U.S. patent application number 14/529589 was filed with the patent office on 2015-02-26 for encoding of microcarriers.
This patent application is currently assigned to MyCartis NV. The applicant listed for this patent is MyCartis NV. Invention is credited to Stefaan Cornelis De Smedt, Joseph Demeester, Rudi Wilfried Jan Pauwels, Christiaan Hubert Simon Roelant.
Application Number | 20150057190 14/529589 |
Document ID | / |
Family ID | 22440543 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150057190 |
Kind Code |
A1 |
De Smedt; Stefaan Cornelis ;
et al. |
February 26, 2015 |
ENCODING OF MICROCARRIERS
Abstract
Encoded microcarriers, and more specifically microcarriers
having codes written on them. Methods for writing the codes on the
microcarriers, methods of reading the codes, and methods of using
the encoded microcarriers. A preferred method of encoding the
microcarriers involves exposing microcarriers containing a
bleachable substance to a high spatial resolution light source to
bleach the codes on the microcarriers. The encoded microcarriers
may be used, for example, as support materials in chemical and
biological assays and syntheses.
Inventors: |
De Smedt; Stefaan Cornelis;
(Gent, BE) ; Demeester; Joseph; (Gent, BE)
; Roelant; Christiaan Hubert Simon; (Leuven, BE) ;
Pauwels; Rudi Wilfried Jan; (Champery, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MyCartis NV |
Zwijnaarde / Ghent |
|
BE |
|
|
Assignee: |
MyCartis NV
Zwijnaarde / Ghent
BE
|
Family ID: |
22440543 |
Appl. No.: |
14/529589 |
Filed: |
October 31, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09958655 |
Jan 9, 2002 |
|
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PCT/EP2000/003280 |
Apr 12, 2000 |
|
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14529589 |
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60129551 |
Apr 16, 1999 |
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Current U.S.
Class: |
506/18 ;
428/402 |
Current CPC
Class: |
B01J 2219/00542
20130101; B01J 2219/00547 20130101; B01J 2219/0054 20130101; B01J
19/0046 20130101; C12Q 1/6837 20130101; G06K 1/126 20130101; B01J
2219/00596 20130101; Y10T 428/2982 20150115; C40B 70/00 20130101;
G01N 33/54313 20130101; B01J 2219/005 20130101; G01N 33/587
20130101 |
Class at
Publication: |
506/18 ;
428/402 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. An encoded microcarrier, comprising: a body having a surface,
wherein said body has a spherical, cylindrical or oval shape; a
chemically etched code written on the surface of the body or at an
internal depth thereof, said chemically etched code identifying the
microcarrier; and one or more ligands bound to the microcarrier,
wherein each ligand binds or reacts with one or more target
analytes, wherein the identity of the ligands are correlated with
the chemically etched code written on the surface of the body or at
the internal depth thereof to determine the presence of or absence
of one or more target analytes.
2. An encoded microcarrier according to claim 1, wherein said body
is a sphere having a diameter of 1 to 200 .mu.m.
3. An encoded microcarrier according to claim 1, wherein said body
is a bead.
4. An encoded microcarrier according to claim 1, wherein the body
is comprised of a material selected from the group consisting of: a
solid, a semi-solid, and a combination of a solid and
semi-solid.
5. An encoded microcarrier according to claim 1, wherein the
chemically etched code is written at a center plane of the
body.
6. An encoded microcarrier according to claim 1, wherein said
chemically etched code takes the form of at least one number,
letter, symbol, picture, bar code, ring code, or three-dimensional
code.
7. An encoded microcarrier according to claim 1, wherein the one or
more target analytes are selected from the group consisting of the
following: antigens, antibodies, receptors, haptens, enzymes,
proteins, peptides, nucleic acids, drugs, hormones, pathogens, and
toxins.
8. A chemical library, comprising: a plurality of encoded
microcarriers, each encoded microcarrier including: a body having a
surface, wherein said body has a spherical, cylindrical or oval
shape, a chemically etched code written on the surface of the body
or at an internal depth thereof, said chemically etched code
identifying the microcarrier, and one or more ligands bound to the
surface of the microcarrier; and a plurality of individual members
of said chemical library, wherein said individual members are bound
to said plurality of encoded microcarriers.
9. A chemical library according to claim 8, wherein the chemical
library is a combinatorial chemical library.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/958,655, filed Jan. 9, 2002, which is the U.S. National
Stage of International Application No. PCT/EP00/03280, filed Apr.
12, 2000, which claims the benefit of U.S. Provisional Application
No. 60/129,551, filed on Apr. 16, 1999, said patent applications
fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to encoded microcarriers, and more
specifically to microcarriers having codes written on them. 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. This invention also relates to methods for writing
codes on microcarriers, methods of reading the codes, and methods
of using the encoded microcarriers. A preferred method of encoding
the microcarriers involves exposing microcarriers that carry a
bleachable substance to a high spatial resolution light source to
bleach the codes on the microcarriers. The encoded microcarriers
may be used, for example, as support materials in chemical and
biological assays and syntheses.
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 reactions 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 what 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 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] The practice of random processing described above requires
accurate encoding of each of the microcarriers separately, and
requires accurate 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 quality 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). The disclosures of the patents cited above are
incorporated by reference herein.
[0009] These known methods identified above for coding
microcarriers each carry disadvantages. For example, microcarriers
that are differentiated solely on the basis of their size, shape,
color, fluorescence intensity, or combinations thereof often cannot
provide enough unique readable combinations of those variables to
create the massive number of unique codes necessary to accompany
the testing of a correspondingly large number of different
molecules. In addition, any microcarriers carrying foreign bodies
on their surface to serve as the codes, such as detachable tags or
fluorescent markers, run the risk that the attached moieties may
interfere with the binding or reaction of the ligand-bound
molecules on the microcarriers that target the analytes in the
assays. After the separation of the microcarriers of interest that
exhibit a favorable reaction, methods of encoding microcarriers
with detachable tags also often involve the additional step of
cleaving and analyzing the tags to ultimately learn the identity of
the underlying ligands on the microcarriers that produced the
favorable reactions. This cleaving step naturally extends the time
and effort necessary to determine the results of the tests.
[0010] In light of the above, there remains in the art a need for
simple ways for identifying single microcarriers in a massive
population of otherwise identical microcarriers, especially ways
for encoding a larger number of unique codes that need not be
attached as foreign bodies to the surfaces of the
microcarriers.
SUMMARY OF THE INVENTION
[0011] An object of the invention is to provide a microcarrier that
is encoded without the need for attaching a foreign object to the
surface of the microcarrier to serve as the code. Another object of
the present invention is to provide a method of encoding
microcarriers that may provide essentially unlimited possibilities
as to the varieties of unique codes that may be written and read on
the microcarriers.
[0012] The present invention fulfills these objectives by providing
microcarriers having codes written on them. Preferred microcarriers
are microcarriers containing bleachable substances, for example,
fluorescent molecules. A preferred method of encoding the
microcarriers involves exposing microcarriers carrying a bleachable
substance to a high spatial resolution light source to bleach the
codes on the microcarriers. This method may preferably involve
bleaching codes on fluorescent microcarriers, where the bleaching
produces either the same or different levels of fluorescent
intensity within the bleached portions of the code. A further
preferred method of encoding the microcarriers is writing the codes
at an internal depth of the microcarriers.
[0013] In another preferred embodiment, large numbers of chemical
compounds or biological molecules are bound to a correspondingly
large number of microcarriers of the invention, the
microcarrier-bound ligands are mixed and reacted simultaneously
according to a screening or assay protocol, and those ligands that
react are identified by reading the code on the microcarriers to
which they are bound.
[0014] The encoded microspheres of the invention allow for the
simultaneous analysis of a large number of analytes in a single
reaction vessel using a single sample aliquot. Use of the
microcarriers of the invention in high-throughput assays and
reactions is therefore far superior compared to the use of
conventional microtiter plate technology.
[0015] The microcarriers of the invention also provide a virtually
unlimited number of codes that may be written and read on the
microspheres, and are therefore superior to known microcarriers
coded with color or fluorescent tags, which carry a more limited
number of coding possibilities. The microcarriers of the invention
are also superior to microcarriers coded with moieties attached to
the surfaces of microcarriers. This is because the writings on the
microcarriers of the invention do not carry the risk associated
with those known microcarriers of potentially interfering with the
analyte/ligand interactions that take place on the surfaces of the
microcarriers.
[0016] Additional features and advantages of the invention are set
forth in the description that follows, and in part will be apparent
from the description or may be learned from practice of the
invention. The advantages of the invention will be realized and
attained by the encoded microcarriers and methods particularly
pointed out in the written description and claims. Both the
foregoing general description and the following detailed
description of the invention are exemplary and explanatory only and
are not restrictive of the claimed invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates a number of principles of conventional
microphotolysis and SCAMP.
[0018] FIGS. 2a and 2b illustrate a bar code and ring code using
different intensities, with each intensity being denoted by the
different colors shown in the Figures.
[0019] FIGS. 3a and 3b illustrate confocal images of a middle plane
of an FD148-dex-ma microsphere before (upper) and after bleaching
(under) a stripe of 3 .mu.m at approximately 10 .mu.m under the
surface of the microsphere.
[0020] FIG. 4 illustrates fluorescence recovery curves of FD148 in
148-dex-ma microspheres (A) and FITC in dex-ma microspheres loaded
with FITC by submersion in a FITC solution (B).
[0021] FIG. 5 illustrates a confocal image of a middle plane of an
FD 148-dex-ma microsphere after bleaching an arbitrary geometry by
SCAMP.
[0022] FIGS. 6a and 6b illustrate confocal images of the middle
plane in a 45 .mu.m FITC-labeled latex bead one hour after
bleaching of a barcode (FIG. 6a) and barcode plus number (FIG.
6b).
[0023] FIG. 7 illustrates a confocal image of the middle plane in a
45-.mu.m FITC-labeled latex bead one hour after bleaching of the
code R1247.
[0024] FIG. 8 illustrates a confocal image of the middle plane in a
45 .mu.m FITC-labeled latex bead one hour after bleaching of the
logo of Ghent University.
[0025] FIG. 9 illustrates a confocal image of the middle plane in a
45 .mu.m FITC-labeled latex bead one hour after bleaching of the
logo of the Tibotec company.
[0026] FIG. 10a illustrates confocal images of codes bleached to
different intensities, and FIGS. 10b to 10d graphically illustrate
the different intensities within the codes.
[0027] FIGS. 11a and 12a illustrate confocal images of codes
bleached to different intensities, and FIGS. 11b and 12b
graphically illustrate the different intensities within the
respective codes.
DETAILED DESCRIPTION OF THE INVENTION
[0028] In one embodiment, the present invention relates to
microcarriers having codes written on them. The microcarriers of
the invention may be made from, for example, any materials that are
routinely used 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.
Non-limiting examples of these materials include latex,
polystyrene, cross-linked dextrans, methylstyrene, polycarbonate,
polypropylene, cellulose, polyacrylamide, and dimethylacrylamide.
Preferred materials include latex, polystyrene, and cross-linked
dextrans. The microcarriers may also be prokaryotic or eukaryotic
cells.
[0029] The microcarriers may be of any shapes and sizes that lend
themselves to the encoding and use of the microcarriers. 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 in shape. When spherical
in shape, the microcarriers may have, for example, a diameter of 1
to 200 .mu.m.
[0030] The codes written on the microcarriers 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.
[0031] The microcarriers of the invention 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. The bleachable substances may be, more specifically,
fluorescein isothiocyanate ("FITC"), phycoerythrines, coumarins,
lucifer yellow, and rhodamine. The bleachable substances should be
chosen so that, when bleaching occurs, the code remains on the
microcarrier for the period of time that is desired for the use of
the microcarriers and any necessary reading of the codes. Thus, a
certain amount of diffusion of non-bleached molecules into the
bleached areas is acceptable as long as the useful life of the code
is preserved.
[0032] 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.
[0033] In another embodiment, the invention relates to a method for
writing codes on microcarriers. The method may be used to write the
codes either on the surfaces of the microcarriers or at an internal
depth of the microcarriers. 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, .alpha. and .beta. rays, ion beams, or any form of
electromagnetic radiation. The codes can also be written on the
microcarriers through photochroming or chemical etching. A
preferred 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. Another preferred
method for writing the codes is by bleaching the code in a
bleachable substance on the microcarrier. Preferred bleachable
substances in this method include those substances identified above
in the description of the microcarriers, and include fluorescent
molecules. With regard to the volume of material that may be
bleached within the microcarriers, one example of such a volume is
between one cubic nanometer and eight cubic millimeters of the
microcarrier.
[0034] One preferred method for writing the codes on the
microcarriers is 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. The above article discloses the use of SCAMP for the
bleaching and visualization of patterns in a thin fluorescent layer
of nail polish. The article does not suggest the use of SCAMP for
encoding microcarriers.
[0035] We have used SCAMP for writing codes on the microcarriers by
bleaching fluorescent molecules within the microcarriers.
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.
[0036] For many years, fluorescence microphotolysis ("MP")
techniques, also called fluorescence recovery after photobleaching
("FRAP") were used to study the mobility of fluorescent molecules
in both biological media, like cells and tissues, and
non-biological media. Peters and Scholtz, "Fluorescence
photobleaching techniques," in New Techniques of Optical Microscopy
and Microspectroscopy, R. J. Cherry (ed.), MacMillan, New York, pp.
199-228 (1991); De Smedt et al., "Structural Information on
Hyaluronic Acid Solutions as Studied by Probe Diffusion
Experiments," Macromolecules, vol. 27, pp. 141-146 (1994); De Smedt
et al., "Diffusion of Macromolecules in Dextran Methacrylate
Solutions and Gels as Studied by Confocal Scanning Laser
Microscopy," Macromolecules, vol. 30, pp. 4863-4870 (1997).
[0037] The mobility of fluorescent molecules can be measured by
bleaching (photolyzing) the fluorescent molecules moving in the
focal area of a light beam, which can be particularly a laser beam
(FIG. 1: A, B). Immediately after a short bleaching process,
typically about ten milliseconds, a highly attenuated laser beam
measures the recovery of the fluorescence in the photobleached area
due to the diffusion of fluorescent molecules from the surrounding
unbleached areas into the bleached area (FIG. 1: B, C). The
characteristic diffusion time, a measure for the diffusion
coefficient, and the fractions of respectively immobile and mobile
fluorescent molecules can be derived from the fluorescence recovery
in the bleached area (FIG. 1: D).
[0038] The mobile fraction, R, is defined as:
R = F ( .infin. ) - F ( 0 ) F ( i ) - F ( 0 ) ##EQU00001##
where F(i) is the fluorescence intensity of the bleach spot before
bleaching, F(0) is fluorescence intensity of the bleach spot just
after bleaching and F(.infin.) is the fluorescence intensity of the
bleach spot at a long time after bleaching.
[0039] In photobleaching experiments using a conventional
(non-scanning) light microscope, a stationary (laser) light beam is
focused on the sample during both the bleaching process as well as
the recovery period. 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 typically 10 .mu.m-20 .mu.m in diameter or even
larger, as schematically illustrated in FIG. 1: B-II.
[0040] 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 of less
than a micrometer in the sample.
[0041] FIG. 1 illustrates schematically how SCAMP proceeds to
measure the mobility of fluorescent molecules. First, the
fluorescence along one x-line of the plane of interest in the
sample is measured by scanning this line (FIG. 1: A-dotted line).
Second, a small segment (e.g. 3 .mu.m) on this x-line, in which
diffusion has to be investigated, is selected to be bleached (FIG.
1: B-I). The length, position, as well as the number of segments
are freely selectable by the SCAMP software. The photobleaching of
this segment occurs at the time the laser beam scans over this
segment accompanied by a temporarily strong increase in the
intensity of the laser beam. Typically, the ratio between
photobleaching and monitoring intensity levels of the laser beam is
larger than 100.
[0042] As SCAMP makes use of a confocal microscope, fluorescence
detection is not only allowed at the surface of the sample, but
also at an arbitrary depth in the sample with little interference
by scattered radiation from out-of-focus levels of the specimen (as
encountered in a conventional microscope). In contrast, when a
fluorescence lamp for illumination and a conventional (non-focal)
microscope is used, only the surface of the beads is typically
observed. An encoding at an internal depth is therefore generally
difficult to observe with an ordinary microscope but becomes well
visible with confocal optics. Both the confocal and scanning
features of the microscope allow photolyzing and reading
microregions at well-defined locations within a microcarrier. This
invention is clearly distinguished from all other applications
described thus far in the art in that, for example, the use of a
high spatial resolution of SCAMP can irreversibly mark microspheres
inside at specific depths and to read that encoding by confocal
techniques.
[0043] The methods of the invention for writing codes on
microcarriers may also involve bleaching the microcarriers to
produce different levels of intensity in the substances bleached in
the 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 according to the invention 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. FIGS. 2a and 2b are two
examples of codes bleached using different intensities, one with a
bar pattern, the other with a ring pattern. The different
intensities in the codes are represented by different colors in the
Figures. Different levels of intensity can also be combined with
different breadths of the coding elements, such as bars in bar
codes.
[0044] Another embodiment of the invention relates to reading the
codes on the encoded microspheres of the invention. 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. 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.
[0045] Another embodiment of the invention relates to methods of
using the encoded microspheres of the invention. The microcarriers
may be used as supports for the measurement of biomolecular
interactions, for drug discovery, receptor binding assays,
therapeutics, medical diagnostics, combinatorial chemistry,
isolation and purification of target molecules, capture and
detection of macromolecules for analytical purposes, selective
removal of contaminants, enzymatic catalysis, chemical
modification, hybridization reactions and forensic
applications.
[0046] The microcarriers may preferably serve as supports for
chemical and biological assays and syntheses. In this capacity, the
microcarriers may contain one or more ligands bound to the surface
of the microcarriers. The ligand-bound microcarriers may then be
contacted with target analytes to determine the presence or absence
of particular analytes of interest, or may serve as supports for
combinatorial chemistry reactions performed on the
microcarrier-bound ligand. 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. Whether or not a microcarrier-bound ligand binds or
reacts with a target analyte may be determined by conventional
techniques used in the art for that determination. For example, the
reaction may be indicated by a luminometric response. The reaction
may be indicated by a colorimetric, chemiluminometric, or
fluorinometric response. The ligand bound to the microcarrier of
interest may be designed so that, in the presence of the analytes
of interest to which it is targeted, an optical signature of the
microsphere is changed. For example, such a change in optical
signature may be the result of a photochemical reaction that occurs
when the binding or reaction takes place between the ligand and
analyte. The microcarriers may then be observed under the
microscope to detect a fluorescence associated with the
photochemical reaction.
[0047] A large spectrum of chemical and biological functionalities
may be attached as ligands to the microcarriers of the invention.
These functionalities include all functionalities that are
routinely used in high-throughput screening technology and
diagnostics. The ligands 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 functionalized in a variety of ways to allow
attachment of an initial reactant.
[0048] The microcarriers of the invention may be used in methods of
detecting the presence or absence of one or more target analytes in
a sample, which 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.
[0049] More specifically, the invention relates to a method of
detecting the presence or absence of one or more target analytes in
a sample, which comprises choosing one or more ligands which bind
or react with the one or more analytes, binding the ligands to a
plurality of microcarriers of the invention, 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.
[0050] A preferred embodiment of the above method is where the
target analyte is a nucleic acid, particularly DNA or RNA, and
wherein at least one microcarrier-bound ligand is the reverse
compliment of the nucleic acid. The microcarriers of the invention
are thus useful in DNA hybridization. The microcarriers are also
useful for enzyme-based assays and immunoassays. The microcarriers
may also be used in assays conducted to screen for certain
compounds in samples, and also for detecting and isolating
compounds from those samples. The microcarriers may also be used as
supports for creating or for reacting members of a combinatorial
chemistry library.
[0051] The microcarriers of the invention may also be used in
methods and devices employed for the efficient and rapid screening
of large numbers of components, where the variety may be in either
or both of a ligand bound to a microcarrier or a soluble analyte
component, where one is interested in determining the occurrence of
an interaction between the two components. The devices include a
microarray such as, for instance, a solid support upon which the
bound ligands have been placed in a predetermined registry and a
reader for detecting the interaction between the components. The
method may involve preparing the microarray such as, for instance,
the solid support for attachment of the ligand, then combining the
ligand and analyte to effect any interaction between the components
and subsequently determining the presence of an interaction between
the components and particular sites.
[0052] 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 10.sup.5. While the number of
components will usually not exceed 10.sup.5, the number of
individual encoded microcarriers may be substantially larger.
[0053] The bound ligand may for instance be an organic entity, such
as a single molecule or assemblages of molecules, ligands and
receptors, nucleic acid bound components, RNA, single strand and
double strand binding proteins, which do not require that there be
a binding ligand attached to the nucleic acid, oligonucleotides,
proteins.
[0054] The encoded microcarriers in the microarray may be arranged
in tracks. Headers are 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 have a cross-section in the range of 5 to
5000 .mu.m. Various modifications are possible, such as
pre-prepared segments which may then be attached to the disk for
assaying.
[0055] The present invention is further illustrated by the
following examples that further teach those of ordinary skill in
the art how to practice the invention. The following examples are
merely illustrative of the invention and disclose various
beneficial properties of certain embodiments of the invention. The
following examples should not be construed as limiting the
invention as claimed.
EXAMPLE 1
[0056] Dextran-methacrylate ("dex-ma"), used to prepare dex-ma
microspheres, was synthesized and characterized as described in
detail in W. N. E. van Dijk-Wolthius et al, "Reaction of Dextran
with Glycidyl Methacrylate: An Unexpected Transesterification,"
Macromolecules, vol. 30, pp. 3411 to 3413 (1997), the disclosure of
which is incorporated by reference herein. Dex ma microspheres were
prepared by radical polymerization, using
N,N,N',N'-tetramethylene-ethylenediamine and potassium persulfate,
from a dex-ma/polyethyleneglycol (PEG) emulsion. See Stenekes et
al., "The Preparation of Dextran Microspheres in an All-Aqueous
System: Effect of the Formulation Parameters on Particle
Characteristics," Pharmaceutical Research, vol. 15, pp. 557-561
(1998), the disclosure of which is incorporated by reference
herein. The concentration of the dex-ma solution (in phosphate
buffer at pH 7) was 10% (w/w). The degree of substitution of
dex-ma, being the number of methacrylate molecules per 100
glycopyranosyl units, was 4. The concentration of PEG solution in
phosphate buffer at pH 7 was 24% w/w, while the average molecular
weight of PEG was 10,000 g/mol (Merck). One batch of microspheres
(FD 148-dex-ma microspheres) was prepared in the presence of
fluorescein isothiocyanate labeled dextran (having a molecular
weight of 148.000 g/mol). A second batch of microspheres was loaded
with fluorescein isothiocyanate ("FITC") by submersion of the
dex-ma microspheres, after complete preparation, in an FITC
solution (0.01 mg/ml in phosphate buffer at pH 7.2). Both FD 148
and FITC were obtained from Sigma. SCAMP experiments were performed
on both batches of microspheres as explained in Example 2.
EXAMPLE 2
[0057] SCAMP was installed on a Bio-Rad MRC1024 confocal laser
scanning microscope ("CLSM") following of the work of 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) and Wedekind et al., "Line-Scanning Microphotolysis for
Diffraction-Limited Measurements of Lateral Diffusion," Biophysical
Journal, vol. 71, pp. 1621-1632 (1996), the disclosures of which
are incorporated by reference herein. A 40.times. oil immersion
objective and a powerful 2 W (representing the maximum possible
output) argon laser (Spectra Physics 2017), used for obtaining
sufficient photobleaching during the extremely short photobleaching
times, were used in the SCAMP experiments on the dex-ma
microspheres made according to Example 1. The wavelength of the
laser beam, also during bleaching, was 488 nm.
[0058] In this example, SCAMP experiments were performed at
approximately 10 .mu.m below the surface of the dex-ma
microspheres. It occurred experimentally as follows. First, the
fluorescence along one x-line of a middle plane of a dex-ma
microsphere was measured by scanning this line in 400 milliseconds
(FIG. 1: A-dotted line). Second, a 3 .mu.m segment on this x-line
was selected to be bleached (FIG. 1: B-I). The length, position, as
well as the number of segments are freely selectable by the SCAMP
software. The photobleaching of this segment occurred at the time
the laser beam scanned over this segment accompanied by a
temporarily strong increase in the intensity of the laser beam. The
ratio between monitoring and photobleaching intensity levels of the
laser beam was 1:500. To measure the fluorescence recovery in the
bleached stripe, a strongly attenuated laser beam scanned along the
selected x-line for approximately 4 seconds.
[0059] FIGS. 3a and 3b shows the confocal images of a middle plane
in an FD148-dex-ma microsphere respectively before and 2 minutes
after bleaching the 3 .mu.m segment. The diameter of the
microsphere is approximately 25 .mu.m. The latter image shows the
bleach spot remains black indicating that, after 2 minutes, no
fluorescence recovery occurred in the bleached region of the
microsphere. FIG. 4 (curve A) shows the fluorescence in the
bleached segment of this experiment did not recover, which allowed
us to conclude that, within the time scale of the experiment, the
"large" FD148 chains were completely immobilized in the region of
the dex-ma microsphere under investigation.
[0060] While FD148 chains could be sterically entrapped in the
dex-ma polymer network as they were present during the formation of
the microspheres, this could not occur for small FITC molecules
when loaded into dex-ma microspheres by submersion of the fully
polymerized dex-ma spheres into a FITC solution. In this case, a
complete fluorescence recovery was expected and experimentally
confirmed. FIG. 4 (curve B) shows that FITC molecules located
around 10 .mu.m under the surface of the microsphere remain
mobile.
[0061] Besides the technical ability of photobleaching small
segments in a sample, using scanning microscopes is straightforward
to specifically select the microregions in the sample where
bleaching has to occur as the laser beam can be locally positioned.
Moreover, as the length, position as well as the number of segments
are freely selectable in SCAMP experiments, any kind of geometry in
the sample can be photobleached. FIG. 5 shows the confocal image in
a middle plane of an FD 148-dex-ma microsphere 2 minutes after
bleaching a cross, a circle and a rectangle in the
microspheres.
EXAMPLE 3
[0062] SCAMP experiments were also performed on 45 .mu.m FITC
labeled latex beads purchased from PolylaB in Antwerp, Belgium.
SCAMP was installed on a Bio-Rad MRC1024 CSLM following the work of
Wedekind et al. (1994) and Wedekind et al. (1996). A 100.times.
objective and a powerful argon laser (Spectra Physics 2017), used
for obtaining sufficient photobleaching during the extremely short
photobleaching times, were used in the SCAMP experiments on the 45
.mu.m FITC labeled latex beads. The intensity of the Spectra
Physics laser was installed at 300 mW, which resulted into a
monitoring and photobleaching laser intensity of respectively 75 _W
and 20 mW (measured at the end of the optic fiber which launches
the laser beam into the confocal scanning laser microscope).
Consequently the ratio between monitoring and photobleaching laser
intensity equaled 1:266. The wavelength of the laser beam, also
during bleaching, was 488 nm.
[0063] SCAMP measurements were performed in the middle plane of the
45 .mu.m FITC labeled latex beads. It occurred as follows. First,
the image was zoomed in until the latex bead totally covered the
picture. Second, the label of interest was defined and, by SCAMP
software, it was indicated where the label had to be bleached on
the latex bead. The labeling occurred at the time the laser beam
scanned over the middle plane of the 45 .mu.m FITC labeled latex
beads. A temporarily strong increase in the intensity of the laser
beam (from 75 _W to 20 mW) occurred when the laser beam scanned
over the segments that had to be bleached to create the label of
interest. As the scan equaled 6 ms per "x-line" (FIG. 1 A) and as
one image of the confocal plane (i.e. the middle plane of the latex
bead) consists of 512 "x-lines" it took 3.072 seconds to label a
latex bead.
[0064] FIGS. 6 to 9 show the confocal images of a middle plane in
the 45 .mu.m FITC labeled latex beads one hour after bleaching of a
barcode (FIG. 6a), a barcode plus number (FIG. 6b), the number
R1247 (FIG. 7), the logo of Ghent University (FIG. 8) and the logo
of Tibotec Company (FIG. 9). The images show the bleached segments
remain black indicating that no significant fluorescence recovery
occurred in the bleached segments of the latex beads.
[0065] The values of the zoom option of the Bio-Rad MRC1024
confocal scanning laser microscope were as follows: 1.61 in FIG.
6a, 1.00 in FIG. 6b, 3.07 in FIG. 7, 1.00 in FIG. 8 and 1.00 in
FIG. 9. The high spatial resolution of SCAMP to bleach labels is
observed in FIGS. 6a and 6b. The label in FIG. 6a is composed of 3
different line types: one with a large width (2.5 .mu.), one with a
medium width (1.25 .mu.m) and one with a small width (0.62 .mu.m).
All lines are positioned at 1.25 .mu.m from each other.
EXAMPLE 4
[0066] The following experiments demonstrate methods for bleaching
codes in fluorescent microcarriers, where the codes contain
different levels of intensity due to different degrees of
bleaching. The microcarriers used in the following experiments were
45 .mu.m FITC labeled latex beads purchased from PolylaB in
Antwerp, Belgium.
[0067] For one set of bleaching experiments, a 60.times.
magnification and 300 mW laser power were used to produce the
patterns shown in FIG. 10a. The squares in the top row of the
figure have a breadth of 32 pixels (=2.46 .mu.m) in the software
and are separated by another 32 pixels. Bleaching enlarged them in
reality. The squares in the bottom row of the figure are half this
size.
[0068] As shown in FIG. 10a, the bleaching of several intensities
is possible. Assuming, for example, a plateau-level of 200 analog
to digital units ("ADU"), bleaching is possible from at least
levels 50 to 200 ADU approximately. Therefore, levels can be
bleached over an interval of about 25% of the original fluorescent
intensity. The photograph of FIG. 10a reveals that, for example, 6
levels of bleaching are certainly possible. Those six levels of
bleaching are apparent from the six squares bleached in the top row
of FIG. 10a. The fluorescent intensities of those squares are shown
in the graph of FIG. 10c. The six squares of different fluorescent
intensity were obtained by repeated bleaching (1 to 6 times). Using
six coding sites having six different levels of fluorescent
intensity allows for 6.sup.6, or 46656 different codes.
[0069] The series of the ten smaller bleached squares shown in FIG.
10a reveals clearly that bleaching by repeated scanning is not
linear. These squares are only half as broad as the previous ones,
but remain clearly distinguishable. The intensity levels for those
markings are shown in the graph of FIG. 10d. Lastly, the intensity
of a single bleach spot between the two rows of six and ten coding
sites is shown in FIG. 10b.
[0070] A second experiment was conducted to bleach a code
effectively with 8 coding sites (software-breadth of 1 bar=24
pix.times.0.069 .mu.m/pix=1.66 .mu.m; separated by another 24
pixels) and 8 different intensities (allowing for 8.sup.8=16777216
different codes). The laser output was selected to be 200 mW. The
photograph of the microcarrier of this experiment is illustrated in
FIG. 11a, and the graph indicating the different intensity levels
of the individual codes is shown in FIG. 11b. The different
intensities are clearly visible. This code of FIG. 11a is the
number 1 5 2 6 3 7 4 8.
[0071] FIGS. 12a and 12b illustrate another example of eight
encoding sites having eight different intensities. In this case,
bleaching was performed with 250 mW instead of 200 mW as in the
previous case, and was performed using 0.056 mm/pix. The code in
this example was 1 3 2 6 5 8 74. The photograph of the codes, and a
graph of the different intensities of the codes, is shown in FIGS.
12a and 12b, respectively.
[0072] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *