinations thereof
are acceptable as well.
[0073] Preferred materials include latex, polystyrene, and
cross-linked dextrans. The microcarriers may also be prokaryotic or
eukaryotic cells or even some viruses. Said microcarriers may be of
any shapes and sizes that should be suitable for encoding,
positioning and orienting and further identification thereof. For
example, the microcarriers may be in the form of spheres, or in the
form of beads that are not necessarily spherical. The microcarriers
may be, for example, cylindrical or oval. When spherical in shape,
the microcarriers may have, for example, a diameter of 0.5 to 300
.mu.m. The microcarrier may also have a diameter of 1 to 200 .mu.m.
Other examples of suitable sizes for said microcarrier could range
from 10 to 90 .mu.m. [0074] The microcarrier can have a net
electric charge or an electric dipole moment. [0075] The
microcarrier can be magnetic or have a magnetic dipole moment.
[0076] The microcarrier can have a certain anisotropy in its shape.
For example, the microcarrier can have an axial symmetric shape,
e.g. rod shaped, ellipsoidal or cylindrical. [0077] The
microcarrier can have a certain anisotropy in its mass
distribution. For example, one region of the particle can be more
dense so that one side is heavier than the other. Also, when a
microcarrier has an asymmetric shape, this will be reflected by an
asymmetric mass distribution as well. [0078] The encoded
microcarrier according to the teaching in PCT/EP00/03280. [0079]
The microcarrier can be a combination of some or all of the above
mentioned features.
[0080] The microcarrier may have different properties such as
optical transparency, ferromagnetism, and can have functional
surface group for binding ligands such as proteins. The
microcarrier may also contain one or more dyes such as
fluorophores, luminophores and the like, or a combination thereof.
The ferromagnetism can be introduced by either in situ
precipitation of ferromagnetic material or coating with a polymer
containing ferromagnetic nanoparticles. Examples of ferromagnetic
materials include but are not limited to Cr.sub.2O.sub.3,
Fe.sub.2O.sub.3, Fe.sub.3O.sub.4, Ni- and Co-metals, other metal
oxides and metals. The compounds can be introduced during the
microcarrier preparation or in a post modification step such as
soaking or coating. The ferromagnetic material can be present in
said microcarrier at a concentration ranging from 0.1 to 50% by
weight, or at a concentration ranging from 0.5 to 40%, or for
example at a concentration ranging from 1 to 30%.
[0081] The codes written on the microcarriers according to the
teaching in PCT/EP00/03280 may be of any geometry, design, or
symbol that can be written and read on the microcarriers. For
example, the codes may be written as numbers or letters, or as
codes in the form of symbols, pictures, bar codes, ring codes, or
three-dimensional codes. Ring codes are similar to bar codes,
except that concentric circles are used rather than straight lines.
A ring may contain, for example, the same information as one bar.
The codes may be written on the surface of the microcarriers or at
an internal depth of the microcarriers. For example, the codes may
be written at an internal depth of the microcarriers, and more
particularly in the center plane of the microcarriers. Depending on
the shape of the microcarriers, the center plane may be a
preferable location for writing the code because it may provide the
largest surface area available for writing. Furthermore, for
microcarriers having curved surfaces, it may be more advantageous
to write the codes at an internal depth rather than on the curved
surfaces. This is because it may often be more convenient to write
and read the codes on a flat plane rather than on a curved
surface.
[0082] The codes can be written on the microcarriers, for example,
by using a high spatial resolution light source, such as a laser, a
lamp, or a source that emits X-rays, a and 13 rays, ion beams, or
any form of electromagnetic radiation. The codes can also be
written on the microcarriers through photochroming or chemical
etching. A convenient method for writing the codes is through the
use of a high spatial resolution light source, and in particular a
laser or a lamp in combination with a confocal microscope. The
codes may also be written at an internal depth of the microcarrier
by using the abovedescribed methods.
[0083] The codes can also be written by deposition of material on
or in said microcarrier. Examples of method of deposition include
but are not limited to laser deposition and electrochemical
deposition. Examples of material which can be used for said
deposition include but is not limited to any organic compound or
material; any inorganic compound or material; a particulate layer
of material or a composite material; polymeric materials;
crystalline or non-crystalline materials; amorphous materials or
glasses; carbonaceous material such as, for example, graphite
particles or carbon nanotubes; metallic material, such as, for
example, gold, silver, copper, nickel, palladium, platinum, cobalt,
rhodium, iridium; any metal chalcognide; metal oxide such as for
example, cupric oxide, titanium dioxide; metal sulfide, metal
selenide, metal telluride, metal alloy, metal nitride, metal
phosphide, metal antimonide, semiconductor, semi-metal. Said
material can be deposited in the form of particles such as micro or
nanoparticles. For example, the particles are nano-particles, that
is, typically, particles in the size range of 10 nm to 1000 nm.
[0084] Knowledge on the position and orientation of the
microcarrier is essential to facilitate the writing and/or reading
of the above written codes involves, in particular when these
identification purpose steps are performed in a high throughput
application. Knowledge on position and orientation of the
microcarrier will improve even more the identification purpose
steps.
[0085] The microcarriers may contain a photosensitive substance.
For example, the microcarrier may contain a bleachable substance,
and the codes on the microcarriers may be in the form of bleached
patterns within the bleachable portions of the microcarriers. The
microcarriers may contain the bleachable substance either on the
surface of the microcarrier or also within the body of the
microcarrier. Any reference in this application to the bleaching of
substances "on" the microcarriers includes bleaching at the surface
of the microcarrier as well as bleaching at. an internal depth of
the microcarriers. Preferred bleachable substances include
bleachable fluorescent or electromagnetic radiation absorbing
substances. The microcarriers may contain bleachable luminophores.
Examples of luminophores that can be used include fluorescers,
phosphorescers, or scintillators. Bleachable chemiluminescent,
bioluminescent, or colored substances may be used. Non-limiting
examples of bleachable substances are listed herein:
3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine,
5-Hydroxy Tryptamine (5-HT), Acid Fuhsin, Acridine Orange, Acridine
Red, Acridine Yellow, Acriflavin, AFA (Acriflavin Feulgen SITSA),
Alizarin Complexon, Alizarin Red, Allophycocyanin, ACMA,
Aminoactinomycin D, Aminocoumarin, Anthroyl Stearate, Aryl- or
Heteroaryl-substituted Polyolefin, Astrazon Brilliant Red 4G,
Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL,
Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9
(Bisaminophenyloxadiazole), BCECF, Berberine Sulphate,
Bisbenzamide, BOBO 1, Blancophor FFG Solution, Blancophor SV,
Bodipy Fl, BOPRO 1, Brilliant Sulphoflavin FF, Calcien Blue,
Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor
White ABT Solution, Calcophor White Standard Solution,
Carbocyanine, Carbostyryl, Cascade Blue, Cascade Yellow,
Catecholamine, Chinacrine, Coriphosphine O, Coumarin,
Coumarin-Phalloidin, CY3.1 8, CYS. 1 8, CY7, Dans (1-Dimethyl Amino
Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic
Acid), Dansyl NH--CH3, DAPI, Diamino Phenyl Oxydiazole (DAO),
Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride,
Diphenyl Brilliant Flavine 7GFF, Dopamine, Eosin, Erythrosin ITC,
Ethidium Bromide, Euchrysin, FIF (Formaldehyde Induced
Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, fluorescein
isothiocyanate ("FITC"), Fura-2, Genacryl Brilliant Red B, Genacryl
Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow SGF,
Gloxalic Acid, Granular Blue, Haematoporphyrin, Hoechst 33258,
Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF,
Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH,
Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant
Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green
Pyronine Stilbene), Mithramycin, NBD Amine, Nile Red,
Nitrobenzoxadidole, N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) diethyl
amine (NODDI, Noradrenaline, Nuclear Fast Red, Nuclear Yellow,
Nylosan Brilliant Flavin EBG, Oregon Green, Oxazine, Oxazole,
Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR
Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,
Phthalocyanine, phycoerythrines, Phycoerythrin R, Polyazaindacene
Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow,
Propidium Iodide, Pyronine, Pyronine B, Pyrozal Brilliant Flavin
7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine
6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB,
Rhodamine BG, Rhodamine WT, Rose Bengal, Serotonin, Sevron
Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B,
Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene
Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can
C, Sulpho Rhodamine G Extra, Tetracycline, Texas Red, Thiazine Red
R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol
Orange, Tinopol CBS, TOTO 1, TOTO 3, True Blue, Ultralite, Uranine
B, Uvitex SFC, Xylene Orange, XRITC, YO PRO 1, or combinations
thereof. Optionally such bleachable substances will contain
functional groups capable of forming a stable fluorescent product
with functional groups typically found in biomolecules or polymers
including activated esters, isothiocyanates, amines, hydrazines,
halides, acids, azides, maleimides, alcohols, acrylamides,
haloacetamides, phenols, thiols, acids, aldehydes and ketones. With
regard to the volume of substance that may be bleached within the
microcarriers, one example of such a volume is between 0.01 cubic
nanometer and 0.01 cubic millimeter of the microcarrier, another
example of such a volume is between 1 cubic nanometer and 100 000
cubic micrometer, yet another example of such a volume is between
10 000 and 10 000 cubic micrometer, another example of such a
volume is between 0.01 cubic micrometer and 1000 cubic micrometer.
The bleachable substances should be chosen so that, when bleaching
occurs, the code remains on the microcarrier at least for the
period of time that is desired for the use of the microcarriers and
any necessary reading of the codes. Said code should at least be
preserved for the duration of the assay, wherein the microcarrier
is used. This functional life of the code may be from several
minutes up to several months, even up to several years depending on
the assay to be performed. Thus, a certain amount of diffusion of
nonbleached molecules into the bleached areas is acceptable as long
as the useful life of the code is preserved. As used hereinafter
the terms fluorescent dye, fluorescer, fluorochrome, or fluorophore
are used interchangeably and bear equivalent meanings.
[0086] Codes bleached on microcarriers may also be written to have
different intensities of fluorescence or color within bleached
areas of the microcarriers. For example, a bleached coding may
contain several different degrees of bleaching, thereby having
several different intensities of fluorescence within the bleached
region as a whole. Thus, microcarriers may be encoded not only by
the geometry of the pattern bleached on the microcarriers, but also
by the use of different fluorescent intensities within the
pattern.
[0087] The codes may be written on the microcarriers through the
use of scanning microphotolysis ("SCAMP"). The technical features
of SCAMP were first described in P. Wedekind et al., "Scanning
microphotolysis: a new photobleaching technique based on fast
intensity modulation of a scanned laser beam and confocal imaging,"
Journal of Microscopy, vol. 176, pp. 23-32 (1994), the content of
which is incorporated by reference herein. Photobleaching is a
well-known phenomenon referring to the fading of colors due to the
fact that certain wavelengths of light when shone on a given
pigment will cause the pigment's molecules to resonate and
eventually break down. This is also the reason why fluorescent
molecules often tend to bleach when excited by a powerful laser
beam of specific wavelength. The codes may be photobleached using a
conventional (non-scanning) light microscope, wherein a stationary
(laser) light beam is focused on the sample during the bleaching
process. The stationary position of the (laser) light beam during
the bleaching process results in a photobleached area that has a
circular geometry. Although non-scanning light microscopes
technically yield an irradiated area of 2 .mu.m or less in
diameter, broadening of the bleach spot often occurs due to the
stationary laser beam. This results in large circular bleached
spots that are from one .mu.m to 35 .mu.m, typically from 10 .mu.m
to 20 .mu.m in diameter or even larger such as 15 .mu.m-35 .mu.m.
The availability of laser light scanning microscopes opened new
opportunities for microphotolysis methods. The combination of
photolysis, beam scanning, and confocal microscopy lead to the
development of SCAMP. In SCAMP, bleaching occurs during scanning a
sample by switching between low monitoring and high photobleaching
laser intensity levels in less than a microsecond using an
intensity modulation device such as an acousto-optical modulator
("AOM"). The combination of bleaching during scanning and the use
of the AOM, which generates extremely short bleaching pulses,
prevents the broadening of the bleach spot that occurs in
conventional microphotolysis due to longer photobleaching times and
the stationary laser beam. SCAMP allows for bleaching spots at the
resolution limit of the objective lens used.
[0088] Writing codes on microcarriers may also involve bleaching
the microcarriers to produce different levels of intensity in the
bleached code. In addition to conveying the information in the
design of the code itself, information can also be conveyed by
different intensities within the bleached patterns. The ability to
encode the microcarriers with different intensities may permit
smaller codes on the microcarriers, thus saving space, but still
conveying the same number or more of unique identifiers to code
microcarriers. As an example, it is possible to bleach four
different intensities in the beads. This can be accomplished in a
number of ways, for example, by repeated bleaching over some
portions of the bead relative to others, or by dissipating
different levels of acoustic power into an AOM to produce a
plurality of different laser powers that will create bleached
patterns having different intensities based on the power of laser
light used for each portion of the code.
[0089] The code may also be written by photochroming. Photochromic
materials of interest undergo an irreversible change in light
absorption that is induced by electromagnetic radiation, most
common applications involve irreversible changes in color or
transparency on exposure to visible or ultraviolet light. This is
often seen as a change in the visible spectrum (400-700 nm), and
can be rapid or very slow. A code could then be written in the
inside of a bead that contains a photochromic dye, with focused UV
light. There are two major classes of photochromic materials,
inorganic and organic. Examples of the inorganic type are the
silver halides. The organic photochromic systems can be subdivided
according to the type of reaction. The photochromic compounds can
be soluble in normal organic solvents such as hexane, toluene,
acetone and DMSO. A non-limiting example is the use of a dispersion
in polystyrene at concentration as high as 99%. Said compounds are
also stable in low as well as high pH and are stable over a wide
range of temperature. The photochromic compounds of interest are
irreversible, wherein the color change is not reversed when the
illumination is absent. Most of the interesting compounds are
thermally irreversible, i.e. they do not change back to the
original colorless state at room temperature. Advantageous
photochromic dyes are those that cannot be bleached back to their
original state. Non-limiting examples of photochromic compounds of
interest include derivatives of diarylethenes with heterocyclic
aryl groups such as furan, indole, thiophene, selenophene, thiazole
aryl groups, monomeric and polymer forms of said compounds and the
like. Examples of compounds include 1, 2-dicyano-1, 2-bis
(2,4,5-trimethylthiophen-3-yl) ethene, 2,3-bis
(2,4,5-trimethylthiophen-3-yl) maleic anhydride, 1, 2-bis
(2,4-dimethyl-5-phenylthiophen-3-yl) perfluorocyclopentene, 1,2-bis
(3-methyl-2-thienyl) perfluorocyclopentene, 1, 2-di
(2-dimethyl-5-phenylthiophen-3-yl) perfluorocylopentene, 1,2-bis
(2-methyl-3-thienyl) perfluorocyclopentene, 1,2-bis
(2,5-dimethyl-3-thienyl) perfluorocyclopentene,
2-(1-octyl-2-methyl-3-indolyl)3-(2, 3,5-trimethyl-3-thienyl) maleic
anhydride, 2-(2'-methoxybenzo [b]
thiophen-3-yl)3-(2-dimethyl-3-indolyl) maleic anhydride, 1, 2-bis
(2-methyl-5-phenyl-3-thienyl)perfluoro cyclopentene, 1,2-bis
(2,4-dimethyl-5-phenyl-3-thienyl) perfluoro cyclopentene, 1, 2-bis
(2-methyl-6-nitro-1-benzothiophen-3-yl) perfluorocyclopentene, 1,
2-bis (2-methoxy-5-phenyl-3-thienyl) perfluorocyclopentene and the
like. The photochromic compounds can be added to the microsphere in
an amount ranging from 0.1 to 100%. In another embodiment, the
photochromic compounds can be added to the microsphere in an amount
ranging from 0.1 to 80%. In yet another embodiment, the
photochromic compounds can be added to the microsphere in an amount
ranging from 0.1 to 50%. The photochromic compound can also be
added in an amount ranging from 1 to 3%. Photochroming is
potentially faster and easier to control than the bleaching of
fluorescent dye, because the coloration is normally linear with
incident power. Readout is simplified because it is sufficient to
take an image that reveals the code on a transparent background. A
pattern written by localized bleaching in a fluorescent bead, on
the other hand, would require a confocal microscope to detect it.
It is possible to encode up to several tens of thousand
microcarriers per second by photochroming.
[0090] Other methods for writing codes can also be used, such as
code writing by changing the refractive index or by selective
spectral photobleaching. In the case of spectral photobleaching the
microcarriers may contain one or more different dyes each dye
having unique spectral characteristics, and wherein one or more of
these dyes may be bleached at different intensities.
[0091] Moreover, the microcarriers may be functionalized, i.e. said
microcarrier may contain one or more ligands or functional units
bound to the surface of the microcarriers. A large spectrum of
chemical and biological functionalities may be attached as ligands
to said microcarriers. These functionalities include all
functionalities that are routinely used in high-throughput
screening technology and diagnostics. The choice of the ligand will
vary according to the analytes to target. The ligand may for
instance be an organic entity, such as a single molecule or an
assemblage of molecules. Examples of functionalization include the
attachment, often via a linker, to an antibody or antibody
fragment, to an oligonucleotide or to a detectable tag. In some
embodiments, the microcarrier can have multiple functionalities. As
used herein, the term functional unit is meant to define any
species that modifies, attaches to, appends from, coats or is
covalently or non-covalently bound to the surface of said
microcarrier. Functionalized, as defined herein, includes any
modification of the surface of the microcarrier as covalently or
non-covalently modified, derivatized, or otherwise coated with an
organic, inorganic, organometallic or composition monolayer,
multilayer, film, polymer, glass, ceramic, metal, semi-metal,
semiconductor, metal oxide, metal chalcoginide, or combinations
thereof. While such functionalization may occur most commonly at
the outer surface of the microcarrier, it also may occur at
interior surfaces of the microcarrier, as it might in the case in a
porous or hollow microcarrier. Examples of target analytes for the
microcarrier-bound ligands include antigens, antibodies, receptors,
haptens, enzymes, proteins, peptides, nucleic acids, drugs,
hormones, pathogens, toxins, or any other chemicals or molecules of
interest. The ligands or functional units may be attached to the
microcarriers by means conventionally used for attaching ligands to
microcarriers in general, including by means of a covalent bound
and through direct attachment or attachment through a linker.
Furthermore, the microcarriers can be further functionalized in a
variety of ways to allow attachment of an initial reactant with
inorganic or organic functional group, including but not limited
to, acids, amines, thiols, ethers, esters, thioesters, thioethers,
carbamates, amides, thiocarbonates, dithiocarbonates, imines,
alkenes, alkanes, alkynes, aromatic groups, alcohols, heterocycles,
cyanates, isocyanates, nitriles, isonitriles, isothiocyanates, and
organocyanides, or combinations thereof; any inorganic coordination
complex, including but not limited to 2-, 3-, 4-, 5-, 6-, 7-, 8-
and 9-coordinate complexes; any organometallic complex, including
but not limited to species containing one or more metal-carbon,
metal-silicon, or metal nitrogen bonds.
[0092] In another embodiment, the functional unit or
functionalization of the microcarrier comprises a detachable tag. A
detachable tag is any species that can be used for detection,
identification, enumeration, tracking, location, positional
triangulation, and/or quantitation. Such measurements can be
accomplished based on absorption, emission, generation and/or
scattering of one or more photons; absorption, emission generation
and/or scattering of one or more particles; mass; charge; faradaic
or non-faradaic electrochemical properties; electron affinity;
proton affinity; neutron affinity; or any other physical or
chemical property, including but not limited to solubility,
polarizability, melting point, boiling point, triple point, dipole
moment, magnetic moment, size, shape, acidity, basicity,
isoelectric point, diffusion coefficient, or sedimentary
coefficient. Such molecular tag could be detected or identified via
one or any combination of such properties.
[0093] The present invention further relates to a method for the
manipulation for an identification purpose of a microcarrier,
comprising the steps of
[0094] a) positioning and orienting said microcarrier and
[0095] b) encoding said microcarrier by writing a code thereon,
[0096] c) allowing a target-analyte reaction on or in said
microcarrier,
[0097] d) positioning and orienting said microcarrier, and
[0098] e) identifying said microcarrier,
[0099] whereby step (c) may also preceed step a).
[0100] Said method may conveniently also include a step whereby
selectively those microcarriers are identified on which a
target-analyte reaction of particular interest occurred. For
instance, microcarriers with a target-analyte reaction of interest
may be separated from the rest of the microcarriers, and those
microcarriers may then be subjected to steps d) and e) of the above
method.
[0101] According to another embodiment the present invention
relates to a method, wherein the positioning and orientation step
results from a physical, mechanical, chemical or biological
interaction on or near said microcarrier. Another embodiment
according to the invention is a method, whereby the positioning and
orientation step restricts the rotational movement of the
microcarrier as a result of a magnetic field imposed on the
microcarrier. Another embodiment according to the invention is a
method, whereby the positioning and orientation step restricts the
rotational movement of the microcarrier as a result of an
electrical or a magnetic field imposed on the microcarrier. Another
embodiment according to the invention is a method, whereby the
positioning and orientation step results from the non-spherical
configuration of the microcarrier, and more in particular by the
ellipsoidal or cylindrical configuration of the microcarrier.
Another embodiment according to the invention is a method, whereby
the positioning and orientation step results from the anisotropy in
the mass distribution of the microcarrier. In such a case, an axial
positioning and orientation in a gravitational as well as in a
centrifugal manner may be obtained. Another embodiment according to
the invention is a method, whereby the positioning and orientation
step results from one or more combination of the above-described
features. For example, a combination of magnetic forces and
anisotropy in shape, combination of magnetic forces and anisotropy
in weight, etc.
[0102] According to another embodiment, the positioning and
orientation step can occur in a flow cell in a flow cytometer. The
term flow cytometer is used herein for any apparatus that creates a
single file flow of particles within a fluid and measures
fluorescence from the particles. The sample fluid can be
constrained within a narrow flow channel or by hydrodynamic
focusing within a sheath fluid. For example, to position and orient
particles in the flow, it is possible to employ the principle of
hydrodynamic focusing in a so-called sheath flow cell or chamber.
The sample fluid containing the particles can be injected into the
center of a faster surrounding flow, the sheath flow, in front of a
convergent nozzle. As the liquid passes through the convergence
into the observation area, the sample flow is accelerated,
stretched out and centered to pass through the focus of the
observation system. The fluid may be air, water, solvent, buffer
and the like. Different type of flow cells can be used,
non-limiting examples are cited herein: cells with a closed optical
chamber which can be used to detect fluorescence, scattering or
light extinction, particles sorters using open-ended flow cells
that divide the flow into electrically charged droplets, which can
be deflected by an electrical field into containers to sort
particles according to their fluorescent signal for example, flow
cells that can have asymmetric nozzles or have asymmetric
constrictions in the flow chamber to orient non-spherical particles
onto the optical axis. Another example includes a flow cell
apparatus as described in U.S. Pat. No. 5,690,895 incorporated
herein by reference.
[0103] According to another embodiment, the positioning and
orientation step may also occur by the dielectrophoretic caging of
microcarriers. Dielectric particles, such as polystyrene
microcarrier, suspended in a liquid can be manipulated by a
high-frequency electrical field in a microelectrode cage. For
example, microcarrier may be brought into a specially designed flow
cell with a number of electrodes; by modifying the amplitude,
frequency and phase of the fields, the microcarrier can be
positioned and oriented.
[0104] According to another embodiment, the positioning and
orienting of the microcarriers may also occur in a semi-liquid or a
liquid support, wherein said semi-liquid or liquid support may have
a differential viscosity or density or can be composed of two or
more semi-liquid or liquid layer with different viscosity or
density. The microcarrier may then float or be positioned on or in
the support at the interface of a viscosity change. The position
and orientation may vary according to the microcarrier density. The
absence of a flow in said distribution of the microcarrier results
in the possibility that the detection means could be mobile.
[0105] According to another embodiment, the positioning and
orientation step may also occur by for example: trapping the
microcarrier in strongly focused laser beams, so-called "laser
tweezers." The positioning and orientation step may also occur
using acoustic waves such as ultrasonic trapping, wherein the
microcarrier is trapped in standing waves in a liquid. A
"microlathe" which is usually used to modify the shape of particles
with a UV laser, can also be used to position and orient the
microcarrier for the identification step.
[0106] According to another embodiment, the positioning and
orientation step may also occur using two or more combination of
the above-described method for positioning and orienting.
[0107] According to an embodiment, the encoding step can be
performed as described above in the description of the
microcarrier. The encoding process, thus, can be selected from the
group comprising photochroming, chemical etching, material
deposition, photobleaching, or exposing said microcarrier to a high
spatial resolution light source, such as a UV laser. According to
another embodiment, the encoding step is performed by
photochroming. According to another embodiment, the encoding step
is performed by photobleaching.
[0108] According to an embodiment, the encoding comprises the
writing of a code on a microcarrier whereby the code is generated
by spatial modulation created inside the microcarrier or on its
outer surface. According to yet another embodiment, said spatial
modulation is a known arrangement of a finite number of distinct
volume elements located inside or on the surface of the
microcarrier. According to another embodiment, said spatial
modulation can be generated by one or more steps comprising (i)
changing one or more properties of the material in an individual
volume element, (ii) removing material from an individual volume
element, (iii) depositing material on an individual volume element
or (iv) leaving an individual volume element unchanged, or a
combination thereof.
[0109] According to an embodiment, the target analyte reaction step
can consist of contacting a solution that may contain said analyte
with a composition comprising a molecule, species or material that
interacts with said analyte bound to an encoded microcarrier or a
microcarrier and in the identification step further detecting
whether an interaction has occurred. Said step also includes
allowing a target analyte reaction for analytes in gas, vapor,
semi-liquid or solid phase.
[0110] According to another embodiment, the present invention
relates to a method wherein the identification step is performed by
any physical or chemical means of interrogation, including but not
limited to electromagnetic, magnetic, optical, spectrometric,
spectroscopic and mechanical means. The identification step relates
to the interpretation of the information coded within a
microcarrier and may also be referred a as "interrogation step" or
"reading step" or "differentiation step." The identification step
may be performed using identification means including but not
limited to visual inspection means, digital (CCD) cameras, video
cameras, photographic film, or current instrumentation such as
laser scanning devices, fluorometers, luminometers, photodiodes,
quantum counters, plate readers, epifluorescence microscopes,
scanning microscopes, confocal microscopes, capillary
electrophoresis detectors, or by other means for amplifying the
signal such as a photomultiplier tube or other light detector
capable of detecting the presence, location, intensity, excitation
and emission spectra, fluorescence polarization, fluorescence
lifetime, and other physical properties of the fluorescent
signal.
[0111] In another embodiment, the identification step is performed
using an optical identification mean. The reading of the codes may
be performed with an ordinary microscope if the code is on the
surface of the microcarrier or, if the microcarrier is sufficiently
translucent, at an internal depth of the microcarrier. Reading of
the codes may also be performed using a confocal microscope, a
transmission microscope or a fluorescence microscope, In
particular, the codes may be read by suspending the microcarriers
in an aqueous environment, placing the microcarriers between two
glass slides or placing them in microcapillaries, and observing the
codes through a microscope or confocal microscope. The reading may
also be performed by using a laser beam scanning instrument. The
reading may also be performed in a flow cell. A myriad of light
sources and photodetectors are known in the flow cytometer art.
[0112] According to another embodiment, during the identification
step, the microcarrier can be 3D-positioned in individual wells, in
such a way that all microcarrier successively pass the stationary
scanning beam of an identification mean. The reading velocity could
also be increased if the microcarriers themselves pass the scan
beam. The limiting factors in such a case would be the response
time of the detector and the time required by the decoding
algorithm. For examples, the wells could be positioned on a disc
according to a spiral with a linear increasing radius. Therefore,
the disc would merely need to rotate with a constant angular
velocity during which the scanner moves with a constant velocity in
a radial direction and the microcarriers will pass one by one the
scan beam.
[0113] Said method can be useful for performing a target analyte
assay. Example of target analyte assay include but are not limited
to DNA hybridization, enzyme-based assays, immunoassays,
combinatorial chemistry assays, assays conducted to screen for
certain compounds in samples, and also assay for detecting and
isolating compounds from those samples.
[0114] The present invention further relates to a method for
encoding a microcarrier, wherein the encoding comprises the writing
of a code on a microcarrier whereby the code is generated by
spatial modulation created inside the microcarrier or on its outer
surface. According to an embodiment, the spatial modulation is a
known arrangement of a finite number of distinct volume elements
located inside or on the surface of the microcarrier. According to
another embodiment, said spatial modulation is a known arrangement
of a finite number of distinct volume elements located inside or on
the surface of the microcarrier. According to yet another
embodiment, said spatial modulation can be generated by one or more
steps comprising (i) changing one or more properties of the
material in an individual volume element, (ii) removing material
from an individual volume element, (iii) depositing material on an
individual volume element or (iv) leaving an individual volume
element unchanged, or a combination thereof.
[0115] The present invention further relates to an encoded
microcarrier obtainable by the method above described method,
wherein the code on said encoded microcarrier is generated by
spatial modulation created inside the microcarrier or on its outer
surface. According to an embodiment, the spatial modulation is a
known arrangement of a finite number of distinct volume elements
located inside or on the surface of the microcarrier. According to
another embodiment, said spatial modulation is a known arrangement
of a finite number of distinct volume elements located inside or on
the surface of the microcarrier. According to yet another
embodiment, said spatial modulation can be generated by one or more
steps comprising (i) changing one or more properties of the
material in an individual volume element, (ii) removing material
from an individual volume element, (iii) depositing material on an
individual volume element or (iv) leaving an individual volume
element unchanged, or a combination thereof.
[0116] The present invention further relates to the use of a
microcarrier as described herein in a high-throughput screening
assay. The assay may consist for example of detecting the presence
or absence of one or more target analytes in a sample. Said assay
may comprise contacting a microcarrier-bound ligand with at least
one analyte, detecting whether the analyte has reacted or bound to
the ligand, and reading the code of any microcarrier upon which any
reaction or binding has occurred. Said assay may comprise choosing
one or more ligands which bind or react with the one or more
analytes, binding the ligands to a plurality of microcarriers,
correlating the identity of the ligands with the codes on the
microcarriers to which the ligands are bound, contacting the one or
more analytes with the ligand-bound microcarriers, observing any
microcarriers upon which the analyte has bound or reacted with the
microcarrier-bound ligand, and reading the codes on the
microcarriers to identify any ligands with which the one or more
analytes have reacted, thereby determining the presence or absence
of the one or more analytes. Said high-throughput screening assay
using the encoded microcarriers can be carried out in water, in
solvent, in buffer or in any biological fluid, including separated
or unfiltered biological fluids such as urine, cerebrospinal fluid,
pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid,
gastric fluid, blood, serum, plasma, lymph fluid, interstitial
fluid, tissue homogenate, cell extracts, saliva, sputum, stool,
physiological secretions, tears, mucus, sweat, milk, semen, vaginal
secretions, fluid from ulcers and other surface eruptions,
blisters, and abscesses, and extracts of tissues including biopsies
of normal, malignant, and suspect tissues or any other constituents
of the body which may contain the analyte of interest. Other
similar specimens such as cell or tissue culture or culture broth
are also of interest. Alternatively, the sample is obtained from an
environmental source such as soil, water, or air; or from an
industrial source such as taken from a waste stream, a water
source, a supply line, or a production lot. Industrial sources also
include fermentation media, such as from a biological reactor or
food fermentation process such as brewing; or foodstuff, such as
meat, game, produce, or dairy products. The test sample can be
pretreated prior to use, such as preparing plasma from blood,
diluting viscous fluids, or the like; methods of treatment can
involve filtration, distillation, concentration, inactivation of
interfering compounds, and the addition of reagents.
[0117] The present invention further encompasses a report
comprising information obtained from the high-throughput assays
described above.
[0118] The present invention further relates to a method for the
preparation of an encoded microcarrier as described above
comprising the step of writing a code on said microcarrier.
Examples of processes for writing said codes include photochroming,
chemical etching, material deposition, photobleaching, or exposing
said microcarrier to a high spatial resolution light source.
According to an embodiment, said code is written by photochroming.
According to another embodiment, said code is written by
photobleaching.
[0119] According to another aspect, the present invention relates
to a computer for monitoring a high-throughput target-analyte assay
with a microcarrier as described herein, wherein said computer is
linked to an apparatus as described above.
[0120] According to an embodiment the present invention further
relates to a device for high-throughput target-analyte assay,
comprising a computer for monitoring said assay and an apparatus as
described above. The device may comprise a microarray and an
identification mean. Examples of identification means include but
are not limited to optical means, electronic means, physical means,
chemical means and magnetic means.
The microarray will normally involve a plurality of different
components. In theory there need by only one component, but there
may be as many as 105. While the number of components will usually
not exceed 105, the number of individual encoded microcarriers used
may be substantially larger.
[0121] The encoded microcarriers in the microarray may be arranged
in tracks. Headers can be provided for defining sites, so that
particular interactions can be rapidly detected. Particularly,
disks having circular tracks with headers defining sites on the
tracks, so that positive signals can be interpreted in relation to
the information provided by the header. The circular tracks are
preferably concentric and may have a cross-section in the range of
5 to 5000 .mu.m, or for example in the range of 100 to 1000 .mu.m
or from 500 to 2000 .mu.m. Various modifications are possible, such
as pre-prepared segments that may then be attached to the disk for
assaying.
[0122] The above and other objects, features and advantages of the
present invention will be more readily understood from the
following description when taken in conjunction with the
accompanying drawing, in which FIGS. 1-3 are cross-sectional
views.
[0123] In order that those skilled in the art will better
understand the practice of the present invention, examples of the
present invention are given below by way of illustration and not by
way of limitation.
1. Examples of Positioning and Orienting a Microcarrier Using a
Solid Support.
[0124] Using such preferred microcarriers as mentioned above, two
preferred embodiments to position and orient a microcarrier are
disclosed. Firstly, the microcarriers are collected on and
transported by a solid support, and in the second preferred
embodiment, the microcarriers are transported by the flow of a
fluid or semi-solid medium.
[0125] The solid support has wells with such a shape that the
microcarrier fits in it in only a particular or a limited way thus
obtaining a certain orientation. The wells can be magnetic in order
to hold a magnetic microcarrier into place and to orient it in a
certain way. One configuration is given as an example in FIG. 1.
Other possibilities are the chemical and/or biological interactions
between the solid support and the microcarrier.
[0126] The wells in the support can further be provided with vacuum
channels in order to keep the microcarriers into place. The support
can be flat and magnetic/electrically charged if only collection of
magnetic/charged microcarriers is needed. The microcarrier can be a
combination of the possibilities mentioned above. The wells
mentioned above, can be ordered in a certain pattern on the solid
support, e.g. one row, 2D array, spiral, concentric rings, etc.
[0127] The wells can also have a non-spherical configuration for
example conical or ellipsoidal such that non-spherical
microcarriers will be housed in the wells in a specific
orientation.
2. Examples of Positioning and Orienting a Microcarrier Using Means
for Transportation Such as a Flow of a Fluid.
[0128] The microcarrier can be transported by a fluid flowing
through a channel, e.g. a capillary. In literature (ref. 1-6) it is
shown theoretically by 2D computer simulations that a spherical or
ellipsoidal particle will be positioned at a certain depth when
flowing through a channel. It is also theoretically shown that an
ellipsoidal particle will have a precise orientation depending on
the exact circumstances. It is for example possible that an
ellipsoidal particle of the right ellipticity flowing in a
capillary with the right diameter, will be positioned on or near
the central line with its longest axis parallel to the flow. In
that way, the only remaining freedom of movement, is a rotation
about its longest axis.
[0129] The next paragraph explains a preferred embodiment of the
method in detail, wherein this positioning and orienting of
microcarriers is obtained by a fluid flow.
[0130] If an ellipsoidal particle is additionally provided with a
dipole moment perpendicular to its longest axis, the rotational
freedom about this axis can be eliminated as well when applying an
electric or magnetic field perpendicular to the flow direction.
[0131] If a spherical microcarrier is transported in a fluid, it
will normally rotate. This rotation can be eliminated using
microcarriers with a magnetic or electric dipole moment and
applying a suitable magnetic/electric field. This is illustrated in
FIG. 2. Spherical microcarriers with a magnetic dipole moment are
transported by a fluid through a capillary. Due to its movement
relative to the flow, a rotational force will act on the carrier.
When the carriers pass through the region between the two coils,
the rotational force will be compensated by the magnetic force
acting upon the dipole moment and trying to position the
microcarrier as illustrated (dipole moment antiparallel to the
magnetic field B). Thus the spherical microcarrier is positioned by
the flow and oriented by the magnetic field, leaving only
rotational freedom about its dipole axis.
[0132] The situations described above can additionally be provided
with an asymmetric mass distribution in order to eliminate the last
degree of rotational freedom making use of the gravitational or
centrifugal force.
Experimental Investigation of the Positioning of Spherical
Microcarriers Flowing in a Fluid, for Example Water Through a
Capillary Tube.
[0133] Using a confocal microscope as detection means, the
inventors have examined the movement of particles in a laminar flow
inside a capillary.
[0134] Spherical fluorescently labeled polystyrene microparticles
(in this first experimental part not magnetic) of 15 um diameter
suspended in distilled water were used and imaged by a confocal
microscope such fluorescent particles can be easily viewed.
[0135] A capillary system was made that fitted onto the confocal
microscope. The capillary itself is made of glass and has a square
shape. The internal dimensions are 80 .mu.m.times.80 .mu.m and the
outer dimensions are 180 .mu.m.times.180 .mu.m. In FIG. 3 this
setup is schematically shown. The capillary is at both ends
connected to a reservoir. The suspended microparticles can be
brought in this reservoir. A constant pressure can be applied to
the reservoir thus causing the suspension to flow to the other
side. A low pressure (<0.2 atm) is preferably used in order to
create a laminar flow in the capillary.
[0136] The particles are imaged when passing the objective lens of
a scanning laser confocal microscope. In this first experiment no
electric or magnetic fields were applied. The objective lens used
has a numerical aperture of 0.45 and a ten fold magnification. This
lens was used in order to have a large field of view.
[0137] When the particles are flowing, images are taken during a
certain time interval, typically about 1 minute, thus obtaining a
time series. Afterwards these images are added together into one
picture showing all the particles that have passed during that time
interval. That way it is possible to see whether or not the
microcarriers are flowing along the same path.
[0138] These experiments show that the particles are indeed
positioned along a constant path when being transported by a
laminar flow inside a capillary, as predicted by 2D computer
simulations. More testing at a higher resolution is of course
necessary to examine the magnitude of possible fluctuations. More
experiments are needed to see the effect of changing the particle
size.
[0139] In a further experiment microcarriers having a magnetic
dipole moment were used in the previously described setup and a
magnetic field B was imposed on the transported microcarriers via
the two coils in the Helmholtz configuration (FIG. 3).
[0140] The magnetic field extends parallel to the capillary tube. A
rotation restriction was observed as explained in FIG. 2.
[0141] These experiments prove that the method of the invention can
provide a specific defined position of a microcarrier useful for
identification purposes.
3. Examples of Magnetic Microcarriers.
[0142] Green fluorescent 40, um microspheres coated with a
ferromagnetic coating (CrO.sub.2) were used. Ferromagnetic
microspheres or microcarriers are the primary candidates to obtain
a correct orientation in the fluid flow by using an external
magnetic field parallel to the flow, as previously explained. This
experiment determines if said microcarriers are still transparent
enough to "see" the central plane and to write patterns inside by
for example photobleaching.
[0143] In this experiment, the ferromagnetic microspheres were
suspended in de-ionized water, deposited on a microscope slide, and
covered with a cover slip. The particles were then imaged using a
Bio-Rad MRC1024 UV confocal microscope. The objective lens used was
a Nikon Plan Apochromat 60.times.N.A. 1.4 water immersion lens. The
light source for excitation of the green fluorescent microspheres
was a Spectra Physics Stabilite 2017 Ar-ion laser tuned to the 488
nm line.
[0144] FIG. 8 shows a confocal image focused on the top of a
ferromagnetic coated microsphere. It can be seen that the coating
consists of a layer of submicron CrO.sub.2 particles deposited on
the surface of the microspheres. Since the CrO.sub.2 particles are
non-fluorescent and non-transparent, they show up as dark specks
against the bright fluorescent microsphere.
[0145] To evaluate the transparency of the microspheres coated with
the ferromagnetic particles, a confocal image of the central plane
of some microspheres was taken (FIG. 9). The central plane was
imaged very clearly, indicating that the coated microspheres were
still transparent. Compared to uncoated microspheres, the recorded
fluorescent signal was less homogeneous across the central
plane.
[0146] Next, the encoding by photobleaching of the
ferromagnetic-coated microspheres was evaluated. The microscope was
focused on the central plane of a coated microsphere and a
bleaching pattern with basic geometric forms was drawn using
dedicated software especially designed for bleaching experiments.
The light power in the sample was about 20 mW. The geometry of the
bleaching pattern was of no importance in this experiment since the
sole purpose of the experiment was to check if the coated particles
could still be bleached or not. FIG. 10 shows that the pattern
could easily be written inside the coated microsphere.
[0147] In conclusion, green fluorescent 40, um polystyrene
microspheres were coated with ferromagnetic Cr0.sub.2 particles.
Despite the non-transparency of those ferromagnetic particles, the
coated microspheres still proved to be transparent. The recorded
fluorescence was less homogeneous when compared to uncoated
particles because the ferromagnetic particles at the surface of the
microspheres blocked part of the light pathway. However, the
concentration of ferromagnetic particles could be altered thus
improving the homogeneity of the recorded fluorescence. The minimal
concentration could be determined by the magnetic force needed to
orient the microcarriers flowing through the positioning device.
The ferromagnetic microspheres were easily encoded by bleaching a
pattern at the central plane.
4. Examples of Photochromic Microcarriers
[0148] An alternative to encoding by photobleaching is encoding by
photochroming. Polystyrene microspheres were loaded with the
photochromic compound 1, 2-Bis (2methoxy-5-phenyl-3-thienyl)
perfluorocyclopentene (Extraordinary Low Cycloreversion Quantum
Yields of Photochromic Diarylethenes with Methoxy Substituents,
Shibata K, Kobatake S, Irie M, Chem. Lett. (2001), vol. 7, 618-619)
which has initially no absorption in the visible range, but develop
an absorption band extending from about 450 nm to 750 nm after UV
illumination, with an absorption maximum near 600 nm. Consequently,
the loaded microspheres were initially transparent, and turned blue
upon UV illumination.
[0149] Encoding a microsphere by photochroming is considered as an
alternative to encoding by photobleaching mainly because it makes
it easier to read the code, requiring less stringent demands about
the precision of the positioning device. In fact, when a bleached
code is used, we have a completely fluorescent microcarrier in
which only a small region has no signal. Consequently, the
identification of said encoded microsphere requires the use of a
confocal microscope and also requires bringing the plane where the
code was bleached exactly into focus. When encoding by
photochroming, we obtain a completely transparent microcarrier in
which a colored pattern is written. This is much easier to detect
and there is therefore no need for a confocal microscope, a
standard light microscope is sufficient. Moreover, using the same
laser power, the process of photochroming is faster than
photobleaching.
[0150] In this example, polystyrene microspheres were loaded with
the photochromic compound. Next, a first attempt is made to write a
pattern inside the photochromic microspheres.
[0151] All the experiments were performed under dark room
conditions. Unloaded transparent 28 .mu.m polystyrene microspheres
(5% crosslinking degree) where loaded with the photochromic
compound 1, 2-Bis
(2-methoxy-5-phenyl-3-thienyl)-perfluorocyclopentene. First, 5 mg
of dry microspheres were suspended in a 2% (w/v) solution of the
photochromic compound in CH.sub.2Cl.sub.2 and were incubated
overnight. The suspension was then centrifuged during 5 minutes at
12000 rpm. After the centrifugation, the floating spheres were
isolated by removing the underlying liquid.
The spheres were then suspended in de-ionized water and applied on
a microscopic slide for observation under the microscope.
[0152] The microscope was setup as described in example 3. A
Coherent Enterprise laser was further used to color the
microspheres (357 nm UV line) and the 647 nm line of a Bio Rad
Ar/Kr laser was used for imaging the microspheres. Red light was
used because it is absorbed by the blue regions resulting in a gray
scale image where the blue regions are dark and the transparent
regions bright. The images were recorded in transmission light mode
by using a transmission light detector.
[0153] To design an encoding pattern, a partial region of the
microsphere was scanned with an UV light by zooming into that
region and performing a regular image scan. This resulted in a
written square (vide infra). FIG. 11 shows a 2811m photochromic
microsphere imaged in transmission light mode using a red laser
line (647 nm). At this stage, the microsphere was not exposed to UV
light. The darkening along the edges was due to lens effects of the
spherical microsphere (refraction index 1.59) suspended in water
(refraction index 1.33).
[0154] After zooming into a small region (3.times.3, um) of the
central plane of the microsphere, a scan was performed using the
357 nm UV line at 0.5 mW. An image was then taken with the red
laser line (FIG. 12). A dark square was clearly visible indicating
that the microspheres were successfully loaded with the
photochromic dye that turned blue upon UV illumination. The blue
square only transmits 50% of the red light compared to the
transparent surroundings. As illustrated FIG. 12, upon UV
illumination, the microsphere became darker then before UV
illumination (FIG. 11). For comparison purposes, FIG. 13 shows two
microspheres, one completely colored (dark sphere) after UV
illumination and a second one that was not exposed to LTV
light.
[0155] In conclusion, the microspheres were successfully loaded
with the photochromic compound. Less laser power (.about.40.times.)
was needed to color said microspheres, when compared to
photobleaching. The advantage of this encoding method is that the
codes can be written faster.
5. Examples of Fluorescent Microcarriers
[0156] According to an embodiment of the positioning and orienting
device, the microcarriers are transported by a fluid flow and pass
the writing beam only once and in one direction only. Therefore,
the preferred code is a one-dimensional "dotcode" rather than a
barcode (which is a one-dimensional code as well, but written in
two dimensions). This experiment determines if it to write such a
"dotcode" by photobleaching at the central plane of a 40 um
microsphere, and check whether the bleaching process is fast enough
for a code to be written by scanning only once.
[0157] In this experiment, 28 pm polystyrene microspheres (5%
cross-linking degree) loaded with the fast bleaching green
fluorescent dye NODD (N-(7-Nitrobenz-2-oxa-1, 3diazol-4-yl) diethyl
amine) were used.
[0158] To simulate the flowing of a microsphere past a writing
beam, the microsphere was positioned under a confocal microscope
that was focused on the central plane of said microsphere. Next,
the instrument was set to scan only one line across the sphere thus
simulating a positioned microsphere passing a stationary writing
beam. Using dedicated software, the instrument was programmed to
switch the laser power on and off a couple of times during the
linescan in order to obtain a "dotcode."
[0159] The scanned line consists of 512 pixels and in this
experiment 1 pixel corresponds to 0.038 .mu.m. One linescan takes
1.2 ms which means a scanspeed of 16.35 .mu.m/ms. This experiment
simulates the situation where a microsphere is flowing past a
stationary writing beam at a speed of 16.35 .mu.m/ms, or a maximum
of 584 spheres (28 tam) per second. Using a dedicated software the
instrument was programmed to switch the laser 5 times to 20 mW (in
sample) during 8 pixels (i.e. 0.304 .mu.m) and 80 pixels between
each flash. A code was obtained consisting of five dots of 3.04
.mu.m apart. The result is shown in FIG. 14, as a dotted line in
the middle of a microsphere. Under the conditions used, about 55%
bleaching was obtained. The top and bottom line were obtained by
bleaching respectively 40 and 16 pixels resulting in a bleaching
level of 80% and 70%.
[0160] In conclusion, 20 mW laser power in sample was sufficient to
create a `dotcode` at the central plane of a NODD loaded
microsphere with a bleaching level of over 50%. The scanspeed was
16.35 .mu.m/ms. This experiment demonstrates the feasibility of the
encoding of a dotcode by photobleaching by using one linescan. It
is also possible to increase the scanspeed and obtain the same
amount of bleaching, by increasing the laser power in sample.
[0161] The next step in the experiment, consisted in checking the
stability of the bleached code inside a fluorescent microcarrier
under solvent conditions, more specifically when the microcarriers
are suspended in a DMSO (dimethylsulfoxide) solution.
[0162] The NODD loaded 28 .mu.m microspheres of the previous
experiment were used. First they were suspended in de-ionized
water, then a drop of this suspension was applied to a microscope
cover slip and air dried, leaving the spheres attached to the
coverslip. The microspheres were then covered with a drop of DMSO
(>99.7%) and placed under a confocal microscope.
[0163] A simple pattern was bleached at the central plane of a
microsphere and was imaged again after three hours to check for any
difference in the fluorescence of the microsphere or the bleached
pattern. The confocal microscope was focused at the central plane
of a microsphere surrounded by DMSO. A simple pattern, consisting
of three lines, was bleached. After three hours, the pattern was
imaged again. The results are illustrated FIG. 15. No difference
could be found in either fluorescence or bleached pattern. The left
image was less sharp because of a slight misfocus on the
pattern.
[0164] No difference could be found in either fluorescence of the
microsphere or bleached pattern after being suspended in 99.7% DMSO
for three hours. This demonstrates the high stability of the
written pattern, which is independent of the assay conditions.
6. Examples of Positioning and Orienting a Microcarrier in a Liquid
or Semi-Liquid Support.
[0165] FIG. 16 represent microcarriers positioned in a semi-liquid
or a liquid support, wherein said semi-liquid or liquid support is
composed of two semi-liquids or liquid media with different
density. The microcarrier are positioned at the interface of the
two media. Two coils are provided that can induce a magnetic field.
Inside the coils, the magnetic field will try to align the dipole
moment antiparallel to itself, thus orienting the microcarrier in a
specific manner, allowing thereby the easy detection of the codes.
The absence of a flow in said distribution of the microcarrier
results in the possibility that the detection means could be
mobile.
7. Examples of Different Types of Codes.
[0166] Performing bead based assays on very large numbers of
compounds or molecules in drug discovery and drug screening,
requires labeling of each of the microcarriers according to the
particular ligand bound to its surface. This allows the further
mixing of the uniquely encoded microcarriers and subjecting them to
an assay simultaneously. Those microcarriers that show a favorable
reaction of interest between the attached ligand and target analyte
may then have their code read, thereby leading to the identity of
the ligand that produced the favorable reaction. Two different ways
of encoding microcarriers are presented here, providing a virtually
unlimited amount of unique codes.
[0167] Photobleaching: According to the first method, a pattern is
written in a homogeneously fluorescently dyed microcarrier by means
of photobleaching. This is a photo-induced process through which
the fluorescent molecules lose their fluorescent properties
resulting in a fading of the color. This can be done by first
focussing a Confocal Laser Scanning Microscope (CLSM) at a certain
depth into the microcarrier where the pattern, designed in
dedicated software, is going to be written. The CLSM is modified by
adding a powerful laser combined with a fast optical switch, which
controls the power of the laser light reaching the microcarrier.
Low power is used for mere imaging, while high power is used for
fast bleaching. The apparatus set up is schematically represented
FIG. 17.
[0168] While subsequently taking an image, which is done by
scanning the laser light in a raster pattern, dedicated software
controls the optical switch in such a way that low and high power
laser light reaches the microsphere according to the designed
pattern. Since the fluorescent molecules are virtually immobile in
the microcarrier matrix, the bleached regions will stay, resulting
in a bright background and a darker permanently bleached pattern.
FIG. 18 shows a confocal image of a bleached barcode, using three
widths and two intensity levels, in the central plane of a 45
micron polystyrene fluorescent microsphere. FIG. 19 shows a
confocal image of a bleached barcode, using 8 different intensity
levels, in the central plane of a polystyrene fluorescent
microsphere (right), and a normalized intensity profile measured
through the middle code (right).
[0169] The second method uses the same technique, except that the
process of photochroming is used instead of photobleaching. Here
the microcarriers are homogeneously dyed with a photochromic
compound which changes color upon radiation of light with the
appropriate wavelength. For example, the microcarriers can be
initially colorless and transparent, but will carry a colored
pattern inside at a certain depth after radiation using essentially
the same instrument as described above.
[0170] The amount of codes depends on a number of factors: the
resolution of the writing beam, the amount of intensity levels
used, the available space in the microcarrier, the dimensions of
the code design, etc. For example, using a 60.times.NA1.4 objective
lens, we have proved that it was possible to create at least
263-106 different codes over a length of only 16 micron with just a
one dimensional code using two different widths and 4 intensity
levels (results not shown). This code could easily be written in
the central plane of a 30 micron microsphere. Many more codes can
be generated if e.g. larger microspheres are used or if the code
design is extended to two or three space dimensions. Examples are
shown FIG. 20 wherein bar codes of different geometry e.g. letters
or numbers, were bleached on two microspheres. Therefore, it is
fair to state that the amount of codes that can be generated using
this technique is virtually unlimited.
8. Experimental Investigation of the Positioning and Orientation of
Ferromagnetic Fluorescent Beads Flowing in a Fluid in a Flow
Cell.
[0171] In this example, 40 micron ferromagnetic fluorescent beads
were flowing in a flow cell. The flow speed was around 6 m/sec. The
pressure was between 0.30 and 0.24 bar.
[0172] Images of said beads are shown FIG. 21. The light source for
excitation of the fluorescent beads was a laser tuned to the 488 nm
line. The objective lens used had a twenty-fold magnification. The
flowing beads were imaged using a camera having a shutter time of
50 milliseconds, and a one microsecond light-pulse illuminated the
flow every 25 microseconds. Because of this setting, each flowing
bead can be seen two or three times in each image. In FIG. 21, on
the bottom-right, is an image of a bead passing twice while the
shutter was open. The image above the aforementioned image
represents a cluster of two beads.
9. Encoding Beads by Photobleaching and Further Positioning and
Orienting of Said Beads.
[0173] Fluorescent beads can be encoded by means of photobleaching
under a microscope. Information can be written in 3 dimensions by
scanning the focus of the writing/reading beam along the X, Y, Z
axis with the Z axis being parallel to the optical axis of the
microscope. a maximum of 60 bits of information may be found along
1 axis, assuming that in practice there is 32 bits, this provides
with the possibility to write 4.times.109 different codes.
[0174] A mechanism is then provided to orient and position bead in
its original write position. When the beads are spherically
symmetric, the codes may be written as concentric spheres of equal
levels of bleaching. The same information is obtained when a line
is read through the center of the bead.
[0175] When a bead is a cylindrically symmetric bead (rotation
symmetry along the Z') the codes can be written in a circle around
the Z' axis. This allows the reading and writing essentially to 1D
((p-angle in polar coordinates). A control pattern can be added,
which indicates the beginning of the code as shown in FIG. 22.
[0176] Code bits can be written along the symmetry axis of a
spherical bead as shown in FIG. 23. This axis is uniquely defined,
once the bead is oriented. Thus, the reading can be done only along
this line. The Z' axis of the bead can be either parallel or
perpendicular to the optical axis of the microscope. In the case
that there is no mirror plane symmetry perpendicular to the Z' axis
(magnetic beads, mass anisotropy . . . ), the line can only be read
in one direction. It is also possible to mark both the start and
the end of the code bits.
[0177] When the reading is performed in a one-dimensional plane, it
is possible to read 1000 beads per second when the following
parameters are met: assuming that there are 50 bits per bead, and
that 5 positions are measured per bit, this give 250 measurements
per code. Since a line of 514 points can be measured in 1.2 ms
(based on the specifications of the Bio-Rad MRC1024 confocal
microscope), this gives 0.6 ms for reading the code of one bead,
hence 1667 beads can be red per second, if the reading process is
the time limiting factor.
[0178] Supplying the beads and scanning them through the focus of a
laser beam can be done with the same steady motion. The steady
motion can be realized in different ways. The beads can be carried
along with a fluid flowing through a capillary tube. Alternatively,
a capillary tube, containing the beads and (index matching) fluid,
can be moved by a translation stage through the focus.
[0179] FIG. 24 shows magnetic beads, which are carried along with a
fluid flow through a capillary, and are moved through the focus of
a laser beam in a direction perpendicular to the optical axis of
the microscope. Coils, carrying an electric current, create a
magnetic field and orient the beads along the direction of motion.
While moving, the codes on the beads can be written or red. Coils
may also serve as indicators for arriving beads and as a velocity
meter, since the moving magnetic field of the beads induces a
current in the coils. The signal from the coils may thus be used to
trigger the reading/writing of a bead, and as a feedback signal for
controlling the bead flow.
[0180] If the flow tube is mounted vertically, the beads can also
be oriented with their symmetry axis parallel to their motion, when
their center of gravity does not corresponds with their geometric
center. The motion of the beads can also be monitored optically. A
control pattern can be added at the beginning and at the end of
each code, in order to reduce the requirements on the steady flow
and the precise knowledge of the velocity. The number of read/write
positions can also be reduced, by using different levels of
photobleaching, e.g. 0% bleached, 33% bleached, 66% bleached, 100%
bleached. With this system, the flow speed can be increased and the
constraints on focusing and precise positioning and orienting can
be reduced. A non-limiting example of a coding scheme, using 4
different intensities, is shown in FIG. 25. Each intensity is
represented by a color and a number from 0 to 3. This coding scheme
has 28 characters, symbolically represented by the 26 letters of
the Roman alphabet and two extra punctuation marks. Each character
consists of 4 coding elements (i.e. 4 possible intensities (or
colors)) with the extra condition that no two identical elements
may follow each other, not even when two characters are placed next
to each other.
10. Example of Positioning and Orientation of Beads Flowing in a
Capillary Submitted to a Variable Magnetic Field Parallel to the
Flow Direction.
[0181] The beads in this experiment contain a small closed
conductor. The capillary through which the beads may flow is placed
in a coil as illustrated FIG. 26. Upon passing the appropriate
amount of electric current through the coil a homogeneous magnetic
field B is generated. Upon variation of the electric current, the
magnetic field B becomes variable.
[0182] As the bead flows through the increasing magnetic field, a
current will be generated in the small conductor which in turn will
generate a magnetic field B' in the bead which will act against the
changes of the external magnetic field. Because a magnetic field B'
is generated, a force couple will act upon the bead which aims at
orienting B' antiparallel to B. An axial positioning and
orientation of the bead is thus obtained, according to the
direction of the magnetic field B and this without having the
disadvantageous effect from the beads sticking together as a result
of the permanent magnetic field. The movement of the bead is not
necessary for this orientation method.
11. Example of Positioning and Orientation of Beads Flowing in a
Capillary Submitted to a Magnetic Field Perpendicular to the Flow
Direction.
[0183] The beads in this experiment contain a small conductor. The
capillary in this case is positioned between two polar plates,
which generate a homogenous magnetic field B, as illustrated in
FIG. 27. The beads are moving through the capillary with a velocity
v within the magnetic B as shown FIG. 27. A Lorentz force F will
act upon the electrons of the small conductor in the beads by which
they will move towards one side of the conductor. The force will
continue to exist as long as the beads continue to flow and as such
the plane of the small conductor will be pulled in parallel with F.
An axial positioning and orientation of the beads is obtained
according to the direction of F. This example presents a simplified
view of the forces at work in this experimental set-up.
12. Examples on the Orientation and Positioning of Ferromagnetic
Microspheres
[0184] These experiments showed that the ferromagnetic 40, um
microspheres could be magnetized and oriented in an external
magnetic field. In these experiments a pattern has been bleached at
the central plane of a magnetized ferromagnetic microsphere while
said microsphere was being exposed to and oriented by an external
magnetic field. Then the pattern has been imaged while the sphere
was exposed to a moving external magnetic field. It was tested
whether the original orientation-known from the bleached
pattern-could be found again after random movement of the
microsphere when said microsphere was subjected again to the
original magnetic field.
[0185] Green Fluorescent ferromagnetic polystyrene microspheres of
40 um diameter were prepared. A 0.01% v/v solution of NP40 (a
neutral detergent) in de-ionized water was made and used to make a
0.1% suspension of the microspheres. The neutral detergent was
added to minimize the interaction of the polymer bead with the
glass microscope cover glass (vide infra).
[0186] A reservoir was made by gluing a plastic cylinder of 0.5 cm
diameter onto a microscope cover glass. The reservoir was filled
with 80, u1 of the microsphere suspension and the microspheres were
allowed to sediment on the cover glass. The reservoir was then
placed above a strong permanent magnet for 1 minute to allow the
microspheres to be magnetized. Next the reservoir was placed on a
Bio-Rad MRC1024 confocal microscope which was attached to an
inverted microscope so that it was possible to use a Nikon
60.times.water immersion lens to look at the beads through the
bottom cover glass. A strong permanent magnet was placed at a 20 cm
distance from the reservoir in order to orient the beads without
changing their magnetic polarization (FIG. 28).
[0187] An arrow was bleached at the central plane of a
ferromagnetic microsphere oriented by the external magnetic field
from the first strong magnet, thus indicating its original
orientation (FIG. 29a). Next, the confocal microscope was set to
take a series of 50 images with a 1.2 second interval between each
image. While taking this series of images, a second magnet was used
to move around the reservoir: first 90 to one side, then 180 in the
opposite direction (FIG. 29b-i) with the first magnet still in
place. Finally the second magnet was taken away and a return from
the microsphere to its original orientation was observed (FIG.
29j-1). The images in FIG. 29 were selected from a series of 50
images taken at a 1.2 second interval recording the movement of the
microsphere. In some images, the arrow was not clearly visible
because of a tilt of the original central plane while moving the
second magnetic field and due to the fact that a confocal
microscope makes optical sections.
[0188] In next experiment, the same microsphere as in the previous
experiment was used. The microsphere was initially oriented in the
magnetic field of the first magnet (FIG. 30a). After having
carefully marked the position of this magnet, it was used to rotate
the microsphere (FIG. 30b-j) by moving the magnet 360 around the
reservoir and finally placing it back in its original position. The
microsphere did not return to its original orientation due to a
relatively strong interaction between the polymer bead and the
glass cover slip. A second magnet was used to loosen the
microsphere by quickly moving it once near the reservoir. It was
observed that the microsphere returned immediately to its exact
original orientation (FIG. 30k-1).
[0189] The ferromagnetic-coated particles could be easily
magnetized using a strong magnet. The microspheres could be
oriented in an external magnetic field. The orientation of the
microspheres in a certain external magnetic field was exactly
reproducible after random movement of the spheres when the initial
field was applied again. No difference in orientation could be
observed within pixel accuracy (0.7 .mu.m/pixel).
13. Example of the Use of Spherical Microcarriers with a Single
Axis of Symmetry for Identification Purposes i.e. Encoding and
Reading in a Flow Cell.
[0190] The necessity of an orientation and a positioning for
identification purposes will be elucidated hereunder. The code on
said spherical microcarrier is written along the symmetry axis,
whereby the code is encoded (written) or identified (read) by means
of a high spatial resolution light source, more in particular by
using fluorescence bleaching.
[0191] Spherical microcarriers are oriented with their symmetry
axis along the flow. The laser beam for fluorescence bleaching has
a stationary position in the confocal microscope, and the code on
said microcarrier is written along the symmetry axis. The flow
itself served as the scanning motion along the symmetry axis. A
code written as described above (along the symmetry axis), may be
read by a laser beam having a stationary position. FIGS. 31a, 31b,
31i, 31j represent schematic field of views of a microcarrier
flowing in front of a microscope objective. FIGS. 31a, 31i show the
field of view before the microcarrier arrives into the focused
laser beam for reading/writing the code. FIGS. 31b, 31j show the
field of view with a microcarrier at the focus position.
[0192] In the case of a stationary writing/reading laser beam, and
accurate flowing of the microcarrier, the code may be written/read
along the axis of symmetry of the microcarrier as illustrated in
FIG. 31c. However, in the case where the flow is not sufficiently
reproducible with respect to the microscope focus, the code may not
be read correctly as illustrated in FIG. 31f, wherein the code is
written/read below the axis of symmetry.
[0193] As illustrated in FIG. 31d, 31g, an auxiliary laser beam may
be used to illuminate the passing microcarrier. In this case, a
shadowing effect will be observed, behind the microcarrier, due to
partial absorption or reflection of light by said microcarrier. A
photodiode consisting of two separated cells (bicell photodetector)
is positioned at the opposite side of the flow cell in order to
measure the shadowing effect. In FIG. 31 d, since the center of the
spherical microcarrier crosses the optical axis of the microscope,
the same amount of light is collected by the 2 cells, and the
bicell photodetector measures a difference signal equal to zero,
indicating that the bead passes by at the correct height. In FIG.
31 g, since the center of the spherical microcarrier does not cross
the optical axis of the microscope, the bicell photodetector
measures a difference signal different from zero, indicating that
the microcarrier flows too high. Consequently, the use of a
photodiode permits the detection of a mispositioning of the
microcarriers in the flow and indicates whether said microcarriers
flow too high or too low from the optical axis.
[0194] This photodiode system may be used to measure the position
of the microcarrier before said microcarrier arrives at the focus
of the reading/writing the laser beam. In this case, the position
error signal generated can be used to adjust the focus of the
reading/writing beam. In FIG. 31c, since the position error signal
measured is zero, the position of the beam focus was not changed.
In FIG. 31h, an error signal is measured in this case, and the beam
focus position is moved up. Adjusting the focus of the laser beam
can be done be changing the direction of incidence of the
writing/reading beam on the microscope objective. An acousto-optic
beam deflector can be used as a device that can quickly adapt the
direction of the laser beam. The same technique can be used to
generate a position error signal for the Z axis, i.e. the optical
axis of the microscope. Because there will be only a difference
signal at the bicell photodetector, the difference signal can be
used to detect the arrival of the microcarrier and can also be used
as a trigger for reading and writing.
[0195] Obviously, numerous modifications and variations of the
present invention are possible in the light of the above teachings.
It is therefore to be understood that within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
REFERENCES
[0196] Direct simulation of initial value problems for the motion
of solid bodies in a Newtonian Fluid. Part 2. Couette and
Poiseuille flows. J. Feng, H. H. Hu, D. D. Joseph, J. Fluid Mech
(1994), vol. 277, pp. 271-301. [0197] Direct simulation of initial
value problems for the motion of solid bodies in a Newtonian Fluid.
Part 2. Sedimentation. J. Feng, H. H. Hu, D. D. Joseph, J. Fluid
Mech (1994), vol. 261, pp. 95-134. [0198] The turning couples on an
elliptic particle settling in a vertical channel. Peter Y. Huang,
Jimmy Feng, Daniel D. Joseph, J. Fluid Mech (1994), vol. 271, pp.
1-16. [0199] Direct Simulation of the motion of solid particles in
Couette and Poiseuille flows of viscoelastic fluids. P. Y. Huang, J
Feng, H. H. Hu, D. D. Joseph, J. Fluid Mech (1997), vol. 343, pp.
73-94. [0200] Dynamic simulation of the motion of capsules in
pipelines. J. Feng, P. Y. Huang, D. D. Joseph, J. Fluid Mech
(1995), vol. 286, pp. 201-227. [0201] The unsteady motion of solid
bodies in creeping flows. J. Feng, D. D. Joseph, J. Fluid Mech
(1995), vol. 303, pp. 83-102.
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