U.S. patent application number 13/478689 was filed with the patent office on 2012-09-20 for multiplexed assay using encoded solid support matrices.
This patent application is currently assigned to IRORI TECHNOLOGIES, INC.. Invention is credited to Gary S. David, Michael P. Nova, Zahra Parandoosh, Andrew E. Senyei, Xiao-Yi Xiao.
Application Number | 20120238466 13/478689 |
Document ID | / |
Family ID | 27578838 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120238466 |
Kind Code |
A1 |
Nova; Michael P. ; et
al. |
September 20, 2012 |
Multiplexed Assay Using Encoded Solid Support Matrices
Abstract
In a multiplexed assay, each molecule of a plurality of
molecules is attached to a support matrix with a substrate adapted
for attachment and/or synthesis of molecules and an
integrally-formed memory device with an optically-encoded
identifier to uniquely identify the molecule attached to the
substrate. The molecules are exposed to one or more processing
conditions then placed within the path of an optical detector
adapted to read the optically-encoded identifier and measure
biochemical processes on each support matrix. The support matrices
may be singulated to be read by the optical detector one at a
time.
Inventors: |
Nova; Michael P.; (Rancho
Santa Fe, CA) ; Senyei; Andrew E.; (La Jolla, CA)
; Parandoosh; Zahra; (San Diego, CA) ; David; Gary
S.; (La Jolla, CA) ; Xiao; Xiao-Yi; (San
Diego, CA) |
Assignee: |
IRORI TECHNOLOGIES, INC.
San Francisco
CA
|
Family ID: |
27578838 |
Appl. No.: |
13/478689 |
Filed: |
May 23, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11011452 |
Dec 14, 2004 |
7935659 |
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13478689 |
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08945053 |
May 11, 1998 |
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11011452 |
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08639813 |
Apr 2, 1996 |
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08945053 |
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08567746 |
Dec 5, 1995 |
6025129 |
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08639813 |
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08538387 |
Oct 3, 1995 |
5874214 |
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08567746 |
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08473660 |
Jun 7, 1995 |
6331273 |
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08538387 |
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08480147 |
Jun 7, 1995 |
6352854 |
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08473660 |
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08480196 |
Jun 7, 1995 |
5925562 |
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08480147 |
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08484504 |
Jun 7, 1995 |
5751629 |
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08480196 |
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08484486 |
Jun 7, 1995 |
6416714 |
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08484504 |
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08428662 |
Apr 25, 1995 |
5741462 |
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08484486 |
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13099198 |
May 2, 2011 |
8219327 |
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08428662 |
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Current U.S.
Class: |
506/9 |
Current CPC
Class: |
B01J 2219/00461
20130101; B01J 2219/00344 20130101; B82Y 10/00 20130101; C07K 1/047
20130101; C40B 40/10 20130101; B01J 2219/0072 20130101; C40B 40/06
20130101; C40B 60/14 20130101; B01L 3/5027 20130101; G01N
2035/00782 20130101; B01J 2219/00695 20130101; B01J 2219/00722
20130101; B01J 2219/0054 20130101; B01J 2219/00547 20130101; B01L
2300/0609 20130101; C40B 70/00 20130101; G11C 13/025 20130101; G01N
2015/149 20130101; B01J 2219/00299 20130101; B01J 2219/00592
20130101; B01J 2219/00308 20130101; B01J 2219/00502 20130101; C40B
60/08 20130101; B01J 2219/00585 20130101; B01J 2219/00542 20130101;
C07K 1/00 20130101; B01J 2219/00563 20130101; C07K 1/04 20130101;
B01J 2219/00463 20130101; B01J 2219/00567 20130101; B01J 2219/00551
20130101; B01J 2219/0056 20130101; B01J 2219/00689 20130101; B01J
2219/005 20130101; B01J 2219/00569 20130101; B01J 2219/00596
20130101; G11C 13/0019 20130101; B01J 2219/00295 20130101; B01J
19/0046 20130101; G11C 13/0014 20130101; B01J 2219/0059 20130101;
B01J 2219/00725 20130101; B01J 2219/00333 20130101; B01J 2219/00549
20130101; B01L 3/502707 20130101; G01N 35/00732 20130101; C40B
30/04 20130101; G01N 35/00871 20130101 |
Class at
Publication: |
506/9 |
International
Class: |
C40B 30/04 20060101
C40B030/04 |
Claims
1. A multiplexed assay, comprising: attaching each molecule of a
plurality of molecules to one of a plurality of separate support
matrices, each support matrix comprising a substrate adapted for
attachment of a molecule and an integrally-formed pre-encoded
memory device comprising an optically-encoded identifier to
uniquely identify the molecule attached to the substrate; exposing
the plurality of molecules to one or more processing conditions in
suspension; placing the plurality of support matrices into a vessel
having a constricted section feeding into an optically-transparent
tube disposed within the path of an optical detector, the optical
detector adapted to read the optically-encoded identifier as the
support matrix passes through the optically-transparent tube; and
measuring biochemical processes on each support matrix.
2. The assay of claim 1, wherein the support matrix substrate is
formed from a material having a fluor incorporated therein.
3. The assay of claim 1, wherein the optically-transparent tube
causes the support matrices to flow past the detector one at a
time.
4. The assay of claim 1, wherein the optical detector comprises a
laser.
5. The assay of claim 1, wherein the optical detector comprises a
plurality of lasers, each laser emitting at a different
wavelength.
6. The assay of claim 1, wherein the one or more processing
conditions comprises attaching a fluorescent label to each
molecule.
7. The assay of claim 6, wherein the step of measuring comprises
detecting an emission from the fluorescent label.
8. The assay of claim 1, wherein the molecules are capture
antibodies and the one or more processing conditions comprise:
reacting the molecules with an analyte; and further reacting the
molecules with a detection antibody, wherein the detection antibody
is conjugated with a detectable label.
9. The assay of claim 8, wherein the analyte comprises a fluid
comprising an antigen.
10. The assay of claim 1, wherein the optically-encoded identifier
is embedded or encased within the substrate.
11. The assay of claim 1, wherein the optically-encoded identifier
is an alphanumeric code or a bar code formed within the support
matrix substrate and a remote memory stores the encoded data in
association with information about the molecule attached to the
support matrix.
12. An assay for evaluating a plurality of molecules, comprising:
linking each of the molecules to a separate support matrix having
an integral optically-readable memory pre-encoded with a unique
identity, wherein the unique identity is associated with the
molecule linked to the support matrix; exposing each molecule and
its linked support matrix to one or more reagents in suspension,
wherein at least one of the one or more reagents is conjugated with
a label; placing the plurality of support matrices into a vessel
having a constricted section feeding into an optically-transparent
tube disposed within the path of an optical detector, the optical
detector adapted to read the optically-readable memory as the
support matrix passes through the optically-transparent tube; and
directing the support matrices past an analytical instrument
adapted to detect the label for measuring biochemical processes on
each support matrix.
13. The assay of claim 12, wherein the support matrix has a fluor
incorporated therein.
14. The assay of claim 12, wherein the optically-readable memory is
embedded or encased within the support matrix.
15. The assay of claim 12, wherein the optically-transparent tube
directs the support matrices past two or more laser detectors one
support matrix at a time.
16. The assay of claim 12, wherein the one or more processing
conditions comprises attaching a fluorescent label to each of the
molecules.
17. The assay of claim 16, wherein the step of measuring comprises
detecting an emission from the fluorescent label.
18. A multiplexed assay, comprising: linking each molecule of a
plurality of molecules to a separate support matrix comprising a
bead or particle having a unique optically-encoded identifier
embedded or encased within the support matrix, wherein the
optically-encoded identifier is associated with the molecule linked
to the support matrix; exposing the plurality of molecules and
their corresponding support matrices to one or more processing
steps in suspension to produce a plurality of processed molecules,
wherein at least one of the one or more processing steps comprises
attaching a detectable label for measuring biochemical activity
within each processed molecule; and placing the plurality of
support matrices into a vessel having a constricted section feeding
into an optically-transparent tube disposed within the path of an
optical detector adapted to read the optically-encoded identifier
and a detection path of an analytical instrument adapted to detect
the detectable label for measuring biochemical activity on each
support matrix, wherein the measured biochemical activity is
associated with the optically-encoded identifier for the
corresponding support matrix and, hence, with the processed
molecule that is associated with the optically-encoded
identifier.
19. The assay of claim 18, wherein the molecules are capture
antibodies and the one or more processing conditions comprise:
reacting each molecule with an analyte; and further reacting the
molecule with a second antibody, wherein the second antibody is
conjugated with the detectable label.
20. The assay of claim 18, wherein the detectable label comprises a
fluorescent label and the analytical instrument comprises a second
optical detector.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
13/099,198 filed May 2, 2011, which is a continuation of
application Ser. No. 11/011,452, filed on Dec. 14, 2004, issued May
3, 2011 as U.S. Pat. No. 7,935,659, which is a continuation of Ser.
No. 08/945,053, filed on May 11, 1998, now abandoned, filed as a
371 of international application No. PCT/US96/06145, filed on Apr.
25, 1996, and which is a continuation-in-part of application Ser.
No. 08/639,813, filed on Apr. 2, 1996, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 08/567,746, filed
on Dec. 5, 1995, now U.S. Pat. No. 6,025,129, Feb. 15, 2000, which
is a continuation-in-part of application Ser. No. 08/538,387, filed
on Oct. 3, 1995, now U.S. Pat. No. 5,874,214, Feb. 23, 1999, which
is a continuation-in-part of each of application Ser. No.
08/473,660, filed on Jun. 7, 1995, now U.S. Pat. No. 6,331,273,
Dec. 18, 2001, application Ser. No. 08/480,147, filed on Jun. 7,
1995, now U.S. Pat. No. 6,352,854, Mar. 5, 2002, application Ser.
No. 08/480,196, filed on Jun. 7, 1995, now U.S. Pat. No. 5,925,562,
Jul. 20, 1999, application Ser. No. 08/484,504, filed on Jun. 7,
1995, now U.S. Pat. No. 5,751,629, May 12, 1998, application Ser.
No. 08/484,486, filed on Jun. 7, 1995, now U.S. Pat. No. 6,416,714,
Jul. 9, 2002, and application Ser. No. 08/428,662, filed on Apr.
25, 1995, now U.S. Pat. No. 5,741,462, Apr. 21, 1998. The subject
matter of each of the above-identified applications is incorporated
herein by reference its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of encoded beads
for molecular tracking and identification during multiplexed
assays.
BACKGROUND OF THE INVENTION
[0003] Drug discovery relies on the ability to identify compounds
that interact with a selected target, such as cells, an antibody,
receptor, enzyme, transcription factor or the like. Traditional
drug discovery relied on collections or "libraries" obtained from
proprietary databases of compounds accumulated over many years,
natural products, fermentation broths, and rational drug design.
Recent advances in molecular biology, chemistry and automation have
resulted in the development of rapid, high throughput screening
(HTS) protocols to screen these collections. In connection with
HTS, methods for generating molecular diversity and for detecting,
identifying and quantifying biological or chemical material have
been developed. These advances have been facilitated by fundamental
developments in chemistry, including the development of highly
sensitive analytical methods, solid state chemical synthesis, and
sensitive and specific biological assay systems.
[0004] Analyses of biological interactions and chemical reactions,
however, require the use of labels or tags to track and identify
the results of such analyses. Typically biological reactions, such
as binding, catalytic, hybridization and signaling reactions, are
monitored by labels such as radioactive, fluorescent,
photoabsorptive, luminescent and other such labels, or by direct or
indirect enzyme labels. Chemical reactions are also monitored by
direct or indirect means, such as by linking the reactions to a
second reaction in which a colored, fluorescent, chemiluminescent
or other such product results. These analytical methods, however,
are often time consuming, tedious and, when practiced in vivo,
invasive. In addition, each reaction is typically measured
individually, in a separate assay. There is, thus, a need to
develop alternative and convenient methods for tracking and
identifying analytes in biological interactions and the reactants
and products of chemical reactions.
[0005] High Throughput Screening:
[0006] In addition, exploitation of this diversity requires
development of methods for rapidly screening compounds. Advances in
instrumentation, molecular biology and protein chemistry and the
adaptation of biochemical activity screens into microplate formats,
has made it possible to screen of large numbers of compounds. Also,
because compound screening has been successful in areas of
significance for the pharmaceutical industry, high throughput
screening (HTS) protocols have assumed importance. Presently, there
are hundreds of HTS systems operating throughout the world, which
are used, not only for compound screening for drug discovery, but
also for immunoassays, cell-based assays and receptor-binding
assays.
[0007] An essential element of high throughput screening for drug
discovery process and areas in which molecules are identified and
tracked, is the ability to extract the information made available
during synthesis and screening of a library, identification of the
active components of intermediary structures, and the reactants and
products of assays. While there are several techniques for
identification of intermediary products and final products,
nanosequencing protocols that provide exact structures are only
applicable on mass to naturally occurring linear oligomers such as
peptides and amino acids. Mass spectrographic (MS) analysis is
sufficiently sensitive to determine the exact mass and
fragmentation patterns of individual synthesis steps, but complex
analytical mass spectrographic strategies are not readily automated
nor conveniently performed. Also, mass spectrographic analysis
provides at best simple connectivity information, but no
stereoisomeric information, and generally cannot discriminate among
isomeric monomers. Another problem with mass spectrographic
analysis is that it requires pure compounds; structural
determinations on complex mixtures are either difficult or
impossible. Finally, mass spectrographic analysis is tedious and
time consuming. Thus, although there are a multitude of solutions
to the generation of libraries and to screening protocols, there
are no ideal solutions to the problems of identification, tracking
and categorization.
[0008] These problems arise in any screening or analytical process
in which large numbers of molecules or biological entities are
screened. In any system, once a desired molecule(s) has been
isolated, it must be identified. Simple means for identification do
not exist. Because of the problems inherent in any labeling
procedure, it would be desirable to have alternative means for
tracking and quantitating chemical and biological reactions during
synthesis and/or screening processes, and for automating such
tracking and quantitating.
[0009] Therefore, it is an object herein to provide methods for
identification, tracking and categorization of the components of
complex mixtures of diverse molecules. It is also an object herein
to provide products for such identification, tracking and
categorization and to provide assays, diagnostics and screening
protocols that use such products. It is of particular interest
herein to provide means to track and identify compounds and to
perform HTS protocols.
SUMMARY OF THE INVENTION
[0010] Combinations of matrix materials with programmable data
storage or recording devices, herein referred to as memories, and
assays using these combinations are provided. These combinations
are referred to herein as matrices with memories. By virtue of this
memory with matrix combination, molecules, such as antigens,
antibodies, ligands, proteins and nucleic acids, and biological
particles, such as phage and viral particles and cells, that are
associated with, such as in proximity to or in physical contact
with the matrix combination, can be electromagnetically tagged by
programming the memory with data corresponding to identifying
information. Programming and reading the memory is effected
remotely, preferably using electromagnetic radiation, particularly
radio frequency or radar. Memories may also be remote from the
matrix, such as instances in which the memory device is precoded
with a mark or identifier or the matrix is encoded with a bar code.
The identity, i.e., the mark or code, of each device is written to
a memory, which may be a computer or a piece of paper or any
recording device, and information associated with each matrix is
stored in the remote memory and linked to the code or other
identifier.
[0011] The molecules and biological particles that are associated
with the matrix combination, such as in proximity to or in physical
contact or with the matrix combination, can be identified and the
results of the assays determined by retrieving the stored data
points from the memories. Querying the memory will identify
associated molecules or biological particles that have reacted.
[0012] The combinations provided herein thus have a multiplicity of
applications, including combinatorial chemistry, isolation and
purification of target macromolecules, capture and detection of
macromolecules for analytical purposes, high throughput screening,
selective removal of contaminants, enzymatic catalysis, drug
delivery, chemical modification, information collection and
management and other uses. These combinations are particularly
advantageous for use in multianalyte analyses, assays in which a
electromagnetic signal is generated by the reactants or products in
the assay, for use in homogeneous assays, and for use in
multiplexed protocols.
[0013] Of particular interest herein, are multiprotocol
applications (such as multiplexed assays or coupled synthetic and
assay protocols) in which the matrices with memories are used in a
series (more than one) of reactions, a series (more than one) of
assays, and/or a series of more or more reactions and one or more
assays, typically on a single platform or coupled via automated
analysis instrumentation. As a result synthesis is coupled to
screening.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 depicts combinatorial synthesis of chemical libraries
on matrix supports with memories.
[0015] FIG. 2 depicts combinatorial synthesis of peptides on a
matrix with memory.
[0016] FIG. 3 depicts combinatorial synthesis of oligonucleotides
on matrix supports with memories.
[0017] FIG. 4 depicts generation of a chemical library, such as a
library of organic molecules, according to the present
invention.
[0018] FIG. 5 is a block diagram of the data storage means and
supporting electrical components of a preferred embodiment.
[0019] FIG. 6 is a diagrammatic view of the memory array within the
recording device, and the corresponding data stored in the host
computer memory.
[0020] FIG. 7 is an illustration of an exemplary apparatus for
separating the matrix particles with memories for individual
exposure to an EM signal.
[0021] FIG. 8 is an illustration of a second exemplary embodiment
of an apparatus for separating matrix particles for individual
exposure to an optical signal.
[0022] FIG. 9 is a diagrammatic view of the memory array within the
recording device, the corresponding data stored in the host
computer memory, and included photodetector with amplifier and
gating transistor.
[0023] FIG. 10 illustrates an exemplary scheme for the synthesis of
the 8 member RF encoded combinatorial decameric peptide
library.
[0024] FIG. 11 illustrates a sequence using fluorescent solid
supports in an application in solid phase synthesis of direct
SPA.
[0025] FIG. 12 illustrates an exemplary sequence using coded macro
"beads".
[0026] FIG. 13 illustrates an exemplary sequence for preparation
and use of a tubular microvessel in which the container is
radiation grafted with monomers.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications referred to herein are, unless noted otherwise,
incorporated by reference in their entirety.
[0028] As used herein, a matrix refers to any solid or semisolid or
insoluble support to which the memory device and/or the molecule of
interest, typically a biological molecule, organic molecule or
biospecific ligand is linked or contacted. Typically a matrix is a
substrate material having a rigid or semi-rigid surface. In many
embodiments, at least one surface of the substrate will be
substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
polymers with, for example, wells, raised regions, etched trenches,
or other such topology. Matrix materials include any materials that
are used as affinity matrices or supports for chemical and
biological molecule syntheses and analyses, such as, but are not
limited to: polystyrene, polycarbonate, polypropylene, nylon,
glass, dextran, chitin, sand, pumice, TEFLON.RTM., agarose,
polysaccharides, dendrimers, buckyballs, polyacrylamide,
Kieselguhr-polyacrlamide non-covalent composite,
polystyrene-polyacrylamide covalent composite, polystyrene-PEG
(polyethyleneglycol) composite, silicon, rubber, and other
materials used as supports for solid phase syntheses, affinity
separations and purifications, hybridization reactions,
immunoassays and other such applications. The matrix herein may be
particulate or may be in the form of a continuous surface, such as
a microtiter dish or well, a glass slide, a silicon chip, a
nitrocellulose sheet, nylon mesh, or other such materials. When
particulate, typically the particles have at least one dimension in
the 5-10 mm range or smaller. Such particles, referred collectively
herein as "beads", are often, but not necessarily, spherical. Such
reference, however, does not constrain the geometry of the matrix,
which may be any shape, including random shapes, needles, fibers,
elongated, etc. The "beads" may include additional components, such
as magnetic or paramagnetic particles (see, e.g., Dyna beads
(Dynal, Oslo, Norway)) for separation using magnets, fluophores and
other scintillants, as long as the additional components do not
interfere with chemical reactions, data entry or retrieval from the
memory.
[0029] As used herein, scintillants include, 2,5-diphenyloxazole
(PPO), anthracene,
2-(4'-tert-butylphenyl)-5-(4''-biphenyl)-1,3,4-oxadiazole
(butyl-PBD); 1-phenyl-3-mesityl-2-pyrazoline (PMP), with or without
frequency shifters, such as 1,4-bis(5-phenyl(oxazolyl)benzene)
(POPOP); p-bis-o-methylstyrylbenzene (bis-MSB). Combinations of
these fluors such as PPO and POPOP or PPO and bis-MSB, in suitable
solvents, such as benzyltoluene (see, e.g., U.S. Pat. No.
5,410,155), are referred to as scintillation cocktails.
[0030] As used herein a luminescent moiety refers to a scintillant
or fluophor used in scintillation proximity assays or in
non-radioactive energy transfer assays, such as HTRF assays.
[0031] As used herein, matrix particles refer to matrix materials
that are in the form of discrete particles. The particles have any
shape and dimensions, but typically have at least one dimension
that is 100 mm or less, preferably 50 mm or less, more preferably
10 mm or less, and typically have a size that is 100 mm.sup.3 or
less, preferably 50 mm.sup.3 or less, more preferably 10 mm.sup.3
or less, and most preferably 1 mm.sup.3 or less. The matrices may
also be continuous surfaces, such as microtiter plates (e.g.,
plates made from polystyrene or polycarbonate or derivatives
thereof commercially available from Perkin Elmer Cetus and numerous
other sources, and Covalink trays (Nunc), microtiter plate lids or
a test tube, such as a 1 ml Eppendorf tube. Matrices that are in
the form of containers refers to containers, such as test tubes and
microplates and vials that are typically used for solid phase
syntheses of combinatorial libraries or as pouches, vessels, bags,
and microvessels for screening and diagnostic assays. Thus, a
container used for chemical syntheses refers to a container that
typically has a volume of about 1 liter, generally 100 ml, and more
often 10 ml or less, 5 ml or less, preferably 1 ml or less, and as
small as about 50 .mu.l-500 .mu.l, such as 100 .mu.l or 250 .mu.l.
This also refers to multi-well plates, such as microtiter plates
(96 well, 384 well or other density format). Such microtiter plate
will typically contain a recording device in, on, or otherwise in
contact with in each of a plurality of wells.
[0032] As used herein, a matrix with a memory refers to a
combination of a matrix with a miniature recording device that
stores multiple bits of data by which the matrix may be identified,
preferably in a non-volatile memory that can be written to and read
from by transmission of electromagnetic radiation from a remote
host, such as a computer. By miniature is meant of a size less than
about 10-20 mm.sup.3 (or 10-20 mm in the largest dimension).
Preferred memory devices or data storage units are miniature and
are preferably smaller than 10-20 mm.sup.3 (or 10-20 mm in its
largest dimension) dimension, more preferably less than 5 mm.sup.3,
most preferably about 1 mm.sup.3 or smaller.
[0033] As used herein, a microreactor refers to combinations of
matrices with memories with associated, such as linked or
proximate, biological particles or molecules. It is produced, for
example, when the molecule is linked thereto or synthesized
thereon. It is then used in subsequent protocols, such as
immunoassays and scintillation proximity assays.
[0034] As used herein, a combination herein called a microvessel
(e.g., an MICROKAN.TM.) refers to a combination in which a single
device (or more than one device) and a plurality of particles are
sealed in a porous or semi-permeable inert material, such as teflon
or polypropylene or membrane that is permeable to the components of
the medium, but retains the particles and memory, or are sealed in
a small closable container that has at least one dimension that is
porous or semi-permeable. Typically such microvessels, which
preferably have at least one end that can be opened and sealed or
closed tightly, has a volume of about 200-500 mm.sup.3, with
preferred dimensions of about 1-10 mm in diameter and 5 to 20 mm in
height, more preferably about 5 mm by 15 mm. The porous wall should
be non-collapsible with a pore size in the range of 70 .mu.M to
about 100 .mu.M, but can be selected to be semi-permeable for
selected components of the reaction medium.
[0035] As used herein, a memory is a data storage unit (or medium)
with programmable memory, preferably a non-volatile memory.
[0036] As used herein, programming refers to the process by which
data or information is entered and stored in a memory. A memory
that is programmed is a memory that contains retrievable
information.
[0037] As used herein, remotely programmable, means that the memory
can be programmed without direct physical or electrical contact or
can be programmed from a distance, typically at least about 10 mm,
although shorter distances may also be used, such as instances in
which the information comes from surface or proximal reactions or
from an adjacent memory or in instances, such as embodiments in
which the memories are very close to each other, as in microtiter
plate wells or in an array.
[0038] As used herein, a recording device (or memory device) is an
apparatus that includes the data storage unit with programmable
memory, and, if necessary, means for receiving information and for
transmitting information that has been recorded. It includes any
means needed or used for writing to and reading from the memory.
The recording devices intended for use herein, are miniature
devices that preferably are smaller than 10-20 mm.sup.3 (or 10-20
mm in their largest dimension), and more preferably are closer in
size to 1 mm.sup.3 or smaller that contain at least one such memory
and means for receiving and transmitting data to and from the
memory.
[0039] As used herein, a data storage unit with programmable memory
includes any data storage means having the ability to record
multiple discrete bits of data, which discrete bits of data may be
individually accessed (read) after one or more recording
operations. Thus, a matrix with memory is a combination of a matrix
material with a miniature data storage unit.
[0040] As used herein, programmable means capable of storing unique
data points. Addressable means having unique locations that may be
selected for storing the unique data points.
[0041] As used herein, a host computer or decoder/encoder
instrument is an instrument that has been programmed with or
includes information (i.e., a key) specifying the code used to
encode the memory devices. This instrument, or one linked thereto,
transmits the information and signals to the recording device and
it, or another instrument, receives the information transmitted
from the recording device upon receipt of the appropriate signal.
This instrument thus creates the appropriate signal to transmit to
the recording device and can interpret transmitted signals. For
example, if a "1" is stored at position 1,1 in the memory of the
recording device means, upon receipt of this information, this
instrument or computer can determine that this means the linked
molecule is, for example, a peptide containing alanine at the
N-terminus, an organic group, organic molecule, oligonucleotide, or
whatever this information has been predetermined to mean.
Alternatively, the information sent to and transmitted from the
recording device can be encoded into the appropriate form by a
person.
[0042] As used herein, an electromagnetic tag is a recording device
that has a memory that contains unique data points that correspond
to information that identifies molecules or biological particles
linked to, directly or indirectly, in physical contact with or in
proximity (or associated with) to the device. Thus, electromagnetic
tagging is the process by which identifying or tracking information
is transmitted (by any means and to any recording device memory,
including optical and magnetic storage media) to the recording
device.
[0043] As used herein, proximity means within a very short
distance, generally less than 0.5 inch, typically less than 0.2
inches. In particular, stating that the matrix material and memory,
or the biological particle or molecule and matrix with memory are
in proximity means that, they are at least or at least were in the
same reaction vessel or, if the memory is removed from the reaction
vessel, the identity of the vessel containing the molecules or
biological particles with which the memory was proximate or linked
is tracked or otherwise known.
[0044] As used herein, associated with means that the memory must
remain in proximity to the molecule or biological particle or must
in some manner be traceable to the molecule or biological particle.
For example, if a molecule is cleaved from the support with memory,
the memory must in some manner be identified as having been linked
to the cleaved molecule. Thus, a molecule or biological particle
that had been linked to or in proximity to a matrix with memory is
associated with the matrix or memory if it can be identified by
querying the memory.
[0045] As used herein, electromagnetic (EM) radiation refers to
radiation understood by skilled artisans to be EM radiation and
includes, but is not limited to radio frequency (RF), infrared
(IR), visible, ultraviolet (UV), radiation, sonic waves, X-rays,
and laser light.
[0046] As used herein, information identifying or tracking a
biological particle or molecule refers to any information that
identifies the molecule or biological particle, such as, but not
limited to the identity particle (i.e., its chemical formula or
name), its sequence, its type, its class, its purity, its
properties, such as its binding affinity for a particular ligand.
Tracking means the ability to follow a molecule or biological
particle through synthesis and/or process steps. The memory devices
herein store unique indicators that represent any of this
information.
[0047] As used herein, combinatorial chemistry is a synthetic
strategy that produces diverse, usually large, chemical libraries.
It is the systematic and repetitive, covalent connection of a set,
the basis set, of different monomeric building blocks of varying
structure to each other to produce an array of diverse molecules
(see, e.g., Gallop et al. (1994) J. Medicinal Chemistry
37:1233-1251). It also encompasses other chemical modifications,
such as cyclizations, eliminations, cleavages, etc., that are
carried in manner that generates permutations and thereby
collections of diverse molecules.
[0048] As used herein, a biological particle refers to a virus,
such as a viral vector or viral capsid with or without packaged
nucleic acid, phage, including a phage vector or phage capsid, with
or without encapsulated nucleotide acid, a single cell, including
eukaryotic and prokaryotic cells or fragments thereof, a liposome
or micellar agent or other packaging particle, and other such
biological materials.
[0049] As used herein, the molecules in the combinations include
any molecule, including nucleic acids, amino acids, other
biopolymers, and other organic molecules, including peptidomimetics
and monomers or polymers of small organic molecular constituents of
non-peptidic libraries, that may be identified by the methods here
and/or synthesized on matrices with memories as described
herein.
[0050] As used herein, the term "bio-oligomer" refers to a
biopolymer of less than about 100 subunits. A bio-oligomer
includes, but is not limited to, a peptide, i.e., containing amino
acid subunits, an oligonucleotide, i.e., containing nucleoside
subunits, a peptide-oligonucleotide chimera, peptidomimetic, and a
polysaccharide.
[0051] As used herein, the term "sequences of random monomer
subunits" refers to polymers or oligomers containing sequences of
monomers in which any monomer subunit may precede or follow any
other monomer subunit.
[0052] As used herein, the term "library" refers to a collection of
substantially random compounds or biological particles expressing
random peptides or proteins or to a collection of diverse
compounds. Of particular interest are bio-oligomers, biopolymers,
or diverse organic compounds or a set of compounds prepared from
monomers based on a selected pharmacophore.
[0053] As used herein, an analyte is any substance that is analyzed
or assayed in the reaction of interest. Thus, analytes include the
substrates, products and intermediates in the reaction, as well as
the enzymes and cofactors.
[0054] As used herein, multianalyte analysis is the ability to
measure many analytes in a single specimen or to perform multiple
tests from a single specimen. The methods and combinations herein
provide means to identify or track individual analytes from among a
mixture of such analytes.
[0055] As used herein, a fluophore or a fluor is a molecule that
readily fluoresces; it is a molecule that emits light following
interaction with radiation. The process of fluorescence refers to
emission of a photon by a molecule in an excited singlet state. For
scintillation assays, combinations of fluors are typically used. A
primary fluor that emits light following interaction with radiation
and a secondary fluor that shifts the wavelength emitted by the
primary fluor to a higher more efficiently detected wavelength.
[0056] As used herein, a peptidomimetic is a compound that mimics
the conformation and certain stereochemical features of the
biologically active form of a particular peptide. In general,
peptidomimetics are designed to mimic certain desirable properties
of a compound but not the undesirable features, such as flexibility
leading to a loss of the biologically active conformation and bond
breakdown. For example, methylenethio bioisostere (CH2S) has been
used as an amide replacement in enkephalin analogs (see, e.g.,
Spatola, A. F. Chemistry and Biochemistry of Amino Acids, Peptides,
and Proteins (Weinstein, B, Ed., Vol. 7, pp. 267-357, Marcel
Dekker, New York (1983); and Szelke et al. (1983) In Peptides:
Structure and Function. Proceedings of the Eighth American Peptide
Symposium. Hruby and Rich, Eds., pp. 579-582, Pierce Chemical Co.,
Rockford, Ill.).
[0057] As used herein, complete coupling means that the coupling
reaction is driven substantially to completion despite or
regardless of the differences in the coupling rates of individual
components of the reaction, such as amino acids In addition, the
amino acids, or whatever is being coupled, are coupled to
substantially all available coupling sites on the solid phase
support so that each solid phase support will contain essentially
only one species of peptide.
[0058] As used herein, the biological activity or bioactivity of a
particular compound includes any activity induced, potentiated or
influenced by the compound in vivo or in vitro. It also includes
the abilities, such as the ability of certain molecules to bind to
particular receptors and to induce (or modulate) a functional
response. It may be assessed by in vivo assays or by in vitro
assays, such as those exemplified herein.
[0059] As used herein, pharmaceutically acceptable salts, esters or
other derivatives of the compounds include any salts, esters or
derivatives that may be readily prepared by those of skill in this
art using known methods for such derivatization and that produce
compounds that may be administered to animals or humans without
substantial toxic effects and that either are pharmaceutically
active or are prodrugs. For example, hydroxy groups can be
esterified or etherified.
[0060] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), mass spectrometry (MS), size exclusion
chromatography, gel electrophoresis, particularly agarose and
polyacrylamide gel electrophoresis (PAGE) and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound may, however,
be a mixture of stereoisomers. In such instances, further
purification might increase the specific activity of the
compound.
[0061] As used herein, adequately pure or "pure" per se means
sufficiently pure for the intended use of the adequately pure
compound.
[0062] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures.
[0063] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound will be
regenerated by metabolic processes. The prodrug may be designed to
alter the metabolic stability or the transport characteristics of a
drug, to mask side effects or toxicity, to improve the flavor of a
drug or to alter other characteristics or properties of a drug. By
virtue of knowledge of pharmacodynamic processes and drug
metabolism in vivo, those of skill in this art, once a
pharmaceutically active compound is known, can design prodrugs of
the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A
Biochemical Approach. Oxford University Press, New York, pages
388-392).
[0064] As used herein, amino acids refer to the naturally-occurring
amino acids and any other non-naturally occurring amino acids, and
also the corresponding D-isomers. It is also understood that
certain amino acids may be replaced by substantially equivalent
non-naturally occurring variants thereof, such as D-Nva, D-Nle,
D-Alle, and others listed with the abbreviations below or known to
those of skill in this art.
[0065] As used herein, hydrophobic amino acids include Ala, Val,
Leu, lie, Pro, Phe, Trp, and Met. the non-naturally occurring amino
acids and the corresponding D isomers of the hydrophobic amino
acids, that have similar hydrophobic properties; the polar amino
acids include Gly, Ser, Thr, Cys, Tyr, Asn, Gin, the non-naturally
occurring amino acids and the corresponding D isomers of the polar
amino acids, that have similar properties, the charged amino acids
include Asp, Glu, Lys, Arg, His, the non-naturally occurring amino
acids and the corresponding D isomers of these amino acids.
[0066] As used herein, Southern, Northern, Western and dot blot
procedures refer to those in which DNA, RNA and protein patterns,
respectively, are transferred for example, from agarose gels,
polyacrylamide gels or other suitable medium that constricts
convective motion of molecules, to nitrocellulose membranes or
other suitable medium for hybridization or antibody or antigen
binding are well known to those of skill in this art.
[0067] As used herein, a receptor refers to a molecule that has an
affinity for a given ligand. Receptors may be naturally-occurring
or synthetic molecules. Receptors may also be referred to in the
art as anti-ligands. As used herein, the terms "receptor" and
"anti-ligand" are interchangeable. Receptors can be used in their
unaltered state or as aggregates with other species. Receptors may
be attached, covalently or noncovalently, or in physical contact
with, to a binding member, either directly or indirectly via a
specific binding substance or linker. Examples of receptors,
include, but are not limited to: antibodies, cell membrane
receptors, surface receptors and internalizing receptors,
monoclonal antibodies and antisera reactive with specific antigenic
determinants (such as on viruses, cells, or other materials),
drugs, polynucleotides, nucleic acids, peptides, cofactors,
lectins, sugars, polysaccharides, cells, cellular membranes, and
organelles.
[0068] Examples of receptors and applications using such receptors,
include but are not restricted to:
[0069] a) enzymes: specific transport proteins or enzymes essential
to survival of microorganisms, which could serve as targets for
antibiotic (ligand) selection;
[0070] b) antibodies: identification of a ligand-binding site on
the antibody molecule that combines with the epitope of an antigen
of interest may be investigated; determination of a sequence that
mimics an antigenic epitope may lead to the development of vaccines
of which the immunogen is based on one or more of such sequences or
lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune diseases
[0071] c) nucleic acids: identification of ligand, such as protein
or RNA, binding sites;
[0072] d) catalytic polypeptides: polymers, preferably
polypeptides, that are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products; such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, in which the
functionality is capable of chemically modifying the bound reactant
(see, e.g., U.S. Pat. No. 5,215,899);
[0073] e) hormone receptors: determination of the ligands that bind
with high affinity to a receptor is useful in the development of
hormone replacement therapies; for example, identification of
ligands that bind to such receptors may lead to the development of
drugs to control blood pressure; and
[0074] f) opiate receptors: determination of ligands that bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0075] As used herein, antibody includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain.
[0076] As used herein, complementary refers to the topological
compatibility or matching together of interacting surfaces of a
ligand molecule and its receptor. Thus, the receptor and its ligand
can be described as complementary, and furthermore, the contact
surface characteristics are complementary to each other.
[0077] As used herein, a ligand-receptor pair or complex formed
when two macromolecules have combined through molecular recognition
to form a complex.
[0078] As used herein, an epitope refers to a portion of an antigen
molecule that is delineated by the area of interaction with the
subclass of receptors known as antibodies.
[0079] As used herein, a ligand is a molecule that is specifically
recognized by a particular receptor. Examples of ligands, include,
but are not limited to, agonists and antagonists for cell membrane
receptors, toxins and venoms, viral epitopes, hormones (e.g.,
steroids), hormone receptors, opiates, peptides, enzymes, enzyme
substrates, cofactors, drugs, lectins, sugars, oligonucleotides,
nucleic acids, oligosaccharides, proteins, and monoclonal
antibodies.
[0080] As used herein, a sensor is a device or apparatus that
monitors external parameters (i.e., conditions), such as ion
concentrations, pH, temperatures. Biosensors are sensors that
detect biological species. Sensors encompass devices that rely on
electrochemical, optical, biological and other such means to
monitor the environment.
[0081] As used herein, multiplexing refers to performing a series
of synthetic and processing steps and/or assaying steps on the same
platform (e.g., solid support or matrix) or coupled together as
part of the same automated coupled protocol, including one or more
of the following, synthesis, preferably accompanied by writing to
the linked memories to identify linked compounds, screening,
including using protocols with matrices with memories, and compound
identification by querying the memories of matrices associated with
the selected compounds. Thus, the platform refers system in which
all manipulations are performed. In general it means that several
protocols are coupled and performed sequentially or
simultaneously.
[0082] As used herein, a platform refers to the instrumentation or
devices in which a reaction or series of reactions is(are)
performed.
[0083] As used herein a protecting group refers to a material that
is chemically bound to a monomer unit that may be removed upon
selective exposure to an activator such as electromagnetic
radiation and, especially ultraviolet and visible light, or that
may be selectively cleaved. Examples of protecting groups include,
but are not limited to: those containing nitropiperonyl,
pyrenylmethoxy-carbonyl, nitroveratryl, nitrobenzyl, dimethyl
dimethoxybenzyl, 5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl
cinnamoyl, and 2-oxymethylene anthraquinone.
[0084] Also protected amino acids are readily available to those of
skill in this art. For example, Fmoc and Boc protected amino acids
can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma,
Cambridge Research Biochemical, Bachem, or Peninsula Labs or other
chemical companies familiar to those who practice this art.
[0085] As used herein, the abbreviations for amino acids and
protective groups are in accord with their common usage and the
IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 11:942-944). Each naturally occurring L-amino acid is
identified by the standard three letter code or the standard three
letter code with or without the prefix "L-"; the prefix "D-"
indicates that the stereoisomeric form of the amino acid is D. For
example, as used herein, Fmoc is 9-fluorenylmethoxycarbonyl; BOP is
benzotriazol-1-yloxytris(dimethylamtno) phosphonium
hexafluorophosphate, DCC is dicyclohexylcarbodiimide; DDZ is
dimethoxydimethylbenzyloxy; DMT is dimethoxytrityl; FMOC is
fluorenylmethyloxycarbonyl; HBTU is
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;
hexafluorophosphate NV is nitroveratryl; NVOC is
6-nitroveratryloxycarbonyl and other photoremovable groups; TFA is
trifluoroacetic acid; DMF for N,N-dimethylformamide; Boc is
tert-butoxycarbonyl; HF for hydrogen fluoride; HFIP for
hexafluoroisopropanol; HPLC for high performance liquid
chromatography; FAB-MS for fast atom bombardment mass spectrometry;
DCM is dichloromethane, Bom is benzyloxymethyl; Pd/C is palladium
catalyst on activated charcoal; DIC is diisopropylcarbodiimide; DCC
is N,N'-dicyclohexylcarbodiimide; (For) is formyl; PyBop is
benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium
hexafluorophosphate; POPOP is 1,4-bis(5-phenyl(oxazolyl)benzene);
PPO is 2,5-diphenyloxazole; butyl-PBD is
(2-(4'-tert-butylphenyl)-5-(4''-biphenyl)-1,3,4-oxadiazole); PMP is
(1-phenyl-3-mesityl-2-pyrazoline) DIEA is diisopropylethylamine;
EDIA is ethyldiiso-propylethylamine; NMP is N-methylpyrrolidone; NV
is nitroveratryl PAL is pyridylalanine; HATU is
O(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate; THF is tetrahydrofuran; and EDT is
1,2-ethanedithiol.
[0086] A. Matrices
[0087] Matrices, which are generally insoluble materials used to
immobilize ligands and other molecules, have application in many
chemical syntheses and separations. Matrices are used in affinity
chromatography, in the immobilization of biologically active
materials, and during chemical syntheses of biomolecules, including
proteins, amino acids and other organic molecules and polymers. The
preparation of and use of matrices is well known to those of skill
in this art; there are many such materials and preparations thereof
known. For example, naturally-occurring matrix materials, such as
agarose and cellulose, may be isolated from their respective
sources, and processed according to known protocols, and synthetic
materials may be prepared in accord with known protocols.
[0088] Matrices include any material that can act as a support
matrix for attachment of the molecules or biological particles of
interest and can be in contact with or proximity to or associated
with, preferably encasing or coating, the data storage device with
programmable memory. Any matrix composed of material that is
compatible with and upon or in which chemical syntheses are
performed, including biocompatible polymers, is suitable for use
herein. The matrix material should be selected so that it does not
interfere with the chemistry or biological reaction of interest
during the time which the molecule or particle is linked to, or in
proximity therewith (see, e.g., U.S. Pat. No. 4,006,403). These
matrices, thus include any material to which the data storage
device with memory can be attached, placed in proximity thereof,
impregnated, encased or otherwise connected, linked or physically
contacted. Such materials are known to those of skill in this art,
and include those that are used as a support matrix. These
materials include, but are not limited to, inorganics, natural
polymers, and synthetic polymers, including, but are not limited
to: cellulose, cellulose derivatives, acrylic resins, glass, silica
gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of
vinyl and acrylamide, polystyrene cross-linked with divinylbenzene
or the like (see, Merrifield (1964) Biochemistry 3:1385-1390),
polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,
rubber, silicon, plastics, nitrocellulose, celluloses, natural
sponges, and many others.
[0089] Among the preferred matrices are polymeric beads, such as
the TENTAGEL resins and derivatives thereof (sold by Rapp Polymere,
Tubingen, Germany; see, U.S. Pat. No. 4,908,405 and U.S. Pat. No.
5,292,814.) Matrices that are also contemplated for use herein
include fluophore-containing or -impregnated matrices, such as
microplates and beads (commercially available, for example, from
Amersham, Arlington Heights, Ill.; plastic scintillation beads from
NE (Nuclear Technology, Inc., San Carlos, Calif.), Packard,
Meriden, Conn.). It is understood that these commercially available
materials will be modified by combining them with memories, such as
by methods described herein.
[0090] The matrix may also be a relatively inert polymer, which can
be grafted by ionizing radiation (see, e.g., FIG. 13, which depicts
a particular embodiment) to permit attachment of a coating of
polystyrene or other such polymer that can be derivatized and used
as a support. Radiation grafting of monomers allows a diversity of
surface characteristics to be generated on plasmid supports (see,
e.g., Maeji et al. (1994) Reactive Polymers 22:203-212; and Berg et
al. (1989) J. Am. Chem. Soc. 111:8024-8026). For example,
radiolytic grafting of monomers, such as vinyl monomers, or
mixtures of monomers, to polymers, such as polyethylene and
polypropylene, produce composites that have a wide variety of
surface characteristics. These methods have been used to graft
polymers to insoluble supports for synthesis of peptides and other
molecules, and are of particular interest herein. The recording
devices, which are often coated with a plastic or other insert
material, can be treated with ionizing radiation so that selected
monomers can be grafted to render the surface suitable for chemical
syntheses.
[0091] Where the matrix particles are macroscopic in size, such as
about at least 1 mm in at least one dimension, such matrix may
contain one or more memories. Where the matrix particles are
smaller, such as NE particles (PVT-based plastic scintillator
microsphere), which are about 1 to 10 .mu.m in diameter, more than
one such particle will generally be associated with one memory.
Also, the bead may include additional material, such as scintillant
or a fluophore impregnated therein. In preferred embodiments, the
solid phase chemistry and subsequent assaying may be performed on
the same bead or matrix with memory combination. All procedures,
including synthesis on the bead and assaying and analysis, can be
automated.
[0092] The matrices are typically insoluble substrates that are
solid, porous, deformable, or hard, and have any required structure
and geometry, including, but not limited to: beads, pellets, disks,
capillaries, hollow fibers, needles, solid fibers, random shapes,
thin films and membranes. Typically, when the matrix is
particulate, the particles are at least about 10-2000 .mu.M, but
may be smaller, particularly for use in embodiments in which more
than one particle is in proximity to a memory. For purposes herein,
the support material will typically encase or be in contact with
the data storage device, and, thus, will desirably have at least
one dimension on the order of 1 mm (1000 .mu.M) or more, although
smaller particles may be contacted with the data storage devices,
particularly in embodiments in which more than one matrix particle
is associated, linked or in proximity to one memory or matrix with
memory, such as the microvessels (see, e.g., FIGS. 11-16). Each
memory will be in associated with, in contact with or proximity to
at least one matrix particle, and may be in contact with more than
one. As smaller semiconductor and electronic or optical devices
become available, the capacity of the memory can be increased
and/or the size of the particles can be decreased. For example,
presently, 0.5 micron semiconductor devices are available.
Integrated circuits 0.25-micron in size have been described and are
being developed using a technology called the Complementary Metal
Oxide-Semiconductor process (see, e.g., Investor's Business Daily
May 30, 1995).
[0093] Also of interest herein, are devices that are prepared by
inserting the recording device into a "tube" (see, e.g., FIG. 13)
or encasing them in an inert material (with respect to the media in
which the device will be in contact). This material is fabricated
from a plastic or other inert material. Preferably prior to
introducing (and preferably sealing) the recording device inside,
the tube or encasing material is treated with ionizing radiation to
render the surface suitable for grafting selected monomers, such as
styrene (see, e.g., Maeji et al. (1994) Reactive Polymers
22:203-212; and Berg et al. (1989) J. Am. Chem. Soc.
111:8024-8026).
[0094] Recording device(s) is(are) introduced inside the material
or the material is wrapped around the device and the resulting
memory with matrix "tubes" (MICROTUBES.TM.) are used for chemical
synthesis or linkage of selected molecules or biological particles.
These "tubes" are preferably synthesized from an inert resin, such
as a polypropylene resin (e.g., a Moplen resin, V29G PP resin from
Montell, Newark Del., a distributor for Himont, Italy). Any inert
matrix that can then be functionalized or to which derivatizable
monomers can be grafted is suitable. Preferably herein,
polypropylene tubes are grafted and then formed into tubes or other
suitable shape and the recording device inserted inside. These
tubes (MICROTUBES.TM.) with grafted monomers are then used as
synthesis, and/or for assays or for multiplexed processes,
including synthesis and assays or other multistep procedures.
[0095] Also larger matrix particles, which advantageously provide
ease of handling, may be used and may be in contact with or
proximity to more than one memory (i.e., one particle may have a
plurality of memories in proximity or linked to it; each memory may
programmed with different data regarding the matrix particle,
linked molecules, synthesis or assay protocol, etc. Thus, so-called
macro-beads (Rapp Polymere, Tubingen, Germany), which have a
diameter of 2 mm when swollen, or other matrices of such size, are
also contemplated for use herein. Particles of such size can be
readily manipulated and the memory can be readily impregnated in or
on the bead. These beads (available from Rapp) are also
advantageous because of their uniformity in size, which is useful
when automating the processes for electronically tagging and
assaying the beads.
[0096] Selection of the matrices will be governed, at least in
part, by their physical and chemical properties, such as
solubility, functional groups, mechanical stability, surface area
swelling propensity, hydrophobic or hydrophilic properties and
intended use.
[0097] The data storage device with programmable memory may be
coated with a material, such as a glass or a plastic, that can be
further derivatized and used as the support or it may be encased,
partially or completely, in the matrix material, such as during or
prior to polymerization of the material. Such coating may be
performed manually or may be automated. The coating can be effected
manually or by using instruments designed for coating such devices.
Instruments for this purpose are available (see, e.g., the Series
C3000 systems for dipping available from Specialty Coating Systems,
Inc., Indianapolis, Ind.; and the Series CM 2000 systems for spray
coating available from Integrated Technologies, Inc. Acushnet,
Mass.).
[0098] The data storage device with memory may be physically
inserted into the matrix material or particle. It also can be
manufactured with a coating that is suitable for use as a matrix or
that includes regions in the coating that are suitable for use as a
matrix. If the matrix material is a porous membrane, it may be
placed inside the membrane. It is understood that when the memory
device is encased in the matrix or coated with protective material,
such matrix or material must be transparent to the signal used to
program the memory for writing or reading data. More than one
matrix particle may be linked to each data storage device.
[0099] In some instances, the data storage device with memory is
coated with a polymer, which is then treated to contain an
appropriate reactive moiety or in some cases the device may be
obtained commercially already containing the reactive moiety, and
may thereby serve as the matrix support upon which molecules or
biological particles are linked. Materials containing reactive
surface moieties such as amino silane linkages, hydroxyl linkages
or carboxysilane linkages may be produced by well established
surface chemistry techniques involving silanization reactions, or
the like. Examples of these materials are those having surface
silicon oxide moieties, covalently linked to
gamma-aminopropylsilane, and other organic moieties;
N-(3-(triethyoxysilyl)propyl)phthelamic acid; and
bis-(2-hydroxyethyl) aminopropyltriethoxysilane. Exemplary of
readily available materials containing amino group reactive
functionalities, include, but are not limited to,
para-aminophenyltriethyoxysilane. Also derivatized polystyrenes and
other such polymers are well known and readily available to those
of skill in this art (e.g., the TENTAGEL.RTM. Resins are available
with a multitude of functional groups, and are sold by Rapp
Polymere, Tubingen, Germany.
[0100] The data storage device with memory, however, generally
should not or cannot be exposed to the reaction solution, and,
thus, must be coated with at least a thin layer of a glass or
ceramic or other protective coating that does not interfere with
the operation of the device. These operations include electrical
conduction across the device and transmission of remotely
transmitted electromagnetic radiation by which data are written and
read. It is such coating that may also serve as a matrix upon which
the molecules or biological particles may be linked.
[0101] The data storage devices with memory may be coated either
directly or following coating with a ceramic, glass or other
material, may then be coated with agarose, which is heated, the
devices are dipped into the agarose, and then cooled to about room
temperature. The resulting glass, silica, agarose or other coated
memory device, may be used as the matrix supports for chemical
syntheses and reactions.
[0102] The combinations herein are matrix materials with recording
devices that contain data storage units that include remotely
programmable memories; the recording devices used in solution must
be coated with a material that prevents contact between the
recording device and the medium, such as the solution or air or gas
(e.g., nitrogen or oxygen or CO.sub.2). The information is
introduced into the memory by addressing the memory to record
information regarding molecules or biological particles linked
thereto. Except in the reaction detecting (verifying) embodiment,
in which the memory can be encoded upon reaction of a linked
molecule or biological particle, solution parameters are not
recorded in the memory.
[0103] 1. Natural Matrix Support Materials
[0104] Naturally-occurring supports include, but are not limited to
agarose, other polysaccharides, collagen, celluloses and
derivatives thereof, glass, silica, and alumina. Methods for
isolation, modification and treatment to render them suitable for
use as supports is well known to those of skill in this art (see,
e.g., Hermanson et al. (1992) Immobilized Affinity Ligand
Techniques, Academic Press, Inc., San Diego). Gels, such as
agarose, can be readily adapted for use herein. Natural polymers
such as polypeptides, proteins and carbohydrates; metalloids, such
as silicon and germanium, that have semiconductive properties, as
long as they do not interfere with operation of the data storage
device may also be adapted for use herein. Also, metals such as
platinum, gold, nickel, copper, zinc, tin, palladium, silver, again
as long as the combination of the data storage device with memory,
matrix support with molecule or biological particle does not
interfere with operation of the device with memory, may be adapted
for use herein. Other matrices of interest include oxides of the
metal and metalloids such as Pt--PtO, Si--SiO, Au--AuO, TiO2,
Cu--CuO, and the like. Also compound semiconductors, such as
lithium niobate, gallium arsenide and indium-phosphide, and
nickel-coated mica surfaces, as used in preparation of molecules
for observation in an atomic force micro-scope (see, e.g., Ill et
al. (1993) Biophys J. 64:91 91 may be used as matrices. Methods for
preparation of such matrix materials are well known.
[0105] For example, U.S. Pat. No. 4,175,183 describes a water
insoluble hydroxyalkylated cross-linked regenerated cellulose and a
method for its preparation. A method of preparing the product using
near stoichio-metric proportions of reagents is described. Use of
the product directly in gel chromatography and as an intermediate
in the preparation of ion exchangers is also described.
[0106] 2. Synthetic Matrices
[0107] There are innumerable synthetic matrices and methods for
their preparation known to those of skill in this art. Synthetic
matrices are typically produced by polymerization of functional
matrices, or copolymerization from two or more monomers of from a
synthetic monomer and naturally occurring matrix monomer or
polymer, such as agarose. Before such polymers solidify, they are
contacted with the data storage device with memory, which can be
cast into the material or dipped into the material. Alternatively,
after preparation of particles or larger synthetic matrices, the
recording device containing the data storage unit(s) can be
manually inserted into the matrix material. Again, such devices can
be pre-coated with glass, ceramic, silica or other suitable
material.
[0108] Synthetic matrices include, but are not limited to:
acrylamides, dextran-derivatives and dextran co-polymers,
agarose-polyacrylamide blends, other polymers and co-polymers with
various functional groups, methacrylate derivatives and
co-polymers, polystyrene and polystyrene copolymers (see, e.g.,
Merrifield (1964) Biochemistry 3:1385-1390; Berg et al. (1990) in
Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp.,
1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) in Pept.,
Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;
Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al.
(1979) Isr. J. Chem. 17:243-247; Kent et al. (1978) J. Org. Chem.
43:2845-2852; Mitchell et al. (1976) Tetrahedron Lett.
42:3795-3798; U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and
U.S. Pat. No. 5,389,449). Methods for preparation of such matrices
are well-known to those of skill in this art.
[0109] Synthetic matrices include those made from polymers and
co-polymers such as polyvinylalcohols, acrylates and acrylic acids
such as polyethylene-co-acrylic acid, polyethylene-co-methacrylic
acid, polyethylene-co-ethylacrylate, polyethylene-co-methyl
acrylate, polypropylene-co-acrylic acid,
polypropylene-co-methyl-acrylic acid,
polypropylene-co-ethylacry-late, polypropylene-co-methyl acrylate,
polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and
those containing acid anhydride groups such as
polyethylene-co-maleic anhydride, polypropylene-co-maleic anhydride
and the like. Liposomes have also been used as solid supports for
affinity purifications (Powell et al. (1989) Biotechnol. Bioeng.
33:173).
[0110] 3 Immobilization and Activation
[0111] Numerous methods have been developed for the immobilization
of proteins and other biomolecules onto solid or liquid supports.
Among the most commonly used methods are absorption and adsorption
or covalent binding to the support, either directly or via a
linker, such as the numerous disulfide linkages, thioether bonds,
hindered disulfide bonds, and covalent bonds between free reactive
groups, such as amine and thiol groups, known to those of skill in
art (see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog &
Handbook, 1992-1993, which describes the preparation of and use of
such reagents and provides a commercial source for such reagents.
To effect immobilization, a solution of the protein or other
biomolecule is contacted with a support material such as alumina,
carbon, an ion-exchange resin, cellulose, glass or a ceramic.
Fluorocarbon polymers have been used as supports to which
biomolecules have been attached by adsorption (see, U.S. Pat. No.
3,843,443; Published International PCT Application WO/86
03840).
[0112] A large variety of methods are known for attaching
biological molecules, including proteins and nucleic acids,
molecules to solid supports (see, e.g., U.S. Pat. No. 5,451,683).
For example, U.S. Pat. No. 4,681,870 describes a method for
introducing free amino or carboxyl groups onto a silica matrix.
These groups may subsequently be covalently linked to other groups,
such as a protein or other anti-ligand, in the presence of a
carbodiimide. Alternatively, a silica matrix may be activated by
treatment with a cyanogen halide under alkaline conditions. The
anti-ligand is covalently attached to the surface upon addition to
the activated surface. Another method involves modification of a
polymer surface through the successive application of multiple
layers of biotin, avidin and extenders (see, e.g., U.S. Pat. No.
4,282,287); other methods involve photoactivation in which a
polypeptide chain is attached to a solid substrate by incorporating
a light-sensitive unnatural amino acid group into the polypeptide
chain and exposing the product to low-energy ultraviolet light
(see, e.g., U.S. Pat. No. 4,762,881). Oligonucleotides have also
been attached using a photochemically active reagents, such as a
psoralen compound, and a coupling agent, which attaches the
photoreagent to the substrate (see, e.g., U.S. Pat. No. 4,542,102
and U.S. Pat. No. 4,562,157). Photoactivation of the photoreagent
binds a nucleic acid molecule to the substrate to give a
surface-bound probe.
[0113] Covalent binding of the protein or other biomolecule or
organic molecule or biological particle to chemically activated
solid matrix supports such as glass, synthetic polymers, and
cross-linked polysaccharides is a more frequently used
immobilization technique. The molecule or biological particle may
be directly linked to the matrix support or linked via linker, such
as a metal (see, e.g., U.S. Pat. No. 4,179,402; and Smith et al.
(1992) Methods: A Companion to Methods in Enz. 4:73-781. An example
of this method is the cyanogen bromide activation of polysaccharide
supports, such as agarose. The use of perfluorocarbon polymer-based
supports for enzyme immobilization and affinity chromatography is
described in U.S. Pat. No. 4,885,250). In this method the
biomolecule is first modified by reaction with a
perfluoroalkylating agent such as perfluorooctylpropylisocyanate
described in U.S. Pat. No. 4,954,444. Then, the modified protein is
adsorbed onto the fluorocarbon support to effect
immobilization.
[0114] The activation and use of matrices are well known and may be
effected by any such known methods (see, e.g., Hermanson et al.
(1992) Immobilized Affinity Ligand Techniques, Academic Press,
Inc., San Diego). Molecules may also be attached to matrices
through kinetically inert metal ion linkages, such as Co(III),
using, for example, native metal binding sites on the molecules,
such as IgG binding sequences, or genetically modified proteins
that bind metal ions.
[0115] Other suitable methods for linking molecules and biological
particles to solid supports are well known to those of skill in
this art (see, e.g., U.S. Pat. No. 5,416,193). These linkers
include linkers that are suitable for chemically linking molecules,
such as proteins and nucleic acid, to supports include, but are not
limited to, disulfide bonds, thioether bonds, hindered disulfide
bonds, and covalent bonds between free reactive groups, such as
amine and thiol groups. These bonds can be produced using
heterobifunctional reagents to produce reactive thiol groups on one
or both of the moieties and then reacting the thiol groups on one
moiety with reactive thiol groups or amine groups to which reactive
maleimido groups or thiol groups can be attached on the other.
Other linkers include, acid cleavable linkers, such as
bismaleimideothoxy propane, acid labile-transferrin conjugates and
adipic acid diihydrazide, that would be cleaved in more acidic
intracellular compartments; cross linkers that are cleaved upon
exposure to UV or visible light and linkers, such as the various
domains, such as C.sub.H1, C.sub.H2, and C.sub.H3, from the
constant region of human IgG, (see, Batra et al. (1993) Molecular
Immunol. 30:379-386).
[0116] Presently preferred linkages are direct linkages effected by
adsorbing the molecule or biological particle to the surface of the
matrix. Other preferred linkages are photocleavable linkages that
can be activated by exposure to light. The selected linker will
depend upon the particular application and, if needed, may be
empirically selected.
[0117] B. Data Storage Units with Memory
[0118] Any remotely programmable data storage device that can be
linked to or used in proximity to the solid supports and molecules
and biological particles as described herein is intended for use
herein. Preferred devices are rapidly and readily programmable
using penetrating electromagnetic radiation, such as radio
frequency or visible light lasers, operate with relatively low
power, have fast access, and are remotely programmable so that
information can be stored or programmed and later retrieved from a
distance, as permitted by the form of the electromagnetic signal
used for transmission. Presently preferred devices are on the order
of 1-10 mm in the largest dimension and are remotely programmable
using RF or radar.
[0119] Recording devices may be active, which contain a power
source, such as a battery, and passive, which does not include a
power source. In a passive device, which has no independent power
source, the transmitter/receiver system, which transfers the data
between the recording device and a host computer and which is
preferably integrated on the same substrate as the memory, also
supplies the power to program and retrieve the data stored in the
memory. This is effected by integrating a rectifier circuit onto
the substrate to convert the received signal into an operating
voltage. Alternatively, an active device can include a battery.
[0120] The remotely programmable device can be programmed
sequentially to be uniquely identifiable during and after stepwise
synthesis of macromolecules or before, or during, or after
selection of screened molecules. In certain embodiments herein, the
data storage units are information carriers in which the functions
of writing data and reading the recorded data are empowered by an
electromagnetic signal generated and modulated by a remote host
controller. Thus, the data storage devices are inactive, except
when exposed to the appropriate electromagnetic signal. In an
alternative embodiment, the devices may be optically or
magnetically programmable read/write devices.
Electromagnetically Programmable Devices
[0121] The programmable devices intended for use herein, include
any device that can record or store data. The preferred device will
be remotely programmable and will be small, typically on the order
of 10-20 mm.sup.3 (or 10-20 mm in its largest dimension) or,
preferably smaller. Any means for remote programming and data
storage, including semiconductors and optical storage media are
intended for use herein.
[0122] Also intended for use herein, are commercially available
precoded devices, such as identification and tracking devices for
animals and merchandise, such those used with and as security
systems (see, e.g., U.S. Pat. Nos. 4,652,528, 5,044,623, 5,099,226,
5,218,343, 5,323,704, 4,333,072, 4,321,069, 4,318,658, 5,121,748,
5,214,409, 5,235,326, 5,257,011 and 5,266,926), and devices used to
tag animals. These devices may also be programmable using an RF
signal. These device can be modified, such as by folding it, to
change geometry to render them more suitable for use in the methods
herein. Of particular interest herein are devices sold by BioMedic
Data Systems, Inc, NJ (see, e.g., the IPTT-100 purchased from
BioMedic Data Systems, Inc., Maywood, N.J.; see, also U.S. Pat.
Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962, 5,250,962, and
see, also, U.S. application Ser. No. 08/322,644, filed Oct. 13,
1994). ID tags available from IDTAG.TM. Inc, particularly the
IDT150 read/write transponder (IDTAG.TM. Ltd. Bracknell, Berks RG12
3XQ, UK. These transponders are packaged in glass or polystyrene or
other such material.
[0123] Devices that rely on other programmable volatile memories
are also intended for use herein. For example, a battery may be
used as to supply the power to provide an operating voltage to the
memory device. When a battery is used the memory can be an EEPROM,
a DRAM, or other erasable memory requiring continuous power to
retain information. It may be advantageous to combine the
antenna/rectifier circuitry with a battery to create a
passive/active device, in which the voltages supplied by each
source supplement each other. For example, the transmitted signal
could provide the voltage for writing and reading, while the
battery, in addition to supplementing this write/read voltage,
provides a refresh voltage for a DRAM memory so that data is
retained when the transmitted signal is removed.
[0124] Electrically-Programmable Memory Devices
[0125] In one embodiment, the recording device utilizes antifuse
technology. An antifuse contains a layer of antifuse material
sandwiched between two conductive electrodes. The antifuse device
is initially an open circuited device in its unprogrammed state and
can be irreversibly converted into an essentially short circuited
device by the application of a programming voltage across the two
electrodes to disrupt the antifuse material and create a low
resistance current path between the two electrodes.
[0126] Examples of the antifuse and its use as a memory cell within
a Read-Only Memory are discussed in Roesner et al., "Apparatus and
Method of Use of Radio frequency Identification Tags", U.S.
application Ser. No. 08/379,923, filed Jan. 27, 1995, Roesner,
"Method of Fabricating a High Density Programmable Read-Only
Memory", U.S. Pat. No. 4,796,074 (1989) and Roesner, "Electrically
Programmable Read-Only Memory Stacked above a Semiconductor
Substrate", U.S. Pat. No. 4,442,507 (1984). A preferred antifuse is
described in U.S. Pat. No. 5,095,362. "Method for reducing
resistance for programmed antifuse" (1992) (see, also U.S. Pat.
Nos. 5,412,593 and 5,384,481).
[0127] Referring to FIG. 5, which depicts a preferred embodiment, a
recording device containing a non-volatile
electrically-programmable read-only memory (ROM) 102 that utilizes
antifuse technology (or EEPROM or other suitable memory) is
combined on a single substrate 100 with a thin-film planar antenna
110 for receiving/transmitting an RF signal 104, a rectifier 112
for deriving a voltage from a received radio frequency (RF) signal,
an analog-to-digital converter (ADC) 114 for converting the voltage
into a digital signal for storage of data in the memory, and a
digital-to-analog converter (DAC) 116 for converting the digital
data into a voltage signal for transmission back to the host
computer is provided. A single substrate 100 is preferred to
provide the smallest possible chip, and to facilitate encapsulation
of the chip with a protective, polymer shell (or shell+matrix or
matrix material) 90. Shell 90 must be non-reactive with and
impervious to the various processes that the recording device is
being used to track in order to assure the integrity of the memory
device components on the chip.
[0128] Referring to FIG. 1, in which chemical building blocks A, C,
and E are added to a molecule linked to a matrix with memory, and
to FIG. 6, an illustrative example of how information is written
onto a particle is provided in Table 1.
TABLE-US-00001 TABLE 1 PROCESS STEP X-REGISTER ADDRESS Y-REGISTER
ADDRESS A 1 8 C 2 4 E 3 2
[0129] For the step in which A is added, the address signal would
increment the x-register 124 one location and increment the
y-register 126 eight locations, and then apply the programming
voltage. The activation of this switch is indicated by an "A" at
the selected address, although the actual value stored will be a
binary "1", indicating ON. (As described, for example, in U.S. Pat.
No. 4,424,579; the manner in which the programming voltage is
applied depends on whether the decoders have depletion or
enhancement transistors.) The host computer 122 would write into
its memory 120 that for process A, the x-, y-address is 1,8. Upon
removal of the RF signal after recording process A, the voltage is
removed and the registers would reset to 0. For the step in which C
is added, the address signal would increment the x-register 124 two
locations and the y-register 126 four locations, then apply the
programming voltage, as indicated by the letter "C". The host
computer 120 would similarly record in memory that an indication of
exposure to process C would be found at x-, y-address 2,4. Again,
upon removal of the RF signal, the registers reset to 0 so that
when the matrix particle's memory is again exposed to RF following
addition of block E, the registers increment 3 and 2 locations,
respectively, and the programming voltage is applied to turn on the
switch, indicated by "E". Desirably all processing steps are
automated.
[0130] Ideally, the tagging of particles that are exposed to a
particular process would be performed in the process vessel
containing all of the particles. The presence, however, of a large
number of particles may result in interference or result in an
inability to generate a sufficiently high voltage for programming
all of the particles simultaneously. This might be remedied by
providing an exposure of prolonged duration, e.g., several minutes,
while stirring the vessel contents to provide the greatest
opportunity for all particles to receive exposure to the RF signal.
On the other hand, since each particle will need to be read
individually, a mechanism for separating the particles may be used
in both write and read operations. Also, in instances in which each
particle will have a different molecule attached, each particle
memory must be addressed separately.
[0131] An apparatus for separating the particles to allow
individual exposure to the RF signal is illustrated in FIG. 7.
Here, the particles are placed in a vessel 140 which has a funnel
142, or other constricted section, which permits only one particle
150 to pass at a time. It is noted that the particles, as
illustrated, are, for purposes of exemplification, depicted as
spherical. The particles, however, can be of any shape, including
asymmetric shapes. Where the particles are asymmetric or of other
shapes, the size of the funnel exit and tube should be selected to
fit the largest diameter of the particles closely. If a particular
orientation of the particle is desired or required for effective
transmission, the tube and funnel exit should be designed and
oriented to permit only particles in the proper alignment with the
tube to exit.
[0132] The RF transmitter 80 is positioned adjacent a tube 144
which receives input from funnel 142. When a particle passes
through tube 144 the RF transmitter provides a signal to write to
or read from the particle's memory. Means for initiating the RF
transmission may include connection to a mechanical gate or shutter
145 in the funnel 142 which controls the admission of the particle
into the tube. As illustrated in FIG. 7, however, optical means for
detecting the presence of the matrix particle with memory to
initiate RF transmission are provided in the form of a laser 146
directed toward the tube 144, which is transparent to the
wavelength of the light emitted by the laser. When the laser light
impinges upon the particle (shown with dashed lines) it is
reflected toward an optical detector 148 which provides a signal to
the host computer 122 to initiate the RF transmission.
Alternatively, magnetic means, or any other means for detecting the
presence of the particle in the tube 144 may be used, with the
limitation that any electromagnetic radiation used does not induce
any reactions in the substances on the particle's surface. After
exposure of the individual particle to the RF signal, the particle
may be received in one or more vessels for further processing. As
illustrated, tube 144 has an exemplary three-way splitter and
selection means, shown here in dashed lines as mechanical gates,
for directing the particles to the desired destination.
[0133] It is understood that the above description of operation and
use of the data storage devices, may be adapted for use with
devices that contain volatile memories, such as EEPROMs, flash
memory and DRAMs.
[0134] Other types of electrically-programmable read-only memories,
preferably non-volatile memories, which are known in the art, may
be used. Preprogrammed remotely addressable identification tags,
such as those used for tracking objects or animals (see, e.g., U.S.
Pat. Nos. 5,257,011, 5,235,326, 5,226,926, 5,214,409, 4,333,072,
available from AVID, Norco, Calif.; see, also U.S. Pat. No.
5,218,189, 5,416,486, 4,952,928, 5,359,250) and remotely writable
versions thereof are also contemplated for use herein.
Preprogrammed tags may be used in embodiments, such as those in
which tracking of linked molecules is desired.
[0135] Alternatively, the matrices or strips attached thereto may
be encoded with a pre-programmed identifying bar code, such as an
optical bar code that will be encoded on the matrix and read by
laser. Such pre-coded devices may be used in embodiments in which
parameters, such as location in an automated synthesizer, are
monitored. The identity of a product or reactant may be determined
by its location or path, which is monitored by reading the chip in
each device and storing such information in a remote computer.
Read/write tags such as the IPTT-100 (BioMedic Data Systems, Inc.,
Maywood, N.J.; see, also U.S. Pat. Nos. 5,422,636, 5,420,579,
5,262,772, 5,252,962, 5,250,962, and U.S. application Ser. No.
08/322,644) are also contemplated for use herein.
[0136] Among the particularly preferred devices are the chips
(particularly, the IPTT-100, Bio Medic Data Systems, Inc., Maywood,
N.J.; see, also U.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772,
5,252,962 and 5,250,962 and U.S. application Ser. No. 08/322,644)
that can be remotely encoded and remotely read. These devices, such
as the IPTT-100 transponders that are about 8 mm long, include a
recording device, an EEPROM, a passive transponder for receiving an
input signal and transmitting an output signal in response. In some
embodiments here, the devices are modified for use herein by
altering the geometry. They are folded in half and the antenna
wrapped around the resulting folded structure. This permits
convenient insertion into the microvessels and formation of other
combinations.
[0137] Another such device is a 4 mm chip with an onboard antenna
and an EEPROM (Dimensional Technology International, Germany). This
device can also be written to and read from remotely.
[0138] Also, ID tags available from IDTAG.TM. Inc, particularly the
IDT150 read/write transponder (ITDAG.TM. Ltd. Bracknell, Berks RG12
3XQ, UK), discussed above, are also preferred herein.
[0139] It is also contemplated herein, that the memory is not
proximate to the matrix, but is separate, such as a remote computer
or other recording device. In these embodiments, the matrices are
marked with a unique code or mark of any sort. The identity of each
mark is saved in the remote memory, and then, each time something
is done to a molecule or biological particle linked to each matrix,
the information regarding such event is recorded and associated
with the coded identity. After completion of, for example, a
synthetic protocol, each matrix is examined or read to identify the
code. Retrieving information from the remote memory that is stored
with the identifying code will permit identification or retrieval
of any other saved information regarding the matrix.
[0140] For example, simple codes, including bar codes, alphanumeric
characters or other visually or identifiable codes or marks on
matrices are also contemplated for use herein. When bar codes or
other precoded devices are used, the information can be written to
an associated but remote memory, such as a computer or even a piece
of paper. The computer stores the bar code that a identifies a
matrix particle or other code and information relating to the
molecule or biological particle linked to the matrix or other
relevant information regarding the linked materials or synthesis or
assay. Instead of writing to an on-board memory, information is
encoded in a remote memory that stores information regarding the
precoded identity of each matrix with bar code and linked molecules
or biological particles. Thus, the precoded information is
associated with, for example, the identity of the linked molecule
or a component thereof, or a position (such as X-Y coordinates in a
grid). This information is transmitted to a memory for later
retrieval. Each treatment or synthetic step that is performed on
the linked molecule or biological particle is transmitted to the
remote memory and associated with the precoded ID.
[0141] For example, an amino acid is linked to a matrix particle
that is encoded with or marked with a bar code or even a letter
such as "A" or other coded mark. The identity the amino acid linked
to the matrix particle "A" is recorded into a memory. This particle
is mixed with other particles, each with a unique identifier or
mark, and this mixture is then treated to a synthetic step. Each
particle is individually scanned or viewed to see what mark is on
each particle and the remote memory is written to describe the
synthetic step, which is then associated with each unique
identifier in the memory, such as the computer or piece of paper.
Thus, in the remote memory the original amino acid linked to
particle A is stored. After the synthetic step, the identity of the
next amino acid is stored in the memory associated with "A" as is
the identity of the next amino acid added. At the end of the
synthesis, the history of each particle can be read by scanning the
particle or visually looking at the particle and noting its bar
code or mark, such as A. The remote memory is then queried to
determine what amino acids are linked to the particle identified as
"A" (see, e.g., FIG. 12).
[0142] For example, many combinatorial libraries contain a
relatively small number of discrete compounds (102-104) in a
conveniently manipulable quantity, rather than millions of members
in minute quantities. These small libraries are ideal for use with
the methods and matrices with memories herein. They may also be
used in methods in which the memory is not in proximity to the
matrix, but is a remote memory, such as a computer or a table of
information stored even on paper. The system depicted in FIG. 12 is
ideal for use in these methods.
[0143] Polypropylene or other inert polymer, including
fluoropolymers or scintillating polymers are molded into a
convenient geometry and size, such an approximately 5 mm.times.5
mm.times.5 mm cube (or smaller or larger) with a unique identifying
code imprinted, preferably permanently, on one side of each cube.
If, for example, a three element code is used, based on all digits
(0 to 9) and all letters of the alphabet, a collection of 46,666
unique three element codes are available for imprinting on the
cubes.
[0144] The cubes are surface grafted with a selected monomer (or
mixture of monomer), such as styrene. Functionalization of the
resulting polymer provides a relatively large surface area for
chemical syntheses and subsequent assaying (on a single platform).
For example, a 5.times.5.times.5 mm.sup.3 cube has a surface area
of 150 mm.sup.2, which is equivalent to about 2-5 .mu.mol
achievable loading, which is about 1-2.5 mg of compounds with a
molecular weight of about 500. A simple computer program or
protocol can direct split and pool during synthesis and the
information regarding each building block of the linked molecules
on each cube conveniently recorded in the memory (i.e., computer)
at each step in the synthesis.
[0145] Since the cubes (herein called MACROCUBES.TM. or
MACROBEADS.TM.) are relatively large, they can be read by the eye
or any suitable device during synthesis and the associated data can
be manually entered into a computer or even written down. The cubes
can include scintillant or fluorophore or label and used in any of
the assay formats described herein or otherwise known to those of
skill in the art.
[0146] For example, with reference to FIG. 12, polypropylene,
polyethylene or fluophore raw material (any such material described
herein, particularly the Moplen resin e.g., V29G PP resin from
Montell, Newark Del., a distributor for Himont, Italy) 1 is molded,
preferably into a cube, preferably about 5.times.5.times.5 mm.sup.3
and engraved, using any suitable imprinting method, with a code,
preferably a three element alphanumeric code, on one side. The cube
can be weighted or molded so that it all cubes will orient in the
same direction. The engraved cubes 2 are then surface-grafted 3 and
functionalized using methods described herein or known to those of
skill in this art, to produce cubes (MACROBEADS.TM. or
MACROCUBES.TM.) or devices any selected geometry 4.
[0147] Optically or Magnetically Programmed Devices
[0148] In addition to electrically-programmable means for storing
information on the matrix particles, optical or magnetic means may
be used. One example of an optical storage means is provided in
U.S. Pat. No. 5,136,572, issued Aug. 4, 1992, of Bradley, which is
incorporated herein by reference. Here, an array of stabilized
diode lasers emits fixed wavelengths, each laser emitting light at
a different wavelength. Alternatively, a tunable diode laser or a
tunable dye laser, each of which is capable of emitting light
across a relatively wide band of wavelengths, may be used. The
recording medium is photochemically active so that exposure to
laser light of the appropriate wavelength will form spectral
holes.
[0149] As illustrated in FIG. 8, an optical write/read system is
configured similar to that of the embodiment of FIG. 7, with a
vessel 212 containing a number of the particles which are separated
and oriented by passing through a constricted outlet into a
write/read path 206 that has an optically-transparent tube (i.e.,
optically transparent to the required wavelength(s)) with a
cross-section that orients the particles as required to expose the
memory surface to the laser 200 which is capable of emitting a
plurality of discrete, stable wavelengths. Gating and detection
similar to that described for the previous embodiment may be used
and are not shown. Computer 202 controls the tuning of laser 200 so
that it emits light at a unique wavelength to record a data point.
Memory within computer 202 stores a record indicating which process
step corresponds to which wavelength. For example, for process A,
wavelength .lamda..sub.1, e.g., 630 nm (red), for process C,
.lamda..sub.2. e.g., 550 nm (green), and for process E, k.sub.3,
e.g., 480 nm (blue), etc. The recording medium 204 is configured to
permit orientation to repeatably expose the recording side of the
medium to the laser beam each time it passes through tube 206. One
possible configuration, as illustrated here, is a disc.
[0150] To write onto the recording medium 204, the laser 200 emits
light of the selected wavelength to form a spectral hole in the
medium. The light is focused by lens 208 to illuminate a spot on
recording medium 204. The laser power must be sufficient to form
the spectral hole. For reading, the same wavelength is selected at
a lower power. Only this wavelength will pass through the spectral
hole, where it is detected by detector 210, which provides a signal
to computer 202 indicative of the recorded wavelength. Because
different wavelengths are used, multiple spectral holes can be
superimposed so that the recording medium can be very small for
purposes of tagging. To provide an analogy to the electrical memory
embodiments, each different wavelength of light corresponds to an
address, so that each laser writes one bit of data. If a large
number of different steps are to performed for which each requires
a unique data point, the recording media will need to be
sufficiently sensitive, and the lasers well-stabilized, to vary
only within a narrow band to assure that each bit recorded in the
media is distinguishable. Since only a single bit of information is
required to tag the particle at any given step, the creation of a
single spectral hole at a specific wavelength is capable of
providing all of the information needed. The host computer then
makes a record associating the process performed with a particular
laser wavelength.
[0151] For reading, the same wavelength laser that was used to
create the spectral hole will be the only light transmitted through
the hole. Since the spectral holes cannot be altered except by a
laser having sufficient power to create additional holes, this type
of memory is effectively nonvolatile. Further, the recording medium
itself does not have any operations occurring within its structure,
as is the case in electrical memories, so its structure is quite
simple. Since the recording medium is photochemically active, it
must be well encased within an optically transmissive (to the
active optical wavelength(s)), inert material to prevent reaction
with the various processing substances while still permitting the
laser light to impinge upon the medium. In many cases, the
photochemical recording media may be erased by exposure to broad
spectrum light, allowing the memory to be reused.
[0152] Writing techniques can also include the formation of pits in
the medium. To read these pits, the detector 210 with be positioned
on the same side of the write/read tube 206 as the laser 200 to
detect light reflected back from the medium. Other types of optical
data storage and recording media may be used as are known in the
art. For example, optical discs, which are typically
plastic-encapsulated metals, such as aluminum, may be miniaturized,
and written to and read from using conventional optical disc
technology. In such a system, the miniature discs must be aligned
in a planar fashion to permit writing and reading. A modification
of the funnel system, described above, will include a flattened
tube to insure the proper orientation. Alternatively, the discs can
be magnetically oriented. Other optical recording media that may be
appropriate for use in the recording devices and combinations
herein include, but are not limited to, magneto-optical materials,
which provide the advantage of erasability, photochromic materials,
photoferroelectric materials, photoconductive electro-optic
materials, all of which utilize polarized light for writing and/or
reading, as is known in the art. When using any form of optical
recording, however, considerations must be made to insure that the
selected wavelength of light will not affect or interfere with
reactions of the molecules or biological particles linked to or in
proximity to matrix particles.
[0153] Three Dimensional Optical Memories
[0154] Another device that is suitable for use in the matrix with
memory combinations are optical memories that employ rhodopsins,
particularly bacteriorhodopsin (BR), or other photochromic
substances that change between two light absorbing states in
response to light of each of two wavelengths (see, e.g., U.S. Pat.
No. 5,346,789, 5,253,198 and 5,228,001; see, also Birge (1990) Ann.
Rev. Phys. Chem. 41:683-733), These substances, particularly BR,
exhibit useful photochromic and optoelectrical properties. BR, for
example, has extremely large optical nonlinearities, and is capable
of producing photoinduced electrical signals whose polarity depends
on the prior exposure of the material to light of various
wavelengths as well as on the wavelength of the light used to
induce the signal. There properties are useful for information
storage and computation. Numerous applications of this material
have been designed, including its use as an ultrafast photosignal
detector, its use for dynamic holographic recording, and its use
for data storage, which is of interest herein.
[0155] The rhodopsins include the visual rhodopsins, which are
responsible for the conversion of light into nerve impulses in the
image resolving eyes of mollusks, anthropods, and vertebrates, and
also bacteriorhodopsin (BR). These proteins also include a class of
proteins that serve photosynthetic and phototactic functions. The
best known BR is the only protein found in nature in a crystalline
membrane, called the "purple membrane" of Halobacterium Halobium.
This membrane converts light into energy via photon-activated
transmembrane proton pumping. Upon the absorption of light, the BR
molecule undergoes several structural transformations in a
well-defined photocycle in which energy is stored in a proton
gradient formed upon absorption of light energy. This proton
gradient is subsequently utilized to synthesize energy-rich
ATP.
[0156] The structural changes that occur in the process of
light-induced proton pumping of BR are reflected in alterations of
the absorption spectra of the molecule. These changes are cyclic,
and under usual physiological conditions bring the molecule back to
its initial BR state after the absorption of light in about 10
milliseconds. In less than a picosecond after BR absorbs a photon,
the BR produces an intermediate, known as the "J" state, which has
a red-shifted absorption maximum. This is the only light-driven
event in the photocycle; the rest of the steps are thermally driven
processes that occur naturally. The first form, or state, following
the photon-induced step is called "K", which represents the first
form of light-activated BR that can be stabilized by reducing the
temperature to 90.degree. K. This form occurs about 3 picoseconds
after the J intermediate at room temperature. Two microseconds
later there occurs an "L" intermediate state which is, in turn,
followed in 50 microseconds by an "M" intermediate state.
[0157] There are two important properties associated with all of
the intermediate states of this material. The first is their
ability to be photochemically converted back to the basic BR state.
Under conditions where a particular intermediate is made stable,
illumination with light at a wavelength corresponding to the
absorption of the intermediate state in question results in
regeneration of the BR state. In addition, the BR state and
intermediates exhibit large two-photon absorption processes which
can be used to induce interconversions among different states.
[0158] The second important property is light-induced vectorial
charge transport within the molecule. In an oriented BR film, such
a charge transport can be detected as an electric signal. The
electrical polarity of the signal depends on the physical
orientation of molecules within the material as well as on the
photochemical reaction induced. The latter effect is due to the
dependence of charge transport direction on which intermediates
(including the BR state) are involved in the photochemical reaction
of interest. For example, the polarity of an electrical signal
associated with one BR photochemical reaction is opposite to that
associated with a second BR photochemical reaction. The latter
reaction can be induced by light with a wavelength around 412 nm
and is completed in 200 ns.
[0159] In addition to the large quantum yields and distinct
absorptions of BR and M, the BR molecule (and purple membrane) has
several intrinsic properties of importance in optics. First, this
molecule exhibits a large two-photon absorption cross section.
Second, the crystalline nature and adaptation to high salt
environments makes the purple membrane very resistant to
degeneration by environmental perturbations and thus, unlike other
biological materials, it does not require special storage. Dry
films of purple membrane have been stored for several years without
degradation.
[0160] Furthermore, the molecule is very resistant to photochemical
degradation. Thus, numerous optical devices, including recording
devices have been designed that use BR or other rhodopsin as the
recording medium (see, e.g., U.S. Pat. Nos. 5,346,789, 5,253,198
and 5,228,001; see, also Birge (1990) Ann. Rev. Phys. Chem.
41:683-733). Such recording devices may be employed in the methods
and combinations provided herein.
[0161] C. The Combinations and Preparation Thereof.
[0162] Combinations of a miniature recording device that contains
or is a data storage unit linked to or in proximity with matrices
or supports used in chemical and biotechnical applications, such as
combinatorial chemistry, peptide synthesis, nucleic acid synthesis,
nucleic acid amplification methods, organic template chemistry,
nucleic acid sequencing, screening for drugs, particularly high
throughput screening, phage display screening, cell sorting, drug
delivery, tracking of biological particles and other such methods,
are provided. These combinations of matrix material with data
storage unit (or recording device including the unit) are herein
referred to as matrices with memories. These combinations have a
multiplicity of applications, including combinatorial chemistry,
isolation and purification of target macromolecules, capture and
detection of macromolecules for analytical purposes, high
throughput screening protocols, selective removal of contaminants,
enzymatic catalysis, drug delivery, chemical modification,
scintillation proximity assays, FET, FRET and HTRF assays,
immunoassays, receptor binding assays, drug screening assays,
information collection and management and other uses. These
combinations are particularly advantageous for use in multianalyte
analyses. These combinations may also be advantageously used in
assays in which a electromagnetic signal is generated by the
reactants or products in the assay. The combination of matrix with
memory is also advantageously used in multiplex protocols, such as
those in which a molecule is synthesized on the matrix, its
identity recorded in the matrix, the resulting combination is used
in an assay or in a hybridization reaction. Occurrence of the
reaction can be detected externally, such as in a scintillation
counter, or can be detected by a sensor that writes to the memory
in the matrix. Thus, combinations of matrix materials, memories,
and linked or proximate molecules and biological materials and
assays using such combinations are provided.
[0163] The combinations contain (i) a miniature recording device
that contains one or more programmable data storage devices
(memories) that can be remotely read and in preferred embodiments
also remotely programmed; and (ii) a matrix as described above,
such as a particulate support used in chemical syntheses. The
remote programming and reading is preferably effected using
electromagnetic radiation, particularly radio frequency or radar.
Depending upon the application the combinations will include
additional elements, such as scintillants, photodetectors, pH
sensors and/or other sensors, and other such elements.
1. Preparation of Matrix-Memory Combinations
[0164] In preferred embodiments, the recording device is cast in a
selected matrix material during manufacture. Alternatively, the
devices can be physically inserted into the matrix material, the
deformable gel-like materials, or can be placed on the matrix
material and attached by a connector, such as a plastic or wax or
other such material. Alternatively, the device or device(s) may be
included in an inert container in proximity to or in contact with
matrix material.
2. Preparation of Matrix-Memory-Molecule or Biological Particle
Combinations
[0165] In certain embodiments, combinations of matrices with
memories and biological particle combinations are prepared. For
example, libraries (e.g., bacteria or bacteriophage, or other virus
particles or other particles that contain genetic coding
information or other information) can be prepared on the matrices
with memories, and stored as such for future use or antibodies can
be linked to the matrices with memories and stored for future
use.
3. Combinations for Use in Proximity Assays
[0166] In other embodiments the memory or recording device is
coated or encapsulated in a medium, such as a gel, that contains
one or more fluophors or one or more scintillants, such as
2,5-diphenyloxazole (PPO) and/or
1,4-bis-(5-phenyl-(oxazolyl))benzene (POPOP) or FlexiScint (a gel
with scintillant available from Packard, Meriden, Conn.) or yttrium
silicates. Any fluophore or scintillant or scintillation cocktail
known to those of skill in the art may be used. The gel coated or
encased device is then coated with a matrix suitable, such as glass
or polystyrene, for the intended application or application(s). The
resulting device is particularly suitable for use as a matrix for
synthesis of libraries and subsequent use thereof in scintillation
proximity assays.
[0167] Similar combinations in non-radioactive energy transfer
proximity assays, such as HTRF, FP, FET and FRET assays, which are
described below. These luminescence assays are based on energy
transfer between a donor luminescent label, such as a rare earth
metal cryptate (e.g., Eu trisbipyridine diamine (EuTBP) or Tb
tribipyridine diamine (TbTBP)) and an acceptor luminescent label,
such as, when the donor is EuTBP, allopycocyanin (APC),
allophycocyanin B, phycocyanin C or phycocyanin R, and when the
donor is TbTBP, a rhodamine, thiomine, phycocyanin R,
phycoerythrocyanin, phycoerythrin C, phycoerythrin B or
phycoerythrin R. Instead of including a scintillant in the
combination, a suitable fluorescent material, such as
allopycocyanin (APC), allophycocyanin B, phycocyanin C, phycocyanin
R; rhodamine, thiomine, phycocyanin R, phycoerythrocyanin,
phycoerythrin C, phycoerythrin B or phycoerythrin R is included.
Alternatively, a fluorescent material, such a europium cryptate is
incorporated in the combination.
4. Other Variations and Embodiments
[0168] The combination of memory with matrix particle may be
further linked, such as by welding using a laser or heat, to an
inert carrier or other support, such as a TEFLON.RTM. strip. This
strip, which can be of any convenient size, such as 1 to 10 mm by
about 10 to 100 .mu.M will render the combination easy to use and
manipulate. For example, these memories with strips can be
introduced into 10 cm culture dishes and used in assays, such as
immunoassays, or they can be used to introduce bacteria or phage
into cultures and used in selection assays. The strip may be
encoded or impregnated with a bar code to further provide
identifying information.
[0169] Microplates containing a recording device in one or a
plurality of wells are provided. The plates may further contain
embedded scintillant or a coating of scintillant (such as
FlashPlate.TM., available from DuPont NEN.RTM., and plates
available from Packard, Meriden, Conn.) FLASHPLATE.TM. is a 96 well
microplate that is precoated with plastic scintillant for detection
of .beta.-emitting isotopes, such as .sup.1251, .sup.3H, .sup.35S,
.sup.14C and .sup.33P. A molecule is immobilized or synthesized in
each well of the plate, each memory is programmed with the identify
of each molecule in each well. The immobilized molecule on the
surface of the well captures a radiolabeled ligand in solution
results in detection of the bound radioactivity. These plates can
be used for a variety of radioimmmunoassays (RIAs), radioreceptor
assays (RRAs), nucleic acid/protein binding assays, enzymatic
assays and cell-based assays, in which cells are grown on the
plates.
[0170] Another embodiment is depicted in FIG. 11. The reactive
sites, such as amines, on a support matrix (1 in the figure) in
combination with a memory (a MICROKAN.TM., a MICROTUBE.TM., a
MACROBEAD.TM., a MICROCUBE.TM. or other matrix with memory
combination) are differentiated by reacting them with a selected
reaction of Fmoc-glycine and Boc-glycine, thereby producing a
differentiated support (2). The Boc groups gropus on 2 are then
deprotected with a suitable agent such as TFA, to produce 3. The
resulting fee amine groups are coupled with a fluophore (or mixture
A and B, to produce a fluorescent support 4, which can be used in
subsequent syntheses or for linkage of desired molecules or
biological particles, and then used in fluorescence assays and
SPAs.
[0171] D. The Recording and Reading and Systems
[0172] Systems for recording and reading information are provided.
The systems include a host computer or decoder/encoder instrument,
a transmitter, a receiver and the data storage device. The systems
also can include a funnel-like device or the like for use in
separating and/or tagging single memory devices. In practice, an EM
signal, preferably a radio frequency signal is transmitted to the
data storage device. The antenna or other receiver means in the
device detects the signal and transmits it to the memory, whereby
the data are written to the memory and stored in a memory
location.
[0173] Mixtures of the matrix with memory-linked molecules or
biological particles may be exposed to the EM signal, or each
matrix with memory (either before, after or during linkage of the
biological particles or molecules) may be individually exposed,
using a device, such as that depicted herein, to the EM signal.
Each matrix with memory, as discussed below, will be linked to a
plurality of molecules or biological particles, which may be
identical or substantially identical or a mixture of molecules or
biological particles depending, upon the application and protocol
in which the matrix with memory and linked (or proximate) molecules
or biological particles is used. The memory can be programmed with
data regarding such parameters.
[0174] The location of the data, which when read and transmitted to
the host computer or decoder/encoder instrument, corresponds to
identifying information about linked or proximate molecules or
biological particles. The host computer or decoder/encoder
instrument can either identify the location of the data for
interpretation by a human or another computer or the host computer
or the decoder/encoder can be programmed with a key to interpret or
decode the data and thereby identify the linked molecule or
biological particle.
[0175] As discussed above, the presently preferred system for use
is the IPTT-100 transponder and DAS-5001 CONSOLE.TM. (Bio Medic
Data Systems, Inc., Maywood, N.J.; see, e.g., U.S. Pat. Nos.
5,422,636, 5,420,579, 5,262,772, 5,252,962 and 5,250,962, 5,252,962
and 5,262,772). These systems may be automated or may be
manual.
[0176] E. Tools and Applications Using Matrices with Memories
[0177] 1. Tools
[0178] The matrix with memory and associated system as described
herein is the basic tool that can be used in a multitude of
applications, including any reaction that incorporates a
functionally specific (i.e., in the reaction) interaction, such as
receptor binding. This tool is then combined with existing
technologies or can be modified to produce additional tools.
[0179] For example, the matrix with memory combination, can be
designed as a single analyte test or as a multianalyte test and
also as a multiplexed assay that is readily automated. The ability
to add one or a mixture of matrices with memories, each with linked
or proximate molecule or biological particle to a sample, provides
that ability to simultaneously determine multiple analytes and to
also avoid multiple pipetting steps. The ability to add a matrix
with memory and linked molecules or particles with additional
reagents, such as scintillants, provides the ability to multiplex
assays.
[0180] As discussed herein, in one preferred embodiment the
matrices are particulate and include adsorbed, absorbed, or
otherwise linked or proximate, molecules, such as peptides or
oligonucleotides, or biological particles, such as cells. Assays
using such particulate memories with matrices may be conduced "on
bead" or "off bead". On bead assays are suitable for multianalyte
assays in which mixtures of matrices with linked molecules are used
and screened against a labeled known. Off bead assays may also be
performed; in these instances the identity of the linked molecule
or biological particle must be known prior to cleavage or the
molecule or biological particle must be in some manner associated
with the memory.
[0181] In other embodiments the matrices with memories use matrices
that are continuous, such as microplates, and include a plurality
of memories, preferably one memory/well. Of particular interest
herein are matrices, such as Flash Plates.TM. (NEN, Dupont), that
are coated or impregnated with scintillant or fluophore or other
luminescent moiety or combination thereof, modified by including a
memory in each well. The resulting memory with matrix is herein
referred to as a luminescing matrix with memory. Other formats of
interest that can be modified by including a memory in a matrix
include the Multiscreen Assay System (Millipore) and gel permeation
technology.
[0182] 2. Scintillation Proximity Assays (SPAs) and
Scintillant-Containing Matrices with Memories
[0183] Scintillation proximity assays are well known in the art
(see, e.g., U.S. Pat. No. 4,271,139; U.S. Pat. No. 4,382,074; U.S.
Pat. No. 4,687,636; U.S. Pat. No. 4,568,649; U.S. Pat. No.
4,388,296; U.S. Pat. No. 5,246,869; International PCT Application
No. WO 94/26413; International PCT Application No. WO 90/03844;
European Patent Application No. 0 556 005 A1; European Patent
Application No. 0 301 769 A1; Hart et al. (1979) Molec. Immunol.
16:265-267; Udenfriend et al. (1985) Proc. Natl. Acad. Sci. U.S.A.
82:8672-8676; Nelson et al. (1987) Analyt. Biochem 165:287-293;
Heath, et al. (1991) Methodol. Surv. Biochem. Anal. 21:193-194;
Mattingly et al. (1995) J. Memb. Sci. 98:275-280; Pernelle (1993)
Biochemistry 32:11682-116878: Bosworth et al. (1989) Nature
341:167-168; and Hart et al. (1989) Nature 341:2651. Beads
(particles) and other formats, such as plates and membranes have
been developed.
[0184] SPA assays refer to homogeneous assays in which quantifiable
light energy produced and is related to the amount of radioactively
labelled products in the medium. The light is produced by a
scintillant that is incorporated or impregnated or otherwise a part
of a support matrix. The support matrix is coated with a receptor,
ligand or other capture molecule that can specifically bind to a
radiolabeled analyte, such as a ligand.
[0185] a. Matrices
[0186] Typically, SPA uses fluomicrospheres, such as
diphenyloxazole-latex, polyacrylamide-containing a fluophore, and
polyvinyltoluene (PVT) plastic scintillator beads, and they are
prepared for use by adsorbing compounds into the matrix. Also
fluomicrospheres based on organic phosphors have been developed.
Microplates made from scintillation plastic, such as PVT, have also
been used (see, e.g., International PCT Application No. WO
90/03844). Numerous other formats are presently available, and any
format may be modified for use herein by including one or more
recording devices.
[0187] Typically the fluomicrospheres or plates are coated with
acceptor molecules, such as receptors or antibodies to which ligand
binds selectively and reversibly. Initially these assays were
performed using glass beads containing fluors and functionalized
with recognition groups for binding specific ligands (or
receptors), such as organic molecules, proteins, antibodies, and
other such molecules. Generally the support bodies used in these
assays are prepared by forming a porous amorphous microscopic
particle, referred to as a bead (see, e.g., European Patent
Application No. 0 154,734 and International PCT Application No. WO
91/08489). The bead is formed from a matrix material such as
acrylamide, acrylic acid, polymers of styrene, agar, agarose,
polystyrene, and other such materials, such as those set forth
above. Cyanogen bromide has been incorporated into the bead into to
provide moieties for linkage of capture molecules or biological
particles to the surface. Scintillant material is impregnated or
incorporated into the bead by precipitation or other suitable
method. Alternatively, the matrices are formed from scintillating
material (see, e.g., International PCT Application No. WO 91/08489,
which is based on U.S. application Ser. No. 07/444,297; see, also
U.S. Pat. No. 5,198,670), such as yttrium silicates and other
glasses, which when activated or doped respond as scintillators.
Dopants include Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce. These materials
can be formed into particles or into continuous matrices. For
purposes herein, the are used to coat, encase or otherwise be in
contact with one or a plurality of recording devices.
[0188] Assays are conducted in normal assay buffers and requires
the use of a ligand labelled with an isotope, such as .sup.3H and
.sup.1251, that emits low-energy radiation that is readily
dissipated easily an aqueous medium. Because .sup.3H .beta.
particles and .sup.1251 Auger electrons have average energies of 6
and 35 keV, respectively, their energies are absorbed by the
aqueous solutions within very small distances (.about.4 .mu.m for
.sup.3H .beta. particles and 35 .mu.m for .sup.1251 Auger
electrons). Thus, in a typical reaction of 0.1 ml to 0.4 ml the
majority of unbound labelled ligands will be too far from the
fluomicrosphere to activate the fluor. Bound ligands, however, will
be in sufficiently close proximity to the fluomicrospheres to allow
the emitted energy to activate the fluor and produce light. As a
result bound ligands produce light, but free ligands do not. Thus,
assay beads emit light when they are exposed to the radioactive
energy from the label bound to the beads through the
antigen-antibody linkage, but the unreacted radioactive species in
solution is too far from the bead to elicit light. The light from
the beads will be measured in a liquid scintillation counter and
will be a measure of the bound label.
[0189] Memories with matrices for use in scintillation proximity
assays (SPA) are prepared by associating a memory with a matrix
that includes a scintillant. In the most simple embodiment, matrix
particles with scintillant (fluomicrospheres) are purchased from
Amersham, Packard, NE Technologies ((formerly Nuclear Enterprises,
Inc.) San Carlos, Calif.) or other such source and are associated
with a memory, such as by including one or more of such beads in a
MICROKAN.TM. microvessel with a recording device. Typically, such
beads as purchased are derivatized and coated with selected
moieties, such as streptavidin, protein A, biotin, wheat germ
agglutinin (WGA), and polylysine. Also available are inorganic
fluomicrospheres based on cerium-doped yttrium silicate or
polyvinyltoluene (PVT). These contain scintillant and may be coated
and derivatized.
[0190] Alternatively, small particles of PVT impregnated with
scintillant are used to coat recording devices, such as the
IPTT-100 devices (Bio Medic Data Systems, Inc., Maywood, N.J.; see,
also U.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962,
5,250,962, 5,074,318, and RE 34,936) that have been coated with a
protective material, such as polystyrene, teflon, a ceramic or
anything that does not interfere with the reading and writing EM
frequency(ies). Such PVT particles may be manufactured or purchased
from commercial sources such as NE TECHNOLOGY, INC. (e.g., catalog
#191A, 1-10 .mu.m particles). These particles are mixed with
agarose or acrylamide, styrene, vinyl or other suitable monomer
that will polymerize or gel to form a layer of this material, which
is coated on polystyrene or other protective layer on the recording
device. The thickness of the layers may be empirically determined,
but they must be sufficiently thin for the scintillant to detect
proximate radiolabels. To make the resulting particles resistant to
chemical reaction they may be coated with polymers such as
polyvinyltoluene or polystyrene, which can then be further
derivatized for linkage and/or synthesis of molecules and
biological particles. The resulting beads are herein called
luminescening matrices with memories, and when used in SPA formats
are herein referred to as scintillating matrices with memories.
[0191] The scintillating matrices with memories beads can be formed
by manufacturing a bead containing a recording device and including
scintillant, such as 2,5-diphenyloxazole (PPO) and/or
1,4-bis-(5-phenyl-(oxazolyl))benzene (POPOP) as a coating. These
particles or beads are then coated with derivatized polyvinyl
benzene or other suitable matrix on which organic synthesis,
protein synthesis or other synthesis can be performed or to which
organic molecules, proteins, nucleic acids, biological particles or
other such materials can be attached. Attachment may be effected
using any of the methods known to those of skill in the art,
including methods described herein, and include covalent,
non-covalent, direct and indirect linkages.
[0192] Molecules, such as ligands or receptors or biological
particles are covalently coupled thereto, and their identity is
recorded in the memory. Alternatively, molecules, such as small
organics, peptides and oligonucleotides, are synthesized on the
beads as described herein so that history of synthesis and/or
identity of the linked molecule is recorded in the memory. The
resulting matrices with memory particles with linked molecules or
biological particles may be used in any application in which SPA is
appropriate. Such applications, include, but are not limited to:
radioimmunoassays, receptor binding assays, enzyme assays and cell
biochemistry assays.
[0193] For use herein, the beads, plates and membranes are either
combined with a recording device or a plurality of devices, or the
materials used in preparing the beads, plates or membranes is used
to coat, encase or contact a recording device. Thus, microvessels
(MICROKANS.TM.) containing SPA beads coated with a molecule or
biological particle of interest; microplates impregnated with or
coated with scintillant, and recording devices otherwise coated
with, impregnated with or contacted with scintillant are
provided.
[0194] To increase photon yield and remove the possibility of loss
of fluor, derivatized fluomicrospheres based on yttrium silicate,
that is doped selectively with rare earth elements to facilitate
production of light with optimum emission characteristics for
photomultipliers and electronic circuitry have been developed (see,
e.g., European Patent Application No. 0 378 059 B1; U.S. Pat. No.
5,246,869). In practice, solid scintillant fibers, such as
cerium-loaded glass or based on rare earths, such as yttrium
silicate, are formed into a matrix. The glasses may also include
activators, such as terbium, europium or lithium. Alternatively,
the fiber matrix may be made from a scintillant loaded polymer,
such as polyvinyltoluene. Molecules and biological particles can be
adsorbed to the resulting matrix.
[0195] For use herein, these fibers may be combined in a
microvessel with a recording device (i.e., to form a MICROKAN.TM.).
Alternatively, the fibers are used to coat a recording device or to
coat or form a microplate containing recording devices in each
well. The resulting combinations are used as supports for synthesis
of molecules or for linking biological particles or molecules. The
identity and/or location and/or other information about the
particles is encoded in the memory and the resulting combinations
are used in scintillation proximity assays.
[0196] Scintillation plates (e.g., FlashPlates.TM., NEN Dupont, and
other such plates) and membranes have also been developed (see,
Mattingly et al. (1995) J. Memb. Sci. 98:275-280) that may be
modified by including a memory for use as described herein. The
membranes, which can contain polysulfone resin M.W. 752 kD,
polyvinylpyrrolidone MW 40 kDA, sulfonated polysulfone, fluor, such
as p-bis-o-methylstyrylbenzene, POP and POPOP, may be prepared as
described by Mattingly, but used to coat, encase or contact a
recording device. Thus, instead of applying the polymer solution to
a glass plate the polymer solution is applied to the recording
device, which, if need is pre-coated with a protective coating,
such as a glass, teflon or other such coating.
[0197] Further, as shown in the Examples, the recording device may
be coated with glass, etched and the coated with a layer of
scintillant. The scintillant may be formed from a polymer, such as
polyacrylamide, gelatin, agarose or other suitable material,
containing fluophors, a scintillation cocktail, FlexiScint (Packard
Instrument Co., Inc., Downers Grove, Ill.) NE Technology beads
(see, e.g., U.S. Pat. No. 4,588,698 for a description of the
preparation of such mixtures). Alternatively, microplates that
contain recording devices in one or more wells may be coated with
or impregnated with a scintillant or microplates containing
scintillant plastic may be manufactured with recording devices in
each well. If necessary, the resulting bead, particle or continuous
matrix, such as a microplate, may be coated with a thin layer
polystyrene, teflon or other suitable material. In all embodiments
it is critical that the scintillant be in sufficient proximity to
the linked molecule or biological particle to detect proximate
radioactivity upon interaction of labeled molecules or labeled
particles with the linked molecule or biological particle.
[0198] The resulting scintillating matrices may be used in any
application for which scintillation proximity assays are used.
These include, ligand identification, single assays, multianalyte
assays, including multi-ligand and multi-receptor assays,
radioimmunoassays (RIAs), enzyme assays, and cell biochemistry
assays (see, e.g., International PCT Application No. WO 93/19175,
U.S. Pat. No. 5,430,150, Whitford et al. (1991) Phyto-chemical
Analysis 2: 134-136; Fenwick et al. (1994) Anal. Proc. Including
Anal. Commun. 31: 103-106; Skinner et al. (1994) Anal. Biochem.
223:259-265; Matsumura et al. (1992) Life Sciences 51: 1603-1611;
Cook et al. (1991) Structure and Function of the Aspartic
Proteinases. Dunn, ed., Penum Press, NY, pp. 525-528; Bazendale et
al. in (1990) Advances in Prostaglandin, Thromboxane and
Leukotriene Research. Vol. 21, Samuelsson et al., eds., Raven
Press, NY, pp 302-306).
[0199] b. Assays
[0200] (1) Receptor Binding Assays
[0201] Scintillating matrices with memories beads can be used, for
example, in assays screening test compounds as agonists or
antagonists of receptors or ion channels or other such cell surface
protein. Test compounds of interest are synthesized on the beads or
linked thereto, the identity of the linked compounds is encoded in
the memory either during or following synthesis, linkage or
coating. The scintillating matrices with memories are then
incubated with radiolabeled (.sup.1251, .sup.3H, or other suitable
radiolabel) receptor of interest and counted in a liquid
scintillation counter. When radiolabeled receptor binds to any of
the structure(s) synthesized or linked to the bead, the
radioisotope is in sufficient proximity to the bead to stimulate
the scintillant to emit light. In contrast By contrast, if a
receptor does not bind, less or no radioactivity is associated with
the bead, and consequently less light is emitted. Thus, at
equilibrium, the presence of molecules that are able to bind the
receptor may be detected. When the reading is completed, the memory
in each bead that emits light (or more light than a control)
queried and the host computer, decoder/encoder, or scanner can
interpret the memory in the bead and identify the active
ligand.
[0202] (a) Multi-Ligand Assay
[0203] Mixtures of scintillating matrices with memories with a
variety of linked ligands, which were synthesized on the matrices
or linked thereto and their identities encoded in each memory, are
incubated with a single receptor. The memory in each light-emitting
scintillating matrix with memory is queried and the identity of the
binding ligand is determined
[0204] (b) Multi-Receptor Assays
[0205] Similar to conventional indirect or competitive receptor
binding assays that are based on the competition between unlabelled
ligand and a fixed quantity of radiolabeled ligand for a limited
number of binding sites, the scintillating matrices with memories
permit the simultaneous screening of a number of ligands for a
number of receptor subtypes.
[0206] Mixtures of receptor coated beads (one receptor type/per
bead; each memory encoded with the identity of the linked receptor)
are reacted with labeled ligands specific for each receptor. After
the reaction has reached equilibrium, all beads that emit light are
reacted with a test compound. Beads that no longer emit light are
read.
[0207] For example receptor isoforms, such as retinoic acid
receptor isoforms, are each linked to a different batch of
scintillating matrix with memory beads, and the identity of each
isoform is encoded in the memories of linked matrices. After
addition of the radiolabeled ligand(s), such as .sup.3H-retinoic
acid, a sample of test compounds (natural, synthetic,
combinatorial, etc.) is added to the reaction mixture, mixed and
incubated for sufficient time to allow the reaction to reach
equilibrium. The radiolabeled ligand binds to its receptor, which
has been covalently linked to the bead and which the emitted short
range electrons will excite the fluophor or scintillant in the
beads, producing light. When unlabelled ligand from test mixture is
added, if it displaces the labeled ligand it will diminish or stop
the fluorescent light signal. At the end of incubation period, the
tube can be measured in a liquid scintillation counter to
demonstrate if any of the test material reacted with receptor
family. Positive samples (reduced or no fluorescence) will be
further analyzed for receptor subtyping by querying their memories
with the RF detector. In preferred embodiments, each bead will be
read with a fluorescence detector and RF scanner. Those that have a
reduced fluorescent signal will be identified and the linked
receptor determined by the results from querying the memory.
[0208] The same concept can be used to screen for ligands for a
number of receptors. In one example. FGF receptor, EGF receptor,
and PDGF receptor are each covalently linked to a different batch
of scintillating matrix with memory beads. The identity of each
receptor is encoded in each memory. After addition of the
.sup.1251-ligands (.sup.125I-FGF, .sup.125I-EGF, and
.sup.125I-PDGF) a sample of test compounds (natural, synthetic,
combinatorial, etc.) is added to the tube containing
.sup.1251-ligand-receptor-beads, m mixed and incubated for
sufficient time to allow the reaction to reach equilibrium. The
radiolabeled ligands bind to their respective receptors receptor
that been covalently linked to the bead. By virtue of proximity of
the label to the bead, the emitted short range electrons will
excite the fluophor in the beads. When unlabelled ligand from test
mixture is added, if it displaces the any of the labeled ligand it
will diminish or stop the fluorescent signal. At the end of
incubation period, the tube can be measured in a liquid
scintillation counter to demonstrate if any of the test material
reacted with the selected receptor family. Positive samples will be
further analyzed for receptor type by passing the resulting
complexes, measuring the fluorescence of each bead, and querying
the memories by exposing them to RF or the selected EM radiation.
The specificity of test ligand is determined by identifying beads
with reduced fluorescence and determining the identity of the
linked receptor by querying the memory.
[0209] (c) Other Formats
[0210] Microspheres, generally polystyrene typically about 0.3
.mu.m-3.9 .mu.m, are synthesized with scintillant inside can either
be purchased or prepared by covalently linking scintillant to the
monomer prior to polymerization of the polystyrene or other
material. They can then be derivatized (or purchased with chemical
functional groups), such as --COOH, and --CH.sub.2OH. Selected
compounds or libraries are synthesized on the resulting
microspheres linked via the functional groups, as described herein,
or receptor, such as radiolabeled receptor, can be coated on the
microsphere. The resulting "bead" with linked compounds, can used
in a variety of SPA and related assays, including immunoassays,
receptor binding assays, protein, protein interaction assays, and
other such assays in which the ligands linked to the
scintillant-containing microspheres are reacted with memories with
matrices that are coated with a selected receptor.
[0211] For example, .sup.1251-labeled receptor is passively coated
on the memory with matrix and then mixed with ligand that is linked
to a the scintillant-containing microspheres. Upon binding the
radioisotope into is brought into close proximity to the
scintillant in which effective energy transfer from the .beta.
particle will occur, resulting in emission of light.
[0212] Alternatively, the memory with matrix (containing
scintillant) can also be coated with .sup.3H-containing polymer on
which the biological target (i.e., receptor, protein, antibody,
antigen) can be linked (via adsorption or via a functional group).
Binding of the ligand brings the scintillant into close proximity
to the label, resulting in light emission.
[0213] (2) Cell-Based Assays
[0214] Cell-based assays, which are fundamental for understanding
of the biochemical events in cells, have been used with increasing
frequency in biology, pharmacology, toxicology, genetics, and
oncology (see, e.g., Benjamin et al. (1992) Mol. Cell. Biol.
12:2730-2738) Such cell lines may be constructed or purchased (see,
e.g., the Pro-Tox Kit available from Xenometrix, Boulder Colo.;
see, also International PCT Application No. WO 94/7208 cell lines).
Established cell lines, primary cell culture, reporter gene systems
in recombinant cells, cells transfected with gene of interest, and
recombinant mammalian cell lines have been used to set up
cell-based assays. For example Xenometrix, Inc. (Boulder, Colo.)
provides kits for screening compounds for toxicological endpoints
and metabolic profiles using bacteria and human cell lines.
Screening is effected by assessing activation of regulatory
elements of stress genes fused to reporter genes in bacteria, human
liver or colon cell lines and provide information on the
cytotoxicity and permeability of test compounds.
[0215] In any drug discovery program, cell-based assays offer a
broad range of potential targets as well as information on
cytotoxicity and permeability. The ability to test large numbers of
compounds quickly and efficiently provides a competitive advantage
in pharmaceutical lead identification.
[0216] High throughput screening with cell-based assays is often
limited by the need to use separation, wash, and disruptive
processes that compromise the functional integrity of the cells and
performance of the assay. Homogeneous or mix-and-measure type
assays simplify investigation of various biochemical events in
whole cells and have been developed using scintillation microplates
(see, e.g., International PCT Application No. WO 94/26413, which
describes scintillant plates that are adapted for attachment and/or
growth of cells and proximity assays using such cells). In certain
embodiment herein, cell lines such as those described in
International PCT Application No. WO 94/17208 are be plated on
scintillant plates, and screened against compounds synthesized on
matrices with memories. Matrices with memories encoded with the
identity of the linked molecule will be introduced into the plates,
the linkages cleaved and the effects of the compounds assessed.
Positive compounds will be identified by querying the associated
memory.
[0217] The scintillant base plate is preferably optically
transparent to selected wavelengths that allow cells in culture to
be viewed using an inverted phase contrast microscope, and permit
the material to transmit light at a given wavelength with maximum
efficiency. In addition the base retains its optical properties
even after exposure to incident beta radiation from radioisotopes
as well as under stringent radiation conditions required for
sterilization of the plates. The base plate can be composed of any
such optically transparent material containing scintillant, e.g., a
scintillant glass based on lanthanide metal compounds. Typically,
the base plate is composed of any plastic material, generally
formed from monomer units that include phenyl or naphthyl moieties
in order to absorb incident radiation energy from radionuclides
which are in close proximity with the surface. Preferably the
plastic base plate is composed of polystyrene or polyvinyltoluene,
into which the scintillant is incorporated. The scintillant
includes, but is not limited to: aromatic hydrocarbons such as
p-terphenyl, p-quaterphenyl and their derivatives, as well as
derivatives of the oxazoles and 1,3,4-oxadiazoles, such as
2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole and
2,5-diphenyloxazole. Also included in the polymeric composition may
be a wavelength shifter such as
1,4-bis(5-phenyl-2-oxazolyl)benzene, 9,10-diphenylanthracene,
1,4-bis(2-methylstyryl)-benzene, and other such compounds. The
function of the wavelength shifter is to absorb the light emitted
by the scintillant substance and re-emit longer wavelength light
which is a better match to the photo-sensitive detectors used in
scintillation counters. Other scintillant substances and polymer
bodies containing them are known to those of skill in this art
(see, e.g., European Patent Application No. 0 556 005 A1).
[0218] The scintillant substances can be incorporated into the
plastic material of the base by a variety of methods. For example,
the scintillators may be dissolved into the monomer mix prior to
polymerization, so that they are distributed evenly throughout the
resultant polymer. Alternatively, the scintillant substances may be
dissolved in a solution of the polymer and the solvent removed to
leave a homogeneous mixture. The base plate of disc may be bonded
to the main body of the well or array of wells, which itself may be
composed of a plastic material including polystyrene,
polyvinyltoluene, or other such polymers. In the case of the
multi-well array, the body of the plate may be made opaque, i.e.,
non-transparent and internally reflective, in order to completely
exclude transmission of light and hence minimize "cross-talk." This
is accomplished by incorporating into the plastic at the
polymerization stage a white dye or pigment, for example, titanium
dioxide. Bonding of the base plate to the main body of the device
can be accomplished by any suitable bonding technique, for example,
heat welding, injection molding or ultrasonic welding.
[0219] For example, a 96-well plate is constructed to the standard
dimensions of 96-well microtiter plates 12.8 cm.times.8.6
cm.times.1.45 cm with wells in an array of 8 rows of 12 wells each.
The main body of the plate is constructed by injection molding of
polystyrene containing a loading of white titanium oxide pigment at
12%. At this stage, the wells of the microtiter plate are
cylindrical tubes with no closed end. A base plate is formed by
injection molding of polystyrene containing
2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole (2%) and
9,10-diphenylanthracene (0.5%). The base plate has been silk screen
printed with a grid array to further reduce crosstalk. The base
plate is then fused in a separate operation to the body by
ultrasonic welding, such that the grid array overlies the portions
of the microtiter plate between the wells.
[0220] A 24-well device is constructed to the dimensions
12.8.times.8.6.times.1.4 cm with 24 wells in an array of 4 rows of
6 wells. The main body of the plate (not including the base of each
well) is constructed by injection molding of polystyrene containing
12% white titanium oxide pigment. The base 24 of each well is
injection molded with polystyrene containing
2-(4-t-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadizaole (2%) and
9,10-diphenylanthracene (0.5%). The heat from the injected base
plastic results in fusion to the main body giving an optically
transparent base to the well.
[0221] The plates may contain multiple wells that are continuous or
that are each discontinuous from the other wells in the array, or
they may be single vessels that have, for example, an open top,
side walls and an optically transparent scintillant plastic base
sealed around the lower edge of the side walls.
[0222] In another format, the plate is a single well or tube. The
tube may be constructed from a hollow cylinder made from optically
transparent plastic material and a circular, scintillant
containing, plastic disc. The two components are welded together so
as to form a single well or tube suitable for growing cells in
culture. As in the plate format, bonding of the circular base plate
to the cylindrical portion is achieved by any conventional bonding
technique, such as ultrasonic welding. The single well or tube may
be any convenient size, suitable for scintillation counting. In
use, the single well may either be counted as an insert in a
scintillation vial, or alternatively as an insert in a
scintillation vial, or alternatively as an insert in a multi-well
plate of a flat bed scintillation counter. In this latter case, the
main body of the multi-well plate would need to be opaque for
reasons given earlier.
[0223] The various formats are selected according to use. They may
be used for growing cells and studying cellular biochemical
processes in living cells or cell fragments. The 96-well plate is a
standard format used in experimental cell biology and one that is
suitable for use in a flat bed scintillation counter (e.g., Wallac
Microbeta or Packard Top Count). In the multi-well format, it is an
advantage to be able to prevent "cross talk" between different
wells of the plate that may be used for monitoring different
biological processes using different amounts or types of
radioisotope. Therefore the main body of the plate can be made from
opaque plastic material. The 24-well plate format is commonly used
for cell culture. This type of plate is also suitable for counting
in a flat bed scintillation counter. The dimensions of the wells
will be larger.
[0224] As an alternative format, the transparent, scintillant
containing plastic disc is made to be of suitable dimensions so as
to fit into the bottom of a counting vessel. The counting vessel is
made from non-scintillant containing material such as glass or
plastic and should be sterile in order to allow cells to grow and
the corresponding cellular metabolic processes to continue. Cells
are first cultured on the disc, which is then transferred to the
counting vessel for the purposes of monitoring cellular biochemical
processes.
[0225] The culture of cells on the scintillation plastic base plate
of the wells (or the disc) involves the use of standard cell
culture procedures, e.g., cells are cultured in a sterile
environment at 37.degree. C. in an incubator containing a
humidified 95% air/5% CO.sub.2 atmosphere. Various cell culture
media may be used including media containing undefined biological
fluids such as fetal calf serum, or media which is fully defined
and serum-free. For example, MCDB 153 is a selective medium for the
culture of human keratinocytes (Tsao et al. (1982) J. Cell.
Physiol. 110:219-2291.
[0226] These plates are suitable for use with any adherent cell
type that can be cultured on standard tissue culture plasticware,
including culture of primary cells, normal and transformed cells
derived from recognized sources species and tissue sources. In
addition, cells that have been transfected with the recombinant
genes may also be cultured using the invention. There are
established protocols available for the culture of many of these
diverse cell types (see, e.g., Freshney et al. (1987) Culture of
Animal Cells: A Manual of Basic Technique. 2nd Edition, Alan R.
Liss Inc.). These protocols may require the use of specialized
coatings and selective media to enable cell growth and the
expression of specialized cellular functions.
[0227] The scintillating base plate or disc, like all plastic
tissue culture ware, requires surface modification in order to be
adapted for the attachment and/or growth of cells. Treatment can
involves the use of high voltage plasma discharge, a well
established method for creating a negatively charged plastic
surface (see, e.g., Amstein et al. (1975) Clinical Microbiol.
2:46-54). Cell attachment, growth and the expression of specialized
functions can be further improved by applying a range of additional
coatings to the culture surface of the device. These can include:
(i) positively or negatively charged chemical coatings such as
poly-lysine or other biopolymers (McKeehan et al. (1976) J. Cell
Biol. 21:727-734 (1976)); (ii) components of the extracellular
matrix including collagen, laminin, fibronectin (see, e.g.,
Kleinman et al. (1987) Anal. Biochem. 166: 1-13); and (iii)
naturally secreted extracellular matrix laid down by cells cultured
on the plastic surface (Freshney et al. (1987) Culture of Animal
Cells: A Manual of Basic Technique, 2nd Edition, Alan R. Liss
Inc.). Furthermore, the scintillating base plate may be coated with
agents, such as lectins, or adhesion molecules for attachment of
cell membranes or cell types that normally grow in suspension.
Methods for the coating of plasticware with such agents are known
(see, e.g., Boldt et al. (1979) J. Immunol. 123:808).
[0228] In addition, the surface of the scintillating layer may be
coated with living or dead cells, cellular material, or other
coatings of biological relevance. The interaction of radiolabeled
living cells, or other structures with this layer can be monitored
with time allowing processes such as binding, movement to or from
or through the layer to be measured.
[0229] Virtually all types of biological molecules can be studied.
A any molecule or complex of molecules that interact with the cell
surface- or that can be taken up, transported and metabolized by
the cells, can be examined using real time analysis. Examples of
biomolecules will include receptor ligands, protein and lipid
metabolite precursors (e.g., amino acids, fatty acids), nucleosides
and any molecule that can be radiolabeled. This would also include
ions such as calcium, potassium, sodium and chloride, that are
functionally important in cellular homeostasis, and which exist as
radioactive isotopes. Furthermore, viruses and bacteria and other
cell types, which can be radiolabeled as intact moieties, can be
examined for their interaction with monolayer adherent cells grown
in the scintillant well format.
[0230] The type of radioactive isotope that can be used with this
system will typically include any of the group of isotopes that
emit electrons having a mean range up to 2000 .mu.m in aqueous
medium. These will include isotopes commonly used in biochemistry
such as (.sup.3H), (.sup.1251), (.sup.14C), (.sup.35S),
(.sup.45Ca), (.sup.33p), and (.sup.32p), but does not preclude the
use of other isotopes, such as (.sup.55Fe), (.sup.109 Cd) and
(.sup.51Cr) that also emit electrons within this range. The wide
utility of the invention for isotopes of different emission energy
is due to the fact that the current formats envisaged would allow
changes to the thickness of the layer containing a scintillant
substance, thereby ensuring that all the electron energy is
absorbed by the scintillant substance. Furthermore, cross-talk
correction software is available which can be utilized with all
high energy emitters. Applications using these plates include
protein synthesis, Ca.sup.2+ transport, receptor-ligand binding,
cell adhesion, sugar transport and metabolism, hormonal
stimulation, growth factor regulation and stimulation of motility,
thymidine transport, and protein synthesis.
[0231] For use in accord with the methods herein, the scintillant
plates can include a memory in each well, or alternatively, memory
with matrix-linked compounds will be added to each well. The
recording device with memory may be impregnated or encased or
placed in wells of the plate, typically during manufacture. In
preferred embodiments, however, the memories are added to the wells
with adsorbed or linked molecules.
[0232] In one embodiment, matrices with memories with linked
molecules are introduced into scintillant plates in which cells
have been cultured (see, e.g., International PCT Application No. WO
94/26413). For example, cells will be plated on the transparent
scintillant base 96-well microplate that permits examination of
cells in culture by inverted phase contrast microscope and permits
the material to transmit light at a given wavelength with maximum
efficiency. Matrices with memories to which test compounds linked
by preferably a photocleaveable linker are added to the wells. The
identity of each test compound is encoded in the memory of the
matrix during synthesis if the compound is synthesized on the
matrix with memory or when the compound is linked to the
matrix.
[0233] Following addition of matrix with memory to the well and
release of chemical entities synthesized on the beads by exposure
to light or other procedures, the effects of the chemical released
from the beads on the selected biochemical events, such as signal
transduction, cell proliferation, protein or DNA synthesis, in the
cells can be assessed. In this format receptor binding Such events
include, but are not limited to: whole cell receptor-ligand binding
(agonist or antagonist), thymidine or uridine transport, protein
synthesis (using, for example, labeled cysteine, methionine,
leucine or proline), hormone and growth factor induced stimulation
and motility, and calcium uptake.
[0234] In another embodiment, the memories are included in the
plates either placed in the plates or manufactured in the wells of
the plates. In these formats, the identities of the contents of the
well is encoded into the memory. Of course it is understood, that
the information encoded and selection of encased or added memories
depends upon the selected protocol.
[0235] In another format, cells will be plated on the tissue
culture plate, after transferring the matrices with memories and
release of compounds synthesized on the beads in the well.
Cytostatic, cytotoxic and proliferative effects of the compounds
will be measured using colorimetric (MTT, XTT, MTS, Alamar blue,
and Sulforhodamine B), fluorimetric (carboxyfluorescein diacetate),
or chemiluminescent reagents (i.e., CytoLite.TM., Packard
Instruments, which is used in a homogeneous luminescent assay for
cell proliferation, cell toxicity and multi-drug resistance).
[0236] For example, cells that have been stably or transiently
transfected with a specific gene reporter construct containing an
inducible promoter co-operatively linked to a reporter gene that
encodes an indicator protein can be colorimetrically monitored for
promoter induction. Cells will be plated on the tissue culture
96-well microtiter plate and after addition of memories with
matrices in the wells and release of chemical entities synthesized
on the matrices, the effect of the compound released from the beads
on the gene expression will be assessed. The Cytosensor
Microphysiometer (Molecular Devices) evaluates cellular responses
that are mediated by G protein-linked receptors, tyrosine
kinase-linked receptors, and ligand-gated ion channels. It measures
extracellular pH to assess profiles of compounds assessed for the
ability to modulate activities of any of the these cell surface
proteins by detecting secretion of acid metabolites as a result of
altered metabolic states, particularly changes in metabolic rate.
Receptor activation requires use of ATP and other energy resources
of the cell thereby leading to increased in cellular metabolic
rate. For embodiments herein, the memories with matrices,
particularly those modified for measuring pH, and including linked
test compounds, can be used to track and identify the added test
compound added and also to detect changes in pH, thereby
identifying linked molecules that modulate receptor activities.
[0237] 3. Memories with Matrices for Non-Radioactive Energy
Transfer Proximity Assays
[0238] Non-radioactive energy transfer reactions, such as FET or
FRET, FP and HTRF assays, are homogeneous luminescence assays based
on energy transfer are carried out between a donor luminescent
label and an acceptor label (see, e.g., Cardullo et al. (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:8790-8794; Peerce et al. (1986)
Proc. Natl. Acad. Sci. U.S.A. 83:8092-8096; U.S. Pat. No.
4,777,128; U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S.
Pat. No. 5,279,943; and International PCT Application No. WO 92/01
225). The donor label is usually a rare earth metal cryptate,
particularly europium trisbipyridine diamine (EuTBP) or terbium
trisbipyridine diamine (TbTBP) and an acceptor luminescent,
presently fluorescent, label. When the donor is EuTBP, the acceptor
is preferably allopycocyanin (APC), allophycocyanin B, phycocyanin
C or phycocyanin R, and when the donor is TbTBP, the acceptor is a
rhodamine, thiomine, phycocyanin R, phycoerythrocyanin,
phycoerythrin C, phycoerythrin B or phycoerythrin R.
[0239] Energy transfer between such donors and acceptors is highly
efficient, giving an amplified signal and thereby improving the
precision and sensitivity of the assay. Within distances
characteristic of interactions between biological molecules, the
excitation of a fluorescent label (donor) is transferred non
radiatively to a second fluorescent label (acceptor). When using
europium cryptate as the donor, APC, a phycobiliprotein of 5 kDa,
is presently the preferred acceptor because it has high molar
absorptivity at the cryptate emission wavelength providing a high
transfer efficiency, emission in a spectral range in which the
cryptate signal is insignificant, emission that is not quenched by
presence of sera, and a high quantum yield. When using Eu.sup.3+
cryptate as donor, an amplification of emitted fluorescence is
obtained by measuring APC emission.
[0240] The rare earth cryptates are formed by the inclusion of a
luminescence lanthanide ion in the cavity of a macropolycyclic
ligand containing 2,2'-biphyridine groups as light absorbers (see,
e.g., U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat.
No. 5,279,943; and International PCT Application No. WO 92/01225).
Preferably the Eu3* trisbypryidine diamine derivative, although the
acceptor may be used as the label, is cross-linked to antigens,
antibodies, proteins, peptides, and oligonucleotides and other
molecules of interest.
[0241] For use herein, matrices with memories are prepared that
incorporate either the donor or, preferably the acceptor, into or
on the matrix. In practice, as with the scintillating matrices with
memories, the matrices may be of any format, i.e., particulate, or
continuous, and used in any assay described above for the
scintillating matrices. For example, the recording device is coated
with a protective coating, such as glass or polystyrene. If glass
it can be etched. As with preparation of the scintillating matrices
with memories, compositions containing the donor or preferably
acceptor, such as APC, and typically a polymer or gel, are coated
on the recording device or the device is mixed with the composition
to produce a fluorescing matrix with memory. To make these matrices
resistant to chemical reaction, if needed, they may be coated with
polymers such as polyvinylbenzene or polystyrene. Molecules, such
as the constituents of combinatorial libraries, are synthesized on
the fluorescing matrices with memories, or molecules or biological
particles are linked thereto, the identity of the synthesized
molecules or linked molecules or biological particles is encoded in
memory, and the resulting matrices with memories employed in any
suitable assay, including any of those described for the
scintillating memories with matrices. In particular, these
homogeneous assays using long-lived fluorescence rare earth
cryptates and amplification by non radiative energy transfer have
been adapted to use in numerous assays including assays employing
ligand receptor interaction, signal transduction, transcription
factors (protein-protein interaction), enzyme substrate assays and
DNA hybridization and analysis (see, Nowak (1993) Science 270:368;
see, also, Velculescu et al. (1995) Science 270:484-487, and Schena
et al. (1995) Science 270:467-470, which describe methods
quantitative and simultaneous analysis of a large number of
transcripts that are particularly suited for modification using
matrices with memories). Each of these assays may be modified using
the fluorescing matrices with memories provided herein.
[0242] For example, a receptor will be labeled with a europium
cryptate (where the matrices with memories incorporate, for example
allophycocyanin (APC)) or will be labeled with APC, where the
matrices incorporate a europium cryptate. After mixing receptor and
mixtures of matrices with different ligands, the mixture is exposed
to laser excitation at 337 nm, and, if reaction has occurred,
typical signals of europium cryptate and APC over background are
emitted. Measurement with an interference filter centered at 665 nm
selects the signal of the APC labeled receptor from that of
europium cryptate labeled ligand on the beads. If particulate, the
memories of matrices that emit at 665, can be queried to identify
linked ligands.
[0243] 4. Other Applications Using Memories with Matrices and
Luminescing Memories with Matrices
[0244] a. Natural Product Screening
[0245] In the past, the vast majority of mainline pharmaceuticals
have been isolated form natural products such as plants, bacteria,
fungus, and marine microorganisms. Natural products include
microbials, botanicals, animal and marine products. Extracts of
such sources are screened for desired activities and products.
Selected products include enzymes (e.g., hyaluronidase), industrial
chemicals (e.g., petroleum emulsifying agents), and antibiotics
(e.g., penicillin). It is generally considered that a wealth of new
agents still exist within the natural products pool. Large mixtures
of natural products, even within a fermentation broth, can be
screened using the matrices with memory combinations linked, for
example, to peptides, such as antigens or antibody fragments or
receptors, of selected and known sequences or specificities, or to
other biologically active compounds, such as neurotransmitters,
cell surface receptors, enzymes, or any other identified biological
target of interest. Mixtures of these peptides linked to memory
matrices can be introduced into the natural r product mixture.
Individual binding matrices, detected by an indicator, such as a
fluorometric dye, can be isolated and the memory queried to
determine which linked molecule or biological particle is bound to
a natural product.
[0246] b. Immunoassays and Immunodiagnostics
[0247] The combinations and methods provided herein represent major
advances in immunodiagnostics Immunoassays (such as ELISAs, RIAs
and EIAs (enzyme immunoassays)) are used to detect and quantify
antigens or antibodies.
(1) Immunoassays
[0248] Immunoassays detect or quantify very small concentrations of
analytes in biological samples. Many immunoassays use solid
supports in which antigen or antibody is covalently,
non-covalently, or otherwise, such as via a linker, attached to a
solid support matrix. The support-bound antigen or antibody is then
used as an analyte in the assay. As with nucleic acid analysis, the
resulting antibody-antigen complexes or other complexes, depending
upon the format used, rely on radiolabels or enzyme labels to
detect such complexes.
[0249] The use of antibodies to detect and/or quantitate reagents
("antigens") in blood or other body fluids has been widely
practiced for many years. Two methods have been most broadly
adopted. The first such procedure is the competitive binding assay,
in which conditions of limiting antibody are established such that
only a fraction (usually 30-50%) of a labeled (e.g., radioisotope,
fluophore or enzyme) antigen can bind to the amount of antibody in
the assay medium. Under those conditions, the addition of unlabeled
antigen (e.g., in a serum sample to be tested) then competes with
the labeled antigen for the limiting antibody binding sites and
reduces the amount of labeled antigen that can bind. The degree to
which the labeled antigen is able to bind is inversely proportional
to the amount of unlabeled antigen present. By separating the
antibody-bound from the unbound labeled antigen and then
determining the amount of labeled reagent present, the amount of
unlabeled antigen in the sample (e.g., serum) can be
determined.
[0250] As an alternative to the competitive binding assay, in the
labeled antibody; or "immunometric" assay (also known as "sandwich"
assay), an antigen present in the assay fluid is specifically bound
to a solid substrate and the amount of antigen bound is then
detected by a labeled antibody (see, e.g., Miles et al. (1968)
Nature 29:186-189; U.S. Pat. No. 3,867,517; U.S. Pat. No.
4,376,110). Using monoclonal antibodies two-site immunometric
assays are available (see, e.g., U.S. Pat. No. 4,376,110). The
"sandwich" assay has been broadly adopted in clinical medicine.
With increasing interest in "panels" of diagnostic tests, in which
a number of different antigens in a fluid are measured, the need to
carry out each immunoassay separately becomes a serious limitation
of current quantitative assay technology.
[0251] Some semi-quantitative detection systems have been developed
(see, e.g., Buechler et al. (1992) Clin. Chem. 38:1678-1 684; and
U.S. Pat. No. 5,089,391) for use with immunoassays, but no good
technologies yet exist to carefully quantitate a large number of
analytes simultaneously (see, e.g., Ekins et al. (1990) J. Clin.
Immunoassay 13:169-181) or to rapidly and conveniently track,
identify and quantitate detected analytes.
[0252] The methods and memories with matrices provided herein
provide a means to quantitate a large number of analytes
simultaneously and to rapidly and conveniently track, identify and
quantitate detected analytes.
(2) Multianalyte Immunoassays
[0253] The combinations of matrix with memories provided herein
permits the simultaneous assay of large numbers of analytes in any
format. In general, the sample that contains an analyte, such as a
ligand or any substance of interest, to be detected or quantitated,
is incubated with and bound to a protein, such as receptor or
antibody, or nucleic acid or other molecule to which the analyte of
interest binds. In one embodiment, the protein or nucleic acid or
other molecule to which the analyte of interest binds has been
linked to a matrix with memory prior to incubation; in another
embodiment, complex of analyte or ligand and protein, nucleic acid
or other molecule to which the analyte of interest binds is linked
to the matrix with memory after the incubation; and in a third
embodiment, incubation to form complexes and attachment of the
complexes to the matrix with memory are simultaneous. In any
embodiment, attachment is effected, for example, by direct covalent
attachment, by kinetically inert attachment, by noncovalent
linkage, or by indirect linkage, such as through a second binding
reaction (i.e., biotin-avidin, Protein A-antibody, antibody-hapten,
hybridization to form nucleic acid duplexes of oligonucleotides,
and other such reactions and interactions). The complexes are
detected and quantitated on the solid phase by virtue of a label,
such as radiolabel, fluorescent label, luminophore label, enzyme
label or any other such label. The information that is encoded in
the matrix with memory depends upon the selected embodiment. If,
for example, the target molecule, such as the protein or receptor
is bound to the solid phase, prior to complexation, the identity of
the receptor and/or source of the receptor may be encoded in the
memory in the matrix.
[0254] For example, the combinations provided herein are
particularly suitable for analyses of multianalytes in a fluid, and
particularly for multianalyte immunoassays. In one example,
monoclonal antibodies very specific for carcinoembryonic antigen
(CEA), prostate specific antigen (PSA), CA-1 25, alphafetoprotein
(AFP), TGF-13, IL-2, IL-8 and IL-10 are each covalently attached to
a different batch of matrices with memories using well-established
procedures and matrices for solid phase antibody assays. Each
antibody-matrix with memory complex is given a specific
identification tag, as described herein.
[0255] A sample of serum from a patient to be screened for the
presence or concentration of these antigens is added to a tube
containing two of each antibody-matrix with memory complex (a total
of 16 beads, or duplicates of each kind of bead). A mixture of
monoclonal antibodies, previously conjugated to fluorescent dyes,
such as fluorescein or phenyl-EDTA-Eu chelate, reactive with
different epitopes on each of the antigens is then added. The tubes
are then sealed and the contents are mixed for sufficient time
(typically one hour) to allow any antigens present to bind to their
specific antibody-matrix with memory-antigen complex to produce
antibody-matrix with memory-antigen-labeled antibody complexes. At
the end of the time period, these resulting complexes are briefly
rinsed and passed through an apparatus, such as that set forth in
FIG. 7, but with an additional light source. As each complex passes
through a light source, such as a laser emitting at the excitation
wavelength of fluorescein, about 494 nm, or 340 nm for the Eu
chelate complex, its fluorescence is measured and quantitated by
reading the emitted photons at about 518 nm for fluorescein or 613
nm for phenyl-EDTA-Eu, and as its identity is determined by the
specific signal received by the RF detector. In this manner, eight
different antigens are simultaneously detected and quantitated in
duplicate.
[0256] In another embodiment, the electromagnetically tagged
matrices with recorded information regarding linked antibodies can
be used with other multianalyte assays, such as those described by
Ekins et al. ((1990) J. Clin. Immunoassay 13:169-181; see, also
International PCT Applications Nos. 89/01157 and 93/08472, and U.S.
Pat. Nos. 4,745,072, 5,171,695 and 5,304,498). These methods rely
on the use of small concentrations of sensor-antibodies within a
few .mu.m2 area. Individual memories with matrices, or an array of
memories embedded in a matrix are used. Different antibodies are
linked to each memory, which is programmed to record the identity
of the linked antibody. Alternatively, the antibody can be linked,
and its identity or binding sites identified, and the information
recorded in the memory. Linkage of the antibodies can be effected
by any method known to those of skill in this art, but is
preferably effected using cobalt-iminodiacetate coated memories
(see, Hale (1995) Analytical Biochem. 231:46-49, which describes
means for immobilization of antibodies to cobalt-iminodiacetate
resin) mediated linkage particularly advantageous. Antibodies that
are revesibly bound to a cobalt-iminodiacetate resin are attached
in exchange insert manner when the cobalt is oxidized from the +2
to +3 state. In this state the antibodies are not removed by metal
chelating regents, high salt, detergents or chaotropic agents. They
are only removed by reducing agents. In addition, since the metal
binding site in antibodies is in the C-terminus heavy chain,
antibodies so-bound are oriented with the combining site directed
away from the resin.
[0257] In particular antibodies are linked to the matrices with
memories. The matrices are either in particular form or in the form
of a slab with an array of recording devices linked to the matrices
or microtiter dish or the like with a recording device in each
well. Antibodies are then linked either to each matrix particle or
to discrete "microspots" on the slab or in the microtiter wells. In
one application, prior to use of these matrices with memories, they
are bound to a relatively low affinity anti-idiotype antibody (or
other species that specifically recognizes the antibody binding
site, such as a single chain antibody or peptidomimetic) labeled
with a fluophore (e.g., Texas Red, see, Ekins et al. (1990) J.
Clin. Immunoassay 13: 169-181) to measure the concentration of and
number of available binding sites present on each matrix with
memory particle or each microspot, which information is then
encoded into each memory for each microspot or each particle. These
low affinity antibodies are then eluted, and the matrices can be
dried and stored until used.
[0258] Alternatively or additionally, the memories in the particles
or at each microspot could be programmed with the identity or
specificity of the linked antibody, so that after reaction with the
test sample and identification of complexed antibodies, the
presence and concentration of particular analytes in the sample can
be determined. They can be used for multianalyte analyses as
described above.
[0259] After reaction with the test sample, the matrices with
memories are reacted with a second antibody, preferably, although
not necessarily, labeled with a different label, such as a
different fluophore, such as fluorescein. After this incubation,
the microspots or each matrix particle is read by passing the
particle through a laser scanner (such as a confocal microscope,
see, e.g., Ekins et al. (1990) J. Clin. Immunoassay 13:169-181; see
also U.S. Pat. No. 5,342,633) to determine the fluorescence
intensity. The memories at each spot or linked to each particle are
queried to determine the total number of available binding sites,
thereby permitting calculation of the ratio of occupied to
unoccupied binding sites.
[0260] Equilibrium dialysis and modifications thereof has been used
to study the interaction of antibody or receptor or other protein
or nucleic acid with low molecular weight dialyzable molecules that
bind to the antibody or receptor or other protein or nucleic acid.
For applications herein, the antibody, receptor, protein or nucleic
acid is linked to solid support (matrix with memory) and is
incubated with the ligand.
[0261] In particular, this method may be used for analysis of
multiple binding agents (receptors), linked to matrices with
memories, that compete for available ligand, which is present in
limiting concentration. After reaction, the matrices with memories
linked to the binding agents (receptors) with the greatest amount
of bound ligand, are the binding agents (receptors) that have the
greatest affinity for the ligand.
[0262] The use of matrices with memories also permits simultaneous
determination of K.sub.a values of multiple binding agents
(receptors) or have multiple ligands. For example, a low
concentration of labeled ligand is mixed with a batch of different
antibodies bound to matrices with memories. The mixture is flowed
through a reader (i.e., a Coulter counter or other such instrument
that reads RF and the label) could simultaneously measure the
ligand (by virtue of the label) and identity of each linked binding
agent (or linked ligand) as the chip is read. After the reaction
equilibrium (determined by monitoring progress of the reaction)
labeled ligand is added and the process of reading label and the
chips repeated. This process is repeated until all binding sites on
the binding agent (or ligand) approach saturation, thereby
permitting calculation of K.sub.a values and binding sites that
were available.
[0263] c. Selection of Antibodies and Other Screening Methods
[0264] (1) Antibody Selection
[0265] In hybridoma preparation and selection, fused cells are
plated into, for example, microtiter wells with the matrices with
memory-tagged antibody binding reagent (such as protein A or
Co-chelate (see, e.g., Smith et al. (1992) Methods: A Companion to
Methods in Enzymology 4:73 (1992); III et al. (1993) Biophys J.
64:919; Loetscher et al. (1992) J. Chromatography 595:113-199; U.S.
Pat. No. 5,443,816; Hale (1995) Analytical Biochem. 231:46-49). The
solid phase is removed, pooled and processed batchwise to identify
the cells that produce antibodies that are the greatest binders
(see, e.g., U.S. Pat. No. 5,324,633 for methods and device for
measuring the binding affinity of a receptor to a ligand; or the
above method by which phage libraries are screened for highest
K.sub.A phage, i.e., limiting labeled antigen).
[0266] (2) Antibody Panning
[0267] Memories with matrices with antibody attached thereto (e.g.
particularly embodiments in which the matrix is a plate) may be
used in antibody panning (see, e.g., Wysocki et al. (1978) Proc.
Natl. Acad. Sci. U.S.A. 25:2844-48; Basch et al. (1983) J. Immunol.
Methods 56:269; Thiele et al. (1986) J. Immunol. 136:1038-1048;
Mage et al. (1981) Eur. J. Immunol. 11:226; Mage et al. (1977) J.
Immunol. Methods 15:47-56; see, also, U.S. Pat. Nos. 5,217,870 and
5,087,570, for descriptions of the panning method). Antibody
panning was developed as a means to fractionate lymphocytes on the
basis of surface phenotype based on the ability of antibody
molecules to adsorb onto polystyrene surfaces and retain the
ability to bind antigen. Originally (Wysocki et al. (1978) Proc.
Natl. Acad. Sci. U.S.A. 75: 2844-2848) polystyrene dishes coated
with antibodies specific for cell surface antigens and permit cells
to bind to the dishes, thereby fractionating cells. In embodiments
herein, polystyrene or other suitable matrix is associated with a
memory device and coated with an antibody, whose identity is
recorded in the memory. Mixtures of these antibody coated memories
with matrices can be mixed with cells, and multiple cell types can
be sorted and identified by querying the memories to which cells
have bound. d.
[0268] d. Phage Display
[0269] Phage, viruses, bacteria and other such manipulable hosts
and vectors (referred to as biological particles) can be modified
to express selected antigens (peptides or polypeptides) on their
surfaces by, for example, inserting DNA encoding the antigen into
the host or vector genome, at a site such as in the DNA encoding
the coat protein, such that upon expression the antigen (peptide or
polypeptide) is presented on the surface of the virus, phage or
bacterial host. Libraries of such particles that express diverse or
families of proteins on their surfaces have been prepared. The
resulting library is then screened with a targeted antigen
(receptor or ligand) and those viruses with the highest affinity
for the targeted antigen (receptor or ligand) are selected (see,
e.g., U.S. Pat. Nos. 5,403,484, 5,395,750, 5,382,513, 5,316,922,
5,288,622, 5,223,409, 5,223,408 and 5,348,867).
[0270] Libraries of antibodies expressed on the surfaces of such
packages have been prepared from spleens of immunized and
unimmunized animals and from humans. In the embodiment in which a
library of phage displaying antibodies from unimmunized human
spleens is prepared, it is often of interest to screen this library
against a large number of different antigens to identify a number
of useful human antibodies for medical applications. Phage
displaying antibody binding sites derived from single or small
numbers of spleen cells can be separately produced, expanded into
large batches, and bound to matrices with memories, such as
programmable PROM or EEPROM memories, and identified according to
phage batch number recorded in the memory. Each antigen can then be
exposed to a large number of different phage-containing memory
devices, and those that bind the antigen can be identified by one
of several means, including radiolabeled, fluorescent labeled,
enzyme labeled or alternate (e.g., mouse) tagged antibody labeled
antigen. The encoded information in the thus identified
phage-containing devices, relates to the batch of phage reactive
with the antigen.
[0271] Libraries can also be prepared that contain modified binding
sites or synthetic antibodies. DNA molecules, each encoding
proteins containing a family of similar potential binding domains
and a structural signal calling for the display of the protein on
the outer surface of a selected viral or bacterial or other
package, such as a bacterial cell, bacterial spore, phage, or virus
are introduced into the bacterial host, virus or phage. The protein
is expressed and the potential binding domain is displayed on the
outer surface of the particle. The cells or viruses bearing the
binding domains to which target molecules bind are isolated and
amplified, and then are characterized. In one embodiment, one or
more of these successful binding domains is used as a model for the
design of a new family of potential binding domains, and the
process is repeated until a novel binding domain having a desired
affinity for the target molecule is obtained. For example,
libraries of de novo synthesized synthetic antibody library
containing antibody fragments expressed on the surface have been
prepared. DNA encoding synthetic antibodies, which have the
structure of antibodies, specifically Fab or Fv fragments, and
contain randomized binding sequences that may correspond in length
to hypervariable regions (CDRs) can be inserted into such vectors
and screened with an antigen of choice.
[0272] Synthetic binding site libraries can be manipulated and
modified for use in combinatorial type approaches in which the
heavy and light chain variable regions are shuffled and exchanged
between synthetic antibodies in order to affect specificities and
affinities. This enables the production of antibodies that bind to
a selected antigen with a selected affinity. The approach of
constructing synthetic single chain antibodies is directly
applicable to constructing synthetic Fab fragments which can also
be easily displayed and screened. The diversity of the synthetic
antibody libraries can be increased by altering the chain lengths
of the CDRs and also by incorporating changes in the framework
regions that may affect antibody affinity. In addition, alternative
libraries can be generated with varying degrees of randomness or
diversity by limiting the amount of degeneracy at certain positions
within the CDRs. The synthetic binding site can be modified further
by varying the chain lengths of the CDRs and adjusting amino acids
at defined positions in the CDRs or the framework region which may
affect affinities. Antibodies identified from the synthetic
antibody library can easily be manipulated to adjust their affinity
and or effector functions. In addition, the synthetic antibody
library is amenable to use in other combinatorial type approaches.
Also, nucleic acid amplification techniques have made it possible
to engineer humanized antibodies and to clone the immunoglobulin
(antibody) repertoire of an immunized mouse from spleen cells into
phage expression vectors and identify expressed antibody fragments
specific to the antigen used for immunization (see, e.g., U.S. Pat.
No. 5,395,750).
[0273] The phage or other particles, containing libraries of
modified binding sites, can be prepared in batches and linked to
matrices that identify the DNA that has been inserted into the
phage. The matrices are then mixed and screened with labeled
antigen (e.g., fluorescent or enzymatic) or hapten, using an assay
carried out with limiting quantities of the antigen, thereby
selecting for higher affinity phage. Thus, libraries of phage
linked to matrix particles with memories can be prepared. The
matrices are encoded to identify the batch number of the phage, a
sublibrary, or to identify a unique sequence of nucleotides or
amino acids in the antibody or antibody fragment expressed on its
surface. The library is then screened with labeled antigens. The
antigens are labeled with enzyme labels or radiolabels or with the
antigen bound with a second binding reagent, such as a second
antibody specific for a second epitope to which a fluorescent
antigen binds.
[0274] Following identification of antigen bound phage, the matrix
particle can be queried and the identity of the phage or expressed
surface protein or peptide determined. The resulting information
represents a profile of the sequence that binds to the antigen.
This information can be analyzed using methods known to those of
skill in this art.
[0275] e. Anti-microbial Assays and Mutagenicity Assays
[0276] Compounds are synthesized or linked to matrix with memory.
The linkage is preferably a photocleavable linkage or other readily
cleavable linkage. The matrices with memories with linked
compounds, whose identities are programmed into each memory are the
placed on, for example, 10-cm culture plates, containing different
bacteria, fungi, or other microorganism. After release of the test
compounds the anti-microbial effects of the chemical will be
assessed by looking for lysis or other indicia of anti-microbial
activity. In preferred embodiments, arrays of memories with
matrices can be introduced into plates. The memories are encoded
with the identity of the linked or associated test compound and the
position on the array.
[0277] The AMES test is the most widely used mutagen/carcinogen
screening assay (see, e.g., Ames et al. (1975) Mutation Res.
31:347-364; Ames et al. (1973) Proc. Natl. Acad. Sci. U.S.A.
20:782-786; Maron et al. (1983) Mutation Research 113:173; Ames
(1971) in Chemical Mutagens. Principles and Methods for their
Detection, Vol. 1, Plenum Press, NY, pp 267-282). This test uses
several unique strains of Salmonella typhimurium that are
histidine-dependent for growth and that lack the usual DNA repair
enzymes. The frequency of normal mutations that render the bacteria
independent of histidine (i.e., the frequency of spontaneous
revertants) is low. The test evaluates the impact of a compound on
this revertant frequency. Because some substances are converted to
a mutagen by metabolic action, the compound to be tested is mixed
with the bacteria on agar plates along with the liver extract. The
liver extract serves to mimic metabolic action in an animal.
Control plates have only the bacteria and the extract. The mixtures
are allowed to incubate. Growth of bacteria is checked by counting
colonies. A test is positive where the number of colonies on the
plates with mixtures containing a test compound significantly
exceeds the number on the corresponding control plates.
[0278] A second type of Ames test (see, International PCT
Application No. WO 95/10629, which is based on U.S. application
Ser. No. 08/011,617; and Gee et al. (1994) Proc. Natl. Acad. Sci.
U.S.A. 91:11606-11610; commercially avail from Xenometrix, Boulder
Colo.) is of interest herein. This test provides a panel of
Salmonella typhimurium strains for use as a detection system for
mutagens that also identifies mutagenic changes. Although a direct
descendant of the traditional Ames Salmonella reverse mutation
assay in concept, the Ames II assay provides the means to rapidly
screen for base mutations through the use of a mixture of six
different Salmonella strains.
[0279] These new strains carry his mutations listed in the table
below. All are deleted for uvrB and are deficient therefore in
excision repair. In addition, all six have lipopolysaccharide (rfa)
mutations rendering them more permeable, and all contain the
pKM.sup.101 plasmid conferring enhanced mutability.
TABLE-US-00002 STRAIN BASE CHANGE MUTATION TA7001 A:T .fwdarw. G:C
hisG1775 TA7002 T:A .fwdarw. A:T hisC9138 TA7003 T:A .fwdarw. G:C
hisG9074 TA7004 G:C .fwdarw. A:T hisG9133 TA7005 G:C .fwdarw. A:T
hisG9130 TA7006 G:C .fwdarw. C:G hisC9070
[0280] These strains, which revert at similar spontaneous
frequencies (approximately 1 to 10.times.10.sup.8) can be exposed
and plated separately for determining mutational spectra, or mixed
and exposed together to assess broad mutagenic potential. The assay
takes 3 days from start to finish and can be performed in 96 well
or 384 well-microtiter plates. Revertant colonies are scored using
bromo-creosol purple indicator dye in the growth medium. The mixed
strains can be assayed first as part of a rapid screening program.
Since this six strain mixture is slightly less sensitive than
individual strains tested alone, compounds which are negative for
the mix can be retested using all six strains. For all but the
weakest mutagens, the Ames II strain mixture appears to be capable
of detecting reversion events even if only one strain is induced to
revert. The mixed strains provide a means to perform rapid initial
screening for genotoxins, while the battery of base-specific tester
strains permit mutational spectra analysis.
[0281] As modified herein, the test compounds are linked to
matrices with memories, that have been encoded with the identity of
the test compounds. The assays can be performed on multiple test
compounds simultaneously using arrays of matrices with memories or
multiple matrices with memories encoded with the identity of the
linked test compound and the array position or plate number into
which the compound is introduced.
[0282] f. Hybridization Assays and Reactions
[0283] (1) Hybridization Reactions
[0284] It is often desirable to detect or quantify very small
concentrations of nucleic acids in biological samples. Typically,
to perform such measurements, the nucleic acid in the sample (i.e.,
the target nucleic acid) is hybridized to a detection
oligonucleotide. In order to obtain a detectable signal
proportional to the concentration of the target nucleic acid,
either the target nucleic acid in the sample or the detection
oligonucleotide is associated with a signal generating reporter
element, such as a radioactive atom, a chromogenic or fluorogenic
molecule, or an enzyme (such as alkaline phosphatase) that
catalyzes a reaction that produces a detectable product. Numerous
methods are available for detecting and quantifying the signal.
[0285] Following hybridization of a detection oligonucleotide with
a target, the resulting signal-generating hybrid molecules must be
separated from unreacted target and detection oligonucleotides. In
order to do so, many of the commonly used assays immobilize the
target nucleic acids or detection oligonucleotides on solid
supports. Presently available solid supports to which
oligonucleotides are linked include nitrocellulose or nylon
membranes, activated agarose supports, diazotized cellulose
supports and non-porous polystyrene latex solid microspheres.
Linkage to a solid support permits fractionation and subsequent
identification of the hybridized nucleic acids, since the target
nucleic acid may be directly captured by oligonucleotides
immobilized on solid supports. More frequently, so-called
"sandwich" hybridization systems are used. These systems employ a
capture oligonucleotide covalently or otherwise attached to a solid
support for capturing detection oligonucleotide-target nucleic acid
adducts formed in solution (see, e.g., EP 276,302 and Gingeras et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173). Solid supports with
linked oligonucleotides are also used in methods of affinity
purification. Following hybridization or affinity purification,
however, if identification of the linked molecule or biological
material is required, the resulting complexes or hybrids or
compounds must be subjected to analyses, such as sequencing. The
combinations and methods herein eliminate the need for such
analyses.
[0286] Use of matrices with memories in place of the solid support
matrices used in the prior hybridization methods permits rapid
identification of hybridizing molecules. The identity of the linked
oligonucleotide is written or encoded into the memory. After
reaction, hybrids are identified, such as by radioactivity or
separation, and the identify of hybridizing molecules are
determined by querying the memories.
[0287] (2) Hybridization Assays
[0288] Mixtures nucleic acid probes linked to the matrices with
memories can be used for screening in assays that heretofore had to
be done with one probe at a time or with mixtures of probes
followed by sequencing the hybridizing probes. There are numerous
examples of such assays (see, e.g., U.S. Pat. No. 5,292,874,
"Nucleic acid probes to Staphylococcus aureus" to Milliman, and
U.S. Pat. No. 5,232,831, "Nucleic acid probes to Streptococcus
pyogenes" to Milliman, et al.; see, also, U.S. Pat. Nos. 5,216,143,
5,284,747 5,352,579 and 5,374,718). For example, U.S. Pat. No.
5,232,831 provides probes for the detection of particular
Streptococcus species from among related species and methods using
the probes. These probes are based on regions of Streptococcus rRNA
that are not conserved among related Streptococcus species.
Particular species are identified by hybridizing with mixtures of
probes and ascertaining which probe(s) hybridize. By virtue of the
instant matrices with memories, following hybridization, the
identity of the hybridizing probes can be determined by querying
the memories, and thereby identifying the hybridizing probe.
[0289] i. Combinatorial Libraries and Other Libraries and Screening
Methodologies
[0290] The combinations of matrices with memories are applicable to
virtually any synthetic scheme and library preparation and
screening protocol. These include, those discussed herein, and also
methodologies and devices, such as the Chiron "pin" technology
(see, e.g., International PCT application No. WO 94/11388; Geysen
et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:178; and Geysen et
al. (1987) J. Immunol. Meth. 102:259-274) which relies on a support
composed of annular synthesis components that have an active
surface for synthesis of a modular polymer and an inert support rod
that is positioned axially to the annular synthesis components.
This pin technology was developed for the simultaneous synthesis of
multiple peptides. In particular the peptides are synthesized on
polyacrylic acid grafted on the tip of polyethylene pins, typically
arranged in a microtiter format. Amino acid coupling is effected by
immersing the pins in a microtiter plate. The resulting peptides
remain bound to the pins and can be resused.
[0291] As provided herein, "pins" may be linked to a memory or
recording device, preferably encasing the device, or each pin may
be coded and the code and the identity of the associated linked
molecule(s) stored in a remote memory. As a result it will not be
necessary to physically array the pins, rather the pins can be
removed and mixed or sorted.
[0292] Also of interest herein, are DIVERSOMER.TM. technology
libraries produced by simultaneous parallel sythesis schemes for
production of nonoligomeric chemical diversity (see, e.g., U.S.
Pat. No. 5,424,483; Hobbs DeWitt et al. (1994) Drug Devel. Res.
33:116-124; Czarnik et al. (1994) Polvm. Preor. 35:985; Stankovic
et al. (1994) in Innovation Perspect. Solid Phase Synth. Collect.
Pap., Int. Svmp., 3rd Epton, R. (Ed), pp. 391-6; DeWitt et al.
(1994) Drug Dev. Res. 33:116-124; Hobbs DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909-6913). In this technology a
startingmaterial is bonded to a solid phase, such as a matrix
metraial, and is subsequently treated with reagents in a stepwise
fashion. Because the products are linked to the solid support,
multistep syntheses can be automated and multiple reactions can be
performed simultaneously to produce libraries of small molecules.
This technology can be readily improved by combining the matrices
with memories or encoding the matrix supports in accord with the
methods herein.
[0293] The matrices with memories, either those with memories in
proximity or those in which the matrix includes a code stored in a
remote memory, can be used in virtually any combinatorial library
protocol. These protocols or methodologies and libraries, include
but are not limited to those described in any of following
references: Zuckermann et al. (1994) J. Med. Chem. 37:2678; Martin
et al. (1995) J. Med. Chem. 38:1431; Campbell et al. (1995) J. Am.
Chem. Soc. 117:5381; Salmon et al. (1993) Proc. Natl. Acad. Sci.
U.S.A. 90:11708; Patek et al. (1994) Tetrahedron Lett. 35:91 69;
Patek et al. (1995) Tetrahedron Lett. 36:2227; Hobbs DeWitt et al.
(1993) Proc. Natl. Acad. Sci. U.S.A. 90:6906; Baldwin et al. (1995)
J. Am. Chem. Soc. 117:5588; and any others.
[0294] h. Nucleic Acid Sequencing
[0295] Methods of DNA sequencing based on hybridization of DNA
fragments with a complete set of fixed length oligonucleotides
(8-mers) that are immobilized individually as dots in a
2-dimensional matrix is sufficient for computer-assisted
reconstruction of the sequences of fragments up to 200 bases long
(International PCT Application WO92/10588). The nucleic acid probes
are of a length shorter than a target, which is hybridized to the
probes under conditions such that only those probes having an exact
complementary sequence are hybridized maximally, but those with
mismatches in specific locations hybridize with a reduced affinity,
as can be determined by conditions necessary to dissociate the
pairs of hybrids. Alignment of overlapping sequences from the
hybridizing probes reconstructs the complement of the target (see,
EP 0 535 242 A1, International PCT Application WO 95/00530, and
Khrapko et al. (1989) FEBS Lttrs. 256:118-122). The target fragment
with the sequence of interest is hybridized, generally under highly
stringent conditions that tolerate no mismatches or as described
below a selected number of mismatches, with mixtures of
oligonucleotides (typically a mixture of octomers of all possible
sequences) that are each immobilized on a matrix with memory that
is encoded with the sequence of the probe. Upon hybridization,
hybridizing probes are identified by routine methods, such as OD or
using labeled probe, and the sequences of the hybridizing probes
can be determined by retrieving the sequences from the linked
memories. When hybridization is carried out under conditions in
which no mismatches are tolerated, the sequence of the target can
then be determined by aligning overlapping sequences of the
hybridizing probes.
[0296] Previous methods used to accomplish this process have
incorporated microscopic arrays of nucleotide oligomers synthesized
on small silicon based chips. It is difficult to synthesize such
arrays and quality control the large number of spots on each chip
(about 64,000 spots for 8-mer oligonucleotides, that number
necessary to accomplish sequencing by hybridization). In the
present method, each oligomer is independently synthesized on a
batch of individual chips, those chips are tested for accuracy and
purity of their respective oligomers, then one chip from each batch
is added to a large pool containing oligomers having all possible
sequences. After hybridization in batch mode with the gene segment
to be sequenced, usually amplified by a method such as PCR, using
appropriate primers, and labeled with a detectable (such as
fluorescent) tag, the chips can be passed through a detector, such
as described above for processing multiplexed assays, including
multiplexed immunoassays, and the degree of binding to each
oligomer can be determined. After exposing the batch to varying
degrees of dissociating conditions, the devices can again be
assayed for degree of binding, and the strength of binding to
related sequences will relate the sequence of the gene segment
(see, e.g., International PCT Application WO95/00530).
[0297] j. Separations, Physical Mapping and Measurements of
Kinetics of Binding and Binding Affinities
[0298] Multiple blots (i.e., Western, Northern, Southern and/or dot
blots) may be simultaneously reacted and processed. Each memory, in
the form of a rectangle or other suitable, is linked or coated on
one surface with material, such as nitrocellulose, to which or the
analyte of interest binds or with which it reacts. The chips are
arranged in an array, such as in strips that can be formed into
rectangles or suitable other shapes, circles, or in other
geometries, and the respective x-y coordinate or other
position-identifying coordinate(s), and, if needed, sheet number
and/or other identifying information, is programmed into each
memory. Alternatively, they may be programmed with this
identification, then positioned robotically or manually into an
array configuration. They are preferably linked together, such as
by reversible glue, or placing them in agarose, or by any suitable
method as long as the reactive surface is not disturbed. Following
transfer of the material, such as transfer of protein from a
Western Blot, nucleic acid from a Southern or Northern blot, dot
blots, replica plated bacterial culture, or viral plaques, the
memories are separated and mixed for reaction with a traditionally
labeled, such as a fluorescent label, detection nucleic acid,
protein, antibody or receptor of interest. Complexes are
identified, and their origin in the blot determined by retrieving
the stored information in each chip. Quantitation may also be
effected based on the amount of label bound.
[0299] A series of appropriately activated matrices with memories
are arranged in an array, one or, preferably two dimensional. In
one configuration, each chip is pre-programmed and placed in a
specific location that is entered into its memory, such as an x-y
coordinate. At least one surface of the memory with matrix is
treated so that the transferred reagent binds. For example, a piece
of nitrocellulose can be fixed to one side of the memory device.
The resulting array is then contacted with a separation medium
whereby each reagent of interest is transferred to and bound to the
end of the matrix with memory such that the reagent location is
known. The matrices are separated and pooled; multiple arrays may
be pooled as long as source information is recorded in each memory.
All matrices with memories are then contacted with detection agents
that specifically bind to reagents in the mixture. The matrices
with memories are passed through a reading device, either after an
incubation for end point determinations or continuously for kinetic
measurements. The reading device is a device that can detect a
label, such as fluorescence, and a reader, such as an RF reader,
that can query the memory and identify each matrix. The rate of
binding and maximum binding and identify of bound reagents can be
determined.
[0300] Dot blots, for example, can be used in hybridoma analysis to
identify clones that secrete antibodies of desired reactivity and
to determine the relative affinities of antibodies secreted by
different cell lines. Matrices with memories that are activated to
bind immunoglobulins and with on-board information specifying their
relative locations in the array are dipped in an array into the
wells of microplates containing hybridoma cells. After incubation,
they are withdrawn, rinsed, removed and exposed to labeled antigen.
Matrices of desired specificity and affinity are selected and read
thereby identifying the original wells containing the hybridoma
cells that produce the selected antibodies.
[0301] In other embodiments, the transfer medium (i.e., the
nitrocellulose or other such medium) may be part of the surface of
the chip or array of chips that can bind to the separated species
subsequent to separation. For example, the separation system, such
as the agarose or polyacryl-amide gel, can be included on the
surface(s) of the matrix with memories in the array. After
separation the surface will be activated with a photactivatable
linker or suitable activating agent to thereby covalently link,
such as by a photoflash, the separated molecules to the matrices in
the array.
[0302] Alternatively, each matrix with memory may have one or more
specific binding agents, such as an antibody or nucleic acid probe,
attached (adsorbed, absorbed, or otherwise in physical contact) to
matrix with memory. The matrix with memory and linked binding agent
is then contacted with a medium containing the target(s). After
contacting, which permits binding of any targets to which the
linked binding agents specifically bind, the matrix with memory is
processed to identify memories with matrices to which target has
specifically bound via interaction with the binding agent. For
example, the (1) the target is labeled, thereby permitted direct
detection of complexes; (2) the memory with matrix is then
contacted with a developing agent, such as a second antibody or
detection probe, whereby binding agent-target complexes are
detected; or (3) the detection agent is present during the
reaction, such as non-specifically attached to the matrix with
memory or by other method (thin film, coated on the matrix with
memory, coated on nitrocellulose).
[0303] Such support bound analytes may also be used to analyze the
kinetics of binding by continuously passing the supports through a
label reading device during the reaction, and identify the labeled
complexes. The binding agents can be eluted, either in a
kinetically readable manner or in batch. In addition, since the
recording devices may also include components that record reaction
conditions, such as temperature and pH, kinetics, which are
temperature and pH dependent, may be accurately calculated.
[0304] After elution, the support bound analytes may be identified
to analyze kinetics of binding to the binding agent. Such binding
and elution protocols may also be adapted to affinity purification
methodologies.
[0305] k. Cell Sorting
[0306] The devices herein may also be used in methods of cell
sorting. For example, the memory with matrix combinations are
linked to selected antigens, information regarding the antigens is
encoded into the memories, the resulting combinations are used in
multi-analyte analyses of cells.
[0307] It is possible to identify a profile of cells exhibiting
different surface markers (antigens, for example, or other ligands
or receptor molecules) by using combinations of labeled and matrix
memory-bound binding agents. In one embodiment, each agent, such as
an antibody, capable of binding specifically to one of many
different surface markers is bound to a different matrix with a
memory. The nature of the recognized marker is recorded in the
memory of each matrix-binding agent complex, and the mixture of
binding-agent-matrix memory complexes is reacted with a mixture of
cells. The cell-matrix complexes that result from binding agents
attaching cells to the surfaces of the respective matrices are then
reacted with a labeled (for example, fluorescent) reagent or
mixture of reagents which also reacts with the cells. These labeled
reagents can be the same or different from those coupled to the
memory matrices. When the matrices are passed through a reader (to
read the label and the memory), those that have bound cells can be
identified and if necessary isolated. This application is
particularly useful for screening for rare cells, for example stem
cells in a bone marrow or peripheral lymphocyte sample, for
detecting tumor cells in a bone marrow sample to be used for
autologous transplantation, or for fetal cells in a maternal
circulation.
[0308] In these embodiments, the memory with matrices herein can be
counted and read with instruments, such as a device that operates
on the principles of a Coulter counter, that are designed to count
cells or particles. In using a Coulter Counter, a suspension of
cells or particles is sucked through a minute hole in a glass tube.
One electrode is placed within the tube and another is outside of
the tube in the suspension. The passage of a particle through the
hole temporarily interrupts the current; the number of
interruptions is determined by a conventional scaling unit.
[0309] For use herein, such instruments are modified by including
an RF reader (or other reader if another frequency or memory means
is selected) so that the identity of the particle or cell (or
antigen on the cell or other encoded information) can be determined
as the particle or cell passes through the hole and interrupts the
current, and also, if needed, a means to detect label, such as
fluorescent label. As the particle passes through the hole the RF
reader will read the memory in the matrix that is linked to the
particle. The particles also may be counted concurrently with the
determination of the identity of the particle. Among the
applications of this device and method, is a means to sort multiple
types of cells at once.
[0310] 1. Drug Delivery and Detecting Changes in Internal
Conditions in the Body
[0311] Memories may also be combined with biocompatible supports
and polymers that are used internally in the bodies of animals,
such as drug delivery devices (see, e.g., U.S. Pat. Nos. 5,447,533,
5,443,953, 5,383,873, 5,366,733, 5,324,324, 5,236,355, 5,114,719,
4,786,277, 4,779,806, 4,705,503, 4,702,732, 4,657,543, 4,542,025,
4,530,840, 4,450,150 and 4,351,337) or other biocompatible support
(see, U.S. Pat. No. 5,217,743 and U.S. Pat. No. 4,973,493, which
provide methods for enhancing the biocompatibility of matrix
polymers). Such biocompatible polymers include matrices of
poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride
copolymer of a stearic acid dimer and sebacic acid (see, e.g.,
Sherwood et al. (1992) Bio/Technology 10:1446-1449).
[0312] The biocompatible drug delivery device in combination with
the memory is introduced into the body. The device, generally by
virtue of combination with a biosensor or other sensor, also
monitors pH, temperature, electrolyte concentrations and other such
physiological parameters and in response to preprogrammed changes,
directs the drug delivery device to release or not release drugs or
can be queried, whereby the change is detected and drug delivered
or administered.
[0313] Alternatively, the device provided in combination with a
biocompatible support and biosensor, such that the information
determined by the biosensor can be stored in the device memory. The
combination of device and biosensor is introduced into the body and
is used to monitor internal conditions, such as glucose level,
which level is written to memory. The internal condition, such as
glucose level, electrolytes, particularly potassium, pH, hormone
levels, and other such level, can then be determined by querying
the device.
[0314] In one embodiment, the device, preferably one containing a
volatile memory that is read to and written using RF, linked to a
biosensor (see, e.g., U.S. Pat. No. 5,384,028 which provides a
biosensor with a data memory that stores data) that can detect a
change in an internal condition, such as glucose or electrolyte,
and store or report that change via RF to the linked matrix with
memory, which records such change as a data point in the memory,
which can then be queried. The animal is then scanned with RF and
the presence of the data point is indicative of a change. Thus,
instead of sampling the body fluid, the memory with matrix with
linked biosensor is introduced into a site in the body, and can be
queried externally. For example, the sensor can be embedded under
the skin and scanned periodically, or the scanner is worn on the
body, such as on the wrist, and the matrix with memory either
periodically, intermittently, or continuously sends signals; the
scanner is linked to an infusion device and automatically, when
triggered triggers infusion or alters infusion rate.
[0315] m. Multiplexed or Coupled Protocols in which the Synthesis
Steps (the Chemistry) is Coupled to Subsequent Uses of the
Synthesized Molecules
[0316] Multiplexed or multiple step processes in which compounds
are synthesized and then assayed without any intermediate
identification steps are provided herein. Since the memories with
matrices permit identification of linked or proximate or associated
molecules or biological particles, there is no need to identify
such molecules or biological particles during any preparative and
subsequent assaying steps or processing steps. Thus, the chemistry
(synthesis) can be directly coupled to the biology (assaying,
screening or any other application disclosed herein). For purposes
herein this coupling is referred to as multiplexing. Thus, high
speed synthesis can be coupled to high throughput screening
protocols.
[0317] F. Applications of the Memories with Matrices and
Luminescing Matrices with Memories in Combinatorial Syntheses and
Preparation of Libraries
[0318] Libraries of diverse molecules are critical for
identification of new pharmaceuticals. A diversity library has
three components: solid support matrix, linker and synthetic
target. The support is a matrix material as described herein that
is stable to a wide range of reaction conditions and solvents; the
linker is selectively cleavable and does not leave a functionalized
appendage on the synthetic target; and the target is synthesized in
high yield and purity. For use herein, the diversity library
further includes a memory or recording device in combination with
the support matrix. The memory is linked, encased, in proximity
with or otherwise associate with each matrix particle, whereby the
identify of synthesized targets is written into the memory.
[0319] The matrices with memories are linked to molecules and
particles that are components of libraries to electronically tagged
combinatorial libraries. Particularly preferred libraries are the
combinatorial libraries that containing matrices with memories that
employ radio frequencies for reading and writing.
[0320] 1. Oligomer and Polypeptide Libraries
[0321] a. Bio-Oligomer Libraries
[0322] One exemplary method for generating a library (see, U.S.
Pat. No. 5,382,513) involves repeating the steps of (1) providing
at least two aliquots of a solid phase support; separately
introducing a set of subunits to the aliquots of the solid phase
support; completely coupling the subunit to substantially all sites
of the solid phase support to form a solid phase support/new
subunit combination, assessing the completeness of coupling and if
necessary, forcing the reaction to completeness; thoroughly mixing
the aliquots of solid phase support/new subunit combination; and,
after repeating the foregoing steps the desired number of times,
removing protecting groups such that the bio-oligomer remains
linked to the solid phase support. In one embodiment, the subunit
may be an amino acid, and the bio-oligomer may be a peptide. In
another embodiment, the subunit may be a nucleoside and the
bio-oligomer may be an oligonucleotide. In a further embodiment,
the nucleoside is deoxyribonucleic acid; in yet another embodiment,
the nucleoside is ribonucleic acid. In a further embodiment, the
subunit may be an amino acid, oligosaccharide, oligoglycosides or a
nucleoside, and the bio-olig-omer may be a peptide-oligonucleotide
chimera or other chimera. Each solid phase support is attached to a
single bio-oligomer species and all possible combinations of
monomer (or multimers in certain embodiments) subunits of which the
bio-oligomers are composed are included in the collection.
[0323] In practicing this method herein, the support matrix has a
recording device with programmable memory, encased, linked or
otherwise attached to the matrix material, and at each step in the
synthesis the support matrix to which the nascent polymer is
attached is programmed to record the identity of the subunit that
is added. At the completion of synthesis of each biopolymer, the
resulting biopolymers linked to the supports are mixed.
[0324] After mixing an acceptor molecule or substrate molecule of
interest is added. The acceptor molecule is one that recognizes and
binds to one or more solid phase matrices with memory/bio-oligomer
species within the mixture or the substrate molecule will undergo a
chemical reaction catalyzed by one or more solid phase matrix with
memory/bio-oligomer species within the library. The resulting
combinations that bind to the acceptor molecule or catalyze
reaction are selected. The memory in the matrix-memory combination
is read and the identity of the active bio-oligomer species is
determined.
[0325] b. Split Bead Sequential Syntheses
[0326] Various schemes for split bead syntheses of polymers (FIG.
1), peptides (FIG. 2), nucleic acids (FIG. 3) and organic molecules
based on a pharmacophore monomer (FIG. 4) are provided. Selected
matrices with memory particles are placed in a suitable separation
system, such as a funnel (see, FIG. 5). After each synthetic step,
each particle is scanned (i.e., read) as it passes the RF
transmitter, and information identifying the added component or
class of components is stored in memory. For each type of synthesis
a code can be programmed (i.e., a 1 at position 1, 1 in the memory
could, for example, represent alanine at the first position in the
peptide). A host computer or decoder/encoder is programmed to send
the appropriate signal to a transmitter that results in the
appropriate information stored in the memory (i.e., for alanine as
amino acid 1, a 1 stored at position 1, 1). When read, the host
computer or decoder/encoder can interpret the signal read from and
transmitted from the memory.
[0327] In an exemplary embodiment, a selected number of beads
(i.e., particulate matrices with memories (matrix particles linked
to recording devices), typically at least 10.sup.3, more often
10.sup.4, and desirably at least 105 or more up to and perhaps
exceeding 10.sup.15, are selected or prepared. The beads are then
divided into groups, depending upon the number of choices for the
first component of the molecule. They are divided into a number of
containers equal to or less than (for pooled screening, nested
libraries or the other such methods) the number of choices. The
containers can be microtiter wells, Merrifield synthesis vessels,
columns, test tubes, gels, etc. The appropriate reagents and
monomer are added to each container and the beads in the first
container are scanned with electromagnetic with radiation,
preferably high frequency radio waves, to transmit information and
encode the memory to identify the first monomer. The beads in the
second container are so treated. The beads are then combined and
separated according to the combinatorial protocol, and at each
stage of added monomer each separate group is labeled by inputting
data specific to the monomer. At the end of the synthesis protocol
each bead has an oligomer attached and information identifying the
oligomer stored in memory in a form that can be retrieved and
decoded to reveal the identity of each oligomer.
[0328] An 8-member decapeptide library was designed, synthesized,
and screened against an antibody specifically generated against one
of the library members using the matrices with memories. Rapid and
clean encoding and decoding of structural information using radio
frequency signals, coupling of combinatorial chemical synthesis to
biological assay protocols, and potential to sense and measure
biodata using suitable biosensors, such as a temperature thermistor
or pH electrode, embedded within the devices have been
demonstrated. The "split and pool" method (see, e.g., Furka et al.
(19910 Int. J. Pept. Protein Res. 37:487-493; Lam et al. (1991)
Nature 354:82-84; and Sebestyen et al. (1993) Biooro. Med. Chem.
Lett. 3:413-418) was used to generate the library. An ELISA (see
e.g., Harlow et al. (1988) Antibodies, a laboratory manual, Cold
Spring Harbor, N.Y.) was used to screen the library for the peptide
specific for the antibody.
[0329] 2. "Nested" Combinatorial Library Protocols
[0330] In this type of protocol libraries of sublibraries are
screened, and a sublibrary selected for further screening (see,
e.g., Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; and
Zuckermann et al. (1992) Am. Chem. Soc. 114:10646-106471. In this
method, three sets of monomers were chosen from commercially
available monomers, a set of four aromatic hydrophobic monomers, a
set of three hydroxylic monomers, a set of seventeen diverse
monomers, and three N-termini were selected. The selection was
based on an analysis of the target receptor and known ligands. A
library containing eighteen mixtures, generated from the six
permutations of the three monomer sets, times three N-termini was
prepared. Each mixture of all combinations of the three sets of
amines, four sets of hydrophobic monomers and seventeen diverse
monomers was then assayed. The most potent mixture was selected for
deconvolution by synthesis of pools of combinatorial mixtures of
the components of the selected pool. This process was repeated,
until individual compounds were selected.
[0331] Tagging the mixtures with the matrices with memories will
greatly simplify the above protocol. Instead of screening each
mixture separately, each matrix particle with memory will be
prepared with sets of the compounds, analogous to the mixtures of
compounds. The resulting matrix particles with memories and linked
compounds can be combined and then assayed. As with any of the
methods provided herein, the linked compounds (molecules or
biological particles) can be cleaved from the matrix with memory
prior to assaying or anytime thereafter, as long as the cleaved
molecules remain in proximity to the device or in some manner can
be identified as the molecules or particles that were linked to the
device. The matrix particle(s) with memories that exhibit the
highest affinity (bind the greatest amount of sample at
equilibrium) are selected and identified by querying the memory to
identify the group of compounds. This group of compounds is then
deconvoluted and further screened by repeating this process, on or
off the matrices with memories, until high affinity compounds are
selected.
[0332] 3. Other Combinatorial Protocols
[0333] The matrices with memories provided herein may be used as
supports in any synthetic scheme and for any protocol, including
protocols for synthesis of solid state materials. Combinatorial
approaches have been developed for parallel synthesis of libraries
of solid state materials (see, e.g., Xiang et al. (1995) Science
268:1738-1740). In particular, arrays containing different
combinations, stoichiometries, and deposition sequences of
inorganics, such as BaCO.sub.3, BiO.sub.3, CaO, CuO, PbO,
SrCO.sub.3 and Y.sub.2O.sub.3, for screening as superconductors
have been prepared. These arrays may be combined with memories that
identify position and the array and/or deposited material.
[0334] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Formulation of a Polystyrene Polymer on Glass and Derivatization of
Polystyrene
[0335] A glass surface of any conformation (beads for
exemplification purposes (1)) that contain a selected memory device
that coat the device or that can be used in proximity to the device
or subsequently linked to the device is coated with a layer of
polystyrene that is derivatized so that it contains a cleavable
linker, such as an acid cleavable linker. To effect such coating a
bead, for example, is coated with a layer of a solution of styrene,
chloromethylated styrene, divinyl benzene, benzoyl peroxide
(88/10/1/1/, molar ratio) and heated at 70.degree. C. for 24 h. The
result is a cross-linked chloromethylated polystyrene on glass (2).
Treatment of (2) with ammonia (2 M in 1,4-dioxane, overnight)
produces aminomethylated coated beads (3). The amino group on (3)
is coupled with polyethylene glycol dicarboxymethyl ether (4)
(n=20) under standard conditions (PyBop/DIEA) to yield carboxylic
acid derivatized beads (5). Coupling of (5) with modified PAL (PAL
is pyridylalanine) linker (6) under the same conditions produces a
bead that is coated with polystyrene that has an acid cleavable
linker (7).
[0336] The resulting coated beads with memories are then used as
solid support for molecular syntheses or for linkage of any desired
substrate.
Example 2
Preparation of a Library and Encoding the Matrices with
Memories
[0337] A pool of the matrices with memories was split into two
equal groups. Each group was then addressed and write-encoded with
a unique radio frequency signal corresponding to the building
block, in this instance an amino acid, to be added to that
group.
[0338] The matrices with memories were then pooled, and common
reactions and manipulations such as washing and drying, were
performed. The pool was then re-split and each group was encoded
with a second set of radio frequency signals corresponding to the
next set of building blocks to be introduced, and the reactions
were performed accordingly. This process was repeated until the
synthesis was completed. The semiconductor devices also recorded
temperature and can be modified to record other reaction conditions
and parameters for each synthetic step for storage and future
retrieval.
[0339] Ninety-six matrices with memories were used to construct a
24-member peptide library using a 3.times.2.times.2.times.2 "split
and pool" strategy. The reactions, standard Fmoc peptide syntheses
(see, e.g., Barany et al. (1987) Int. J. Peptide Protein Res.
30:705-7391 were carried out separately with each group. All
reactions were performed at ambient temperature; fmoc deprotection
steps were run for 0.5 h; coupling steps were run for 1 h; and
cleavage for 2 h. This number was selected to ensure the
statistical formation of a 24-member library (see, Burgess et al.
(1994) J. Med. Chem. 37:2985).
[0340] Each matrix with memory in the 96-member pool was decoded
using a specifically designed radio frequency memory retrieving
device (Bio Medic Data Systems Inc. DAS-5001 CONSOLE.TM. System,
see, also U.S. Pat. No. 5,252,962 and U.S. Pat. No. 5,262,772) the
identity of the peptide on each matrix with memory (Table 2). The
structural identity of each peptide was confirmed by mass
spectrometry and .sup.1H NMR spectroscopy. The content of peptide
in each crude sample was determined by HPLC to be higher than 90%
prior to any purification and could be increased further by
standard chromatographic techniques.
TABLE-US-00003 TABLE 2 Radio Frequency Encoded Combinatorial
24-member peptide library # of Entry matrices (SEQ RF with Mass
ID). Code Peptide memories.sup.a,b (Actual).sup.e 1 LAGD
Leu-Ala-Gly-Asp 3 372 (372.2) 2 LEGD Leu-Glu-Gly-Asp 4 432 (432.2)
3 SAGD Ser-Ala-Glv-Asp 5 348 (348.1) 4 SEGD Ser-Giu-Glv-Asp 5 406
(406.1) 5 LAVD Leu-Ala-Val-Asp 4 416 (416.2) 6 LEVD Leu-Glu-Vai-Asp
6 474 (474.2) 7 SAVD Ser-Ala-Val-Asp 2 390 (390.2) 8 SEVD
Ser-Glu-Val-Asp 3 446 (446.2) 9 LAGF Leu-Ala-Gly-Phe 5 406 (406.2)
10 LEGF Leu-Glu-Gly-Phe 5 464 (464.2) 11 SAGF Ser-Ala-Gly-Phe 5 380
(380.2) 12 SEGF Ser-Glu-Gly-Phe 6 438 (438.2) 13 LAVF
Leu-Ala-Val-Phe 6 448 (448.3) 14 LEVF Leu-Glu-Val-Phe 2 XXX 15 SAVF
Ser-Ala-Val-Phe 2 XXX 16 SEVF Ser-Glu-Val-Phe 1 480 (480.2) 17 LAGK
Leu-Ala-Gly-Lys 2 387 (387.3) 18 LEGK Leu-Glu-Gly-Lys 1 445 (445.3)
19 SAGK Ser-Ala-Gly-Lys 4 361 (361.2) 20 SEGK Ser-Glu-Gly-Lys 3 419
(419.2) 21 LAVK Leu-Ala-Val-Lys 4 429 (429.3) 22 LEVK
Leu-Glu-Val-Lys 6 487 (487.3) 23 SAVK Ser-Ala-Val-Lys 6 403 (403.3)
24 SEVK Ser-Glu-Val-Lys 6 461 (461.3) .sup.aThis is the number of
packets of each matrix with memory containing the same peptide. The
ambient temperature was recorded by the sensor device of the chip
in the matrices with memories at various points during the
synthetic pathway. .sup.cMass refers to (M + H) except entry 1 and
8 which refer to (M-H). Since each peptide has a unique mass, the
mass spectrum confirms its structure. .degree. HPLC conditions:
Shimadzu SCL 10A with a MICROSORB-MV .TM. C-1 8 column (5 .mu.M,
100 .ANG.; isocratic elution with acetonittle/water.
Example 3
Synthesis of a Decapeptide Library
[0341] Materials and Methods
[0342] (1) A memory device (IPTT-100, Bio Medic Data Systems, Inc.,
Maywood, N.J.), which is 8.times.1.times.1 mm, and TENTAGEL.RTM.
beads (20 mg) were encapsulated using a porous membrane wall and
sealed (final size.apprxeq.10.times.2.times.2 mm). In particular,
each memory with matrix microvessel 20 mg of TENTAGEL.RTM. resin
carrying the acid-cleavable linker PAL.
[0343] (2) Solvents and reagents (DMF, DCM, MeOH, Fmoc-amino acids,
PyBOP, HATU, DIEA, and other reagents) were used as received. Mass
spectra were recorded on an API I Perkin Elmer SCIEX Mass
Spectrometer employing electrospray sample introduction. HPLC was
performed with a Shimadzu SCI 10A with an AXXiOM C-1 8 column (5
.mu.m, 100 .ANG.; gradient: 0-20 min, 25-100% acetonitrile/water
(0.1% TFA). UV spectra were recorded on a Shimadzu UV-1 601
instrument. Peptide sequencing was performed using a Beckman model
6300 amino acid analyzer. Chemicals, solvents and reagents were
obtained from the following companies: amino acid derivatives
(CalBiochem); solvents (VWR; reagents (Aldrich-Sigma).
[0344] (3) General Procedure for Fmoc-Amino Acid Coupling
[0345] The matrix with memory microvessels were placed in a
flat-bottomed flask. Enough DMF (v, ml, 0.75 ml per microvessel)
was added to completely cover all the matrix with memory
microvessels. Fmoc-amino acid, EDIA, and PyBOP (or HATU for the
hindered amino acids Pro and lie) were added sequentially with
final concentrations of 0.1, 0.2, and 0.1 M, respectively. The
flask was sealed and shaken gently at ambient temperature for 1 h.
The solution was removed and the matrix with memory microvessels
were washed with DMF (4.times.vr), and re-subjected to the same
coupling conditions with half the amount of reagents. They were
finally washed with DMF (4.times.vr), MeOH (4.times.v4), DCM
(4.times.v4), and dried under vacuum at ambient temperature.
[0346] (4) Fmoc-Deprotection
[0347] The matrix with memory microvessels were placed in a flat
bottomed flash. Enough 20% piperidine solution in DMF (vr ml, 0.75
ml/matrix with memory microvessel) was added to completely cover
the microvessels. The flask was sealed and gently shaken at ambient
`temperature for 30 min. Aliquots were removed and the UV
absorption of the solution was measured at 302 nm to determine the
Fmoc number. The matrix with memory microvessels were then washed
with DMF (6.times.vr) and DCM (6.times.vr) and dried under vacuum
at ambient temperature.
[0348] (5) Procedure for Peptide Cleavage from Solid Support
[0349] The TENTAGEL.RTM. beads (20-120 mg) from each matrix with
memory microvessel were treated with 1 ml of TFA cleavage mixture
(EDT:thioanisole;H.sub.2O:PhOH:TFA, 1.5:3:3:4.5:88, w/w) at ambient
temperature for 1.5 hours. The resin beads were removed by
filtration through a glass-wool plug, the solution was
concentrated, diluted with water (2 ml), extracted with diethyl
ether (8.times.2 ml), and lyophilized to yield the peptide as a
white powder (4-20 mg).
[0350] (6) Preparation of Polyclonal Antibodies
[0351] The peptide (SEQUENCE ID No. 25) with a cysteine at the
N-terminus, was synthesized by standard solid phase methods using
an automated Applied Biosystems 430A peptide synthesizer (see,
Sakakibara (1971) Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, Weinstein, ed, Vol. 1, Marcel Dekker, NY,
pp. 51-85). The synthetic peptide was conjugated to keyhole limpet
hemocyanin using maleimidohexanoyl-N-hydroxysuccinimide as a
cross-linking agent (see, Ishikawa et al. (1983) J. Immunoassay
4:209-237). Rabbits were injected at multiple dorsal intradermal
sites with 500 .mu.g peptide emulsified with complete Freund's
adjuvant. The animals were boosted regularly at 3-6 week intervals
with 200 .mu.g of peptide conjugate emulsified in incomplete
Freund's adjuvant. The titer of the antisera after a few booster
injections was approximately 1:50,000 to 1:100,000 as determined by
ELISA using the unconjugated peptide as the antigen.
[0352] (7) Enzyme Linked Immunosorbant Assay (ELISA)
[0353] Plates were coated with 100 .mu.l/well of a 0.5 .mu.g/.mu.l
solution of peptides diluted in phosphate buffered saline (PBS) by
incubating them overnight at 4.degree. C. The plates were washed
extensively with PBS and incubated with 200 .mu.l of 0.1% bovine
serum albumin (BSA) in PBS for 1 h at room temperature. The plates
were then washed with PBS and 100 .mu.l of prebled or rabbit
anti-peptide (peptide of SEQ ID No. 25) antibody (1:100,000) was
added to the duplicate wells. After a 1 h incubation at ambient
temperature, the plates were washed with PBS and 100 .mu.l of
peroxidase-goat-antirabbit IgG diluted in PBS supplemented with
0.1% BSA was added. After incubation for another hour at ambient
temperature, the plates were extensively washed with PBS and 1 00
.mu.l of peroxidase substrate solution was added to each well. The
plates were then incubated for 15 minutes at ambient temperature.
The peroxidase reaction was measured by the increase in absorbance
at 405 nm
[0354] The Library
[0355] The library included the peptide having the sequence
Met-Leu-Asp-Ser-lle-Trp-Lys-Pro-Asp-Leu (MLDSIWKPDL; SEQ ID NO.
25), against which an antibody had been generated in rabbits (the
peptide used for rabbits had an additional N-terminal Cys residue
for linking), and seven other peptides differing at residues L, P,
and/or I (SEQ ID NOs. 26-32 and the Scheme set forth in FIG.
10).
[0356] The matrix with memory microvessels loaded with
TENTAGEL.RTM. beads carrying PAL linkers (20 mg each) were split
into two equal groups. Each group was encoded with the radio
frequency code L or A (the one-letter symbols for amino acids
leucine and alanine, respectively) and the first coupling was
carried out separately using Fmoc-Leu-OH or Fmoc-Ala-OH,
respectively and ByBOP, or HATU for the sterically hindered amino
acids (STEP 1, FIG. 10). The microvessels were then pooled,
deprotected with 20% piperidine in DMF (Fmoc removal), encoded with
the code D and subjected to coupling with Fmoc-Asp(OfBu)-OH and
deprotection as above (STEP 2). The microvessels were then re-split
into two equal and fully randomized groups and encoding was
performed on each group with the codes P or F and amino acid
derivatives Fmoc-Pro-OH or Fmoc-Phe-OH were coupled, respectively
(STEP 3). The microvessels were pooled again and amino acid
derivatives Fmoc-Lys(Boc)-OH and Fmoc-Trp(Boc)-OH were coupled
sequentially with appropriate encoding and deprotection procedures
(STEPS 4 and 5), and then were re-split into two equal groups,
encoded appropriately and the amino acid derivatives Fmoc-lle-OH or
Fmoc-Gly-OH were coupled separately (STEP 6). The matrix with
memory microvessels were pooled, the amino groups deprotected and
the remaining amino acids (Ser, Asp, Leu and Met) were sequentially
introduced with appropriate encoding and deprotections using
suitably protected Fmoc derivatives (STEPS 7-10). The introduction
of each amino acid was performed by double couplings at every step.
The coupling efficiency for each step was generally over 90% as
measured by Fmoc number determination after Fmoc deprotection (UV
spectroscopy).
[0357] Decoding each matrix with memory allowed identification of
identical units. It was observed that a fairly even distribution of
matrix with memories over the entire library space was obtained. It
should be noted that sorting out the matrices with memories at each
split by decoding allows this random process to become an exact,
"one compound--one matrix with memory method."
[0358] TENTAGEL.RTM. beads from matrices with memories with
identical codes were pooled together and the peptides were cleaved
from the resin separately with EDT:thioanisole:H.sub.2O:PhOH:TFA
(1.5:3:3:4.5:88m, w/w). The work-up and isolation procedures
involved filtration, evaporation, dilution with water, thorough
extraction with diethyl ether, and lyophilization. The fully
deprotected peptides were obtained as white solids, their
structures were confirmed by mass spectroscopy, and their purity
was determined by HPLC analysis. The peptide sequence in entry 2,
(SEQ ID NO. 26) was confirmed by peptide amino acid sequence
analysis. Ambient reactor temperature was also measured at specific
synthesis steps by the on-board temperature thermistor.
[0359] Biological Screening of the Peptide Library
[0360] A rabbit polyclonal antibody generated specifically against
the peptide SEQ ID NO. 25 was used to detect this specific sequence
in the REC.TM. peptide library by the ELISA method. The ELISA assay
correctly identified the library member with the SEQ ID NO. 25
(100% binding). The sequence of this peptide was also confirmed by
the radio frequency code, mass spectroscopy, and amino acid
sequence analysis.
[0361] It was also of interest to observe trends in the binding of
the antibody to the other members of the library. It was observed
that the binding of each peptide was dependent on the type,
position, and number of modifications from the parent sequence.
Thus, replacement of I with G did not change significantly the
antigenicity of the peptide. Substitution of L with A reduced
antibody binding by .apprxeq.40% and replacement of P with F
essentially converted a peptide to a non-recognizable sequence.
Replacement of two amino acids resulted in significant loss of
binding. Thus the concurrent substitutions (1.fwdarw.G and
P.fwdarw.F), (1.fwdarw.G and L.fwdarw.A), and (P.fwdarw.F and
L.fwdarw.A) reduced antibody binding by .apprxeq.40, 60, and 92%,
respectively. Finally, the peptide library member in which I, P and
L were replaced with G, F and A, respectively, was not recognized
by the antibody. Collectively, these results suggest that amino
acids at the C-terminus of the peptide, especially P play an
important role in this particular antibody-peptide recognition.
Example 4
Procedures for Coating Glass-Enclosed Memory Devices with Silylated
Polystyrene
[0362] A procedure for coating glass-enclosed memory devices, such
as the IPTT-100, is represented schematically as follows:
[0363] A. Procedure A
[0364] 1. Before coating, the glass surface of the IPTT-100
transponder was cleaned using base, chloroform, ethanol and water,
sequentially, and, then heated to 200.degree. C. (or 300.degree.
C.) to remove water.
[0365] 2. The residue from the solvents in step 1 were removed
under vacuum.
[0366] 3. N-styrylethyltrimethoxy silane HCl, chloromethyl styrene,
divinyl benzene and benzoyl peroxide (9:1:0.1:0.2 mol) is stirred
for 10 minutes.
[0367] 4. The resulting mixture was coated on the cleaned glass,
which was then baked at 150-200.degree. C. for 5 to 10 minutes in
air or under nitrogen.
[0368] 5. The coated glass was then sequentially washed with DCM,
DMF and water. The resulting coating was stable in DCM, DMF, acid
and base for at two weeks at 70.degree. C.
[0369] B. Procedure B
[0370] 1. Before coating, the glass surface is cleaned using base,
chloroform, ethanol and water, sequentially, and, then heating to
200.degree. C. (or 300.degree. C.) to remove water.
[0371] 2. The residue from the solvents in step 1 are removed under
vacuum.
[0372] 3. N-styryjethyltrimethoxy silane HCl (10-15%) is refluxed
in toluene with the cleaned glass surface.
[0373] 4. After reaction, the glass surface is washed with toluene,
DCM, ethanol and water sequentially.
[0374] 5. A mixture of chloromethyl styrene, divinyl benzene and
benzoyl peroxide (molar ratio of N-styrylethyltrimethoxy silane HCl
to the other compounds is 9:1:0.1:0.2 mol) is coated on the glass,
which is then baked at 150-200.degree. C. for 10 to 60 minutes.
[0375] 6. The coated glass is then sequentially washed with
toluene, DCM, DMF and water.
Example 5
Preparation of Scintillant-Encased Glass Beads and Chips
[0376] Materials:
[0377] POPOP (Aldrich) or PPO (concentrations about 5 to 6 g/l),
and/or .mu.-bis-.sigma.-methylstyrylbenzene (bis-MSB) or
di-phenylanthracene (DPA) (concentrations about 1 g/l), or
scintillation wax (FlexiScint from Packard). Precise concentrations
may be determined empirically depending upon the selected mixture
of components;
[0378] Porous glass beads (Sigma); and
[0379] IPTT-100 transponders.
[0380] A. Preparation of Scintillant Coated Beads
[0381] Porous glass beads are soaked in a mixture of PPO (22-25% by
weight) and bis-MSB (up to 1% by weight) in a monomer solution,
such styrene or vinyltoluene, or in hot liquified scintillation wax
(3-5 volume/volume of bead). A layer of polystyrene (about 2 to 4
.mu.M) is then applied. A peptide is either synthesized on the
polystyrene, as described above, or is coated (adsorbed) or linked
via a cleavable linker to the polystyrene.
[0382] B. Preparation of Scintillant Coated Matrix with Memory
Beads
[0383] 1. The porous glass beads are replaced with glass-encased
(etched prior to use) transponders and are treated as in A. The
resulting beads are sealed with polystyrene (2 to 5 .mu.M) and then
coated with a selected acceptor molecule, such as an antigen,
antibody or receptor, to which a radiolabeled ligand or antibody
selectively binds. The identity of the linked peptides or protein
is encoded into each memory. After reaction and counting in a
liquid scintillation counter, the beads that have bound acceptor
molecule are read to identify the linked protein.
[0384] 2. The porous glass beads are replaced with glass encased
(etched prior to use) transponders and are treated as in A and
sealed as in A with polystyrene. A peptide, small organic or other
library is synthesized on the polystyrene surface of each bead, and
the identity of each member of the library encoded into the memory.
The beads with linked molecules are reacted with labeled receptor
and counted in a liquid scintillation counter. After counting in a
liquid scintillation counter, the beads that have bound receptor
are read to identify the molecule that bound to the receptor.
Example 6
Use of the Scintillant Coated or Encased Particles in Assays
[0385] In experiments 1-3, as model system, the binding of biotin
to functional amine groups was detected using
.sup.1251-strepavidin. In experiment 4, the binding of (Met.sup.5)
enkephalin to the functional amine groups was detected using
.sup.1251-antibody.
Experiment #1
[0386] 1. Scintillant (PPO %2 and DPA %0.05) was introduced
(Emerald Diagnostics, Eugene, Oreg.) and incorporated on the
interior surface of polystyrene beads (Bang Laboratories). The
polystyrene beads were 3.1 .mu.M, with 20% crosslinking and were
derivatized with amine groups.
[0387] 2. The concentration of the functional amine groups on the
bead surfaces was estimated to be about 0.04125 .mu.mol/mg. The
amine groups were covalently linked to the N-hydroxy succinimide
derivative of Biotin (Calbiochem 203112) at molecular ratio of
1:10, respectively. This was done by resuspending the beads in a
50% acetonitrile: water, Hepes (pH 8.0) buffered solution
containing biotin for 2 hours at room temperature. After 2 hours,
the beads were washed 6 times with 10 ml of 50% acetonitrile in
water. Beads were resuspended in PBS (pH 7.2) and stored overnight
at 4.degree. C.
[0388] 3. Using an SPA format, biotin was detected using
.sup.125I-streptavidin to the biotin was detected. This was done by
diluting beads to a 20 mg/ml and adding them to 96 well plates at
4, 2, 1, 0.5, 0.25, and 0.125 mg per well. Volumes were adjusted to
100 ul per well. .sup.1251-strepavidin was added to final
concentration of 0.1 .mu.Ci per well. Plates were counted in a
Wallac MicroBeta Trilux scintillation counter after 2 hours. Bound
biotin was detected.
Experiment #2
[0389] 1. Scintillants (pyrenebutyric acid and
9-anthracenepropionic acid) were covalently linked to the
TENTAGEL.RTM. beads, with 0.25 mmol/g available functional amine
groups, at 2%:0.05% ratio, respectively. The fluorophore was linked
to 15% of these sites.
[0390] 2. The functional amine group on the TENTAGEL.RTM. beads
were covalently linked to the N-hydroxy succinimide derivative of
biotin. The free functional amine groups on beads (0.21 .mu.mol/mg)
were covalently linked to biotin (Calbiochem 203112). Briefly,
Biotin was mixed with the beads at a molecular ratio of 10:1 in 6
ml of 50% acetonitrile with Hepes (pH 8.0) and incubated for 2
hours at room temperature. At the end of incubation period, the
beads were washed 3 times with 10 ml of 100% acetonitrile followed
by 3 washes with 50% acetonitrile in water. The beads were
resuspended in PBS (pH 7.2) and stored overnight at 4.degree.
C.
[0391] 3. Biotin was detected using .sup.1251-streptavidin detected
in a SPA format. This was done by diluting beads to a 20 mg/ml, and
introducing them into wells in 96 well plats at 4, 2, 1, 0.5, 0.25,
and 0.125 mg per well. Volumes were adjusted to 100 .mu.l per well.
.sup.1251-streptavidin (Amersham IM236) was added to each well at a
concentration of 0.05 .mu.Ci/well. After approximately 2 hours,
additional .sup.1251-strepavidin was added for a final
concentration of 0.1 uCi per well. Plates were counted in a Wallac
MicroBeta Trilux scintillation counter after 2 hours. Bound biotin
was detected.
Experiment #3
[0392] 1. BMDS chips (and also similar chips ID TAG available from
Identification Technologies Inc.) were coated with scintillant (PPO
%2 and DPA 0.5% in polystyrene (10% in dichloromethane).
[0393] 2. The chip was then coated with a layer of derivatized
silane.
[0394] 3. The functional amine groups were covalently linked to the
N-hydroxy succinimide derivative of Biotin. The free functional
amine groups on the silane (375 nmol/chip) were covalently linked
to Biotin (Calbiochem 203112). Briefly, biotin was dissolved in 1
ml of 30% acetonitrile with Hepes (pH 8.0) and incubated with the
chip for 2 hours at room temperature. At the end of incubation
period, the chip was washed 3 times with 50% acetonitrile in water,
resuspended in PBS (pH 7.2) and stored overnight at 4.degree.
C.
[0395] 4. Biotin was detected in a SPA format by
.sup.1251-streptavidin. The chips were placed in 24-well plate with
500 .mu.l .sup.1251-streptavidin (0.1 .mu.Ci/well, Amersham
IM236)). After a 2 hour incubation, the plates were counted in
Wallac MicroBeta Trilux scintillation counter. Binding was
detected.
Experiment #4
[0396] 1. The chips were coated with scintillant (PPO %2 and DPA)
0.05% in polystyrene (10% in dichloromethane).
[0397] 2. The functional amine group was derivatized for
spontaneous covalent binding to amine group (Xenopore, NJ).
[0398] 3. (Met.sup.5)Enkephalin (tyr-gly-gly-phe-met; SEQ ID No.
33) peptide (R&D Antibodies) were covalently linked to the
amine group by incubating the coagted chip with the peptide in 500
.mu.l of PBS (160 .mu.g peptide/ml, pH 8)) overnight at room
temperature.
[0399] 4. At the end of the incubation, the chips were washed and
then incubated in 3% bovine serum albumin for 2 hours.
[0400] 5. Linked peptide was detected in a SPA format. The chips
were was placed in 24-well plate containing 500 .mu.l of
.sup.1251-anti-(Met5)Enkephalin antibody (0.1 .mu.Ci/well, R&D
Antibodies). The antibody is a rabbit polyclonal against the
C-terminal region of the peptide. After a two hour incubation, the
plates were counted in Wallac MicroBeta Trilux scintillation
counter and linked peptide was detected.
[0401] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
Sequence CWU 1
1
3314PRTartificial sequencerandom coupling in split and pool
synthesis 1Leu Ala Gly Asp124PRTartificial sequencerandom coupling
in split and pool synthesis 2Leu Glu Gly Asp134PRTartificial
sequencerandom coupling in split and pool synthesis 3Ser Ala Gly
Asp144PRTartificial sequencerandom coupling in split and pool
synthesis 4Ser Glu Gly Asp154PRTartificial sequencerandom coupling
in split and pool synthesis 5Leu Ala Val Asp164PRTartificial
sequencerandom coupling in split and pool synthesis 6Leu Glu Val
Asp174PRTartificial sequencerandom coupling in split and pool
synthesis 7Ser Ala Val Asp184PRTartificial sequencerandom coupling
in split and pool synthesis 8Ser Glu Val Asp194PRTartificial
sequencerandom coupling in split and pool synthesis 9Leu Ala Gly
Phe1104PRTartificial sequencerandom coupling in split and pool
synthesis 10Leu Glu Gly Phe1114PRTartificial sequencerandom
coupling in split and pool synthesis 11Ser Ala Gly
Phe1124PRTartificial sequencerandom coupling in split and pool
synthesis 12Ser Glu Gly Phe1134PRTartificial sequencerandom
coupling in split and pool synthesis 13Leu Ala Val
Phe1144PRTartificial sequencerandom coupling in split and pool
synthesis 14Leu Glu Val Phe1154PRTartificial sequencerandom
coupling in split and pool synthesis 15Ser Ala Val
Phe1164PRTartificial sequencerandom coupling in split and pool
synthesis 16Ser Glu Val Phe1174PRTartificial sequencerandom
coupling in split and pool synthesis 17Leu Ala Gly
Lys1184PRTartificial sequencerandom coupling in split and pool
synthesis 18Leu Glu Gly Lys1194PRTartificial sequencerandom
coupling in split and pool synthesis 19Ser Ala Gly
Lys1204PRTartificial sequencerandom coupling in split and pool
synthesis 20Ser Glu Gly Lys1214PRTartificial sequencerandom
coupling in split and pool synthesis 21Leu Ala Val
Lys1224PRTartificial sequencerandom coupling in split and pool
synthesis 22Leu Glu Val Lys1234PRTartificial sequencerandom
coupling in split and pool synthesis 23Ser Ala Val
Lys1244PRTartificial sequencerandom coupling in split and pool
synthesis 24Ser Glu Val Lys12510PRTartificial sequencerandom
coupling in split and pool synthesis 25Met Leu Asp Ser Ile Trp Lys
Pro Asp Leu1 5 102610PRTartificial sequencerandom coupling in split
and pool synthesis 26Met Leu Asp Ser Gly Trp Lys Pro Asp Leu1 5
102710PRTartificial sequencerandom coupling in split and pool
synthesis 27Met Leu Asp Ser Ile Trp Lys Pro Asp Ala1 5
102810PRTartificial sequencerandom coupling in split and pool
synthesis 28Met Leu Asp Ser Ile Trp Lys Phe Asp Leu1 5
102910PRTartificial sequencerandom coupling in split and pool
synthesis 29Met Leu Asp Ser Gly Trp Lys Phe Asp Leu1 5
103010PRTartificial sequencerandom coupling in split and pool
synthesis 30Met Leu Asp Ser Gly Trp Lys Pro Asp Ala1 5
103110PRTartificial sequencerandom coupling in split and pool
synthesis 31Met Leu Asp Ser Ile Trp Lys Phe Asp Ala1 5
103210PRTartificial sequencerandom coupling in split and pool
synthesis 32Met Leu Asp Ser Gly Trp Lys Phe Asp Ala1 5
10335PRTartificial sequencerandom coupling in split and pool
synthesis 33Tyr Gly Gly Phe Met1 5
* * * * *