U.S. patent application number 10/830253 was filed with the patent office on 2004-10-14 for low fluorescence assay platforms and related methods for drug discovery.
This patent application is currently assigned to AURORA DISCOVERY, INC.. Invention is credited to Coassin, Peter J., Harootunian, Alec Tate, Pham, Andrew A., Stylli, Harry, Tsien, Roger Y..
Application Number | 20040202582 10/830253 |
Document ID | / |
Family ID | 26706197 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202582 |
Kind Code |
A1 |
Coassin, Peter J. ; et
al. |
October 14, 2004 |
Low fluorescence assay platforms and related methods for drug
discovery
Abstract
One aspect of the present invention is a multi-well platform for
fluorescence measurements, comprising a plurality of wells within a
frame, wherein the multi-well platform has low fluorescence
background. Another aspect of the present invention is a system for
spectroscopic measurements, comprising reagents for an assay and a
multi-well platform for fluorescence measurements. A further aspect
of the present invention is a method for detecting the presence of
an analyte in a sample contained in a multi-well platform by
detecting light emitted from the sample. Another aspect of the
present invention is a method from identifying a modulator of a
biological process or target in a sample contained in a multi-well
platform by detecting light emitted from the sample. Another aspect
of the present invention is a composition identified by this
method. A further aspect of the present invention is a method to
identify a therapeutic. A further aspect of the present invention
is a method of testing a therapeutic for therapeutic activity and
toxicology by identifying a therapeutic using a method of the
present invention and monitoring the toxicology and efficacy of the
therapeutic in an in vivo model.
Inventors: |
Coassin, Peter J.;
(Encinitas, CA) ; Harootunian, Alec Tate; (Del
Mar, CA) ; Pham, Andrew A.; (Del Mar, CA) ;
Stylli, Harry; (San Diego, CA) ; Tsien, Roger Y.;
(La Jolla, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
AURORA DISCOVERY, INC.
|
Family ID: |
26706197 |
Appl. No.: |
10/830253 |
Filed: |
April 21, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10830253 |
Apr 21, 2004 |
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10120644 |
Apr 9, 2002 |
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6730520 |
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10120644 |
Apr 9, 2002 |
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09476959 |
Jan 3, 2000 |
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6517781 |
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09476959 |
Jan 3, 2000 |
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09030578 |
Feb 24, 1998 |
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6171780 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01J 2219/00317
20130101; C12Q 1/00 20130101; B01J 2219/00315 20130101; B01L
2300/0829 20130101; B01L 2300/12 20130101; G01N 33/542 20130101;
C40B 60/14 20130101; G01N 1/30 20130101; B01L 9/523 20130101; G01N
33/5308 20130101; G01N 21/03 20130101; B01L 2300/021 20130101; B01J
2219/00707 20130101; G01N 21/6428 20130101; G01N 2021/0346
20130101; B01J 2219/0072 20130101; B01J 2219/00659 20130101; C12M
23/12 20130101; G01N 21/6452 20130101; B01L 3/5085 20130101 |
Class at
Publication: |
422/102 |
International
Class: |
B01L 003/00 |
Claims
1 (canceled).
2. A multi-well platform, comprising: a) a plurality of wells, each
well comprising: i) a wall having less fluorescence than a
polystyrene-wall of at least about 90 percent of said wall's
thickness, and ii) a bottom.
3. The multi-well platform of claim 2 further comprising a frame
and wherein said wells are disposed in said frame.
4. The multi-well platform of claim 2 wherein said bottom has less
fluorescence than a polystyrene-bottom of at least about 90 percent
of said bottom's thickness.
5. The multi-well platform of claim 2 wherein said bottom has a
high transmittance and a thickness less than about 450 microns.
6. The multi-well platform of claim 5 wherein said bottom has less
fluorescence than a polystyrene-bottom of at least about 90 percent
of said bottom's thickness.
7. The multi-well platform of claim 6 wherein said bottom produces
about 200 percent or less of the fluorescence compared to fused
silica glass of 100 microns thickness throughout the following
wavelength ranges: excitation wavelengths between about 300 to 400
nm and at emission wavelengths between about 300 to 800 nM.
8. The multi-well platform of claim of claim 2 wherein said
plurality of wells comprises 864 or more wells.
9. The multi-well platform of claim of claim 2 wherein said
plurality of wells comprises between 96 and 864 wells.
10. The multi-well platform of claim 2 wherein said wall of each of
said wells is formed of an opaque material.
11. The multi-well platform of claim 2 wherein said wall of each of
said wells is coated with an optically opaque material.
12. The multi-well platform of claim 2 wherein said wall of each of
said wells is formed of a cycloolefin material.
13. The multi-well platform of claim 2 wherein said bottom is made
of polystyrene.
14. The multi-well platform of claim 2 wherein said wells have a
depth of between 3 millimeters and 20 mm.
15. The multi-well platform of claim 2 wherein said wells have a
volume of 10 microliters or less.
16. The multi-well platform of claim 2 wherein said plurality of
wells is formed by injection molding process.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention generally relates to multi-well
platforms for use in spectroscopic measurements and methods of
using such multi-well platforms. Multi-well platforms are
particularly useful for fluorescence measurements of chemical or
biological samples. The multi-well platforms can be used in
automated and integrated systems and methods for rapidly
identifying chemicals with biological activity in liquid samples,
particularly automated screening of low volume samples for new
medicines, agrochemicals, or cosmetics.
[0003] 2. Background Information
[0004] A number of multi-well platforms are commercially available
for culturing cells or performing chemical or cellular assays.
While many of these multi-well platforms offer the desirable
features of biocompatibility, ease of manufacture and substantial
structural integrity, the inventors of the present invention have
generally found that these multi-well platforms, especially plates
with polymeric bottoms, suffer from a substantially high degree of
fluorescence. The relatively high amount of background fluorescence
inherent in commercially available multi-well platforms with
polymeric bottoms makes such multi-well platforms generally not
suitable for highly sensitive fluorescence measurements associated
with many assays.
[0005] In the course of miniaturizing and automating screening
assays, the inventors of the present invention realized that
existing multi-well platforms were generally not suited for assay
volumes of a microliter or less. The inventors of the present
invention discovered that when existing multi-well platforms were
used for such small volumes, the assay became unpredictable and
sometimes inoperable. Others have used a variety of multi-well
platforms in an attempt to produce a multi-well platform suitable
for miniaturization, but none of these multi-well platforms were
found by the present inventors to be suitable for their
applications. Having discovered this previously unrecognized
problem, the inventors of the present invention set out to make a
multi-well platform compatible with miniaturized assays, such as
fluorescent based assays.
[0006] The inventors prepared selection criteria for suitable
materials for manufacturing multi-well platforms for such
applications. As a key example of the selection criteria, which is
more fully described herein, the inventors investigated the
spectral properties of various materials, including their
fluorescence and transmittance, for compatibility with
spectroscopic measurements of chemical and biological events. Such
materials would also desirably, but not necessarily depending on
the application, have biocompatibility, relative chemical
inertness, and sufficient rigidity for the application at hand, and
ease of manufacture. The inventors selected a variety of materials
for testing based, in part, on the structural features of the
materials, which is more fully described herein. The inventors'
search for materials included searching fields not associated with
spectroscopic measurements, such as the electronics and audio
recording arts. The inventors compared a variety of materials to
glass that has relatively minor inherent fluorescence. The
inventors realized that fused silica would tend to have less
inherent fluorescence than glass.
[0007] As described herein the inventors for the first time have
developed novel multi-well platforms that offer excellent
performance characteristics in fluorescent assays. Such multi-well
platforms can be used in conventional 96-well formats or higher
density formats, such as less than 864 wells per platform or 864 or
more wells per platform. Higher density formats, such as greater
than 3,000 wells per multi-well platform, are also part of the
invention.
[0008] Systems and methods for rapidly identifying chemicals with
biological activity in samples, especially small liquid samples,
can benefit a number of different fields. For instance, the
agrochemical, pharmaceutical, environmental and cosmetic fields all
have applications where large numbers of liquid samples containing
chemicals are processed. Currently, many such fields use various
strategies to reduce processing times, such as simplified
chemistry, semi-automation, and robotics. While such strategies may
improve the processing time for a particular type of liquid sample,
process step or chemical reaction, such methods or apparatuses can
seldom integrate the entire process, especially the generation or
detection of chemical events in small volumes. Such apparatuses are
also often limited in their application, since many of them are
designed for, and dedicated to, a particular type of liquid sample
or chemical reaction.
[0009] In most processes involving liquid samples, as the
complexity of the liquid sample processing increases, the process
time per sample increases. Although some very simple chemical
reactions or liquid processing methods can achieve extremely high
throughput rates, such as in the manufacturing of containerized
liquids, complicated processing of liquids is typically several
orders of magnitude slower. In some instances, the processing of
liquid samples, such as in pharmaceutical arts, which usually
demands complicated liquid processing for drug discovery, can
obtain throughput rates of approximately 3,000 samples per day.
This type of processing in general, however, uses liquid sample
volumes on the order of 100 to 200 microliters, which often
requires relatively large amounts of exotic and expensive reagents,
and does not typically incorporate automated access to large stores
of liquid reagents.
[0010] Consequently, there is a need to provide components, systems
and methods for rapidly processing liquid samples at high
throughput rates, particularly liquid samples of microliter
volumes, one to ten microliters, to identify chemicals with useful
activity. The multi-well platform of the present invention
addresses these concerns and provides additional benefits as
well.
SUMMARY OF THE INVENTION
[0011] One aspect of the present invention is a multi-well platform
that has a plurality of wells within a frame. Each of these wells
has a wall, which exhibits less fluorescence than a polystyrene
wall of at least about 90 percent of the wall's thickness. Each
well also has a bottom, which has a high transmittance portion and
exhibits low background fluorescence. The thickness of the bottom
can be less than about 450 microns, and can be as thin as about 20
to 100 micrometers and can exhibit superior optical properties
compared to existing multi-well platforms. The optical properties
of the multi-well platforms of the present invention can approach
those of glass in the near-uv and visible spectra. The multi-well
platform is particularly useful for measuring fluorescent events
that can take place within the wells. The combination of the
materials for the well wall and the thin bottom material, provides
for particularly low fluorescent background. To make the multi-well
platform compatible with robotics and automation equipment, it can
have a footprint of a standard 96-well microtiter plate. To
miniaturize and increase the throughput of such plates through
automation equipment, the multi-well platform can have a high
density of wells within that footprint. Furthermore, to reduce
costs associated with valuable reagents, the wells of the
multi-well platform can have a small volume, such as less than
three microliters.
[0012] Another aspect of the present invention is a system for
spectroscopic measurements, which includes a reagent for an assay
and a multi-well platform for fluorescence measurements. The
reagents can include, for example, cells, chemicals, solvents,
buffers, and the like. The system can further comprise other
elements, such as a detector to measure spectroscopic events within
the wells, such as fluorescent events. Other elements can include
robotics to retrieve and move the multi-well platform, robotics to
dispense liquids into the multi-well platform, readers to measure
events taking place within the wells of the multi-well platform,
and informatics to store and analyze measurements obtained from
reactions within the wells of the multi-well platform.
[0013] A further aspect of the present invention is a method for
detecting the presence of an analyte in a sample contained in a
multi-well platform. The method can be based on fluorescence, so
that light emitted from a sample within a well of a multi-well
platform is measured. The amount of fluorescence emitted from the
well is indicative of a reaction within the well.
[0014] Another aspect of the present invention is a method for
identifying a modulator of a biological process or target in a
sample contained in a well of a multi-well platform. The method can
use fluorescence, so that light emitted from the sample is
detected. In practicing the method a biological process or target
is contacted with a test chemical. This mixture, contained within a
well of a multi-well platform, is excited with radiation of a first
wavelength. Radiation of a second wavelength emitted from the
sample is measured and can be indicative of the presence of a
modulator within the sample. This method can be used to identify
useful compounds; thus the present invention includes compounds
identified by these methods.
[0015] A further aspect of the present invention is a method to
identify a therapeutic by contacting a test chemical with a
biological process or target. This mixture, contained within a well
of a multi-well platform of the present invention, is excited with
radiation of a first wavelength. Radiation of a second wavelength
emitted from the sample is measured and can be indicative of the
presence of a therapeutic within the sample. This method can be
used to identify therapeutics; thus the present invention includes
therapeutics identified by this method. A therapeutic identified
using this method can be provided in a pharmaceutically acceptable
carrier.
[0016] A further aspect of the present invention is a method of
testing a therapeutic for therapeutic activity and toxicology. The
method identifies a therapeutic using a method of the present
invention. The identified therapeutic is then monitored for
toxicity and efficacy in an in vitro or in vivo model.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A and 1B depict a cross-section of two embodiments of
a multi-well platform.
[0018] FIG. 2 depicts a top plain view of one embodiment of a
multi-well platform.
[0019] FIG. 3 depicts a top plain view of one embodiment of a
covering means.
[0020] FIG. 4 depicts a top plain view of one embodiment of a
holding means.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Definitions
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein, and the laboratory
procedures in spectroscopy, drug discovery, cell culture, molecular
genetics, plastic C manufacture, polymer chemistry, diagnostics,
amino acid and nucleic acid chemistry, and sugar chemistry
described below, are those well known and commonly employed in the
art. Standard techniques are typically used for preparation of
plastics, signal detection, recombinant nucleic acid methods,
polynucleotide synthesis, and microbial culture and transformation
(e.g., electroporation, lipofection). The techniques and procedures
are generally performed according to conventional methods in the
art and various general references (see generally, Sambrook et al.
Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Lakowicz, J.
R. Principles of Fluorescence Spectroscopy, New York: Plenum Press
(1983) for fluorescence techniques, which are incorporated herein
by reference) which are provided throughout this document. Standard
techniques are used for chemical syntheses, chemical analyses, and
biological assays. As employed throughout the disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0023] "Fluorescent donor moiety" refers to the radical of a
fluorogenic compound that can absorb energy and is capable of
transferring the energy to another fluorogenic molecule or part of
a compound. Suitable donor fluorogenic molecules include, but are
not limited to, coumarins and related dyes, xanthene dyes such as
fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes,
bimanes, acridines, isoindoles, dansyl dyes, aminophthalic
hydrazides such as luminol and isoluminol derivatives,
aminophthalimides, aminonaphthalimides, aminobenzofurans,
aminoquinolines, dicyanohydroquinones, europium and terbium
complexes, and related compounds.
[0024] "Quencher" refers to a chromophoric molecule or part of a
compound which is capable of reducing the emission from a
fluorescent donor when attached to the donor. Quenching may occur
by any of several mechanisms including fluorescence resonance
energy transfer, photoinduced electron transfer, paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling, and
excitation coupling such as the formation of dark complexes.
[0025] "Acceptor" refers to a quencher that operates via
fluorescence resonance energy transfer. Many acceptors can re-emit
the transferred energy as fluorescence. Examples of these acceptors
include coumarins and related fluorophores, xanthenes such as x
fluoresceins, rhodols, and rhodamines, resorufins, cyanins,
difluoroboradiazaindacenes, and phthalocyanines. Other chemical
classes of acceptors generally do not re-emit the transferred
energy. Examples of these acceptors include indigos, benzoquinones,
anthraquinones, azo compounds, nitro compounds, indoanilines,
di-and triphenylmethanes.
[0026] "Binding pair" refers to two moieties (e.g., chemical or
biochemical) that have an affinity for one another. Examples of
binding pairs include antigen/antibodies, lectin/avidin, target
polynucleotide/probe oligonucleotide, antibody/anti-antibody,
receptor/ligand, enzyme/ligand and the like. "One member of a
binding pair" refers to one moiety of the binding pair, such as an
antigen or ligand.
[0027] "Dye," "pigment," or "chromophore" refer to a molecule or
part of a compound that absorbs specific frequencies of light,
including but not limited to ultraviolet light.
[0028] "Fluorophore" refers to a chromophore that fluoresces.
[0029] "Membrane-permeant derivative" refers a chemical derivative
of a compound that has enhanced membrane permeability compared to
an underivativized compound. Examples include ester, ether and
carbamate derivatives. These derivatives are made better able to
cross cell membranes, i.e., are membrane permeant, because
hydrophilic groups are masked to provide more hydrophobic
derivatives. Also, masking groups are designed to be cleaved from a
precursor (e.g., fluorogenic substrate precursor) within the cell
to generate the derived substrate intracellularly. Because the
substrate is more hydrophilic than the membrane permeant derivative
becomes trapped within the cells.
[0030] "Alkyl" refers to straight, branched, and cyclic aliphatic
groups, generally of 1 to 8 carbon atoms, preferably 1 to 6 carbon
atoms, and most preferably 1 to 4 carbon atoms. The term "lower
alkyl" refers to straight and branched chain alkyl groups of 1 to 4
carbon atoms.
[0031] "Aliphatic" refers to saturated and unsaturated alkyl
groups, generally of 1 to 10 carbon atoms, preferably 1 to 6 carbon
atoms, and most preferably 1 to 4 carbon atoms.
[0032] "Heat fusion weld" refers to a weld induced by heat. The
source of heat can be m y source sufficient to promote some degree
of attachment between two portions (separate or otherwise) of a
material(s), including a chemical reaction, an external heat source
(e.g., a heated platen, ultrasonic or air), or internal heating
(e.g., radio frequency heating).
[0033] "Isolated polynucleotide" refers a polynucleotide of
genomic, cDNA, or synthetic origin, or some combination there of,
which by virtue of its origin the "isolated polynucleotide" (1) is
not associated with the cell in which the "isolated polynucleotide"
is found in nature, or (2) is operably linked to a polynucleotide
which it is not linked to in nature.
[0034] "Isolated protein" refers a protein of cDNA, recombinant
RNA, or synthetic origin, or some combination thereof, which by
virtue of its origin the "isolated protein" (1) is not associated
with proteins found it is normally found with in nature, or (2) is
isolated from the cell in which it normally occurs, or (3) is
isolated free of other proteins from the same cellular source,
e.g., free of human proteins, or (4) is expressed by a cell from a
different species, or (5) does not occur in nature. "Isolated
naturally occurring protein" refers to a protein, which by virtue
of its origin, the "isolated naturally occurring protein" (1) is
not associated with proteins that it is normally found with in
nature, or (2) is isolated from the cell in which it normally
occurs, or (3) is isolated free of other proteins from the same
cellular source, e.g., free of human proteins.
[0035] "Polypeptide" as used herein as a generic term to refer to
native protein, fragments thereof, active fragments thereof, or
analogs of a polypeptide sequence. Hence, native protein,
fragments, and analogs are species of the polypeptide genus.
[0036] "Naturally-occurring" as used herein, as applied to an
object, refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0037] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0038] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding and non-coding
sequences to which they are ligated. The nature of such control
sequences differs depending upon the host organism. In prokaryotes,
such control sequences generally include promoter, ribosomal
binding site, and transcription termination sequence. In
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, components whose presence can
influence expression and can also include additional components
whose presence is advantageous, for example, leader sequences and
fusion partner sequences.
[0039] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides, or a modified form of either type of nucleotide.
The term includes single, double, and triple stranded forms of
DNA.
[0040] "Corresponds to" refers to a polynucleotide sequence that is
homologous (i.e., is identical, not strictly evolutionarily
related) to all or a portion of a reference polynucleotide
sequence, or that a polypeptide sequence is identical to a
reference polypeptide sequence. In contradistinction, the term
"complementary to" is used herein to mean that the complementary
sequence is homologous to all or a portion of a reference
polynucleotide sequence. For illustration, the nucleotide sequence
"TATAC" corresponds to a reference sequence "TATAC" and is
complementary to a reference sequence "GTATA."
[0041] "Polypeptide fragment" refers to a polypeptide that has an
amino-terminal and/or carboxy-terminal deletion, but where the
remaining amino acid sequence is usually identical to the
corresponding positions in the naturally-occurring sequence
deduced, for example, from a full-length cDNA sequence. Fragments
typically can be at least 5, 6, 8, or 10 amino acids long,
preferably at least 14 amino acids long, more preferably at least
20 amino acids long, usually at least 50 amino acids long, and can
be at least 70 amino acids long.
[0042] "Plate" or "platform" refers to a multi-well platform,
unless otherwise modified in the context of its usage.
[0043] "Cycloolefins" refer generally to cycloolefin polymers,
unless otherwise modified in the context of its usage, and includes
copolymers such as those so specified herein. "Cycloolefin
copolymers" refer generally to cycloolefin copolymers, unless
otherwise modified in the context of its usage.
[0044] "Modulation" refers to the capacity to either enhance or
inhibit a functional property of biological activity or process
(e.g., enzyme activity or receptor binding). Such enhancement or
inhibition may be contingent on the occurrence of a specific event,
such as activation of a signal transduction pathway, and/or may be
manifest only in particular cell types.
[0045] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g., nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian)
cells or tissues. Modulators are evaluated for potential activity
as inhibitors or activators (directly or indirectly) of a
"biological process or processes" (e.g., agonist, partial
antagonist, partial agonist, antagonist, antineoplastic agents,
cytotoxic agents, inhibitors of neoplastic transformation or cell
proliferation, cell proliferation-promoting agents, and the like)
by inclusion in screening assays described herein. The activity of
a modulator may be known, unknown or partially known.
[0046] The term "test chemical" refers to a chemical to be tested
by one or more screening method(s) of the invention as a putative
modulator.
[0047] The term "analyte" refers to a chemical whose presence is to
be tested by one or more screening method(s) of the invention.
[0048] The terms "label" or "labeled" refers to incorporation of a
detectable marker, e.g., by incorporation of a radiolabeled amino
acid or attachment to a polypeptide of biotinyl moieties that can
be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Various methods of labeling genes
and polypeptides and glycoproteins are known in the art and may be
used. Examples of labels include, but are not limited to, the
following: radioisotopes (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.125I, .sup.131), fluorescent labels (e.g., FITC, rhodamine,
lanthanide phosphors), enzymatic labels (or reporter genes) (e.g.,
horseradish peroxidase, .beta.-galactosidase, .beta.-lactamase,
luciferase, alkaline phosphatase), chemiluminescent labels,
biotinyl groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, epitope
tags). In some embodiments, labels are attached by spacer arms of
various lengths to reduce potential steric hindrance.
[0049] "Fluorescent label" refers to incorporation of a detectable
marker, e.g., by incorporation of a fluorescent moiety to a
chemical entity that binds to a target or attachment to a
polypeptide of biotinyl moieties, that can be detected by avidin
(e.g., streptavidin containing a fluorescent label or enzymatic
activity that can be detected by fluorescence detection methods) or
other moieties. Various methods of labeling polypeptides and
glycoproteins are known in the art and may be used. Examples of
labels for polypeptides or other moieties include, but are not
limited to dyes (e.g., FITC and rhodamine), intrinsically
fluorescent proteins, and lanthanide phosphors. In some
embodiments, labels are attached by spacer arms of various lengths
to reduce potential steric hindrance.
[0050] "Reporter gene" refers to a nucleotide sequence encoding a
protein that is readily detectable, either by its presence or
activity, including, but not limited to, luciferase, green
fluorescent protein, chloramphenicol acetyl transferase,
3-galactosidase, secreted placental alkaline phosphatase,
.beta.-lactamase, human growth hormone, and other secreted enzyme
reporters. Generally, reporter genes encode a polypeptide not
otherwise produced by the host cell which is detectable by analysis
of the cell(s), e.g., by the direct fluorometric, radioisotopic or
spectrophotometric analysis of the cell(s) and preferably without
the need to remove the cells for signal analysis of a well.
Preferably, the gene encodes an enzyme that produces a change in
fluorometric properties of the host cell that is detectable by
qualitative, quantitative, or semi-quantitative function of
transcriptional activation. Exemplary enzymes include esterases,
phosphatases, proteases (tissue plasminogen activator or
urokinase), and other enzymes whose function can be detected by
appropriate chromogenic or fluorogenic substrates known to those
skilled in the art. Proteins, particularly enzymes encoded by
reporter genes can also be used as probes in biochemical assays,
for instance after proper conjugation to either the target or a
chemical entity that binds to the target.
[0051] "Transmittance" refers to the fraction of incident light
that passes through a medium at a given wavelength. It can also be
considered the ratio of radiant power transmitted through a medium
to the radiant power incident on the medium at a particular
wavelength.
[0052] "Fluorescent measurement" refers to the measurement of
fluorescence from a sample, such as from a sample in a well of a
multi-well platform, by any appropriate means known in the art or
later developed.
[0053] "High transmittance portion" refers to a portion of a bottom
of a well of a multi-well platform that can transmit radiation,
such as light, in an assay, such as a fluorescence assay. Portion,
in this context, refers to about 0.1% to 100%, preferably about 50%
to greater than 90%, of the surface area of the bottom having high
transmittance properties. Efficiently transmit radiation, in this
context, refers to a transmittance of greater than about 1% of
incident light.
[0054] "Footprint approximately that of a standard 96-well plate"
refers to the dimensions of about 85.5 mm in width by about 127.5
mm in length, or the approximate dimensions of an industry standard
multi-well plate.
[0055] "Chamfered wall" refers to the wall of a well having an
angle of greater than 90.degree. at least a portion between the top
of the well to the bottom of the well. For example, the angle can
be for the entire depth of the well, or only a portion of that
depth. The angle is preferable from the top of the well towards the
bottom of the well so that the area of the top of the well is
larger than the area of the bottom of the well.
[0056] "Well center-to-center distance" refers to the distance
between the center of one well to the center of a neighboring
well.
[0057] "Optically opaque" refers to a material that presents a
substantial barrier to the transmission of light. Substantial
barrier, in this instance, refers to the ability of a material to
block transmittance of greater than about 10% of incident
light.
[0058] "Reflective coating" refers to a coating that renders a
surface capable of reflecting radiation such as light. The
reflective coating can be on the wall of a well and can reduce the
amount of incident radiation that can be transmitted through the
bottom. Such reflective coating is preferred when the wall is
chamfered.
[0059] "Plurality of living cells" refers to one or more cells,
wherein the cells can be prokaryotic, eukaryotic, or a mixture
thereof.
[0060] "Target" refers to any biological entity, such as a protein,
sugar, carbohydrate, nucleic acid, lipid, a cell or population of
cells or an extract thereof, a vesicle, or any combination
thereof.
[0061] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill,
San Francisco, incorporated herein by reference).
[0062] Introduction
[0063] During the course of miniaturizing and automating screening
assays that use multi-well platforms, such as fluorescent assays,
the inventors of the present invention realized that existing
multi-well platforms were generally not suited for use with small
assay volumes, such as a microliter or less. The inventors of the
present invention discovered that when existing multi-well
platforms were used for such small volumes, the assay became
unpredictable and sometimes inoperable. Having discovered this
previously unrecognized problem, the inventors of the present
invention made multi-well platforms suitable for their purposes.
The wall of a multi-well platform of the present invention exhibits
less fluorescence than a polystyrene wall of at least about 90
percent of the wall's thickness. The bottom of the well can be
about 450 micrometers thick, preferably about 20 to 100 micrometers
thick, and can exhibit optical properties superior to existing
multi-well platforms. The multi-well platforms of the represent
invention can have a footprint compatible with robotics, and
automation, and instrumentation. Furthermore, to miniaturize and
increase the throughput of these plates through such automation
equipment, the multi-well platform of the present invention can
have a high density of wells in such a footprint, such as between
about 6 and 5000 wells.
[0064] As a non-limiting introduction to the breadth of the
invention, the invention includes several general and useful
aspects, including:
[0065] 1) multi-well platforms that are useful for performing
fluorescent measurements,
[0066] 2) a system for spectroscopic measurements using (1),
[0067] 3) a method for detecting the presence of an analyte in a
sample contained in (1),
[0068] 4) a method for identifying a modulator of a biological
process or target in a sample using (1),
[0069] 5) a composition identifying a composition using (1) and (3)
or (4),
[0070] 6) a method of testing a therapeutic for therapeutic
activity using (1), and
[0071] 7) a therapeutic identified by (6).
[0072] These aspects of the invention, as well as others described
herein, can be achieved by using the methods, devices, and
compositions of matter described herein. To gain a full
appreciation of the scope of the invention, it will be further
recognized that various aspects of the invention can be combined to
make desirable embodiments of the invent ion.
[0073] Multi-Well Platforms
[0074] The multi-well platforms of the present invention are well
suited for use in fluorescent based assays, but can be used for any
appropriate purpose. These multi-well platforms comprise a frame,
wherein said wells are disposed in said frame. The frame can be of
any thickness, such as between about 0.5, 1, 2, 3, or 5 millimeters
and 2, 3, 5, 10 or 20 millimeters. The frame can be made of any
material, such as polymers, such as polystyrene or cycloolefins, or
other materials, such as glass or quartz. The frame can be of any
shape, and typically defines the footprint of the multi-well
platform.
[0075] The bottom of the frame can be substantially flat, meaning
in this instance that the bottom of the frame does not have
additional structures, such as means to form a band of opaque
material in the bottom when the frame and bottom are sealed
together (see, U.S. Pat. No. 5,319,436 (Mann et al.)). Such bands
of opaque material are preferred when the wall is chamfered,
however, the present invention is useful with or without such bands
of opaque material. The bottom of the frame can also include
structures such as pins, grooves, flanges or other known structures
or those developed in the future to orient the multi-well platform
on another structure, such as a detector or another platform.
[0076] The multi-well platform can have a footprint of any shape or
size, such as square, rectangular, circular, oblong, triangular,
kidney, or other geometric or non-geometric shape. The footprint
can have a shape that is substantially similar to the footprint of
existing multi-well platforms, such as the standard 96-well
microtiter plate, whose footprint is approximately 85.5 mm in width
by 127.75 mm in length or other sizes that represent a current or
future industry standard (see T. Astle, Standards in Robotics and
Instrumentation, J of Biomolecular Screening, Vol. 1 pages 163-168
(1996)). Other standard footprints are presented in Table 1.
Multi-well platforms of the present invention having this footprint
can be compatible with robotics and instrumentation, such as
multi-well platform translocators and readers as they are known in
the art.
1 TABLE 1 Outside Dimensions Wells Mfrs Cat. # Mfrs Name Length
Width Height Wells Shape Color Material Bottom AGTC 128.118 85.319
41.148 Styrene 1 ml AIM 127.762 85.598 41.504 Styrene 1 ml AIM
127.635 85.141 40.945 Propylene 1 ml Beckman 127.93 85.55 41.84 96
round clear Styrene Round Beckman 127.93 85.55 41.84 96 round
translucent Propylene Round 373660 Beckman 127.787 85.573 14.224
clear Styrene Flat 25870 Corning/Costar 127.68 85.12 14.2 96 round
clear Styrene Flat (bezel) 35207 Corning/Costar 127.61 85.166
14.224 clear Styrene Flat 35205 Corning/Costar 127.33 85.014 14.224
clear Styrene U-Bottom Corning/Costar 127.6 85.2 14.3 96 round
clear Styrene Cone 7000003 Corning/Costar 127.1 85.3 14.3 96 round
black Styrene Flat 7000004 Corning/Costar 127.6 85.47 14.2 96 round
black Styrene Flat 7000008 Corning/Costar 126.7 84.62 14.45 96
round translucent Propylene Round 7000010 Corning/Costar 127.83
85.42 14.53 96 round clear Styrene Flat 35203 Corning/Costar
127.508 85.319 14.224 clear Styrene Flat 35202 Corning/Costar 85.42
14.326 clear Styrene Flat A/2 35190 Dynatech 127.889 85.649 14.173
clear Styrene Flat 35189 Dynatech 127.838 85.522 14.097 clear
Styrene V-Bottom 35194 Evergreen 127.483 85.344 14.376 clear
Styrene Flat 35192 Evergreen 127.483 85.217 14.275 clear Styrene
U-Bottom 35191 Evergreen 127.432 85.268 14.3 clear Styrene V-Bottom
35197 Falcon 127.381 85.471 14.351 clear Styrene Flat 7000017
Genetix 128.28 86.31 10.17 384 round clear Styrene Flat 35188
Immulon 127.406 85.344 14.402 clear Styrene Flat 35176 Interlab
127.914 85.852 13.665 clear Styrene V-Bottom Iwaki 127.279 85.065
14.021 Styrene Flat 35181 LabSystems 127.838 85.598 15.291 black
Propylene Flat 35187 MicroFluor 127.406 85.217 12.224 white
Propylene Flat 35184 MicroFluor 127.508 85.42 14.275 black
Propylene Flat 35183 MicroFluor 127.533 85.42 14.224 white
Propylene Flat A/2 35185 MicroLite 127.584 85.369 14.148 white
Propylene Flat 35186 MicroLite 2 127.635 85.471 14.199 white
Propylene Flat Millipore 128.016 85.75 14.859 white Propylene Flat
Millipore 127.813 85.598 14.605 clear Styrene Flat 35177 NBT
127.838 85.598 14.3 clear Styrene Flat 7000001 Nunc 127.6 83.7 14.4
96 round clear Styrene U-Bottom 7000006 Nunc 127.7 85.6 14.5 384
square clear Styrene Flat 63765 Nunc 127.559 85.573 14.351 clear
Styrene Flat 35201 Nunc 127.432 85.344 14.097 clear Styrene
U-Bottom 35200 Nunc 126.314 84.379 14.351 Propylene U-Bottom 35199
Nunc 127.305 85.395 14.402 clear Styrene V-Bottom 35210 Packard
127.762 85.471 14.275 white Propylene GF/B 35209 Packard 127.965
85.776 14.351 white Propylene GF/C 35203 Pall 127.635 85.598 14.325
white Propylene Flat 7000005 Polyfiltronics 127.5 85.8 44.03 96
square translucent Propylene Round 7000009 Polyfiltronics 127.09
85.12 30.43 96 round translucent Propylene Filter 7000011
Polyfiltronics 127.3 85.25 16 96 round translucent Propylene Cone
7000012 Polyfiltronics 127.8 85.69 9.56 384 round translucent
Propylene Cone 35175 Polyfiltronics 127.787 85.552 15.24 white
Propylene Flat 35174 Polyfiltronics 127.483 85.547 15.189 black
Propylene Flat 35173 Polyfiltronics 127.991 85.7 15.24 white
Propylene Clear-flat 35179 Polyfiltronics 127.559 85.344 14.351
white Propylene GF/B 35180 Polymetrics 127.533 85.369 14.097
translucent Propylene Deep V Sumilon 127.33 85.395 14.503 Styrene
Flat 35178 Tilertek 127.381 85.319 14.224 clear Styrene Flat
[0077] Each well comprises a wall having less fluorescence than
polystyrene wall of 100 to 70% of the wall's thickness, preferably
at least 90 percent of the wall's thickness. These determinations
can be made using fluorescent detection methods well known in the
art, such as determining the fluorescence of appropriate sheets of
the materials being compared or as described herein.
[0078] Typically, wells will be arranged in two-dimensional linear
arrays on the multi-well platform. However, the wells can be
provided in any type of array, such as geometric or non-geometric
arrays. The number of wells can be no more than 864 wells, or
greater than 864 wells, on a standard multi-well platform
footprint. Larger numbers of wells or increased well density can
also be easily accomplished using the methods of the claimed
invention. Other commonly used number of wells include 1536, 3456,
and 9600. The number of wells can be between about 50, 100, 200,
500, 700, 800 or 1000 wells and 150, 250, 600, 800, 1000, 2000,
4000, 5000, or 10000 wells. Preferably, the number of wells can be
between about 50 and 10000, more preferable between about 800 and
5000, and most preferably between about 900 and 4000. The number of
wells can be a multiple of 96 within these ranges, preferably the
square of an integer multiplied by 96.
[0079] Well volumes typically can vary depending on well depth and
cross sectional area. Well volumes can range between about 0.5,
1.5, 10, 25, 50, 75, 100 or 200 microliter and about 5, 15, 40, 80,
100, 200, 500, or 1000 microliters. Preferably, the well volume is
between about 500 nanoliters and 500 microliters, more preferably
between about 1 microliter and 200 microliter, and most preferably
between about 0.5 microliters and 10 microliters.
[0080] Wells can be made in any cross sectional shape (in plan
view) including, square, round, hexagonal, other geometric or
non-geometric shapes, and combinations (intra-well and inter-well)
thereof. Wells can be made in any cross sectional shape (in
vertical view) including shear vertical or chamfered walls, wells
with flat or round bottoms, conical walls with flat or round
bottoms, and curved vertical walls with flat or round bottoms, and
combinations thereof.
[0081] As shown in FIG. 1A, the walls can be chamfered (e.g.,
having a draft angle) 14. Chamfered walls can have an angle between
about 1, 2, 3, 4, or 5 degrees and about 2, 3, 4, 5, 6, 7, 8, 10,
or 20 degrees. Preferably, the angle is between about 1 and 10
degrees, more preferably between about 2 and 8 degrees, and most
preferable between about 3 and 5 degrees.
[0082] As shown in FIG. 1, the wells can be placed in a
configuration so that the well center-to well-center distance 12
can be between about 0.5, 1, 2, 5, or 10 millimeters and about 1,
2, 5, 10, 20, 50, or 100 millimeters. The wells can be placed in
any configuration, such as a linear-linear array, or geometric
patterns, such as hexagonal patterns. The well-to-well distance can
be about 9 mm divided by an integer between 1 and 10. Typically,
the multi-well plate has wells with a well-center-to-well-center
distance of less than about 2.5 mm, preferably less than 2 mm and
some times less than about 1 mm. Smaller well-center to well-center
distances are preferred for smaller volumes.
[0083] The wells can have a depth between about 0.5, 1, 2, 3, 4, 5,
10, 20, or 50 millimeters and about 5, 10, 20, 50, or 100
millimeters. Preferably, the well depth is between about 1
millimeter and 100 millimeters, more preferably between about 2
millimeters and 50 millimeters, and most preferably between about 3
millimeters and 20 millimeters.
[0084] The wells can have a diameter (when the wells are circular)
or maximal diagonal distance (when the wells are not circular)
between about 0.2, 0.5, 0.7, 1, 5, 10, or 50 millimeters and about
1, 5, 10, 20, 50, or 100 millimeters. Preferably, the well diameter
is between about 0.5 and 100 millimeters, more preferably between
about 1 and 50 millimeters, and most preferably, between about 2
and 20 millimeters.
[0085] The wells of the multi-well platform can comprise an
optically opaque material that can interfere with the transmission
of radiation, such as light, through the wall of a well or bottom
of a well. Such optically opaque materials can reduce the
background associated with optical detection methods. Optically
opaque materials can be any known in the art or later developed,
such as dyes, pigments or carbon black. The frame can be made of an
optically opaque material, or the walls or bottom, or both, can be
coated with an optically opaque material. The optically opaque
material can prevent radiation from passing from one well to
another, to prevent cross-talk between wells, so that the
sensitivity and accuracy of the assay is increased. The optically
opaque material can also be reflective, such as those known in the
art, such as thin metal layers, mirror coatings, or mirror polish.
Optically opaque materials can be coated onto any surface of the
multi-well platform, or be an integral part of the frame or bottom
as they are manufactured. Optically opaque material can prevent the
transmittance of between about 100% to about 50% of incident light,
preferably between about 80% and greater than 95%, more preferably
greater than 99%.
[0086] Since most measurements will not typically require light to
pass through the wall of the well, materials such as polymers can
include pigments to darken well walls or absorb light. Such
application of pigments will help reduce background fluorescence.
Pigments can be introduced by any means known in the art, such as
coating or mixing during the manufacture of the material or
multi-well platform. Pigment selection can be based on a mixture of
pigments to dampen all background inherent to the polymer, or a
single pigment or ensemble of pigments selected to filter or absorb
light at desired wavelengths. Pigments can include carbon black.
Such pigmentation is generally not desired in embodiments where
light is directed through the well wall as a method for
illuminating the contents of the well.
[0087] Each well also comprises a bottom 11 having a high
transmittance portion and having less fluorescence than a
polystyrene-bottom of at least about 90 percent of said bottom's
thickness. This property can be determined by comparing the
fluorescence of an appropriate control bottom material with the
fluorescence of a test material. These procedures can be performed
using well known methods. The thickness of the bottom can vary
depending on the overall properties required of the plate bottom
that may be dictated by a particular application. Preferably, the
bottom is a plate or film as these terms are known in the art. Such
properties include the amount of intrinsic fluorescence, rigidity,
breaking strength, and manufacturing requirements relating to the
material used in the plate. Well bottom layers typically have a
thickness between about 10, 15, 20, 50, 100, 200, or 300
micrometers and about 20, 50, 100, 200, 300, 450, 500, or 1000
micrometers. Preferably, the well bottom has a thickness between
about 10 micrometers and 450 micrometers, more preferably between
about 15 micrometers and 300 micrometers, and most preferably
between about 20 micrometers and 100 micrometers.
[0088] The bottom of a well can have a high transmittance portion,
typically meaning that either all or a portion of the bottom of a
well can transmit light. As shown in FIG. 1B, the bottom can have
an optically opaque portion 12 and a high transmittance portion 13
of any shape, such as circular, square, rectangular, kidney shaped,
or other geometric or non-geometric shape or combinations thereof.
In applications of the invention that can utilize focused light,
the bottom, or a portion thereof, can be used to form a lens. Lens
will vary in thickness and curvature depending on the application,
such as convex or concave in shape.
[0089] The bottom can produce about 200 percent or less of the
fluorescence compared to glass of 100 microns thickness at
excitation wavelengths between about 290, 300, 310, 320, or 350 to
about 300, 340, 370, 400, or 420 nm and at emission wavelengths
between about 290, 300, 350, 400, or 500 and about 400, 600, 800,
or 1000 nm.
[0090] Preferably, the bottom of the multi-well platform can be
substantially flat, e.g., having a surface texture between about
0.00 1 mm and 2 mm, preferably between about 0.01 mm and 0.1 mm
(see, Surface Roughness, Waviness, and Lay, Am. Soc. of Mech. Eng.
#ANSI ASME B46.1-2985 (1956)). If the bottom is not substantially
flat, then the optical quality of the bottom and wells can decrease
because of altered optical and physical properties of one or both.
Furthermore, the bottom of the frame can be substantially flat
within the meaning set forth in this paragraph.
[0091] One feature of the preferred multi-well platform of the
present invention is their low intrinsic fluorescence. Bottom
layers comprising cycloolefin typically produces about 100 to 200
percent or less of the fluorescence compared to glass of about 130
to 170 micrometers in thickness. Glass, particularly fused silica,
is typically used a "gold standard' for comparison of relative
fluorescence. Fluorescence and relative fluorescence can be
measured using any reliable techniques known or developed in the
art, preferably the techniques described herein are used.
Preferably, the glass standard used herein to show the surprisingly
low fluorescence of polymers such as cycloolefin is used as a
standard. Preferably, the bottom typically produces about 100 to
200 percent or less of the fluorescence compared to glass of about
0.085 to 0.13 millimeters (85 to 130 micrometers) thick (for glass
slides, see Thomas Scientific, MicroCover Glasses, No. 0, product
No. 6661-B40). The amount of intrinsic fluorescence can be
dictated, in part, by the layer thickness. In some applications
that can tolerate particularly thin bottoms, such as applications
where the bottom does not require significant structural strength,
layer thickness can be quite thin (e.g., 20 to 80 microns) in order
to reduce fluorescence arising from the bottom. The thinness of a
bottom is usually also balanced against the difficulty of uniformly
welding or generating thinner layers in manufacturing processes.
The low relative fluorescence of the multi-well platform is usually
present at excitation wavelengths between about 300 to 400 nm and
at emission wavelengths between about 350 to 800 nm.
[0092] The bottom or wells can also include at least one or a
plurality of living cells. The cells can be prokaryotic, such as
bacteria, or eukaryotic, such as plant cells, mammalian cells, or
yeast cells. The cells can include more than one type of cell, such
as a mixture of different mammalian cells, or a mixture of
prokaryotic and eukaryotic cells. Such embodiments are useful for
cell based assays described herein and for growing cells using
culture methods. The multi-well platforms of the invention can
include a coating (e.g., polylysine or fibronectin) to enhance
attachment of cells. Coatings can also include at least one
substrate for a cell adhesion molecule, such as integrins. Such
substrates are known in the art.
[0093] The multi-well platform of the present invention can include
coatings or surface modifications to facilitate various
applications of the plate as described herein and known or
developed in the relevant art. Coatings can be introduced using any
suitable method known in the art, including printing, spraying,
radiant energy, ionization techniques or dipping. Surface
modifications can also be introduced by appropriately derivatizing
a polymer or other material, such as glass or quartz, before,
during, or after the multi-well platform is manufactured and by
including an appropriate derivatized polymer or other material in
the bottom layer or frame. The derivatized polymer or other
material can then be reacted with a chemical moiety that is used in
an application of the plate. Prior to reaction with a chemical
moiety, such polymer or other material can then provide either
covalent or non-covalent attachment sites on the polymer or other
material. Such sites in or on the polymer or other material surface
can be used to attach moieties, such as assay components (e.g., one
member of a binding pair), chemical reaction components (e.g.,
solid synthesis components for amino acid or nucleic acid
synthesis), and cell culture components (e.g., proteins that
facilitate growth or adhesion). Examples of derivatized polymers or
other materials include those described by U.S. Pat. No. 5,583,211
(Coassin et al.) and others known in the art or later developed.
Particularly preferred embodiments are based on polyethylene and
polypropylene derivatives that can be included as cycloolefin
copolymers.
[0094] The materials for manufacturing the multi-well platform will
typically be polymeric, since these materials lend themselves to
mass manufacturing techniques. However, other materials can be used
to make the frame or bottom of the multi-well platform, such as
glass or quartz. The frame and bottom can be made of the same or
different materials and the bottom can comprise polystyrene, or
another material. Preferably, polymers are selected that have low
fluorescence or other properties using the methods described
herein. The methods herein can be used to confirm that selected
polymers possess the desire properties. Polymeric materials can
particularly facilitate plate manufacture by molding methods known
in the art and developed in the future, such as insert or injection
molding.
[0095] The multi-well platform of the present intention can be made
of one or more pieces. For example, the frame and bottom can be
made as one discrete piece Alternatively, the frame can be one
discrete piece, and the bottom can be a second discrete piece,
which are combined to form a multi-well platform. In this instance,
the frame and bottom can be attached to each other by sealing
means, such as adhesives, sonic welding, heat welding, melting,
insert injection molding or other means known in the art or later
developed. The frame and bottom can be made of the same or
different material. For example, the frame can be made of a
polymer, and the bottom made of polystyrene, glass, or quartz.
[0096] Uses for multi-well platforms are known in the relevant arts
and include diagnostic assays, chemical or biochemical binding
assays, filtration assays, chemical synthesis sites, storage sites,
and the like. Such uses can also be applied to the multi-well
platforms of the present invention. It will be recognized that some
types of multi-well platforms for spectroscopic measurements can
often be used for other multi-well platform applications.
Typically, a multi-well platform is used for detecting a signal
from a sample. Different types of signal measurements are discussed
herein.
[0097] The multi-well platform of the present invention can also
include at least one orienting structure, such as holes,
indentations, flanges, grooves, notches, or other such structures
to orient the multi-well platform in robotics or
instrumentation.
[0098] As shown in FIG. 2, the multi-well platform 20 of the
present invention can also include at least one recessed groove 21.
As shown in FIG. 2, the recessed groove can surround the matrix of
wells 22. The recessed groove can be filled with a fluid, such as
water or buffer, to provide a humid atmosphere for the wells. The
multi-well platform can also comprise an identification structure,
such as a barcode, numbering or lettering.
[0099] As shown in FIG. 3, the multi-well platform can also include
a structure 30 to cover at least a portion of the multi-well
platform. This structure can cover the entire multi-well platform,
such as the case of a polymeric bag, or can be a ridged cover or
flexible film that covers at least a portion of the upper face of
the multi-well plate which can include at least a portion of a
recessed groove, which can comprise a fluid to maintain a humid
environment within the wells. Alternatively, this structure can
form a seal by being immersed in a fluid within the at least on
recessed groove. The cover can be a water vapor permeable barrier
made of materials known in the art or a non-aqueous barrier, such
as mineral oil, silicon oil, or paraffin wax. When the multi-well
platform and a ridged covering means are engaged, the distance
between the top of the frame and the bottom of the covering means
is preferably between about 0.1, 0.5, 1, or 5 millimeters and 0.5,
1, 5, or 10 millimeters, but any distance is useful in the present
invention. As the well volume decreases, the effects of evaporation
become greater, and the need for such covering means can increase.
This structure can also comprise an identification structure, such
as a barcode, numbering or lettering.
[0100] As shown in FIG. 4, the multi-well platform of the present
invention can be held in a structure 40 to hold the multi-well
platform in a substantially planar configuration to prevent optical
distortion of the wells of the multi-well platform of the present
invention. The multi-well platform can have a flatness between
about 0.001 mm and 2 mm, preferably between about 0.001 mm and 2
mm, or between about 0.01 mm and 0.1 mm (see, Am. Soc. of Mech.
Eng. #ASME B46.1-1985, supra, (1986)). This structure holding can
comprise at least one orienting structure that can match the
orienting structure of the multi-well platform. Preferably, the
bottom or top of the wells are not obscured by this holding
structure so that the wells can be observed while held by the
holding structure by, for example, optical devices. This holding
structure can also comprise numbering or lettering to indicate a
grid matrix anywhere on the structure 50, and can also include
identification marks, such a bar-codes, to identify the multi-well
platform housed therein. This structure can also comprise a window
or an opening to allow identification marks, such as bar codes, on
the multi-well plate to be observed and read by, for example, a
bar-code reader.
[0101] The multi-well platform, covering structure, and holding
structure can be provided separately, or in any combination, in a
container, such as a hermetically sealed container as is known in
the art. The contents of the hermetically sealed container can be
provided sterile. The contents within the hermetically sealed
container can be sterilized using, for example, ionizing radiation
as is known in the art.
[0102] Materials, Selection Criteria and Testing
[0103] This section describes materials, selection criteria, and
rapid tests to facilitate choosing a material for the multi-well
plates described herein.
[0104] Materials
[0105] The present inventors conducted extensive research on
different polymers in search of polymers that offer the appropriate
properties for detecting spectroscopic signals, particularly
fluorescence signals. Although any suitable material can be used,
such as polymers or other materials such as glass or quartz, some
of the materials used in the present invention have not been used
in the commercially available multi-well platforms listed in Table
1. Surprisingly, these materials offer exceptional properties,
including low intrinsic fluorescence, which was demonstrated herein
for the first time.
[0106] The methods described herein to identify cycloolefin
copolymers as low fluorescent materials can be used to screen other
materials, such as other polymers and other materials such as
glasses and quartz, in a variety of configurations, such as in
plates, sheets, or films, to determine if they possess desirable
optical or fluorescent properties. Thus, these teachings should not
be construed to be limited to cycloolefins.
[0107] Polymers that are compatible with cycloolefin can be used in
regions of the multi-well platform in physical contact with
cycloolefin. In some embodiments, the frame can be manufactured
with a material other than a cycloolefin polymer and the
cycloolefin bonded, welded or otherwise fused to the second
material. Polymers with glass transition temperatures suitable for
heat induced fusion with cycloolefin can be selected for
manufacturing the wells and other portions of the plate.
[0108] Typically, cycloolefins can be used as films, plates, or
resins to make various embodiments of present invention. Resins and
films based on cycloolefin polymers can be used in various
manufacturing processes known in the relevant art and described
herein. Selection criteria for cycloolefin films or resins are
described more fully below.
[0109] Suitable cycloolefins for many embodiments of the present
invention include those described in U.S. Pat. No. 5,278,238 (Lee
B. L. et al.); U.S. Pat. No. 4,874,808 (Minami et al.; U.S. Pat.
No. 4,918,133 (Moriya et al.); U.S. Pat. No. 4,935,475 (Kishimura
et al.); 4,948,856 (Minchak et al.); U.S. Pat. No. 5,115,052
(Wamura et al.); U.S. Pat. No. 5,206,306 (Shen); U.S. Pat. No.
5,270,393 (Sagane et al.); U.S. Pat. No. 5,272,235 (Wakatsuru et
al.); U.S. Pat. No. 5,278,214 (Monya et al.); U.S. Pat. No.
5,534,606 (Bennett et al.); U.S. Pat. No. 5,532,030 (Hirose et
al.); U.S. Pat. No. 4,689,380 (Nahm et al.); and U.S. Pat. No.
4,899,005 (Lane et al.). Cycloolefins available from Hoechst
(Summerville, N.J.) are preferred, especially cycloolefin (e.g.,
cyclopentane, cyclohexane, and cycloheptene) and their polyethylene
copolymers, as well as the thermoplastic olefin polymers of
amorphous structure (TOPAS line).
[0110] Multilayer laminates are preferred when multiple functional
requirements are difficult to obtain from a single laminate (e.g.,
layer or film). The properties of transmittance, rigidity, heat
sealability, fluorescence, moisture penetration can be blended by
the use of films of differing resins. Blended resins known in the
art and developed in the future can be used when multilaminate
films or blended resins have properties consistent with those of
the present invention. For example, U.S. Pat. No. 5,532,030 (Hirose
et al.) describes the manufacture of certain cycloolefin films,
both single and multilaminate, that can be adapted for use in the
devices described herein. The present invention includes
multilaminates of any suitable material, such as polymers and other
materials, such as glass or quartz.
[0111] Selection Criteria and Testing
[0112] Desirable properties for materials used in the present
invention will vary depending on the type of multi-well plate
desired. Generally, the materials are selected to yield a final
product with low fluorescence, high transmittance, sufficient
rigidity to resist deformity, and to allow for substantially single
plane (especially for spectroscopic embodiments), good chemical
inertness to, for example, DMSO, relatively low cytotoxicity, low
water absorption, heat resistance/deflection up to about
150.degree. C., and resistance to acids and bases. Starting
materials with good molding properties are particularly
desirable.
[0113] Fluorescence of the materials or final product can be
readily measured. Such measurements proceed rapidly and a number of
plates or films (e.g., 20 to 80 films), or prototype products, can
be rapidly tested within a matter of hours or days, usually less
than a one person week. Consequently, films or resins used to make
final products can rapidly be selected for the desired properties
that are important in a particular application. The fluorescence
measurements can be used as described herein or those known in the
art, so long as the measurements are comparable (or better) in
sensitivity to the measurements described herein. A standard
reference point for relative fluorescence, such as the standard
described herein, is particularly useful for comparing different
cycloolefins and for determining their applicability to certain
applications. Relative fluorescence properties described herein are
particularly desirable. Similarly, transmittance of films, plates,
or final products can be measured using techniques known in the
relevant art.
[0114] In the final product, layer thicknesses of generally, about
20 to 500 micrometers, are most likely to impart the properties
desirable for use in the devices described herein, especially low
fluorescence and high transmittance. Although thinner or thicker
films, such as about 10 to 1,500 micrometers, can be used in
applications where the demands for extremely low fluorescence and
high transmittance films are less stringent, or when e desired
properties as a function of film thickness. Preferably, film
thickness is between about 30 to 200 micrometers for multi-well
platform applications, and more preferably between about 50 to 150
micrometers, and most preferably between about 80 to 100
micrometers. Preferably, film thickness is between about 30 to 600
micrometers for scaffolding applications where the film typically
contributes to a structural function in the device that usually
demands more strength or rigidity, and more preferably between
about 100 to 500 micrometers, and most preferably between about 120
to 200 micrometers. Preferably, film thickness is between about 75
to 600 micrometers for the thinner regions of injection molded
applications where the film typically contributes to a structural
function, more preferably between about 100 to 500 micrometers and
most preferably between about 120 to 200 micrometers. Film
thickness refers to the thickness of the film used (or material
thickness). Layer thickness is generally about 100 to 200 percent
of film thickness, preferably about 100 to 150 percent of film
thickness and more preferably about 100 to 125 percent of film
thickness.
[0115] In the final product, breaking stresses (Kg/cm.sup.2 at
22.degree. C.) of generally, about 400 to 3,000 Kg/cm.sup.2 are
most likely to impart the properties desirable for use in the
devices described herein, especially rigid devices of low
fluorescence and high transmittance. Although weaker or stronger
films, such as about 200 to 3,500 Kg/cm.sup.2, can be used in
different applications based on the demands for breaking strength
of the device. For example, the breaking strength of the film,
generally need not be as great for the bottoms of multi-well
platforms as compared to applications where the film is part of the
frame in a multi-well platform. Preferably, breaking stress is
between about 500 to 2,000 Kg/cm.sup.2 for multi-well plate
applications, and more preferably between about 800 to 1,600
Kg/cm.sup.2 and most preferably between about 900 to 1,400
Kg/cm.sup.2. Preferably, breaking stress for platform/scaffolding
applications is about 15 to 60 percent higher than for multi-well
platform applications. Breaking stresses can be measured by
standard techniques as known in the art. In addition to
cycloolefins, materials such as other polymers such as polystyrene,
polycarbonate, polypropylene, poly-methyl pentene, copolymers of
and of the above-mentioned polymers, or any other polymer
appropriate for an intended use of a multi-well platform of the
present invention, or other materials, such as glass or quartz, can
be used to make the frame or bottom of a multi-well platform of the
present invention.
[0116] Manufacturing Methods
[0117] The present invention includes a process for making
cycloolefin based multi-well platforms. Other methods appropriate
for other materials, such as other polymers or other materials such
as glasses or quartz, are readily apparent to those skilled in the
art based on the properties of the material or materials
selected.
[0118] A variety of processes can be used, including heat welding,
insert molding, injection molding and other processes described
herein and known in the art. One process comprises heat welding a
frame to a bottom exhibiting low fluorescence and high
transmittance, such as a cycloolefin copolymer. Processes typically
use a cycloolefin copolymer selected from the group of cyclopentane
polyethylene copolymer, cyclohexane polyethylene copolymer, and
cycloheptene polyethylene copolymer. The process can alternatively,
or optionally, comprise the step of exposing the layer and the
polymer to a sufficient amount of radio frequency energy to promote
internal heating of the layer and the polymer, or ultrasonic
welding. Alternatively the process can entail heating the layer and
the polymer that forms the wells to about 320.degree. C. for a
sufficient amount of time to allow fusion of the polymers. Pressure
can be applied to enhance the welding process (e.g., about 100 and
1,000 psi of pressure to the layer and the polymer for low pressure
processes using low viscosity monomer solutions and about 10,000 to
25,000 psi for high pressure processes such as insert molding).
[0119] In another embodiment, the invention provides for a process
for making multi-well plates by injection molding or insert
molding. Injection molding techniques known in the art or developed
in the future can be applied. The process comprises insert molding
at least a well to a bottom of the well of the multi-well plate,
wherein the bottom is a cycloolefin copolymer. Using this method,
cycloolefin films can be heat fused to the supporting structure
(e.g., the frame) to make a multi-well platform. The entire frame
or platform can also be made of a cycloolefin. Inserting molding
can be performed between about 195 and 350.degree. C. degrees,
preferably resins are heated to 260.degree. to 320.degree. C.
Pressures used are typically between 10,000 and 25,000 psi and
preferably about 15,000 to 22,000 psi.
[0120] Methods for preparing of cycloolefins and their polymers
have been described. Other methods and cycloolefins were described
in U.S. Pat. Nos. 4,002,815; 4,069,376; 4,110,528; 4,262,103 and
U.S. Pat. No. 4,380,617 (by Robert J. Minchak and co-workers). A
number of catalysts can be used in the manufacture of cycloolefins
as known in the art or developed in the future and can be used to
manufacture materials for various embodiments of the present
invention. Such catalysts include those described in U.S. Pat. No.
5,278,238 (Lee et al.) and U.S. Pat. No. 5,278,214 (Moriya et al.).
Regardless of the exact type of catalyst system utilized,
cycloolefin monomers can be polymerized in the presence of a
catalyst and the ethylene based functional copolymers to make
embodiments of the invention suitable for injection molding.
Polymerization can carried out preferably in bulk. Bulk
polymerization such as reaction injection molding (RIM), liquid
injection molding (LIM), reinforced reaction injection molding
(RRIIM), and resin transfer molding (RTM), and combinations thereof
are known to the art well as those techniques developed in the
future. Bulk polymerization is polymerization conducted in the
absence of a solvent or a diluent. Reaction injection molding is a
type of bulk polymerization wherein a monomer in a liquid state is
transferred or is injected into a mold where polymerization of the
monomer takes place in the presence of a catalyst system. RIM is
not conventional injection molding for melt polymers and is readily
distinguishable therefrom.
[0121] RIM is a low pressure, one-step or one-shot, mix and
injection of two or more liquid components into a closed mold where
rapid polymerization occurs resulting in a molded plastic product.
RIM differs from conventional injection molding in a number of
important aspects. Conventional injection molding is conducted at
pressures of about 10,000 to 20,000 psi in the mold cavity by
melting a solid resin and conveying it into a mold maintained at a
temperature less than the melt temperature of the resin. At an
injection temperature of about 150.degree. to 350.degree. C.,
viscosity of the molten resin in conventional injection molding
process is generally in the range of 50,000 to 1,000,000 and
typically about 200,000 cps. In the injection molding process,
solidification of the resin occurs in about 10 to 90 seconds,
depending on the size of the molded product, following which, the
molded product is removed from the mold. There is no chemical
reaction occurring in a conventional injection molding process when
the resin is introduced into a mold.
[0122] In a RIM process, the viscosity of the materials fed to a
mix chamber is about 1 to 10,000 cps, preferably 1 to about 1500
cps, at injection temperatures varying from room temperature to
about 100.degree. C. for different cycloolefin monomer systems.
Mold temperatures in a RIM process are in the range of about
50.degree. C. to 150.degree. C. and pressures in the mold are
generally in the range of about 50 to 150 psi. At least one
component in the RIM formulation is a monomer that is polymerized
to a polymer in the mold. The main distinction between conventional
injection molding and RIM resides in the fact that in RIM, a
chemical reaction is initiated on mixing, with optional heating,
and is completed in the mold to transform monomers to a polymeric
state. For practical purposes, the chemical reaction must take
place rapidly in less than about 2 minutes.
[0123] Conventional injection molding can also be used to make
various embodiments of the invention. The term injection molding
refers to both conventional injection molding and the other types
of injection molding described herein and known or developed in the
art.
[0124] A LIM process is similar to a RIM system except that
generally an impingement head is not utilized. Instead, a simple
mixer is utilized such as a static mixer, an agitating mixer, and
the like. Moreover, in a LIM system, the injection molding cycle is
carried out over a longer period of time and thus the chemical
reaction can take place in a period of up to about 5 or 10
minutes.
[0125] Various reinforcing particles can also be utilized, that is
injected with the solution when utilizing either the RIM or the LIM
process. As a practical manner, the RIM process is not always
suitable and hence reinforced particles are generally utilized only
in a LIM process, that is a reinforced liquid injection molding
process. Another alternative is to utilize a mat that already
exists in a mold, for example a fiberglass mat, or the like.
Accordingly, such systems are called RMRIM, RMLIM, or RTM. Due to
the reaction cure times as well as injection molding times, the
RMLIM system is generally preferred for some operations, RMRIM or
RTM for others.
[0126] Hence, the blends or alloys of cycloolefins and suitable
copolymers can he utilized in any of the above noted bulk
polymerization systems as well as variations thereof. In as much as
the above systems are generally conventional or known to the art as
well as to the literature, they have not been discussed in detail,
but rather briefly discussed herein for purposes or brevity.
[0127] U.S. Pat. No. 4,426,502 to Minchak describes bulk (e.g.,
RIM) polymerization of cycloolefins using a modified co-catalyst
with a catalyst whereby polymerization of the cycloolefin monomers
can be conducted in absence of a solvent or a diluent. The
alkylaluminum halide co-catalyst is modified by pre-reacting it
with an alcohol or an active hydroxy-containing compound to form an
alkyoxyalkylaluminum halide or an aryloxyalk-ylaluminum halide that
is then used in the polymerization reaction. The pre-reaction can
be accomplished by using oxygen, an alcohol, or a phenol. Such
modification of the co-catalyst results in lowering of its reducing
potential of the catalyst.
[0128] Regardless of whether the halide metathesis or the
halogen-free metathesis catalyst system is utilized, the reaction
rate is generally slowed down by utilization of the above-described
alcohols. Thus, depending if little or no alcohol is utilized, the
halide metathesis catalyst system can cure the various cycloolefins
in a matter of minutes and even seconds. If high amounts of alcohol
are utilized, the cure can be a matter of hours and even days.
[0129] It is important to lower the reducing power of the
co-catalyst of either metathesis system in order to make such bulk
polymerization reactions practical. When a monomer diluted with
unmodified alkylaluminum co-catalyst is mixed with a
monomer-diluted catalyst to polymerize a cycloolefin, the reaction
is very rapid. In such systems, the polymerization is usually
unacceptable because polymer formed at the interfaces or the two
streams during intermingling prevents thorough mixing and results
in poor conversions. Modifying the co-catalyst by pre-reaction with
hydroxy-containing materials reduces the activity of the
co-catalyst to the point where adequate mixing of the liquid
components can occur and acceptable polymer products can be
produced. Sometimes, a cycloolefinic monomer will contain various
impurities that naturally reduce the activity of the co-catalyst.
In such cases, it is not necessary to add active hydroxy-containing
materials to reduce the activity of the co-catalyst. With the
modified co-catalyst, mixing or the cycloolefins, and other
components, can be carried out at lower temperatures, such as room
temperature, without immediately initiating polymerization. The
co-catalyst can be formulated to allow a reasonable pot life at
room temperature and thermal activation in the mold of the mixed
liquid components. The co-catalyst can also be formulated to give
mixing initiated RIM systems.
[0130] When utilizing a bulk polymerization method, the blend of
the cycloolefin monomers and the ethylene-based functional
copolymers as well as the catalyst and any optional additives
therein can be added to a bulk polymerizing mold having a
temperature well below the Tg of the polymerized cycloolefin
polymers. This is especially desirable since the reaction is
usually exothermic and can result in a temperature increase of the
mold up to about 120.degree. C. The final mold temperature is thus
from about 50.degree. C. to about 200.degree. C., generally from
about 50.degree. C. to about 150.degree. C., and preferably from
about 50.degree. C. to about 90.degree. C. Of course, such
temperatures will vary depending upon the specific type of catalyst
system utilized, the specific type of cycloolefin monomers, and the
like. When utilizing the catalyst systems described herein above,
the cycloolefin monomer and ethylene-based functional co-polymer
mixture has a good shelf life that is up to about 24 hours. Should
longer times be desirable, the catalyst system is not added to the
mixture but kept separate. Thus, upon the point in time of carrying
out the polymerization of the cycloolefin monomers, the catalyst
system is added to the mixture and polymerized in bulk. A preferred
method of polymerization includes the above noted RIM method.
[0131] A System for Spectroscopic Measurements
[0132] The present invention is a system for spectroscopic
measurement, comprising: a reagent for an assay, and a device
comprising at least one multi-well platform of the present
invention, and a second platform to hold said multi-well platform
for detecting a signal from a sample. The system can further
comprise a detector. In this context, a reagent for an assay
includes any reagent useful to perform biochemical or biological in
vitro or in vivo testing procedures, such as, for example, buffers,
proteins, carbohydrates, lipids, nucleic acids, active fragments
thereof, organic solvents such as DMSO, chemicals, analytes,
therapeutics, compositions, cells, antibodies, ligands, and the
like. In this context, an active fragment is a portion of a reagent
that has substantially the activity of the parent reagent. The
choice of a reagent depends on the type of assay to be performed.
For example, an immunoassay would include an immuno-reagent, such
as an antibody, or an active fragment thereof.
[0133] The present invention is directed to systems and methods
that utilize automated and integratable workstations for detecting
the presence of an analyte and identifying modulators or chemicals
having useful activity. The present invention is also directed to
chemical entities and information (e.g., modulators or chemical or
biological activities of chemicals) generated or discovered by
operation of workstations of the present invention.
[0134] The present invention includes automated workstations that
are programmably controlled to minimize processing times at each
workstation and that can be integrated to minimize the processing
time of the liquid samples from the start to finish of the process.
Typically, a system of the present invention would include: A) a
storage and retrieval module comprising storage locations for
storing a plurality of chemicals in solution in addressable
chemical wells, a chemical well retriever and having programmable
selection and retrieval of the addressable chemical wells and
having a storage capacity for at least 100,000 the addressable
wells, B) a sample distribution module comprising a liquid handler
to aspirate or dispense solutions from selected the addressable
chemical wells, the chemical distribution module having
programmable selection of, and aspiration from, the selected
addressable chemical wells and programmable dispensation into
selected addressable sample wells (including dispensation into
arrays of addressable wells with different densities of addressable
wells per centimeter squared), C) a sample transporter to transport
the selected addressable chemical wells to the sample distribution
module and optionally having programmable control of transport of
the selected addressable chemical wells (including adaptive routing
and parallel processing), D) a reaction module comprising either a
reagent dispenser to dispense reagents into the selected
addressable sample wells or a fluorescent detector to detect
chemical reactions in the selected addressable sample wells, and a
data processing and integration module.
[0135] The present invention can be used with systems and methods
that utilize automated and integratable workstations for
identifying modulators, pathways, chemicals having useful activity
and other methods described herein. Such systems are described
generally in the art (see, U.S. Pat. No.: 4,000,976 to Kramer et
al. (issued Jan. 4, 1977), U.S. Pat. No. 5,104,621 to Pfost et al.
(issued Apr. 14, 1992), U.S. Pat. No. 5,125,748 to Bjornson et al.
(issued Jun. 30, 1992), U.S. Pat. No. 5,139,744 to Kowalski (issued
Aug. 18, 1992), U.S. Pat. No. 5,206,568 Bjornson et al. (issued
Apr. 27, 1993), U.S. Pat. No. 5,350,564 to Mazza et al. (Sep. 27,
1994), U.S. Pat. No. 5,589,351 to Harootunian (issued Dec. 31,
1996), and PCT Application Nos: WO 93/20612 to Baxter Deutschland
GMBH (published Oct. 14, 1993), WO 96/05488 to McNeil et al.
(published Feb. 22, 1996) and WO 93/13423 to Agong et al.
(published Jul. 8, 1993).
[0136] The storage and retrieval module, the sample distribution
module, and the reaction module are integrated and programmably
controlled by the data processing and integration module. The
storage and retrieval module, the sample distribution module, the
sample transporter, the reaction module and the data processing and
integration module are operably linked to facilitate rapid
processing of the addressable sample wells. Typically, devices of
the invention can process at least 100,000 addressable wells in 24
hours. This type of system is described in U S. Ser. No. 08/858,016
by Stylli et al., filed May 16, 1997, entitled "Systems and method
for rapidly identifying useful chemicals in liquid samples," which
has attorney docket no. 08366/008001, which is incorporated herein
by reference.
[0137] If desired, each separate module is integrated and
programmably controlled to facilitate the rapid processing of
liquid samples, as well as being operably linked to facilitate the
rapid processing of liquid samples.
[0138] In one embodiment the invention provides for a reaction
module that is a fluorescence detector to monitor fluorescence. The
fluorescence detector is integrated to other workstations with the
data processing and integration module and operably linked with the
sample transporter. Preferably, the fluorescence detector is of the
type described herein and can be used for epi-fluorescence. Other
fluorescence detectors that are compatible with the data processing
and integration module and the sample transporter, if operable
linkage to the sample transporter is desired, can be used as known
in the art or developed in the future. For some embodiments of the
invention, particularly for plates with 96, 192, 384 and 864 wells
per plate, detectors are available for integration into the system.
Such detectors are described in U.S. Pat. No. 5,589,351
(Harootunian), U.S. Pat. No. 5,355,215 (Schroeder), and PCT patent
application WO 93/13423 (Akong). Each well of a multi-well platform
can be "read" sequentially. Alternatively, a portion of, or the
entire plate, can be "read" simultaneously using an imager, such as
a Molecular Dynamics Fluor-Imager 595 (Sunnyvale, Calif.).
[0139] Fluorescence Measurements
[0140] It is recognized that different types of fluorescent
monitoring systems can be used to practice the invention with
fluorescent probes, such as fluorescent dyes or substrates.
Preferably, systems dedicated to high throughput screening, e.g.,
96-well or greater microtiter plates, are used. Methods of
performing assays on fluorescent materials are well known in the
art and are described in, e.g., Lakowicz, J. R., Principles of
Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman,
B., Resonance Energy Transfer Microscopy, in: Fluorescence
Microscopy of Living Cells in Culture, Part B. Methods in Cell
Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego:
Academic Press (1989), pp. 219-243; Turro, N. J., Modern Molecular
Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.
(1978), pp. 296-361 and the Molecular Probes Catalog (1997), OR,
USA.
[0141] Fluorescence in a sample can be measured using a detector
described herein or known in the art for multi-well platforms. In
general, excitation radiation from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics causes the excitation radiation to excite the sample. In
response, fluorescent probes in the sample emit radiation that has
a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage (e.g., a dedicated
X, Y positioner) moves a multi-well platform holding a plurality of
samples in order to position different wells to be exposed. The
multi-axis translation stage, temperature controller, auto-focusing
feature, and electronics associated with imaging and data
collection can be managed by an appropriately programmed digital
computer. The computer also can transform the data collected during
the assay into another format for presentation.
[0142] Preferably, FRET (fluorescence resonance energy transfer) is
used as a way of monitoring probes in a sample (cellular or
biochemical). The degree of FRET can be determined by any spectral
or fluorescence lifetime characteristic of the excited construct,
for example, by determining the intensity of the fluorescent signal
from the donor, the intensity of fluorescent signal from the
acceptor, the ratio of the fluorescence amplitudes near the
acceptor's emission maxima to the fluorescence amplitudes near the
donor's emission maximum, or the excited state lifetime of the
donor. For example, cleavage of the linker increases the intensity
of fluorescence from the donor, decreases the intensity of
fluorescence from the acceptor, decreases the ratio of fluorescence
amplitudes from the acceptor to that from the donor, and increases
the excited state lifetime of the donor.
[0143] Preferably, changes in signal are determined as the ratio of
fluorescence at two different emission wavelengths, a process
referred to as "ratioing." Differences in the absolute amount of
probe (or substrate), cells, excitation intensity, and turbidity or
other background absorbances between addressable wells can affect
the fluorescence signal. Therefore, the ratio of the two emission
intensities is a more robust and preferred measure of activity than
emission intensity alone.
[0144] A ratiometric fluorescent probe system can be used with the
invention. For instance the reporter system described in PCT
publication WO 96/30540 (Tsien and Zlokarnik) has significant
advantages over existing reporters for gene integration analysis,
as it allows sensitive detection and isolation of both expressing
and non-expressing single living cells. This assay system uses a
non-toxic, non-polar fluorescent substrate that is easily loaded
and then trapped intracellularly. Cleavage of the fluorescent
substrate by .beta.-lactamase yields a fluorescent emission shift
as substrate is converted to product. Because the .beta.-lactamase
reporter readout is ratiometric, it is unique among reporter gene
assays in that it controls variables such as the amount of
substrate loaded into individual cells. The stable, easily
detected, intracellular readout simplifies assay procedures by
eliminating the need for washing steps, which facilitates screening
with cells using the invention.
[0145] Methods for Detecting the Presence an Analyte in a
Sample
[0146] A method of the present invention uses targets for detecting
the presence of an analyte, such as chemicals that are useful in
modulating the activity of a target, in a sample. Typically, as
discussed below targets can be proteins such as cell surface
proteins or enzymes. A biological process or a target can be
assayed in either biochemical assays (targets free of cells), or
cell based assays (targets associated with a cell). This method can
also be used to identify a modulator of a biological process or
target in a sample. This method detects the presence of an analyte
in a sample contained in a multi-well platform of the present
invention by detecting light emitted from the sample. The method
comprises the steps of: exciting at least one sample with radiation
of a first wavelength, wherein at least one sample suspected of
containing an analyte is placed into at least one well of a
multi-well platform of the present invention, which can contain a
biological process or target. The sample and biological process or
target can be contacted within the well, or outside of the well and
later placed within the well. The emission of radiation of a second
wavelength emitted from the sample is measured, wherein the amount
of radiation of a second wavelength measured indicates the presence
or absence of the analyte in the sample.
[0147] Targets can be cells, which may be loaded with ion or
voltage sensitive dyes to report receptor or ion channel activity,
such as calcium channels or N-methyl-D-aspartate (NMDA) receptors,
GABA receptors, kainate/AMPA receptors, nicotinic acetylcholine
receptors, sodium channels, calcium channels, potassium channels
excitatory amino acid (EAA) receptors, nicotinic acetylcholine
receptors. Assays for determining activity of such receptors can
also use agonists and antagonists to use as negative or positive
controls to assess activity of tested chemicals. In preferred
embodiments of automated assays for identifying chemicals that have
the capacity to modulate the function of receptors or ion channels
(e.g., agonists, antagonists), changes in the level of ions in the
cytoplasm or membrane voltage will be monitored using an
ion-sensitive or membrane voltage fluorescent indicator,
respectively. Among the ion-sensitive indicators and voltage probes
that may be employed, are those disclosed in the Molecular Probes
1997 Catalog, herein incorporated by reference.
[0148] Other methods of the present invention concern determining
the activity of receptors. Receptor activation can sometimes
initiate subsequent intracellular events that release intracellular
stores of calcium ions for use as a second messenger. Activation of
some G-protein-coupled receptors stimulates the formation of
inositol triphosphate (IP3 a G-protein coupled receptor second
messenger) through phospholipase C-mediated hydrolysis of
phosphatidylinositol, Berridge and Irvine (1984), Nature 312:
315-21. IP3 in turn stimulates the release of intracellular calcium
ion stores. Thus, a change in cytoplasmic calcium ion levels caused
by release of calcium ions from intracellular stores can be used to
reliably determine G-protein-coupled receptor function. Among
G-protein-coupled receptors are muscarinic acetylcholine receptors
(mAChR), adrenergic receptors, serotonin receptors, dopamine
receptors, angiotensin receptors, adenosine receptors, bradykinin
receptors, metabotropic excitatory amino acid receptors and the
like. Cells expressing such G-protein-coupled receptors may exhibit
increased cytoplasmic calcium levels as a result of contribution
from both intracellular stores and via activation of ion channels,
in which case it may be desirable, although not necessary, to
conduct such assays in calcium-free buffer, optionally supplemented
with a chelating agent such 3s EGTA, to distinguish fluorescence
response resulting from calcium release from internal stores.
[0149] Other assays can involve determining the activity of
receptors which, when activated, result in a change in the level of
intracellular cyclic nucleotides, e.g., CAMP, cGMP. For example,
activation of some dopamine, serotonin, metabotropic glutamate
receptors and muscarinic acetylcholine receptors results in a
decrease in the CAMP or cGMP levels of the cytoplasm. Furthermore,
there are cyclic nucleotide-gated ion channels, e.g., rod
photoreceptor cell channels and olfactory neuron channels (see,
Altenhofen. W. et al. (1991) Proc. Natl. Acad. Sci. USA
88:9868-9872 and Dhallan et al. (1990) Nature 347:184-187) that are
permeable to cations upon activation by binding of cAMP or cGMP. In
cases where activation of the receptor results in a decrease in
cyclic nucleotide levels, it may be preferable to expose the cells
to agents that increase intracellular cyclic nucleotide levels,
e.g., forskolin, prior to adding a receptor-activating compound to
the cells in the assay. Cells for this type of assay can be made by
co-transfection of a host cell with DNA encoding a cyclic
nucleotide-gated ion channel and DNA encoding a receptor (e.g.,
certain metabotropic glutamate receptors, muscarinic acetylcholine
receptors, dopamine receptors, serotonin receptors, and the like),
which, when activated, cause a change in cyclic nucleotide levels
in the cytoplasm.
[0150] Any cell expressing a protein target in sufficient quantity
for measurement in a cellular assay can be used with the invention.
Cells endogenously expressing a protein can work as well as cells
expressing a protein from heterologous nucleic acids. For example,
cells may be transfected with a suitable vector encoding one or
more such targets that are known to those of skill in the art or
may be identified by those of skill in the art. Although
essentially any cell which expresses endogenous ion channel or
receptor activity may be used, when using receptors or channels as
targets it is preferred to use cells transformed or transfected
with heterologous DNAs encoding such ion channels and/or receptors
so as to express predominantly a single type of ion channel or
receptor. Many cells that can be genetically engineered to express
a heterologous cell surface protein are known. Such cells include,
but are not limited to, baby hamster kidney (BHK) cells (ATCC No.
CCL10), mouse L cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB 152)
and 153 DG44 cells (see, Chasin (1986) Cell. Molec. Genet. 12: 555)
human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese
hamster ovary (CHO) cells (ATCC Nos. CRL96 18, CCL61, CRL9096), PC
12 cells (ATCC No. CRL 17.21) and COS-7 cells (ATCC No. CRL 1651).
Preferred cells for heterologous cell surface protein expression
are those that can be readily and efficiently transfected.
Preferred cells include Jurkat cells and HEK 293 cells, such as
those described in U.S. Pat. No. 5,024,939 and by Stillman et al.
(1985) Mol. Cell. Biol. 5:2051-2060.
[0151] Exemplary membrane proteins include, but are not limited to,
surface receptors and ion channels. Surface receptors include, but
are not limited to, muscarinic receptors, e.g., human M2 (GenBank
accession #M16404); rat M3 (GenBank accession #M16407); human M4
(GenBank accession #M16405); human M5 (Bonner, et al., (1988)
Neuron 1, pp. 403-410); and the like. Neuronal nicotinic
acetylcholine receptors include, but are not limited to, e.g., the
human .alpha..sub.2, .alpha..sub.3, and .beta..sub.2, subtypes
disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990, which is
hereby expressly incorporated by reference herein in its entirety);
the human .alpha..sub.5 subtype (Chini, et al. (1992) Proc. Natl.
Acad. Sci. USA 89:1572-1 576), the rat .alpha..sub.2 subunit (Wada,
et al. (1988) Science 240, pp. 330-334); the rat .alpha..sub.3
subunit (Boulter, et al. (1986) Nature 319, pp. 368-374); the rat
.alpha..sub.4 subunit (Goldman, et al. (1987) Cell 48, pp.
965-973); the rat .alpha..sub.5 subunit (Boulter, et al. (1990) J.
Biol. Chem. 265, pp. 4472-4482); the chicken .alpha..sub.7 subunit
(Couturier et al. (1990) Neuron 5:847-856); the rat .beta..sub.2
subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat
.beta.3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp.
6268-6272); the rat .beta..sub.4 subunit (Duvoisin, et al. (1989)
Neuron 3, pp. 487-496); combinations of the rat .alpha. subunits,
.beta. subunits and a and p subunits; GABA receptors, e.g., the
bovine n, and p, subunits (Schofield, et al. (1987) Nature 328, pp.
221-227); the bovine n, and a, subunits (Levitan, et al. (1988)
Nature 335, pp-76-79); the .gamma.-subunit (Pritchett, et al.
(1989) Nature 338, pp-582-585); the p, and p, subunits (Ymer, et
al. (1989) EMBO J. 8, pp. 1665-1670); the 6 subunit (Shivers, B. D.
(1989) Neuron 3, pp. 327-337); and the like. Glutamate receptors
include, but are not limited to, e.g., rat GluR1 receptor (Hollman,
et al. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3
receptors (Boulter et al. (1990) Science 249:1033-1037; rat GluR4
receptor (Keinanen et al. (1990) Science 249:556-560); rat GluRS
receptor (Bettler et al. (1990) Neuron 5:583-595) g rat GluR6
receptor (Egebjerg et al. (1991) Nature 351:745-748); rat GluR7
receptor (Bettler et al. (1992) Neuron 8:257-265); rat NMDARI
receptor (Moriyoshi et al. (1991) Nature 354:31-37 and Sugihara et
al. (1992) Biochem. Biophys. Res. Comm. 185:826-832); mouse NMDA e1
receptor (Meguro et al. (1992) Nature 357:70-74): rat NMDAR2A.
NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science
256:1217-1221); rat metabotropic mGluR1 receptor (Houamed et al.
(1991) Science 252:1318-1321); rat metabotropic mGluR2, mGluR3 and
mGluR4 receptors (Tanabe et al. (1992) Neuron 8:169-179); rat
metabotropic mGluR5 receptor (Abe et al. (1992) J. Biol. Chem.
267:13361-13368); and the like. Adrenergic receptors include, but
are not limited to, e.g., human p1 (Frielle, et al. (1987) Proc.
Natl. Acad. Sci. 84, pp. 7920-7924); human .alpha..sub.2 (Kobilka,
et al. (1987) Science 238, pp. 650-656); hamster .beta..sub.2
(Dixon, et al. (1986) Nature 321, pp. 75-79); and the like.
Dopamine receptors include, but are not limited to, e.g., human D2
(Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6); mammalian
dopamine D2 receptor (U S. Patent No. 5,128,254); rat (Bunzow, et
al. (1988) Nature 336, pp. 783-787); and the like. NGF receptors
include, but are not limited to, e.g., human NGF receptors
(Johnson, et al. (1986) Cell 47, pp. 545-554); and the like.
Serotonin receptors include, but are not limited to, e.g., human
5HT1a (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin
5HT1C receptor (U.S. Pat. No. 4,985,352); human 5HT1D (U.S. Pat.
No. 5,155,218); rat 5HT2 (Julius, et al. (1990) PNAS 87, pp.
928-932); rat 5HT1c (Julius, et al. (1988) Science 241, pp.
558-564); and the like.
[0152] Ion channels include, but are not limited to, calcium
channels comprised of the human calcium channel .alpha..sub.2
.beta. and/or .gamma.-subunits disclosed in commonly owned U.S.
application Ser. Nos. 07/745,206 and 07/868,354, filed Aug. 15,
1991 and April 10, 1992, respectively, the contents of which are
hereby incorporated by reference; (see also, WO89/09834; human
neuronal .alpha..sub.2 subunit); rabbit skeletal muscle a1 subunit
(Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal
muscle .alpha..sub.2 subunit (Ellis, et al. (1988) Science 241, pp.
1661-1664); rabbit skeletal muscle p subunit (Ruth, et al. (1989)
Science 245, pp. 1115-1118); rabbit skeletal muscle .gamma. subunit
(Jay, et al. (1990) Science 248, pp. 490-492); and the like.
Potassium ion channels include, but are not limited to, e.g., rat
brain (BK2) (McKinnon, D. (1989) J Biol Chem. 264, pp, 9230-8236);
mouse brain (BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839);
and the like. Sodium ion channels include, but are not limited to,
e.g., rat brain I and II (Noda, et al. (1986) Nature 320, pp.
188-192); rat brain III (Kayano, et al. (1988) FEBS Lett. 228, pp.
187-194); human II (ATCC No. 59742, 59743 and Genomics 5:204-208
(1989); chloride ion channels (Thiemann, et al. (1992), Nature 356,
pp. 57-60 and Paulmichl, et al. (1992) Nature 356, pp. 238-241),
and others known or developed in the art.
[0153] Intracellular receptors may also be used as targets, such as
estrogen receptors, glucocorticoid receptors, androgen receptors,
progesterone receptors, and mineralocorticoid receptors, in the
invention. Transcription factors and kinases can also be used as
targets, as well as plant targets.
[0154] Various methods of identifying activity of chemical with
respect to a target can be applied, including: ion channels (PCT
publication WO 93/13423) and intracellular receptors (PCT
publication WO 96/41013, U.S. Pat. No. 5,548,063, U.S. Pat. No.
5,171,671, U.S. Pat. No. 5,274,077, U.S. Pat. No. 4,981,784, EP 0
540 065 A1, U.S. Pat. No. 5,071,773, and U.S. Pat. No. 5,298,429).
All of the foregoing references are herein incorporated by
reference in their entirety.
[0155] If the analyte is present in the sample, then the target
will exhibit increased or decreased fluorescence. Such fluorescence
can be detected using the methods of the present invention by
exciting the sample with radiation of a first wavelength, which
excites a fluorescent reporter in the sample, which emits radiation
of a second wavelength, which can be detected. The amount of the
emission is measured, and compared to proper control or background
values. The amount of emitted radiation that differs from the
background and control levels: either increased or decreased,
correlates with the amount or potency of the analyte in the sample.
Standard curves can be determined to make the assay more
quantitative.
[0156] Testing a Therapeutic for Therapeutic Activity and
Toxicology
[0157] The present invention also provides a method for testing a
therapeutic for therapeutic activity and toxicology. A therapeutic
is identified by contacting a test chemical suspected of having a
modulating activity of a biological process or target with a
biological process or target in a multi-well platform of the
present invention. If the sample contains a modulator, then the
amount of a fluorescent reporter product in the sample, such as
inside or outside of the cell, will either increase or decrease
relative to background or control levels. The amount of the
fluorescent reporter product is measured by exciting the
fluorescent reporter product with an appropriate radiation of a
first wavelength and measuring the emission of radiation of a
second wavelength emitted from said sample. The amount of emission
is compared to background or control levels of emission If the
sample having the test chemical exhibits increased or decreased
emission relative to that of the control or background levels, then
a candidate modulator has been identified. The amount of emission
is related to the amount or potency of the therapeutic in the
sample. Such methods are described in, for example. Tsien
(PCT/US90/04059) The candidate modulator can be further
characterized and monitored for structure, potency, toxicology, and
pharmacology using well known methods.
[0158] The structure of a candidate modulator identified by the
invention can be determined or confirmed by methods known in the
art, such as mass spectroscopy. For putative modulators stored for
extended periods of time, the structure, activity, and potency of
the putative modulator can be confirmed.
[0159] Depending on the system used to identify a candidate
modulator, the candidate modulator will have putative
pharmacological activity. For example, if the candidate modulator
is found to inhibit T-cell proliferation (activation) in vitro,
then the candidate modulator would have presumptive pharmacological
properties as an immunosuppressant or anti-inflammatory (see,
Suthanthiran et al., Am. J. Kidney Disease 28:159-172 (1996)). Such
nexuses are known in the art for several disease states, and more
are expected to be discovered over time. Based on such nexuses,
appropriate confirmatory in vitro and in vivo models of
pharmacological activity, as well as toxicology, can be selected.
The methods described herein can also be used to assess
pharmacological selectivity and specificity, and toxicity.
[0160] Toxicology of Candidate Modulators
[0161] Once identified, candidate modulators can be evaluated for
toxicological effects using known methods (see, Lu, Basic
Toxicology, Fundamentals, Target Organs, and Risk Assessment,
Hemisphere Publishing Corp., Washington (1985); U.S. Pat. Nos.
5,196,313 to Culbreth (issued Mar. 23, 1993) and U.S. Pat. No.
5,567,952 to Benet (issued Oct. 22, 1996). For example, toxicology
of a candidate modulator can be established by determining in vitro
toxicity towards a cell line, such as a mammalian i.e. human cell
line. Candidate modulators can be treated with, for example, tissue
extracts such as preparations of liver, such as microsomal
preparations, to determine increased or decreased toxicological
properties of the chemical after being metabolized by a whole
organism. The results of these types of studies are often
predictive of toxicological properties of chemicals in animals,
such as mammals, including humans.
[0162] Alternatively, or in addition to these in vitro studies, the
toxicological properties of a candidate modulator in an animal
model, such as mice, rats, rabbits, or monkeys, can be determined
using established methods (see, Lu, supra (1985); and Creasey, Drug
Disposition in Humans, The Basis of Clinical Pharmacology, Oxford
University Press, Oxford (1979)). Depending on the toxicity, target
organ, tissue, locus, and presumptive mechanism of the candidate
modulator, the skilled artisan would not be burdened to determine
appropriate doses, LD.sub.50 values, routes of administration, and
regimes that would be appropriate to determine the toxicological
properties of the candidate modulator. In addition to animal
models, human clinical trials can be performed following
established procedures, such as those set forth by the United
States Food and Drug Administration (USFDA) or equivalents of other
governments. These toxicity studies provide the basis for
determining the efficacy of a candidate modulator in vivo.
[0163] Efficacy of Candidate Modulators
[0164] Efficacy of a candidate modulator can be established using
several art recognized methods, such as in vitro methods, animal
models, or human clinical trials (see, Creasey, supra (1979)).
Recognized in vitro models exist for several diseases or
conditions. For example, the ability of a chemical to extend the
life-span of HIV-infected cells in vitro is recognized as an
acceptable model to identify chemicals expected to be efficacious
to treat HIV infection or AIDS (see, Daluge et al., Antimicro.
Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of
cyclosporin A (CsA) to prevent proliferation of T-cells in vitro
has been established as an acceptable model to identify chemicals
expected to be efficacious as immunosuppressants (see, Suthanthiran
et al., supra, (1996)). For nearly every class of therapeutic,
disease, or condition, an acceptable in vitro or animal model is
available. Such models exist, for example, for gastro-intestinal
disorders, cancers, cardiology, neurobiology, and immunology. In
addition, these in v i m methods can use tissue extracts, such as
preparations of liver, such as microsomal preparations, to provide
a reliable indication of the effects of metabolism on the candidate
modulator. Similarly, acceptable animal models may be used to
establish efficacy of I treat various diseases or conditions. For
example, the rabbit knee is an accepted model for testing chemicals
for efficacy in treating arthritis (see, Shaw and Lacy, J Bone
Joint Surg. (Br) 55:197-205 (1973)). Hydrocortisone, which is
approved for use in humans to treat arthritis, is efficacious in
this model which confirms the validity of this model (see,
McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an
appropriate model to determine efficacy of a candidate modulator,
the skilled artisan can be guided by the state of the art to choose
an appropriate model, dose, and route of administration, regime,
and endpoint and as such would not be unduly burdened.
[0165] In addition to animal models, human clinical trials can be
used to determine the candidate modulator in humans. The USFDA, or
equivalent governmental we established procedures for such
studies.
[0166] Selectivity of Candidate Modulators
[0167] The in vitro and in vivo methods described above also
establish the selectivity of a candidate modulator. It is
recognized that chemicals can modulate a wide variety of biological
processes or be selective. Panels of cells based on the present
invention can be used to determine the specificity of the candidate
modulator. Selectivity is evident, for example, in the field of
chemotherapy, where the selectivity of a chemical to be toxic
towards cancerous cells, but not towards non-cancerous cells, is
obviously desirable. Selective modulators are preferable because
they have fewer side effects in the clinical setting. The
selectivity of a candidate modulator can be established in vitro by
testing the toxicity and effect of a candidate modulator on a
plurality of cell lines that exhibit a variety of cellular pathways
and sensitivities. The data obtained from these in vitro toxicity
studies can be extended animal model studies, including human
clinical trials, to determine toxicity, efficacy, and selectivity
of the candidate modulator.
[0168] Identified Compositions
[0169] The invention includes compositions such as novel chemicals,
and therapeutics identified as having activity by the operation of
methods, systems or components described herein. Novel chemicals,
as used herein, do not include chemicals already publicly known in
the art as of the filing date of this application. Typically, a
chemical would be identified as having activity from using the
invention and then its structure revealed from a proprietary
database of chemical structures or determined using analytical
techniques such as mass spectroscopy.
[0170] One embodiment of the invention is a chemical with useful
activity, comprising a chemical identified by the method described
above. Such compositions include small organic molecules, nucleic
acids, peptides and other molecules readily synthesized by
techniques available in the art and developed in the future. For
example, the following combinatorial compounds are suitable for
screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication No. WO 93/20242, 14 Oct.
1993), random bioooligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et
al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993), vinylogous
polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568
(1992)), nonpeptidyl peptidomimetics with a Beta-D-Glucose
scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc. 114:
9217-9218 (1992)), analogous organic syntheses of small compound
libraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),
oligocarbamates (Cho, C. Y. et al., Science 261:1303 (1993)),
and/or peptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem.
59:658 (1994)). See, generally, Gordon, E. M. et al., J. Med. Chem.
37: 1385 (1994). The contents of all of the aforementioned
publications are incorporated herein by reference.
[0171] The present invention also encompasses the identified
compositions in a pharmaceutical compositions comprising a
pharmaceutically acceptable carrier prepared for storage and
subsequent administration, which have a pharmaceutically effective
amount of he products disclosed above in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,
stabilizers, dyes and even flavoring agents may be provided in the
pharmaceutical composition. For example, sodium benzoate, sorbic
acid and esters of p-hydroxybenzoic acid may be added as
preservatives. In addition, antioxidants and suspending agents may
be used.
[0172] The compositions of the present invention may be formulated
and used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions,
suspensions for injectable administration; and the like.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, saline, dextrose, mannitol,
lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. If desired, absorption enhancing preparations
(e.g., liposomes), may be utilized.
[0173] The pharmaceutically effective amount of the composition
required as a dose will depend on the route of administration, the
type of animal being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize.
[0174] In practicing the methods of the invention, the products or
compositions can be used alone or in combination with one another,
or in combination with other therapeutic or diagnostic agents.
These products can be utilized in vivo, ordinarily in a mammal,
preferably in a human, or in vitro. In employing them in vivo, the
products or compositions can be administered to the mammal in a
variety of ways, including parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, nasally or
intraperitoneally, employing a variety of dosage forms. Such
methods may also be applied to testing chemical activity in
vivo.
[0175] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0176] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage for the products of the present
invention can range broadly depending upon the desired affects and
the therapeutic indication. Typically, dosages may be between about
10 kg/kg and 100 mg/kg body weight, preferably between about 100
.mu.g/kg and 10 mg/kg body weight. Administration is preferably
oral on a daily basis.
[0177] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See, e.g., Fingl et al., in The Pharmacological Basis
of Therapeutics, 1975.) It should be noted that the attending
physician would know how to and when to terminate, interrupt, or
adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the disorder of interest will vary with
the seventy of the condition to be treated and to the route of
administration. The seventy of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary
according to the age, body weight, and response of the individual
patient. A program comparable to that discussed above may be used
in veterinary medicine.
[0178] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,
Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parented delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0179] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0180] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external micro-environment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0181] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration may be in the form
of tablets, drages, capsules, or solutions. The pharmaceutical
compositions of the present invention may be manufactured in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving, granulating, drage-making, levitating, emulsifying,
encapsulating, entrapping, or lyophilizing processes.
[0182] Pharmaceutical formulations for parented administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0183] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or drage cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Drage cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dye-stuffs or pigments may be added to the
tablets or drage coatings for identification or to characterize
different combinations of active compound doses.
EXAMPLES
Example 1
Fluorescence Properties of Cycloolefins Compared to Glass and Other
Polymeric Materials
[0184] To investigate the fluorescence properties of various
selected materials, different polymeric films were tested for
fluorescence emission at predetermined excitation wavelengths and
compared to two types of glass sheets (standard). These experiments
were conducted using a SPEX Fluorolog 111 Fluorimeter with
excitation wavelengths between 315 and 425 nm. The films and glass
materials were disposed on a holder. The sample was positioned with
the excitation beam perpendicular to the sample face. The
fluorescence emission from the sample was collected off angle at
about 12.5 degrees. The material's fluorescence emission was
reflected off of a mirror and onto a monochrometer. The emission
radiation was selected by the monochromatic grating and was
detected by the photomultiplier tube of the instrument. The SPEX
Fluorolog 111 Fluorometer utilizes Raman radiation lines of water
to calibrate and background correct the instrument measurements
from day to day. This background correction was performed each day
before instrument use for calibration. The calibration file is
stored with the measurements made that day and then subsequent
measurements with the SPEX instrument can be compared directly and
corrected for instrument fluctuation.
[0185] The materials tested were 1) glass sheets (Coming Glass
Works cover-slip No 1 (catalog number 2935/58333 1) (average
thickness between about 130 and 170 micrometers), 2) polystyrene
films (ps1, ps2 (Gom Plastic Suppliers) and ps3 (from Dow Chemical
Company), 3) polycarbonate films (pc1 (from General Electric
Corporation) and pc2 (from Plastic Suppliers); 4) non-aromatic,
alkyl polymers (nap; obtained from Mobil Oil Company), 5)
cycloolefin copolymer film (coc; obtained from Hoechst, Topas (2
mil, or 50 micrometers thick)) and 6) Aclar (a fluorocarbon
material from Allied Signal).
[0186] Table 2 shows the fluorescence normalized emission data over
400 to 650 nm at three different excitation wavelengths. The data
is normalized to glass and to correct for instrumentation
fluctuation. Polystyrene, which is often used as a component of
multi-well plates (see Table 1), generated high background
fluorescence levels, consistent with its aromatic structure.
Surprisingly, polycarbonate, which is often a biocompatible
polymer, was generally better than polystyrene, especially at
longer wavelengths. Surprisingly, the non-aromatic, alkyl polymer
was generally the second best polymer across the range of
wavelengths tested. Also surprisingly, the cycloolefin copolymer
produced the best results and nearly approached the extremely low
fluorescence levels of glass.
2TABLE 2 Material Em = 400 Em = 425 Em = 450 Em = 475 Em = 500 Em =
550 Em = 600 Em = 650 Ex = 315 Glass 0.22513 0.25824 0.26817
0.30459 0.33107 0.38735 0.51316 Pc1 - 5 mil 3.31071 2.10230 2.01953
1.78778 1.41036 0.66876 0.60586 Pc2 - 5 mil 11.04128 7.04943
6.11517 5.18091 3.79367 1.70432 1.05317 Ps1 - 2 mil 2.45986 1.96447
1.93714 1.78340 1.52374 1.02494 1.18893 Ps2 - 2 mil 2.20826 1.72697
1.69866 1.64204 1.48633 1.07582 1.18906 Ps3 - 2 mil 4.55807 3.29823
3.00096 2.72352 2.34132 1.57409 1.98743 Nap - 1.5 mil 1.01919
0.75307 0.62850 0.52942 0.50110 0.56622 1.12111 Nap - 1.5 mil
0.52658 0.48978 0.42466 0.37654 0.38220 0.50960 1.00787 Coc - 2 mil
0.40485 0.40485 0.34256 0.31142 0.31142 0.41617 0.83234 Aclar - .75
mil 0.08473 0.08875 0.07864 0.07368 0.07503 0.09701 0.22497 Aclar -
3 mil 0.27245 0.28586 0.26367 0.26522 0.29309 0.44479 1.03199 Ex =
350 Glass 0.30790 0.20526 0.23837 0.17547 0.16222 0.17878 0.25492
Pc1 - 5 mil 0.77802 0.62572 0.60586 0.50323 0.42708 0.31452 0.33769
Pc2 - 5 mil 3.96354 2.74616 2.20826 1.61373 1.24568 0.75024 0.62284
Ps1 - 2 mil 1.28801 1.44858 2.22754 2.06013 1.78340 1.06594 0.84387
Ps2 - 2 mil 1.01919 1.34477 1.85437 1.84021 1.64204 1.08997 0.89180
Ps3 - 2 mil 2.13182 2.68388 3.47092 3.14252 2.68388 1.57692 1.29381
Nap - 1.5 mil 0.95408 0.80120 0.81536 0.59170 0.53508 0.58321
0.79554 Nap - 1.5 mil 0.53791 0.48695 0.55206 0.39918 0.39918
0.48129 0.69079 Coc - 2 mil 0.42466 0.38220 0.43033 0.31142 0.31142
0.38503 0.56056 Aclar - .75 mil 0.08689 0.08710 0.08669 0.07327
0.07224 0.08050 0.10733 Aclar - 3 mil 0.24045 0.23323 0.24974
0.21981 0.23375 0.31373 0.43756 Ex = 400 Glass 0.29134 0.21520
0.25492 0.18540 0.26817 0.43039 Pc1 - 5 mil 0.38073 0.30459 0.32114
0.22844 0.31783 0.48667 Pc2 - 5 mil 0.65115 0.59736 0.62284 0.43033
0.53791 0.77855 Ps1 - 2 mil 0.55347 0.55347 0.67646 0.43731 0.61155
0.91561 Ps2 - 2 mil 0.49544 0.50960 0.60869 0.46996 0.65115 1.00221
Ps3 - 2 mil 0.75873 0.80120 0.97107 0.63417 0.86065 1.24568 Nap -
1.5 mil 0.57754 0.59170 0.67663 0.50110 0.72476 1.08431 Nap - 1.5
mil 0.41900 0.39635 0.50394 0.42466 0.66248 1.05883 Coc - 2 mil
0.32558 0.33407 0.41900 0.37087 0.55489 0.87198 Aclar - .75 mil
0.06295 0.06295 0.07121 0.06966 0.10010 0.15686 Aclar - 3 mil
0.14138 0.14654 0.17750 0.20433 0.32405 0.47988
Example 2
Fluorescence Properties of Cycloolefins Compared to Glass and Other
Polymeric Materials
[0187] To further investigate fluorescence properties of various
selected materials, different polymeric films were tested for
fluorescence emission at predetermined excitation wavelengths and
compared to two types off used silica glass sheets (standard).
These experiments were conducted to simulate biochemical or
cell-based assays that involve aqueous media. Therefore, films were
mounted on a horizontal plastic holder to permit addition of a drop
of aqueous media. Three milliliters of water were dispensed onto
the film and fluorescence recorded using a Zeiss inverted
fluorescence microscope. Background in the absence of a film was
recorded and subtracted from signals in the presence of a film.
[0188] The materials tested were 1) glass sheets (Fisher cover-slip
Number 1 (Fisher Catalog number 12-542B (1996)), 2) polystyrene
films (ps1, ps2 (from Plastic Suppliers) and ps3 (from Dow Chemical
Company), 3) polycarbonate films (pc1 (from General Electric
Corporation) and pc2 (from Plastic Suppliers); 4) non-aromatic,
alkyl polymers (obtained from Mobil), 5) cycloolefin copolymer film
(coc; obtained from Hoechst, Topas), and 6) Aclar (a fluorocarbon
material from Allied Signal) and 7) Syran Wrap.
[0189] Table 3 shows the fluorescence normalized emission data at
460 nm at 350 and 405 nm (excitation wavelengths). The data is
normalized to glass. Polystyrene, which is often used as a
component of multi-well plates (see Table 1), generated high
background fluorescence levels, consistent with its aromatic
structure as in Example 1. In contrast to Example 1, polycarbonate,
which is often a biocompatible polymer, was worse than polystyrene,
especially at longer wavelengths. Generally consistent with Example
1, the non-aromatic, alkyl polymer was generally better than
polystyrene across the range of wavelengths tested. Generally
consistent with Example 1, the cycloolefin copolymer produced the
best results and surprisingly out performed the extremely low
fluorescence levels of glass. Aclar film also surprisingly produced
either low or extremely low fluorescence values relative to glass.
However, Aclar films were later found to have undesirable
manufacturing characteristics, such as bonding to other materials
and suitability for use in injection molding.
3TABLE 3 Material 350ex/460em Rank Material 405ex/460em Rank Fisher
#1 coverslip 1.02 1 Fisher #1 coverslip 1.03 1 Polycarbonate 5 mil
6.91 6 Polycarbonate 5 mil 19.79 6 Polystyrene 2 mil 3.57 5
Polystyrene 2 mil 3.36 4 NAP 1.5 ml 2.06 3 NAP 1.5 ml 5.76 3 NAP
1.5 ml 1.33 3 NAP 1.5 ml 3.51 3 coc#2 2 mil 1.58 2 coc#2 2 mil 2.60
2 coc#1 2 mil 1.22 2 coc#1 2 mil 1.59 2 Aclar sample (>2 yrs
old) 2.62 4 Aclar sample (>2 yrs old) 9.08 5 Fisher #1 coverslip
1.00 5 Fisher #1 coverslip 1.00 1 Polycarbonate 5 mil 5.15 9
Polycarbonate 5 mil 17.75 8 Polystyrene 2 mil 2.01 7 Polystyrene 2
mil 2.53 7 coc#2 A 2 mil 1.09 6 coc#2 A 2 mil 1.71 4 coc#2 B 2 mil
0.89 4 coc#2 B 2 mil 1.65 3 coc#1 2 mil 0.86 3 coc#1 2 mil 1.47 2
Aclar 3 mil (<1 yr old) 0.71 1 Aclar 3 mil (<1 yr old) 2.34 6
Aclar 0.75 mil (<1 yr old) 0.64 1 Aclar 0.75 mil (<1 yr old)
2.14 5 Syran wrap 4.18 8 Syran wrap 22.12 9
Example 3
Cycloolefins are Not CytoToxic To Cultured Cells
[0190] The cytotoxicity of cycloolefin was evaluated by incubating
cells in cycloolefin multi-well plates for 60 hours at 37.degree.
C. 1.8 .mu.L volumes of media containing about 90 Chinese hamster
ovary (CHO) were placed in cycloolefin multi-well plates using a
tapered pipette. A glass cover was placed over the wells to prevent
evaporation. Cells were incubated for 60 hours in a 5% CO.sub.2,
37.degree. C., 90% RH incubator. Cells were then tested for
viability by loading with the vital dye calcein. The CHO cells were
loaded by incubation in a solution containing 4 .mu.M calcein/AM
for 30 minutes at room temperature. Cells were inspected using both
phase contrast microscopy to determine the total number of cells
and fluorescence microscopy to determine the number of live cells.
Approximately, greater than 95% of cells were alive as indicated by
loading with calcein dye (approximately 200 cells/well).
Example 4
Cycloolefins Are Not CytoToxic to Cultured Cells and Can Be Used
for Drug Screening Assays
[0191] To investigate the cytotoxic properties of cyclolefins,
cycloolefin film were tested using an assay for cell viability.
CCF2 a vital dye, as described in PCT publication WO 96/30540
(Tsien), diffuses into cells and is trapped by living cells having
esterase activity that cleaves ester groups on the molecules which
results in a negatively charged molecule that is trapped inside the
cell. Trapped dye appears green inside of living cells and turns
blue in the presence of beta-lactarnase. CCF2 was incubated with
Jurkat cells for at least hour in a 1 microliter well having black
walls and a cycloolefin bottom, and fluorescence was appropriately
monitored. These Jurkat cells were constitutively expressing
.beta.-lactamase. Cells were cultured for 60 hours in the
conditions of Example 3. After 60 hours, .beta.-lactamase activity
was measured using CCF2. Cells appeared blue indicating that
.beta.-lactamase was indeed active in these cells, which normally
do not contain .beta.-lactamase. These results demonstrate that
cycloolefins can be used with sensitive fluorescent assays because
the films yield low fluorescent backgrounds. This is particularly
beneficial because it permits smaller assay volumes (e.g., 2
microliters or less) and the measurement of smaller signals (e.g.,
from fewer cells or fewer number of isolated biochemical
targets).
Example 5
High Density Multi-Well Platforms
[0192] FIG. 2 shows a preferred multi-well platform of the present
invention. A 240 well (5.times.48 wells) injection molded
multi-well platform and a 45 well (three sets of 3.times.5 wells)
multi-well platform, each having a well-center-to-well-center
distance of 1.5 mm, were made.
[0193] Injection Molded Multi-well Platform
[0194] This multi-well platform comprised a frame, wherein the wall
of a well was disposed in the frame. The frame was made of
cycloolefin copolymer, which was made optically opaque with about
2% black pigment (OmniColor.RTM. IM0055 Reed Spectrum, Holden,
Mass.). The frame was about 3.25 mm thick and was made by injection
molding. The bottom of the frame was substantially flat.
[0195] Each well had a bottom, which had a high transmittance
portion. The bottom had a thickness of about 50 micrometers and was
made of clear, flat, cycloolefin copolymer film. The frame and
bottom were joined by heat-sealing to from the wells. The wall of
each well was chamfered at about 2.87 degrees and the
well-center-to-well-center distance was about 1.5 millimeters. The
diameter of the wells at the bottom of the frame was about 0.95
mm.
[0196] The multi-well platform further comprised a groove 21 that
surrounded three of the four sides of the well matrix 22. These
multi-well platforms were used in fluorescent based assays as
described in the following examples.
[0197] Machined Multi-well Platform
[0198] Alternatively, the frame was machined from an acrylic plate
(black Acrylic butyl styrene (black ABS)) made optically opaque
with about 2% to 4% of back pigment, and a bottom. The bottom was a
glass plate 0.01 mm thick (borosilicate glass, Precision Glass)
attached to the bottom of the frame by adhesive silicone. The frame
and the bottom were combined for form a multi-well platform 13 mm
thick. In this multi-well platform the wells were not chamfered and
the well-center-to-well center distance was about 1.5 millimeters.
The diameter of each well was about 0.95 mm.
Example 6
Detection of Protease Activity in a Machined Multi-Well
Platform
[0199] In this example, trypsin activity in the machined multi-well
platform described in Example 5 was detected using a green
fluorescent protein tandem construct comprising two green
fluorescent protein molecules coupled by a linker as reported by
Tsien et al. (WO 97/28261). The two green-fluorescent protein
molecules can exhibit fluorescence resonance energy transfer
between themselves, and the linker comprises a trypsin substrate.
When a sample comprises this intact construct, fluorescence
resonance energy transfer between the green fluorescent protein
molecules causes the sample to fluoresce at 535 nm when excited
with light of about 400 nm. When the linker is cleaved with a
protease such as trypsin, the green fluorescent protein molecules
no longer exhibit fluorescence resonance energy transfer, and the
sample will fluoresce at 460 nm when exited with light of about 400
nm. The increase in the ratio of the emission of 460 nm and 535 nm
correlates with the protease activity in the sample.
[0200] To individual wells, 2.0 .mu.L of the same 1 .mu.M solution
of the tandem construct with or without 0.015 nM trypsin were
added. The bottom of each well was excited with light of 400 nm,
and the emission at 460 nm and 535 nm measured through the bottom
of each well. The samples were incubated at room temperature for
thirty minutes. The bottom of each well was exited again with light
of 400 nm, and the emission at 460 nm arid 535 nm measured through
the bottom of each well. As shown in Table 4, addition of trypsin
to the wells consistently elicited a greater than four fold
increase in the emission ratio.
4 TABLE 4 460/535 Emission Ratio Well Number No Trypsin Trypsin
Added 1 0.20 1.00 2 0.20 0.94 3 0.20 0.94 4 0.20 0.98 5 0.20
0.93
Example 7
Detection of an Activated Reporter Gene in a Cell
[0201] In the example, a concentration response of carbachol in a
Jurkat cell line stably transfected with a plasmid encoding the M1
muscarinic receptor and a NF-AT-.beta.-lactamase reporter gene. In
this transfected cell line, carbachol acts to stimulate the M1
muscarinic receptor so that the NFAT-.beta.-lactamase reporter gene
is expressed. When expressed, this gene produces .beta.-lactamase,
which can then be detected using a fluorescent probe, such as
CCF2/AM, that exhibits different emissions when intact and cleaved
by .beta.-lactamase as reported by, for example, Tsien et al. (WO
96/30540).
[0202] Jurkat cells used in this example were made using the
following procedures. Wild-type Jurkat cells were transfected with
plasmid 3XNFAT-blax by electroporation (regarding the plasmid
3XNFAT-blax, see generally Fiering, Genes and Development,
4:1823-1834 (1990)). This plasmid is driven by the 1L-2 minimum
promoter. A portion of this population of transfected cells was
seeded into 96-well plates with limited dilution and selected by
Zeocin.RTM. (250 .mu.g/ml). The clones in each well were screened
for CCF2AM staining in the presence and the absence of 10 nm PMA/2
.mu.M ionomycin (Calbiochem). FACS sorting was used to isolate
individual clones, which were further transfected with pcDNA3-M1,
which comprises pcDNA3 (Invitrogen) configured such that nucleic
acids encoding M1 can be expressed. These transfected cells were
selected using G418 (1mg/ml) for about 3 weeks. The clones in each
well were screened for CCF2-AM staining in the presence and absence
of 30 .mu.M Carbachol (Calbiochem). FACS sorting was used to
isolate individual clones.
[0203] Transfected Jurkat cells in 1.8 .mu.L of RPMT buffer were
dispensed at approximately 500 cells per well into individual wells
of injection molded multi-well platform described in Example 5.
These wells contained 0.3 to 31 nL of stock carbachol solution.
Cells were incubated for three hours at 37.degree. C. The solution
in each well was made one .mu.M CCF2/AM. The bottom of each well
was excited with light of 400 nm and the emission of light at 460
nm and 535 rim was detected and measured through the bottom of the
well. The ratio of the emission at these wavelengths is correlated
with .beta.-lactamase activity in the cell, which is correlated
with the stimulation of the cell. As shown in Table 5, the
stimulation of the cells, as measured by the ratio of emission at
460 nm and 535 nm, was dependent upon the concentration of
carbachol provided in the well.
5 TABLE 5 Carbachol Concentration Emission Ratio (.mu.M) (460/535)
0.09 1.28 0.17 2.44 0.43 3.38 0.87 5.90 1.70 7.70 4.30 8.90
Example 8
Detection of an Activated Reporter
[0204] In this example, a Jurkat cell line stably transfected with
a CMV-.beta.-lactamase reporter gene. When expressed, this gene
produces .beta.-lactamase, which can then be detected using a
fluorescent probe, CCF2/AM, that exhibits different fluorescent
emissions when intact or cleaved by .beta.-lactamase as reported by
Tsien et al. (WO 96/30540). The same Jurkat cell line without the
CMV-.beta.-lactamase reporter gene was used as a control.
[0205] The Jurkat cells used in this example were obtained in a
similar way as described in the example above. Briefly, pcDNA3-bla,
which encodes .beta.-lactamase operatively linked to the CMV
promoter, was transfected into wild type Jurkat cells. The G418 (1
mg/ml) selected population were stained with CCF2-AM and FACS
sorted.
[0206] Control and transfected Jurkat cells in RPMI buffer were
dispensed at approximately 800 cells per well into individual wells
of the machined multi-well platform described in Example 5. Cells
were incubated for ninety minutes at 23.degree. C. in an RPMI
buffer containing 10 .mu.M CCF2/AM as described above. The bottom
of each well was excited with light of 400 nm and the emission of
light at 460 and 535 nm was detected and measured through the
bottom of the well. The emission at that wavelength is correlated
with .beta.-lactamase activity in the cell, which is correlated
with the expression of .beta.-lactamase cell. As shown in Table 6,
about 800 cells expressing .beta.-lactamase showed at least a
twelve-fold increase in the ration of emission at 460 and 535
compared to control cells.
6TABLE 6 Emission Ratio Well Number Cell Type 460/535 1 Wild Type 1
2 Wild Type 1 3 Wild Type 1 4 Wild Type 1 5 Wild Type 1 6
CMV-.beta.-lactamase 13 7 CMV-.beta.-lactamase 13 8
CMV-.beta.-lactamase 13 9 CMV-.beta.-lactamase 13 10
CMV-.beta.-lactamase 13
[0207] Publications
[0208] All publications, including patent documents and scientific
articles, referred to in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication were individually incorporated by
reference.
[0209] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *