U.S. patent application number 14/602616 was filed with the patent office on 2015-07-16 for method for enhancing transport of semiconductor nanocrystals across biological membranes.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Marcel BRUCHEZ, Aquanette Burt, R. Daniels, Jennifer Dias, Berndt Lagerholm, Hongjian Liu, Danith Ly, Larry Mattheakis.
Application Number | 20150198606 14/602616 |
Document ID | / |
Family ID | 34911195 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150198606 |
Kind Code |
A1 |
BRUCHEZ; Marcel ; et
al. |
July 16, 2015 |
METHOD FOR ENHANCING TRANSPORT OF SEMICONDUCTOR NANOCRYSTALS ACROSS
BIOLOGICAL MEMBRANES
Abstract
Semiconductor nanoparticle complexes comprising semiconductor
nanoparticles in association with cationic polymers are described.
Also described are methods for enhancing the transport of
semiconductor nanoparticles across biological membranes to provide
encoded cells. The methods are particularly useful in multiplex
settings where a plurality of encoded cells are to be assayed. Kits
comprising reagents for performing such methods are also
provided.
Inventors: |
BRUCHEZ; Marcel;
(Pittsburgh, PA) ; Daniels; R.; (Palo Alto,
CA) ; Dias; Jennifer; (Dublin, CA) ;
Mattheakis; Larry; (Cupertino, CA) ; Liu;
Hongjian; (Cupertino, CA) ; Burt; Aquanette;
(San Francisco, CA) ; Lagerholm; Berndt; (Chapel
Hill, NC) ; Ly; Danith; (Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
34911195 |
Appl. No.: |
14/602616 |
Filed: |
January 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12905950 |
Oct 15, 2010 |
8993349 |
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14602616 |
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10735608 |
Dec 12, 2003 |
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12905950 |
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09972744 |
Oct 5, 2001 |
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10735608 |
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60312558 |
Aug 15, 2001 |
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60238677 |
Oct 6, 2000 |
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Current U.S.
Class: |
506/4 ; 435/34;
530/328 |
Current CPC
Class: |
G01N 2500/10 20130101;
G01N 2333/726 20130101; B82Y 15/00 20130101; G01N 33/5014 20130101;
G01N 33/5076 20130101; C07K 7/06 20130101; G01N 33/5008 20130101;
G01N 33/588 20130101; G01N 33/566 20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58; C07K 7/06 20060101 C07K007/06 |
Claims
1. A semiconductor nanoparticle complex comprising a semiconductor
nanoparticle associated with a cationic polymer capable of
enhancing the transport of the semiconductor nanoparticle across a
biological membrane.
2. The semiconductor nanoparticle complex of claim 1, wherein the
semiconductor nanoparticle is a semiconductor nanocrystal.
3. The semiconductor nanoparticle complex of claim 2, wherein the
semiconductor nanocrystal comprises a core is selected from the
group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe,
HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe,
BaTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb,
AlS, Ge, Si, Pb, PbS, PbSe, an alloy thereof, and a mixture
thereof.
4. The semiconductor nanoparticle complex of claim 4, wherein the
semiconductor nanocrystal core is CdSe.
5. The semiconductor nanoparticle complex of claim 3, wherein the
semiconductor nanocrystal core is surrounded by a semiconductor
shell.
6. The semiconductor nanoparticle complex of claim 5, wherein the
semiconductor shell comprises a semiconductor material selected
from the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,
HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,
BaSe, BaTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,
AlSb, AlS, Ge, Si, Pb, PbS, PbSe, an alloy thereof, and a mixture
thereof.
7. The semiconductor nanoparticle complex of claim 6, wherein the
semiconductor shell is ZnS.
8. The semiconductor nanoparticle complex of claim 1, wherein the
cationic polymer is tat peptide from the protein transduction
domain of the HIV tat protein.
9. The semiconductor nanoparticle complex of claim 8, wherein the
tat peptide comprises the sequence RKKRRQRRR (SEQ ID NO: 1).
10. The semiconductor nanoparticle complex of claim 1, wherein the
cationic polymer has from 5 to 25 contiguous Lys and/or Arg
residues.
11. The semiconductor nanoparticle complex of claim 1, wherein the
biological membrane is a cell membrane.
12-16. (canceled)
17. A method of enhancing the transport of a semiconductor
nanoparticle across a biological membrane comprising contacting a
cell with the semiconductor nanoparticle complex of claim 1, under
conditions that provide for the transport of the semiconductor
nanoparticle across the biological membrane.
18-23. (canceled)
24. A method of distinguishably identifying a cell, comprising: (a)
providing a cell; and (b) contacting the cell with a semiconductor
nanoparticle complex according to claim 1 under conditions in which
the semiconductor nanoparticle is transported across the cell
membrane to provide a labeled cell, thereby identifying the
cell.
25-30. (canceled)
31. A method of identifying a cell in a mixed population of cells,
comprising: (a) providing a first cell; (b) contacting the cell
with a semiconductor nanoparticle complex according to claim 1
under conditions in which the semiconductor nanoparticle is
transported across the cell membrane to provide an encoded first
cell; (c) mixing the encoded first cell with a second cell distinct
therefrom to form a mixed population of cells; (d) culturing the
mixed population of cells; (e) exposing the cultured mixed
population of cells to an excitation energy source; and (f)
detecting a semiconductor nanoparticle code to identify the encoded
cell.
32-37. (canceled)
38. A kit comprising a semiconductor nanoparticle complex according
to claim 1 and instructions for preparing encoded cells using the
semiconductor nanoparticle complex.
40-41. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/972,744, filed Oct. 3, 2001, from which
priority is claimed pursuant to 35 U.S.C. .sctn.120 and which is
incorporated herein by reference in its entirety, which in turn
claims the benefit of U.S. Ser. No. 60/238,677, filed Oct. 6, 2000,
and U.S. Ser. No. 60/312,558, filed Aug. 15, 2001, from which
applications priority is claimed under 35 USC .sctn.119(e)(1) and
both of which applications are incorporated herein by reference in
their entireties.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
filed entitled IVGN6204CON.txt which was created Jul. 16, 2007 and
is 2 KB in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0003] The present invention relates to semiconductor nanocrystal
probes for biological applications, and methods of screening
modulators of receptors using encoded cells. The invention also
relates to the use of cationic polymers for enhancing the transport
of semiconductor nanocrystals across biological membranes.
BACKGROUND OF THE INVENTION
[0004] Multiplexed assay formats are necessary to meet the demands
of today's high-throughput screening methods, and to match the
demands that combinatorial chemistry is putting on the established
discovery and validation systems for pharmaceuticals. In addition,
the ever-expanding repertoire of genomic information is rapidly
necessitating very efficient, parallel and inexpensive assay
formats. The requirements for all of these multiplexed assays are
ease of use, reliability of results, a high-throughput format, and
extremely fast and inexpensive assay development and execution.
[0005] For these high-throughput techniques, a number of assay
formats are currently available. Each of these formats has
limitations, however. By far the most dominant high-throughput
technique is based on the separation of different assays into
different regions of space. The 96-well plate format is the
workhorse in this arena. In 96-well plate assays, the individual
wells (which are isolated from each other by walls) are charged
with different components, the assay is performed and then the
assay result in each well measured. The information about which
assay is being run is carried with the well number, or the position
on the plate, and the result at the given position determines which
assays are positive. These assays can be based on
chemiluminescence, scintillation, fluorescence, absorbance,
scattering, or colorimetric measurements, and the details of the
detection scheme depend on the reaction being assayed. Assays have
been reduced in size to accommodate 1536 wells per plate, though
the fluid delivery and evaporation of the assay solution at this
scale are significantly more problematic. High-throughput formats
based on multi-well arraying require complex robotics and fluid
dispensing systems to function optimally. The dispensing of the
appropriate solutions to the appropriate bins on the plate poses a
challenge from both an efficiency and a contamination standpoint,
and pains must be taken to optimize the fluidics for both
properties. Furthermore, the throughput is ultimately limited by
the number of wells that one can put adjacent on a plate, and the
volume of each well. Arbitrarily small wells have arbitrarily small
volumes, resulting in a signal that scales with the volume,
shrinking proportionally to R.sup.3. The spatial isolation of each
well, and thereby each assay, comes at the cost of the ability to
run multiple assays in a single well. Such single-well multiplexing
techniques are not widely used, due in large part to the inability
to "demultiplex" or resolve the results of the different assays in
a single well. However, such multiplexing would obviate the need
for high-density well assay formats.
[0006] Each of the current techniques for ultra-high-throughput
assay formats suffers from severe limitations. The present
invention relates to methods for encoding spectra, which are
readable with a single light source for excitation, into cells,
which can be used in highly multiplexed assays.
[0007] The methods of the invention for encoding spectra can be
used, for example, for screening for drug candidates, such as
agonists or antagonists of receptors, for identifying new
receptors, or for obtaining functional information pertaining to
receptors, such as orphan G-protein coupled receptors (GPCRs).
GPCRs represent one of the most important families of drug targets.
G protein-mediated signaling systems have been identified in many
divergent organisms, such as mammals and yeast. GPCRs respond to,
among other extracellular signals, neurotransmitters, hormones,
odorants and light. GPCRs are thought to represent a large
superfamily of proteins that are characterized by the seven
distinct hydrophobic regions, each about 20-30 amino acids in
length, that forms the transmembrane domain. The amino acid
sequence is not conserved across the entire superfamily, but each
phylogenetically related subfamily contains a number of highly
conserved amino acid motifs that can be used to identify and
classify new members. Individual GPCRs activate particular signal
transduction pathways, although at least ten different signal
transduction pathways are known to be activated via GPCRs. For
example, the beta 2-adrenergic receptor (.E-backward.AR) is a
prototype mammalian GPCR. In response to agonist binding,
.E-backward.AR receptors activate a G protein (G.sub.s) which in
turn stimulates adenylate cyclase and cyclic adenosine
monophosphate production in the cell.
[0008] It has been postulated that members of the GPCR superfamily
desensitize via a common mechanism involving G protein-coupled
receptor kinase (GRK) phosphorylation followed by arrestin binding.
The protein .beta.-arrestin regulates GPCR signal transduction by
binding agonist-activated receptors that have been phosphorylated
by G protein receptor kinases. The .beta.-arrestin protein remains
bound to the GPCR during receptor internalization. The interaction
between a GPCR and .beta.-arrestin can be measured using several
methods. In one example, the .beta.-arrestin protein is fused to
green fluorescent protein to create a protein fusion (Barak et al.
(1997) J. Biol. Chem. 272(44):27497-500). The agonist-dependent
binding of .beta.-arrestin to a GPCR can be visualized by
fluorescence microscopy. Microscopy can also be used to visualize
the subsequent trafficking of the GPCR/.beta.-arrestin complex to
clathrin coated pits. Other methods for measuring binding of
.beta.-arrestin to a GPCR in live cells include techniques such as
FRET (fluorescence resonance energy transfer), BRET (bioluminescent
energy transfer) or enzyme complementation (Rossi et al. (1997)
Proc. Natl Acad. Sci. USA 94(16):8405-10).
[0009] At present, there are nearly 400 GPCRs whose natural ligands
and function are known. These known GPCRs, named for their
endogenous ligands, have been classified into five major
categories: Class-A Rhodopsin-like; Class-B Secretin-like; Class-C
Metabotropic glutamate/pheromone; Class-D Fungal pheromone; Class-E
cAMP (dictyostelium). Representative members of Class-A are the
amine receptors (e.g., muscarinic, nicotinic, adrenergic,
adenosine, dopamine, histamine and serotonin), the peptide
receptors (e.g., angiotensin, bradykinin, chemokines, endothelin
and opioid), the hormone receptors (e.g., follicle stimulating,
lutropin and thyrotropin), and the sensory receptors, including
rhodopsin (light), olfactory (smell) and gustatory (taste)
receptors. Representatives of Class-B include secretin, calcitonin,
gastrin and glucagon receptors. Much less is known about Classes
C-E.
[0010] Many available therapeutic drugs in use today target GPCRs,
as they mediate vital physiological responses, including
vasodilation, heart rate, bronchodilation, endocrine secretion, and
gut peristalsis (Wilson and Bergsma (2000) Pharm. News 7: 105-114).
For example, ligands to .E-backward.-adrenergic receptors are used
in the treatment of anaphylaxis, shock, hypertension, hypotension,
asthma and other conditions. Additionally, diseases can be caused
by the occurrence of spontaneous activation of GPCRs, where a GPCR
cellular response is generated in the absence of a ligand. Drugs
that are antagonists of GPCRs decrease this spontaneous activity (a
process known as inverse agonism) are important therapeutic agents.
Examples of commonly prescribed GPCR-based drugs include Atenolol
(Tenormin.RTM.), Albuterol (Ventolin.RTM.), Ranitidine
(Zantac.RTM.), Loratadine (Claritin.RTM.), Hydrocodone
(Vicodin.RTM.) Theophylline (TheoDur.RTM.), and Fluoxetine
(Prozac.RTM.).
[0011] Due to the therapeutic importance of GPCRs, methods for the
rapid screening of compounds for GPCR ligand activity are
desirable. Additionally, there is a need for methods of screening
orphan GPCRs for interactions with known and putative GPCR ligands
in order to characterize such receptors. The present invention
meets these and other needs.
[0012] Peptides and cationic polymers have been used to transport
various substances across biological membranes. For example,
Tkachenko et al., J. Am. Chem. Soc. (2003) 125:4700-4701 describes
gold nanoparticle-peptide complexes for targeting molecules to the
cell nucleus. U.S. Pat. No. 6,495,663 describes methods for
transporting drugs and macromolecules across biological membranes
using transport polymers, such as poly-Arg polymers, conjugated to
the agent to be transported. U.S. Pat. No. 4,847,240 describes the
use of high molecular weight polymers of lysine for increasing
transport of various drugs across cellular membranes. PCT Pub. No.
WO 94/04686 and Fawell et al., Proc. Natl. Acad. Sci. (1994)
91:664-668 proposed the use of fragments of the tat protein
containing the tat basic region (residues 49-57 having the sequence
RKKRRQRRR (SEQ ID NO: 1).
[0013] However, none of the above-described art pertains to the use
of cationic polymers to transport semiconductor nanoparticles
across biological membranes.
SUMMARY OF THE INVENTION
[0014] Methods and compositions for encoding cells with
semiconductor nanoparticles such as semiconductor nanocrystals,
other fluorescent species, or otherwise detectable species and
combinations thereof are provided. In one aspect, a method is
provided comprising the ability to separately identify individual
populations of cells in a mixture of different types of cells which
is highly advantageous for many applications. This method is
especially useful for identifying a population of cells derived
from an initial sample of one or more cells via its unique spectral
code after several cell divisions. The method facilitates analysis
of many otherwise identical cells which only differ by the presence
or absence of one or more genes and which are subjected to a
functional assay.
[0015] The ability to detect populations of cells derived from a
few precursors by virtue of their spectral code greatly facilitates
the high-throughput analysis of many systems. It allows the
identification of populations that have multiplied in a particular
environment in the absence of any further experimental processing.
The number of cells bearing the diluted code can be determined
using various spectral scanning devices.
[0016] Many specific binding interactions can only occur when at
least one of the binding partners is in its `natural` environment.
This environment is often the membrane of a cell. Therefore to have
a method to simultaneously interrogate multiple populations of cell
that are of different lineages or are expressing different binding
partners for a molecule of interest requires an ability to
separately encode those cells. This invention describes a method by
which this is done using SCNCs, other fluorescent species, or
otherwise detectable species and combinations thereof. This is
useful in, for example, high throughput cell based screening
systems. One example is the analysis of G-protein coupled receptors
and their binding partners--these receptors span lipid bilayers 7
times and can only bind their partners when in this
conformation.
[0017] Another utility for this invention is as a method for
separately coding cells in order to follow the fate of a specific
population of cells while it is in a mixed population.
[0018] The present invention also relates to methods for providing
enhanced transport of semiconductor nanoparticles, such as
semiconductor nanocrystals across cell membranes, thereby encoding
cells which can be used in highly multiplexed assays. The methods
of the invention for encoding spectra can be used, for example, in
multiplex settings where a plurality of different cell types are
encoded and assayed for a phenotype. The large number of
distinguishable semiconductor nanoparticles can be employed to
simultaneously analyze differently spectrally encoded cells. The
methods may be used to enhance transport of semiconductor
nanoparticles across any of a number of biological membranes
including, but not limited to, eukaryotic cell membranes,
prokaryotic cell membranes, and cell walls. The methods are also
useful in single-plex type assays, such as to follow cell mobility,
for cell tracking and in in vivo applications.
[0019] Accordingly, in one aspect, a method is provided for
enhancing the transport of semiconductor nanoparticles, such as
semiconductor nanocrystals and related species across biological
membranes. The method entails the use of these species with
associated cationic polymers. Labeling cells in this way allows
individual populations of cells in a mixture of different types of
cells to be separately identified. This method is especially useful
for identifying a population of cells derived from an initial
sample of one or more cells via its unique spectral code after
several cell divisions. The method facilitates analysis of many
otherwise identical cells which only differ by the presence or
absence of one or more genes and which are subjected to a
functional assay.
[0020] The ability to detect populations of cells derived from a
few precursors by virtue of their spectral code greatly facilitates
the high-throughput analysis of many systems. It allows the
identification of populations that have multiplied in a particular
environment in the absence of any further experimental processing.
The number of cells bearing the diluted code can be determined
using various spectral scanning devices.
[0021] Another utility for this invention is as a method for
separately coding cells in order to follow the fate of a specific
population of cells while it is in a mixed population.
[0022] The invention thus provides a semiconductor nanoparticle
complex comprising a semiconductor nanoparticle associated with a
cationic polymer capable of enhancing the transport of the
semiconductor nanoparticle across a biological membrane.
[0023] In certain embodiments, the semiconductor nanoparticle is a
semiconductor nanocrystal. The semiconductor nanocrystal can
comprise a core and optionally a shell. The core and the shell can
be selected from the group consisting of ZnS, ZnSe, ZnTe, CdS,
CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS,
SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs,
InSb, AlAs, AlP, AlSb, AlS, Ge, Si, Pb, PbS, PbSe, an alloy
thereof, and a mixture thereof.
[0024] In further embodiments, the cationic polymer is tat peptide
from the protein transduction domain of the HIV tat protein, such
as a peptide comprising the sequence RKKRRQRRR (SEQ ID NO: 1). In
other embodiments, the cationic polymer has from 5 to 25 contiguous
Lys and/or Arg residues.
[0025] In yet additional embodiments, the biological membrane is a
cell membrane.
[0026] In a further embodiment, the invention is directed to a
semiconductor nanocrystal complex comprising a semiconductor
nanocrystal associated with a cationic polymer capable of enhancing
the transport of the semiconductor nanocrystal across a cell
membrane, wherein the semiconductor nanocrystal comprises a core
and a shell, wherein the core and the shell are each selected from
the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,
HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,
BaSe, BaTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,
AlSb, AlS, Ge, Si, Pb, PbS, PbSe, an alloy thereof, and a mixture
thereof.
[0027] In certain embodiments, the core is CdSe and the shell is
ZnS.
[0028] In additional embodiments, the cationic polymer is tat
peptide from the protein transduction domain of the HIV tat
protein, such as a peptide comprising the sequence RKKRRQRRR (SEQ
ID NO: 1). In other embodiments, the cationic polymer has from 5 to
25 contiguous Lys and/or Arg residues.
[0029] In yet further embodiments, the invention is directed to a
method of enhancing the transport of a semiconductor nanoparticle
across a biological membrane comprising contacting a cell with any
of the semiconductor nanoparticles described above, under
conditions that provide for the transport of the semiconductor
nanoparticle across the biological membrane.
[0030] In additional embodiments, the invention is directed to a
method of distinguishably identifying a cell, comprising:
[0031] (a) providing a cell; and
[0032] (b) contacting the cell with any of the semiconductor
nanoparticle complexes described above, under conditions in which
the semiconductor nanoparticle is transported across the cell
membrane to provide a labeled cell, thereby identifying the
cell.
[0033] In yet further embodiments, the invention is directed to a
method of identifying a cell in a mixed population of cells,
comprising:
[0034] (a) providing a first cell;
[0035] (b) contacting the cell with any of the semiconductor
nanoparticle complexes described above, under conditions in which
the semiconductor nanoparticle is transported across the cell
membrane to provide an encoded first cell;
[0036] (c) mixing the encoded first cell with a second cell
distinct therefrom to form a mixed population of cells;
[0037] (d) culturing the mixed population of cells;
[0038] (e) exposing the cultured mixed population of cells to an
excitation energy source; and
[0039] (f) detecting a semiconductor nanoparticle code to identify
the encoded cell.
[0040] In certain embodiments of any of the methods above, the cell
is prokaryotic or eukaryotic. Moreover, the cell can be a mammalian
cell selected from the group consisting of a human cell, a mouse
cell, a rat cell, a bovine cell, and a hamster cell.
[0041] Kits comprising reagents useful for performing the methods
of the invention are also provided.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 is a pictorial representation illustrating a method
for introducing semiconductor nanocrystals (SCNCs), to which have
been conjugated cellular target-specific ligands, into live cells
using peptides that facilitate passage into cells.
[0043] FIG. 2 is a pictorial representation illustrating the use
SCNCs as a marker for identifying microinjected cells in which
SCNCs are microinjected either alone or together with other
molecules of interest to allow color-coded identification of a
particular microinjected cell.
[0044] FIG. 3 is a pictorial representation illustrating the use of
SCNCs as markers in multicolor immunofluorescent staining in which
(A) represents a co-injected protein detected by indirect
fluorescence with antibody conjugated to Fluo-4 or SCNC-5, (B)
represents a nucleus stained with Fluo-3 or SCNC-4, (C) represents
the cell marked with SCNC-1, (D) represents actin cytoskeleton
stained with Fluo-1 or SCNC-2 conjugated to phalloidin, and (E)
represents microtubules stained with Fluo-2 or SCNC-3 conjugated to
tubulin.
[0045] FIG. 4 is a pictorial representation illustrating a method
for introducing SCNCs into live cells in which the SCNC is enclosed
in a liposome which contains proteins to trigger receptor mediated
endocytosis and acid-induced fusogenic proteins.
[0046] FIG. 5 depicts a bioluminescence resonance energy transfer
experiment using semiconductor nanocrystals linked to a prospective
binding partner for a protein of interest; this conjugate is
introduced into cells expressing a fusion protein between the
protein of interest and a luciferase to determine if fluorescence
transfer occurs from the luciferase to the semiconductor
nanocrystal in vivo.
[0047] FIG. 6 depicts the conjugation of semiconductor nanocrystals
to different types of proteins for use in affinity targeting of
cells and subcellular structures.
[0048] FIG. 7 depicts the toxicity screening in a single well of a
single compound against a plurality of cell types encoded through
the techniques described herein.
[0049] FIG. 8 depicts a predictive in silico biodistribution and
toxicity model that integrates high throughput histological
information regarding prospective targets with a compound's
proteome-wide selectivity against those targets.
[0050] FIG. 9 lists some of the wide range of applications for
cells encoded with semiconductor nanocrystals.
[0051] FIG. 10A is a fluorescence micrograph of CHO cells and SCNCs
incubated in the presence of Chariot reagent as described in
Example 1.
[0052] FIG. 10B is a fluorescence micrograph of CHO cells and SCNCs
incubated in the absence of Chariot reagent as described in Example
1.
[0053] FIG. 11 is a fluorescence micrograph of CHO cells incubated
with 40 nM noncrosslinked polymer SCNC as described in Example
2.
[0054] FIG. 12 is a fluorescence micrograph of SKBR3 breast cancer
cells and green SCNCs transfected using BioPORTER reagent as
described in Example 3. Cells were also stained with herceptin
antibody.
[0055] FIG. 13A is a fluorescence micrograph of CHO cells
cotransfected with red polymer crosslinked SCNCs and EGFP/rac DNA
as described in Example 4 using a 535 nm emission filter.
[0056] FIG. 13B is a fluorescence micrograph of CHO cells
cotransfected with red polymer crosslinked SCNCs and EGFP/rac DNA
as described in Example 4 using a 625 nm emission filter.
[0057] FIG. 13C is a fluorescence micrograph showing the
fluorescence micrographs depicted in FIG. 13A and FIG. 13B
overlayed.
[0058] FIG. 14A is a graphical representation of raw spectra and of
four individual CHO cells encoded with green SCNCs using Chariot
reagent as described in Example 5.
[0059] FIG. 14B is a graphical representation of normalized spectra
and of four individual CHO cells encoded with green SCNCs using
Chariot reagent as described in Example 5.
[0060] FIG. 15A is a graphical representation of raw spectra of
five individual CHO cells encoded with red SCNCs using Chariot
reagent as described in Example 5.
[0061] FIG. 15B is a graphical representation of normalized spectra
of five individual CHO cells encoded with red SCNCs using Chariot
reagent as described in Example 5.
[0062] FIG. 16A is a graphical representation of raw spectra of
five individual CHO cells encoded with green and red SCNCs using
Chariot reagent as described in Example 5.
[0063] FIG. 16B is a graphical representation of normalized spectra
of five individual CHO cells encoded with green and red SCNCs using
Chariot reagent as described in Example 5.
[0064] FIG. 17 is a pictorial representation illustrating the
simultaneous single-plate screening of a plurality of different
encoded cells for their ability to grow under selective conditions
as described in Example 7.
[0065] FIG. 18 is a graphical representation of isoproterenol dose
responses of encoded or unencoded CHO cells expressing the MI
muscarinic receptor.
[0066] FIG. 19A illustrates the non-competed encoded CHO cells
expressing the b2 adrenergic receptor.
[0067] FIG. 19B illustrates the competition binding of 1 FM CGP
12177 (19B) to encoded CHO cells expressing the b2 adrenergic
receptor.
[0068] FIG. 20 shows the results of peptide mediated uptake of
SCNCs using biotinylated D-Arg9 (nine contiguous D-Arg residues),
and streptavidin conjugated quantum dots. HeLa Cells were labeled
with Qtracker.TM. 655 Reagent (Quantum Dot Corporation, Hayward,
Calif.) and a Leica SP-2 confocal microscope was used to observe
the Qtracker.TM. reagent in cytoplasm at an excitation of 488
nm.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of synthetic organic
chemistry, biochemistry, molecular biology, and the like, which are
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Sambrook, Fritsch & Maniatis,
Molecular Cloning: A Laboratory Manual, Second Edition (1989);
Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid
Hybridization (B. D. Haines & SJ. Higgins, eds., 1984); Methods
in Enzymology (Academic Press, Inc.); Kirk-Othmer's Encyclopedia of
Chemical Technology; and House's Modern Synthetic Reactions.
[0070] All publications mentioned herein are hereby incorporated by
reference for the purpose of disclosing and describing the
particular materials and methodologies for which the reference was
cited. The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
[0071] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a cell" includes two or more
cells, and the like.
[0072] Unless defined otherwise or the context clearly dictates
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. Although any methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the invention, the preferred
methods and materials are now described.
[0073] The following amino acid abbreviations are used throughout
the text: [0074] Alanine: Ala (A) Arginine: Arg (R) [0075]
Asparagine: Asn (N) Aspartic acid: Asp (D) [0076] Cysteine: Cys (C)
Glutamine: Gln (Q) [0077] Glutamic acid: Glu (E) Glycine: Gly (G)
[0078] Histidine: His (H) Isoleucine: Ile (I) [0079] Leucine: Leu
(L) Lysine: Lys (K) [0080] Methionine: Met (M) Phenylalanine: Phe
(F) [0081] Proline: Pro (P) Serine: Ser (S) [0082] Threonine: Thr
(T) Tryptophan: Trp (W) [0083] Tyrosine: Tyr (Y) Valine: Val
(V)
I. DEFINITIONS
[0084] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0085] The term "biological membrane" as used herein refers to a
lipid-containing barrier which separates cells or groups of cells
from the extracellular space. Biological membranes include, without
limitation, plasma membranes, cell walls, intracellular organelle
membranes, such as the mitochondrial membrane, nuclear membranes,
and the like
[0086] By a "enhancing transport" of a semiconductor nanoparticle,
such as a semiconductor nanocrystal or like species across a
biological membrane is meant that the transporting agent, such as
the cationic polymer, is effective to impart to the associated
semiconductor nanoparticle a degree (i.e., amount) and/or rate of
transmembrane transport across a biological membrane that is
greater than the degree and/or rate of transmembrane transport of
the semiconductor nanoparticle when it is not associated with the
transporting agent. Enhanced transport is determined by comparing
the delivery of the semiconductor nanoparticle that is not
associated with the cationic polymer with a semiconductor
nanoparticle so associated, delivered under the same
conditions.
[0087] The term "polymer" is used herein in its conventional sense
to refer to a compound having two or more monomer units, and is
intended to include linear and branched polymers, the term
"branched polymers" encompassing simple branched structures as well
as hyperbranched and dendritic polymers. The term "monomer" is used
herein to refers to compounds that are not polymeric. The term
"polymer" as used herein includes homopolymers and copolymers
(random and block). "Polymers" herein may be naturally occurring,
chemically modified, or chemically synthesized. A peptide is an
example of a polymer that can be composed of identical or
non-identical naturally occurring or non-naturally occurring amino
acid subunits that are joined by peptide linkages.
[0088] The term "cationic polymer" as used herein refers to a
polymer that includes a sequence of positive charges sufficient to
enhance transport of semiconductor nanoparticles across a
biological membrane of choice when in association with the polymer.
The polymer may have a net positive charge. Alternatively, the
multiple positive charges may form an adequate sequence in the
primary structure, or an adequate spacial arrangement in the
tertiary structure, or both, to cause enhanced cellular uptake,
even though the molecule does not have an overall net positive
charge. Preferably, the cationic polymer will include from about 5
to about 50 subunits, more preferably from about 5 to about 25
subunits, or any number within these ranges, such as 6, 7, 8, 9, 10
. . . 15 . . . 20 . . . 25, and so forth, with at least half of the
subunits bearing a positive charge.
[0089] The term "peptide" is used herein to refer to a compound
made up of a single chain of D- or L-amino acids or a mixture of D-
and L-amino acids joined by peptide bonds. Generally, peptides
contain at least two amino acid residues and are less than about 50
amino acids in length.
[0090] The terms "poly-arginine," "poly-lysine," "poly-Arg,"
"poly-Lys" and like terms with reference to other naturally and
non-naturally occurring amino acids refer to a polymeric sequence
composed of contiguous residues of the particular amino acid, such
as arginine, lysine, etc.; poly-L-arginine, poly-L-lysine, etc.,
refers to all L-arginines, L-lysines, etc.; poly-D-arginine,
poly-D-lysine, etc. refers to all D-arginines, D-lysines, etc.
Poly-L-arginine, poly-L-lysine, etc., are also abbreviated by the
three or one letter code for the amino acid shown in the table
herein, followed by the number of like residues in the peptide
(e.g., D-Arg9 represents a 9-mer of contiguous D-arginine
residues).
[0091] The term "guanidyl", "guanidinyl", and "guanidino" are used
interchangeably herein to refer to a moiety having a formula
--HN.dbd.C (NH.sub.2)NH (unprotenated form). As an example,
arginine contains a guanidyl (guanidino) moiety, and is also
referred to as 2-amino-5-guanidinovaleric acid or
.alpha.-amino-.delta.-guanidinovaleric acid. "Guanidinium" refers
to the positively charged conjugate acid form.
[0092] "Amidinyl" and "amidino" refer to a moiety having the
formula --C(.dbd.NH) (NH.sub.2). "Amidinium" refers to the
positively charged conjugate acid form.
[0093] The term "nanoparticle" refers to a particle, generally a
semiconductive or metallic particle, having a diameter in the range
of about 1 nm to about 1000 nm, preferably in the range of about 2
nm to about 50 nm, more preferably in the range of about 2 nm to
about 20 nm (for example about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 nm).
[0094] The terms "semiconductor nanoparticle" and "semiconductive
nanoparticle" refer to a nanoparticle as defined above that is
composed of an inorganic semiconductive material, an alloy or other
mixture of inorganic semiconductive materials, an organic
semiconductive material, or an inorganic or organic semiconductive
core contained within one or more semiconductive overcoat
layers.
[0095] The term "metallic nanoparticle" refers to a nanoparticle as
defined above that is composed of a metallic material, an alloy or
other mixture of metallic materials, or a metallic core contained
within one or more metallic overcoat layers.
[0096] The terms "semiconductor nanocrystal" (SCNC), "quantum dot"
and "Qdot.TM. nanocrystal" are used interchangeably herein to refer
to semiconductor nanoparticles composed of an inorganic crystalline
material that is luminescent (i.e., capable of emitting
electromagnetic radiation upon excitation), and that include an
inner core of one or more first semiconductor materials that is
optionally contained within an overcoating or "shell" of a second
semiconductor material. A semiconductor nanocrystal core surrounded
by a semiconductor shell is referred to as a "core/shell"
semiconductor nanocrystal. The surrounding shell material will
preferably have a bandgap energy that is larger than the bandgap
energy of the core material and may be chosen to have an atomic
spacing close to that of the core substrate. Suitable semiconductor
materials for the core and/or shell include, but are not limited
to, the following: materials comprised of a first element selected
from Groups 2 and 12 of the Periodic Table of the Elements and a
second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CDs,
CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS,
SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of
a first element selected from Group 13 of the Periodic Table and a
second element selected from Group 15 (GaN, GaP, GaAs, GaSb, InN,
InP, InAs, InSb, and the like); materials comprised of a Group 14
element (Ge, Si, and the like); materials such as PbS, PbSe and the
like; and alloys and mixtures thereof. As used herein, all
references to the Periodic Table of the Elements and groups thereof
is with reference to the new IUPAC system for numbering element
groups, as set forth in the Handbook of Chemistry and Physics,
81.sup.st Edition (CRC Press, 2000).
[0097] An SCNC is optionally surrounded by a "coat" of an organic
capping agent. The organic capping agent may be any number of
materials, but has an affinity for the SCNC surface. In general,
the capping agent can be an isolated organic molecule, a polymer
(or a monomer for a polymerization reaction), an inorganic complex,
or an extended crystalline structure. The coat can be used to
convey solubility, e.g., the ability to disperse a coated SCNC
homogeneously into a chosen solvent, functionality, binding
properties, or the like. In addition, the coat can be used to
tailor the optical properties of the SCNC.
[0098] Thus, the terms "semiconductor nanocrystal," "SCNC,"
"quantum dot" and "Qdot.TM. nanocrystal" as used herein include a
coated SCNC core, as well as a core/shell SCNC.
[0099] "Monodisperse" particles include a population of particles
wherein at least about 60% of the particles in the population, more
preferably about 75% to about 90% or more, or any percentage within
these stated ranges, of the particles in the population fall within
a specified particle size range. A population of monodisperse
particles deviates less than 10% rms (root-mean-square) in
diameter, and preferably deviates less than 5% rms.
[0100] The phrase "one or more sizes of SCNCs" is used synonymously
with the phrase "one or more particle size distributions of SCNCs."
One of ordinary skill in the art will realize that particular sizes
of SCNCs are actually obtained as particle size distributions.
[0101] By "luminescence" is meant the process of emitting
electromagnetic radiation (light) from an object. Luminescence
results when a system undergoes a transition from an excited state
to a lower energy state with a corresponding release of energy in
the form of a photon. These energy states can be electronic,
vibrational, rotational, or any combination thereof. The transition
responsible for luminescence can be stimulated through the release
of energy stored in the system chemically or added to the system
from an external source. The external source of energy can be of a
variety of types including chemical, thermal, electrical, magnetic,
electromagnetic, and physical, or any other type of energy source
capable of causing a system to be excited into a state higher in
energy than the ground state. For example, a system can be excited
by absorbing a photon of light, by being placed in an electrical
field, or through a chemical oxidation-reduction reaction. The
energy of the photons emitted during luminescence can be in a range
from low-energy microwave radiation to high-energy x-ray radiation.
Typically, luminescence refers to photons in the range from UV to
IR radiation.
[0102] "Preferential binding" refers to the increased propensity of
one member of a binding pair to bind to a second member as compared
to other components in the sample.
[0103] The terms "polynucleotide," "oligonucleotide," "nucleic
acid" and "nucleic acid molecule" are used interchangeably herein
to refer to a polymeric form of nucleotides of any length, and may
comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or
mixtures thereof. This term refers only to the primary structure of
the molecule. Thus, the term includes triple-, double- and
single-stranded deoxyribonucleic acid ("DNA"), as well as triple-,
double- and single-stranded ribonucleic acid ("RNA"). It also
includes modified, for example by alkylation, and/or by capping,
and unmodified forms of the polynucleotide. More particularly, the
terms "polynucleotide," "oligonucleotide," "nucleic acid" and
"nucleic acid molecule" include polydeoxyribonucleotides
(containing 2-deoxy-D-ribose), polyribonucleotides (containing
D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or
unspliced, any other type of polynucleotide which is an N- or
C-glycoside of a purine or pyrimidine base, and other polymers
containing nonnucleotidic backbones, for example, polyamide (e.g.,
peptide nucleic acids (PNAs)) and polymorpholino (commercially
available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene)
polymers, and other synthetic sequence-specific nucleic acid
polymers providing that the polymers contain nucleobases in a
configuration which allows for base pairing and base stacking, such
as is found in DNA and RNA. There is no intended distinction in
length between the terms "polynucleotide," "oligonucleotide,"
"nucleic acid" and "nucleic acid molecule," and these terms are
used interchangeably herein. These terms refer only to the primary
structure of the molecule. Thus, these terms include, for example,
3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3'-P5'
phosphoramidates, oligodeoxyribonucleotide N3'-P5'
thiophosphoramidates, 2'-O-alkyl-substituted RNA, double- and
single-stranded DNA, as well as double- and single-stranded RNA,
and hybrids thereof including for example hybrids between DNA and
RNA or between PNAs and DNA or RNA, and also include known types of
modifications, for example, labels, alkylation, "caps,"
substitution of one or more of the nucleotides with an analog,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, thiophosphoramidates, carbamates, etc.), with
negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters),
those containing pendant moieties, such as, for example, proteins
(including enzymes (e.g. nucleases), toxins, antibodies, signal
peptides, poly-L-lysine, etc.), those with intercalators (e.g.,
acridine, psoralen, etc.), those containing chelates (of, e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide or oligonucleotide.
[0104] It will be appreciated that, as used herein, the terms
"nucleoside" and "nucleotide" will include those moieties which
contain not only the known purine and pyrimidine bases, but also
other heterocyclic bases which have been modified. Such
modifications include methylated purines or pyrimidines, acylated
purines or pyrimidines, or other heterocycles. Modified nucleosides
or nucleotides can also include modifications on the sugar moiety,
e.g., wherein one or more of the hydroxyl groups are replaced with
halogen, aliphatic groups, or are functionalized as ethers, amines,
or the like. The term "nucleotidic unit" is intended to encompass
nucleosides and nucleotides.
[0105] Furthermore, modifications to nucleotidic units include
rearranging, appending, substituting for or otherwise altering
functional groups on the purine or pyrimidine base that form
hydrogen bonds to a respective complementary pyrimidine or purine.
The resultant modified nucleotidic unit optionally may form a base
pair with other such modified nucleotidic units but not with A, T,
C, G or U. A basic sites may be incorporated which do not prevent
the function of the polynucleotide. Some or all of the residues in
the polynucleotide can optionally be modified in one or more
ways.
[0106] Standard A-T and G-C base pairs form under conditions which
allow the formation of hydrogen bonds between the N3-H and C4-oxy
of thymidine and the N1 and C6-NH.sub.2, respectively, of adenosine
and between the C2-oxy, N3 and C4-NH.sub.2, of cytidine and the
C2-NH.sub.2, N'--H and C6-oxy, respectively, of guanosine. Thus,
for example, guanosine
(2-amino-6-oxy-9-.beta.-D-ribofuranosyl-purine) may be modified to
form isoguanosine (2-oxy-6-amino-9-.beta.-D-ribofuranosyl-purine).
Such modification results in a nucleoside base which will no longer
effectively form a standard base pair with cytosine. However,
modification of cytosine
(1-.beta.-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form
isocytosine (1-.beta.-D-ribofuranosyl-2-amino-4-oxy-pyrimidine)
results in a modified nucleotide, which will not effectively base
pair with guanosine but will form a base pair with isoguanosine.
Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.);
isocytidine may be prepared by the method described by Switzer et
al. (1993) Biochemistry 32:10489-10496 and references cited
therein; 2'-deoxy-5-methyl-isocytidine may be prepared by the
method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and
references cited therein; and isoguanine nucleotides may be
prepared using the method described by Switzer et al. (1993),
supra, and Mantsch et al. (1993) Biochem. 14:5593-5601, or by the
method described in U.S. Pat. No. 5,780,610 to Collins et al. Other
nonnatural base pairs may be synthesized by the method described in
Piccirilli et al. (1990) Nature 343:33-37 for the synthesis of
2,6-diaminopyrimidine and its complement
(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such
modified nucleotidic units which form unique base pairs are known,
such as those described in Leach et al. (1992) J. Am. Chem. Soc.
114:3675-3683 and Switzer et al., supra.
[0107] "Nucleic acid probe" and "probe" are used interchangeably
and refer to a structure comprising a polynucleotide, as defined
above, that contains a nucleic acid sequence that can bind to a
corresponding target. The polynucleotide regions of probes may be
composed of DNA, and/or RNA, and/or synthetic nucleotide
analogs.
[0108] "Complementary" or "substantially complementary" refers to
the ability to hybridize or base pair between nucleotides or
nucleic acids, such as, for instance, between the two strands of a
double stranded DNA molecule or between a polynucleotide primer and
a primer binding site on a single stranded nucleic acid to be
sequenced or amplified. Complementary nucleotides are, generally, A
and T (or A and U), or C and G. Two single-stranded RNA or DNA
molecules are said to be substantially complementary when the
nucleotides of one strand, optimally aligned and compared and with
appropriate nucleotide insertions or deletions, pair with at least
about 80% of the nucleotides of the other strand, usually at least
about 90% to 95%, and more preferably from about 98 to 100%.
[0109] Alternatively, substantial complementarity exists when an
RNA or DNA strand will hybridize under selective hybridization
conditions to its complement. Typically, selective hybridization
will occur when there is at least about 65% complementary over a
stretch of at least 14 to 25 nucleotides, preferably at least about
75%, more preferably at least about 90% complementary. See,
Kanehisa (1984) Nucleic Acids Res. 12:203.
[0110] "Preferential hybridization" as a form of preferential
binding refers to the increased propensity of one polynucleotide to
bind to a complementary target polynucleotide in a sample as
compared to noncomplementary polynucleotides in the sample or as
compared to the propensity of the one polynucleotide to form an
internal secondary structure such as a hairpin or stem-loop
structure under at least one set of hybridization conditions.
[0111] Stringent hybridization conditions will typically include
salt concentrations of less than about 1M, more usually less than
about 500 mM and preferably less than about 200 mM. Hybridization
temperatures can be as low as 5 C, but are typically greater than
22 C, more typically greater than about 30 C, and preferably in
excess of about 37 C. Longer fragments may require higher
hybridization temperatures for specific hybridization. Other
factors may affect the stringency of hybridization, including base
composition and length of the complementary strands, presence of
organic solvents and extent of base mismatching, and the
combination of parameters used is more important than the absolute
measure of any one alone. Other hybridization conditions which may
be controlled include buffer type and concentration, solution pH,
presence and concentration of blocking reagents to decrease
background binding such as repeat sequences or blocking protein
solutions, detergent type(s) and concentrations, molecules such as
polymers which increase the relative concentration of the
polynucleotides, metal ion(s) and their concentration(s),
chelator(s) and their concentrations, and other conditions known in
the art. Less stringent, and/or more physiological, hybridization
conditions are used where a labeled polynucleotide amplification
product cycles on and off a substrate linked to a complementary
probe polynucleotide during a real-time assay which is monitored
during PCR amplification such as a molecular beacon assay. Such
less stringent hybridization conditions can also comprise solution
conditions effective for other aspects of the method, for example
reverse transcription or PCR.
[0112] The terms "aptamer" (or "nucleic acid antibody") is used
herein to refer to a single- or double-stranded polynucleotide that
recognizes and binds to a desired target molecule by virtue of its
shape. See, e.g., PCT Publication Nos. WO 92/14843, WO 91/19813,
and WO 92/05285.
[0113] "Polypeptide" and "protein" are used interchangeably herein
and include a molecular chain of amino acids linked through peptide
bonds. The terms do not refer to a specific length of the product.
Thus, "peptides," "oligopeptides," and "proteins" are included
within the definition of polypeptide. The terms include
polypeptides contain co- and/or post-translational modifications of
the polypeptide, for example, glycosylations, acetylations,
phosphorylations, and sulphations. In addition, protein fragments,
analogs (including amino acids not encoded by the genetic code,
e.g., homocysteine, ornithine, D-amino acids, and creatine),
natural or artificial mutants or variants or combinations thereof,
fusion proteins, derivatized residues (e.g., alkylation of amine
groups, acetylations or esterifications of carboxyl groups) and the
like are included within the meaning of polypeptide.
[0114] The terms "substrate" and "support" are used interchangeably
and refer to a material having a rigid or semi-rigid surface.
[0115] As used herein, the term "binding pair" refers to first and
second molecules that bind specifically to each other with greater
affinity than to other components in the sample. The binding
between the members of the binding pair is typically noncovalent.
Exemplary binding pairs include immunological binding pairs (e.g.,
any haptenic or antigenic compound in combination with a
corresponding antibody or binding portion or fragment thereof, for
example digoxigenin and anti-digoxigenin, fluorescein and
anti-fluorescein, dinitrophenol and anti-dinitrophenol,
bromodeoxyuridine and anti-bromodeoxyuridine, mouse immunoglobulin
and goat anti-mouse immunoglobulin) and nonimmunological binding
pairs (e.g., biotin-avidin, biotin-streptavidin,
biotin-neutravidin, hormone [e.g., thyroxine and cortisol]-hormone
binding protein, receptor-receptor agonist or antagonist (e.g.,
acetylcholine receptor-acetylcholine or an analog thereof)
IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,
enzyme-enzyme-inhibitor, and complementary polynucleotide pairs
capable of forming nucleic acid duplexes) and the like. One or both
member of the binding pair can be conjugated to additional
molecules.
[0116] Terms such as "connected," "attached," "linked," and
"conjugated" are used interchangeably herein and encompass direct
as well as indirect connection, attachment, linkage or conjugation,
including covalent and non-covalent binding, adsorption, physical
immobilization and the like, unless the context clearly dictates
otherwise.
[0117] Where a range of values is recited, it is to be understood
that each intervening integer value, and each fraction thereof,
between the recited upper and lower limits of that range is also
specifically disclosed, along with each subrange between such
values. The upper and lower limits of any range can independently
be included in or excluded from the range, and each range where
either, neither or both limits are included is also encompassed
within the invention. Where a value being discussed has inherent
limits, for example where a component can be present at a
concentration of from 0 to 100%, or where the pH of an aqueous
solution can range from 1 to 14, those inherent limits are
specifically disclosed. Where a value is explicitly recited, it is
to be understood that values which are about the same quantity or
amount as the recited value are also within the scope of the
invention.
[0118] Where a combination is disclosed, each subcombination of the
elements of that combination is also specifically disclosed and is
within the scope of the invention. Conversely, where different
elements or groups of elements are disclosed, combinations thereof
are also disclosed. Where any element of an invention is disclosed
as having a plurality of alternatives, examples of that invention
in which each alternative is excluded singly or in any combination
with the other alternatives are also hereby disclosed; more than
one element of an invention can have such exclusions, and all
combinations of elements having such exclusions are hereby
disclosed.
[0119] The terms "specific-binding molecule" and "affinity
molecule" are used interchangeably herein and refer to a molecule
that will selectively bind, through chemical or physical means to a
detectable substance present in a sample. By "selectively bind" is
meant that the molecule binds preferentially to the target of
interest or binds with greater affinity to the target than to other
molecules. For example, an antibody will selectively bind to the
antigen against which it was raised; A DNA molecule will bind to a
substantially complementary sequence and not to unrelated
sequences. The affinity molecule can comprise any molecule, or
portion of any molecule, that is capable of being linked to a
semiconductor nanocrystal and that, when so linked, is capable of
recognizing specifically a detectable substance. Such affinity
molecules include, by way of example, such classes of substances as
antibodies, as defined below, monomeric or polymeric nucleic acids,
aptamers, proteins, polysaccharides, sugars, and the like. See,
e.g., Haugland, "Handbook of Fluorescent Probes and Research
Chemicals" (Sixth Edition), and any of the molecules capable of
forming a binding pair as described above.
[0120] An "SCNC conjugate" is an SCNC linked to a first member of a
binding pair, as defined above, or an SCNC linked to a cationic
polymer. For example, an SCNC is "linked" or "conjugated" to, or
chemically "associated" with, such molecules when the SCNC is
coupled to, or physically associated with the molecule. Thus, these
terms intend that the SCNC may either be directly linked to the
molecule or may be linked via a linker moiety, such as via a
chemical linker. The terms indicate items that are physically
linked by, for example, covalent chemical bonds, physical forces
such van der Waals or hydrophobic interactions, encapsulation,
embedding, non-covalent interactions, adsorption and the like. For
example, nanocrystals can be associated with biotin which can bind
to the proteins avidin, streptavidin, neutravidin, and the
like.
[0121] When used in relation to a composition comprising a cell and
an SCNC or other detectable moiety, the term "associated" is
intended to include cells in which the SCNC is contained in the
nucleus, in the cytoplasm, in an organelle contained within the
cell, embedded either in whole or in part in the cytoplasmic
membrane, the nuclear membrane or any other membrane within the
cell, is bound to a molecule within the cell or in the cell
membrane, or otherwise fixed to the cell in a manner resistant to
the environment or changes in the environment, such as experimental
manipulations, exposure to candidate pharmacological agents, or the
like.
[0122] The term "antibody" as used herein includes antibodies
obtained from both polyclonal and monoclonal preparations, as well
as: hybrid (chimeric) antibody molecules (see, for example, Winter
et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567);
F(ab')2 and F(ab) fragments; Fv molecules (noncovalent
heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad
Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem
19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston
et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); dimeric and
trimeric antibody fragment constructs; minibodies (see, e.g., Pack
et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J
Immunology 149B:120-126); humanized antibody molecules (see, e.g.,
Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988)
Science 239:1534-1536; and U.K. Patent Publication No. GB
2,276,169, published 21 Sep. 1994); and, any functional fragments
obtained from such molecules, wherein such fragments retain
specific-binding properties of the parent antibody molecule.
[0123] As used herein, the term "monoclonal antibody" refers to an
antibody composition having a homogeneous antibody population. The
term is not limited regarding the species or source of the
antibody, nor is it intended to be limited by the manner in which
it is made. Thus, the term encompasses antibodies obtained from
murine hybridomas, as well as human monoclonal antibodies obtained
using human hybridomas or from murine hybridomas made from mice
expression human immunoglobulin chain genes or portions thereof.
See, e.g., Cote et al. (1985) Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77.
[0124] "Multiplexing" herein refers to an assay or other analytical
method in which multiple cell types can be assayed simultaneously
by using more than spectral code to encode each cell type, each
different code having at least one different fluorescence
characteristic (for example excitation wavelength, emission
wavelength, emission intensity, FWHM (full width at half maximum
peak height), or fluorescence lifetime).
[0125] For example, two different preparations of SCNCs may have
the same composition but different particle sizes, and thus differ
in excitation and/or emission wavelength. Or, two different
preparations may have the same particle size or particle size
distribution but different composition, and thus also differ in
excitation and/or emission wavelength. Different preparations
having different compositions of SCNCs can have different
fluorescent lifetimes, and thus their emission spectra can be
distinguished even when they have the same emission wavelength and
intensity, for example by sampling the emission from the encoded
substance at different times after excitation. Differences in FWHM
can be achieved for example by using SCNCs of different
composition, or of the same composition but which are synthesized
differently, or by mixing different SCNC "preparations" having
overlapping emission peaks together to form a new preparation.
[0126] An SCNC having a known emission wavelength and/or intensity
may be included with the SCNCs used for the encoding to provide an
internal standard for calibrating the wavelength and/or intensity
of the other SCNC(s) used in the conjugate. In addition, other
nanoparticles, e.g., metallic or magnetic nanoparticles, examples
of which are tabulated herein, can be used for the encoding.
[0127] The phenotypic assays of the invention can be performed in
multiplex formats. Multiplex methods are provided employing 2, 3,
4, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000 or more different
encoded cell types which can be used simultaneously to assay for a
phenotype.
[0128] Where different ligands are included in a multiplex assay,
the different ligands can be encoded so that they can be
distinguished. Any encoding scheme can be used; conveniently, the
encoding scheme can employ one or more different fluorescent
species, which can be nanoparticles, e.g., fluorescent
semiconductor nanocrystals and other metallic or magnetic
nanoparticles. For the sake of simplicity, the following discussion
will refer to semiconductor nanocrystals as the encoding species.
However, it is to be understood that this convention is not
intended to be limiting in any way and that other encoding species,
e.g., other nanoparticles, such as metallic or magnetic
nanoparticles, as well as combinations of encoding species such as
SCNCs and other nanoparticles, can be used to encode cells
according to the disclosure that follows.
[0129] Thus, for example, in addition to SCNCs, the nanoparticles
of the invention may also be light-scattering metallic
nanoparticles. Such particles are useful, for example, in
surface-enhanced Raman scattering (SERS), which employs
nanometer-size particles onto which Raman-active moieties (e.g., a
dye or pigment, or a functional group exhibiting a characteristic
Raman spectrum) are adsorbed or attached. Metallic nanoparticles
may be comprised of any metal or metallic alloy or composite,
although for use in SERS, a SERS-active metal is used, e.g.,
silver, gold, copper, lithium, aluminum, platinum, palladium, or
the like. In addition, the particles can be in a core-shell
configuration, e.g., a gold core may be encased in a silver shell;
see, e.g., Freeman et al. (1996) J. Phys. Chem. 100:718-724, or the
particles may form small aggregates in solution. Kneipp et al.
(1998) Applied Spectroscopy 52:1493.
[0130] In addition, organic fluorescent species can be used to
encode cells alone or in combination with nanoparticles. Suitable
fluorescent species include, but are not limited to, fluorescein,
5-carboxyfluorescein (FAM), rhodamine,
5-(2'-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),
anthranilamide, coumarin, terbium chelate derivatives, Reactive Red
4, BODIPY dyes and cyanine dyes. In a preferred aspect, the organic
fluorescent donors include Alexa 488, fluorescein, fluorescein
iso-thiocyanate (FITC), Cy3, Cy5, PE, Texas Red, Cascade Blue,
Bodipy, TMR and tetramethyl rhodamine isothiocyanate (TRITC).
[0131] Other fluorescent species are set forth below in Table 1.
Those of skill in the art will know of other suitable fluorescence
species suitable for use in the present invention.
TABLE-US-00001 TABLE 1 Excitation Emission Fluorochrome Wavelength
Wavelength Acid Fuchsin 540 630 Acridine Orange (Bound to DNA) 502
526 Acridine Red 455-600 560-680 Acridine Yellow 470 550 Acriflavin
436 520 AFA (Acriflavin Feulgen SITSA) 355-425 460 Alizarin
Complexon 530-560 580 Alizarin Red 530-560 580 Allophycocyanin 650
661 ACMA 430 474 AMCA-S, AMC 345 445 Aminoactinomycin D 555 655
7-Aminoactinomycin D-AAD 546 647 Aminocoumarin 350 445 Anthroyl
Stearate 361-381 446 Astrazon Brilliant Red 4G 500 585 Astrazon
Orange R 470 540 Astrazon Red 6B 520 595 Astrazon Yellow 7 GLL 450
480 Atabrine 436 490 Auramine 460 550 Aurophosphine 450-490 515
Aurophosphine G 450 580 BAO 9-(Bisamino-phenyloxadiazole) 365 395
BCECF 505 530 Berberine Sulphate 430 550 Bisbenzamide 360 600-610
BOBO-1, BO-PRO-1 462 481 Blancophor FFG Solution 390 470 Blancophor
SV 370 435 Bodipy F1 503 512 Bodipy TMR 542 574 Bodipy TR 589 617
BOPRO 1 462 481 Brilliant Sulpho-flavin FF 430 520 Calcein 494 517
Calcien Blue 370 435 Calcium Green 505 532 Calcium Orange 549 576
Calcofluor RW Solution 370 440 Calcofluor White 440 500-520
Calcofluor White ABT Solution 380 475 Calcofluor White Standard
Solution 365 435 5-(and 6-)carboxy SNARF-1 indicator 548(low pH)
576(high pH) 587(low pH) 635(high pH) 6-Carboxyrhodamine 6G 525 555
Cascade Blue 400 425 Catecholamine 410 470 Chinacrine 450-490 515
CL-NERF 504(low pH) 514(high pH) 587(low pH) 540(high pH)
Coriphosphine O 460 575 Coumarin-Phalloidin 387 470 CY3.18 554 568
CY5.18 649 666 CY7 710 805 DANS (1-DimethylAmino-Naphthaline-5- 340
525 Sulphonic Acid) DANSA (DiaminoNaphthyl-Sulphonic Acid) 340-380
430 Dansyl NH--CH.sub.3 in water 340 578 DAPI 350 470 DiA 456 590
Diamino Phenyl Oxydiazole (DAO) 280 460 Di-8-ANEPPS 488 605
Dimethylamino-5-Sulphonic Acid 310-370 520 DiI [DiIC.sub.18(3)] 549
565 DiO [DiOC.sub.18(3)] 484 501 Diphenyl Brilliant Flavine 7GFF
430 520 DM-NERF 497(low pH) 510(high pH) 527(low pH) 536(high pH)
Dopamine 340 490-520 ELF-97 alcohol 345 530 Eosin 525 545
Erythrosin ITC 530 558 Ethidium Bromide 510 595 Euchrysin 430 540
FIF (Formaldehyde Induced Fluorescence) 405 435 Flazo Orange
375-530 612 Fluorescein 494 518 Fluorescein Iso-thiocyanate (FITC)
490 525 Fluo 3 485 503 FM1-43 479 598 Fura-2 335 (high 363 (low
[Ca.sup.2+]) [Ca.sup.2+]) 512 (low [Ca.sup.2+]) Fura Red 505 (high
472 (low [Ca.sup.2+]) [Ca.sup.2+]) 436 (high 657 (low [Ca.sup.2+])
[Ca.sup.2+]) 637 (high [Ca.sup.2+]) Genacryl Brilliant Red B 520
590 Genacryl Brilliant Yellow 10GF 430 485 Genacryl Pink 3G 470 583
Genacryl Yellow 5GF 430 475 Gloxalic Acid 405 460 Granular Blue 355
425 Haematoporphyrin 530-560 580 Hoechst 33258, 33342 (Bound to
DNA) 352 461 3-Hydroxypyrene-5,-8,10-TriSulfonic Acid 403 513
7-Hydroxy-4-methylcoumarin 360 455 5-Hydroxy-Tryptamine (5-HT)
380-415 520-530 Indo-1 350 405-482 Intrawhite Cf Liquid 360 430
Leucophor PAF 370 430 Leucophor SF 380 465 Leucophor WS 395 465
Lissamine Rhodamine B200 (RD200) 575 595 Lucifer Yellow CH 425 528
Lucifer Yellow VS 430 535 LysoSensor Blue DND-192, DND-167 374 425
LysoSensor Green DND-153, DND-189 442 505 LysoSensor Yellow/Blue
384(low pH) 329(high pH) 540(low pH) 440(high pH) LysoTracker Green
504 511 LysoTracker Yellow 534 551 LysoTracker Red 577 592 Magdala
Red 524 600 Magnesium Green 506 531 Magnesium Orange 550 575
Maxilon Brilliant Flavin 10 GFF 450 495 Maxilon Brilliant Flavin 8
GFF 460 495 Mitotracker Green FM 490 516 Mitotracker Orange CMTMRos
551 576 MPS (Methyl Green Pyronine Stilbene) 364 395 Mithramycin
450 570 NBD 465 535 NBD Amine 450 530 Nile Red 515-530 525-605
Nitrobenzoxadidole 460-470 510-650 Noradrenaline 340 490-520
Nuclear Fast Red 289-530 580 Nuclear Yellow 365 495 Nylosan
Brilliant Flavin E8G 460 510 Oregon Green 488 fluorophore 496 524
Oregon Green 500 fluorophore 503 522 Oregon Green 514 fluorophore
511 530 Pararosaniline (Feulgen) 570 625 Phorwite AR Solution 360
430 Phorwite BKL 370 430 Phorwite Rev 380 430 Phorwite RPA 375 430
Phosphine 3R 465 565 Phosphine R 480-565 578 Pontochrome Blue Black
535-553 605 POPO-1, PO-PRO-1 434 456 Primuline 410 550 Procion
Yellow 470 600 Propidium Iodide 536 617 Pyronine 410 540 Pyronine B
540-590 560-650 Pyrozal Brilliant Flavin 7GF 365 495 Quinacrine
Mustard 423 503 R-phycoerythrin 565 575 Rhodamine 110 496 520
Rhodamine 123 511 534 Rhodamine 5 GLD 470 565 Rhodamine 6G 526 555
Rhodamine B 540 625 Rhodamine B 200 523-557 595 Rhodamine B Extra
550 605 Rhodamine BB 540 580 Rhodamine BG 540 572 Rhodamine Green
fluorophore 502 527 Rhodamine Red 570 590 Rhodamine WT 530 555
Rhodol Green fluorophore 499 525 Rose Bengal 540 550-600 Serotonin
365 520-540 Sevron Brilliant Red 2B 520 595 Sevron Brilliant Red 4G
500 583 Sevron Brilliant Red B 530 590 Sevron Orange 440 530 Sevron
Yellow L 430 490 SITS (Primuline) 395-425 450 SITS (Stilbene
Isothiosulphonic Acid) 365 460 Sodium Green 507 535 Stilbene 335
440 Snarf 1 563 639 Sulpho Rhodamine B Can C 520 595 Sulpho
Rhodamine G Extra 470 570 SYTOX Green nucleic acid stain 504 523
SYTO Green fluorescent nucleic acid stains 494 .A-inverted. 6 515
.A-inverted. 7 SYTO Green fluorescent nucleic acid stains 515
.A-inverted. 7 543 .A-inverted. 13 SYTO 17 red fluorescent nucleic
acid stain 621 634 Tetracycline 390 560 TRITC (Tetramethyl
Rhodamine 557 576 Isothiocyanate) Texas Red 596 615 Thiazine Red R
510 580 Thioflavin S 430 550 Thioflavin TCN 350 460 Thioflavin 5
430 550 Thiolyte 370-385 477-484 Thiozol Orange 453 480 Tinopol CBS
390 430 TOTO 1, TO-RRO-1 514 533 TOTO 3, TO-PRO-3 642 661 True Blue
365 420-430 Ultralite 656 678 Uranine B 420 520 Uvitex SFC 365 435
X-Rhodamine 580 605 Xylene Orange 546 580 XRITC 582 601 YOYO-1,
YOYO-PRO-1 491 509 YOYO-3, YOYO-PRO-3 612 613
[0132] One or more different populations of spectrally encoded
cells can be created, each population comprising one or more
different semiconductor nanocrystals. Different populations of the
cells, and thus different assays, can be blended together, and the
assay can be performed in the presence of the blended populations.
The individual cells are scanned for their spectral properties,
which allows the spectral code to be decoded and thus identifies
the cell. Because of the large number of different semiconductor
nanocrystals and combinations thereof which can be distinguished,
large numbers of different encoded cells can be simultaneously
interrogated.
[0133] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where the event or circumstance
occurs and instances in which it does not. For example, the phrase
"optionally surrounded by a `coat` of an organic capping agent"
with reference to an SCNC includes SCNCs having such a coat, and
SCNCs lacking such a coat.
II. MODES OF CARRYING OUT THE INVENTION
[0134] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0135] Although a number of compositions and methods similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0136] As explained above, the present invention pertains to
methods of encoding cells with SCNCs and related species and uses
of the encoded cells. In order to further an understanding of the
invention, a more detailed discussion is provided below regarding
SCNCs for use in the subject methods, as well as detailed
descriptions of providing encoded cells and uses of the encoded
cells.
[0137] Production of SCNCs
[0138] SCNCs can be made from any material and by any technique
that produces SCNCs having emission characteristics useful in the
methods, articles and compositions taught herein. The SCNCs have
absorption and emission spectra that depend on their size, size
distribution and composition. Suitable methods of production are
disclosed in, e.g., U.S. Pat. Nos. 6,576,291; 6,207,229; 6,048,616;
5,990,479; 5,690,807; 5,505,928; 5,262,357; PCT Publication No. WO
99/26299 (published May 27, 1999; inventors Bawendi et al.), the
disclosures of all of said patents and publications incorporated
herein by reference in their entireties. Other suitable methods of
manufacture are described in, e.g., Murray et al. (1993) J. Am.
Chem. Soc. 115:8706-8715; Guzelian et al. (1996) J. Phys. Chem.
100:7212-7219; Peng et al. (2001) J. Am. Chem. Soc. 123:183-184;
Hines et al. (1996) J. Phys. Chem. 100:468; Dabbousi et al. (1997)
J. Phys. Chem. B 101:9463; Peng et al. (1997) J. Am. Chem. Soc.
119:7019; Peng et al. (1998) J. Am. Chem. Soc. 120:5343; and Qu et
al. (2001) Nano Lett. 1:333-337.
[0139] Examples of materials from which SCNCs can be formed include
group II-VI, III-V and group IV semiconductors such as ZnS, ZnSe,
ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe,
SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS,
AlP, AlSb, Pb, Ge, Si, and other materials such as PbS, PbSe, and
mixtures of two or more semiconducting materials, and alloys of any
semiconducting material(s).
[0140] The composition, size and size distribution of the
semiconductor nanocrystals affect their absorption and emission
spectra. Exemplary SCNCs that emit energy in the visible range
include CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs. Exemplary SCNCs
that emit energy in the near IR range include InP, InAs, InSb, PbS,
and PbSe. Exemplary SCNCs that emit energy in the blue to
near-ultraviolet include ZnS and GaN. The size of SCNCs in a given
population can be determined by the synthetic scheme used and/or
through use of separation schemes, including for example
size-selective precipitation and/or centrifugation. The separation
schemes can be employed at an intermediate step in the synthetic
scheme or after synthesis has been completed. For a given
composition, larger SCNCs absorb and emit light at longer
wavelengths than smaller SCNCs. SCNCs absorb strongly in the
visible and UV and can be excited efficiently at wavelengths
shorter than their emission peak. This characteristic allows the
use in a mixed population of SCNCs of a single excitation source to
excite all the SCNCs if the source has a shorter wavelength than
the shortest SCNC emission wavelength within the mixture; it also
confers the ability to selectively excite subpopulation(s) of SCNCs
within the mixture by judicious choice of excitation
wavelength.
[0141] The surface of the SCNC is preferably modified to enhance
emission efficiency by adding an overcoating layer to form a
"shell" around the "core" SCNC, because defects in the surface of
the core SCNC can trap electrons or holes and degrade its
electrical and optical properties. Addition of an insulating shell
layer removes nonradiative relaxation pathways from the excited
core, resulting in higher luminescence efficiency. Suitable
materials for the shell include semiconductor materials having a
higher bandgap energy than the core and preferably also having good
conductance and valence band offset. Thus, the conductance band of
the shell is desirably of a higher energy and the valence band is
desirably of a lower energy than those of the core. For SCNC cores
that emit energy in the visible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe,
GaP, GaAs) or near IR (e.g., InP, InAs, InSb, PbS, PbSe), a
material that has a bandgap energy in the ultraviolet may be used
for the shell, for example ZnS, GaN, and magnesium chalcogenides,
e.g., MgS, MgSe, and MgTe. For an SCNC core that emits in the near
IR, materials having a bandgap energy in the visible, such as CdS
or CdSe, or the ultraviolet may be used. Preparation of core-shell
SCNCs is described in, e.g., Dabbousi et al. (1997) J. Phys. Chem.
B 101:9463; Kuno et al. (1997) J. Phys. Chem. 106:9869; Hines et
al. (1996) J. Phys. Chem. 100:468; PCT Publ. No. WO 99/26299; and
U.S. Pat. No. 6,207,229 to Bawendi et al. issued Mar. 27, 2001. The
SCNCs can be made further luminescent through overcoating
procedures as described in Danek et al. (1996) Chem. Mat.
8(1):173-180, and Peng et al. (1997) J. Am. Chem. Soc.
119:7019-7029.
[0142] In a preferred embodiment, the nanocrystals are used in a
core/shell configuration wherein a first semiconductor nanocrystal
forms a core ranging in diameter, for example, from about 20
.DELTA. to about 100 .DELTA., with a shell of another semiconductor
nanocrystal material grown over the core nanocrystal to a thickness
of, for example, 1-10 monolayers in thickness. In a preferred
embodiment, a 1-10 monolayer thick shell of CdS is epitaxially
grown over a core of CdSe.
[0143] Most SCNCs are typically prepared in coordinating solvent,
such as TOPO and trioctyl phosphine (TOP), resulting in the
formation of a passivating organic layer on the surface of SCNCs
with and without a shell. Such passivated SCNCs can be readily
solubilized in organic solvents, for example toluene, chloroform
and hexane. Molecules in the passivating layer can be displaced or
modified to provide an outermost coating that adapts the SCNCs for
use in other solvent systems, for example aqueous systems.
[0144] Alternatively, an outermost layer of an inorganic material
such as silica can be added around the shell to improve the aqueous
dispersibility of the SCNCs, and the surface of the silica can
optionally be derivatized (Bruchez et al. (1998), supra). See,
also, U.S. Pat. No. 6,649,138 for methods of making
water-dispersible SCNCs, incorporated herein by reference in its
entirety.
[0145] A displacement reaction may also be employed to modify the
SCNC to improve the solubility in a particular organic solvent. For
example, if it is desired to associate the SCNCs with a particular
solvent or liquid, such as pyridine, the surface can be
specifically modified with pyridine or pyridine-like moieties which
are soluble or miscible with pyridine to ensure solvation.
Water-dispersible SCNCs can be prepared as described in U.S. Pat.
No. 6,251,303 to Bawendi et al. and PCT Publ. No. WO 00/17655,
published Mar. 30, 2000.
[0146] The surface layer of the SCNCs may be modified by
displacement to render the SCNC reactive for a particular coupling
reaction. For example, displacement of trioctylphosphine oxide
(TOPO) moieties with a group containing a carboxylic acid moiety
enables the reaction of the modified SCNCs with amine containing
moieties to provide an amide linkage. For a detailed description of
these linking reactions, see, e.g., U.S. Pat. No. 5,990,479 to
Weiss et al.; Bruchez et al. (1998), supra, Chan et al. (1998),
supra, Bruchez "Luminescent SCNCs: Intermittent Behavior and use as
Fluorescent Biological Probes" (1998) Doctoral dissertation,
University of California, Berkeley, and Mikulec "SCNC Colloids:
Manganese Doped Cadmium Selenide, (Core)Shell Composites for
Biological Labeling, and Highly Fluorescent Cadmium Telluride"
(1999) Doctoral dissertation, Massachusetts Institute of
Technology. The SCNC may be conjugated to other moieties directly
or indirectly through a linker.
[0147] Examples of suitable spacers or linkers are polyethylene
glycols, dicarboxylic acids, polyamines and alkylenes. The spacers
or linkers are optionally substituted with functional groups, for
example hydrophilic groups such as amines, carboxylic acids and
alcohols or lower alkoxy group such as methoxy and ethoxy groups.
Additionally, the spacers will have an active site on or near a
distal end. The active sites are optionally protected initially by
protecting groups. Among a wide variety of protecting groups which
are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the
like. Various exemplary protecting groups are described in, for
example, Atherton et al., Solid Phase Peptide Synthesis, IRL Press
(1989).
[0148] The Cell
[0149] The cell(s) used in the methods described herein can be of
any origin, including from prokaryotes, eukaryotes, or archeons.
The cell(s) may be living or dead. If obtained from a multicellular
organism, the cell may be of any cell type. The cell(s) may be a
cultured cell line or a primary isolate, the cell(s) may be
mammalian, amphibian, reptilian, plant, yeast, bacterium,
spirochetes, or protozoan. The cell(s) may be, for example, human,
murine, rat, hamster, chicken, quail, goat or dog. The cell may be
a normal cell, a mutated cell, a genetically manipulated cell, a
tumor cell, etc.
[0150] Exemplary cell types from multicellular organisms include
acidophils, acinar cells, pinealocytes, adipocytes, ameloblasts,
astrocytes, basal (stem) cells, basophils, hepatocytes, neurons,
bulging surface cells, C cells, cardiac muscle cells, centroacinar
cells, chief cells, chondrocytes, Clara cells, columnar epithelial
cells, corpus luteal cells, decidual cells, dendrites, endrocrine
cells, endothelial cells, enteroendocrine cells, eosinophils,
erythrocytes, extraglomerular mesangial cells, fetal fibroblasts,
fetal red blood cells, fibroblasts, follicular cells, ganglion
cells, giant Betz cells, goblet cells, hair cells, inner hair
cells, type I hair cells, hepatocytes, endothelial cells, Leydig
cells, lipocytes, liver parenchymal cells, lymphocytes,
lysozyme-secreting cells, macrophages, mast cells, megakaryocytes,
melanocytes, mesangial cells, monocytes, myoepithelial cells, myoid
cells, neck mucous cells, nerve cells, neutrophils,
oligodendrocytes, oocytes, osteoblasts, osteochondroclasts,
osteoclasts, osteocytes, pillar cells, sulcal cells, parathyroid
cells, parietal cells, pepsinogen-secreting cells, pericytes,
pinealocytes, pituicytes, plasma cells, platelets, podocytes,
spermatocytes, Purkinje cells, pyramidal cells, red blood cells,
reticulocytes, Schwann cells, Sertoli cells, columnar cells,
skeletal muscle cells, smooth muscle cells, somatostatin cells,
enteroendocrine cells, spermatids, spermatogonias, spermatozoas,
stellate cells, supporting Deiter cells, support Hansen cells,
surface cells, surface epithelial cells, surface mucous cells,
sweat gland cells, T lymphocytes, theca lutein cells, thymocytes,
thymus epithelial cell, thyroid cells, transitional epithelial
cells, type I pneumonocytes, and type II pneumonocytes. Exemplary
types of tumor cells include adenomas, carcinomas, adenocarcinomas,
fibroadenomas, ameloblastomas, astrocytomas, mesotheliomas,
cholangiocarcinomas, cholangiofibromas, cholangiomas, chondromas,
chondrosarcomas, chordomas, choriocarcinomas, craniopharyngiomas,
cystadenocarcinomas, cystadenomas, dysgerminomas, ependymomas,
epitheliomas, erythroid leukemias, fibroadenomas, fibromas,
fibrosarcomas, gangliogliomas, ganglioneuromas,
ganglioneuroblastomas, gliomas, granulocytic leukemias,
hemangiomas, hemangiopericytomas, hemangiosarcomas, hibernomas,
histiocytomas, keratoacanthomas, leiomyomas, leiomyosarcomas,
lipomas, liposarcomas, luteomas, lymphangiomas, lymphangiosarcomas,
lymphomas, medulloblastomas, melanomas, meningiomas, mesotheliomas,
myelolipomas, nephroblastomas, neuroblastomas, neuromyoblastomas,
odontomas, oligodendrogliomas, osteochondromas, osteomas,
osteosarcomas, papillomas, paragangliomas, pheochromocytomas,
pinealomas, pituicytomas, retinoblastomas, rhabdomyosarcomas,
sarcomas, schwannomas, seminomas, teratomas, thecomas and
thymomas.
[0151] Exemplary bacteria which may be encoded include
Staphylococcus aureus, Legionella pneumophila, Escherichia coli, M.
tuberculosis, S. typhimurium, Vibrio cholera, Clostridium
perfringens, Clostridium tetani, Clostridium botulinum, Clostridium
baratii, Clostridium difficile, M. leprae, Helicobacter pylori,
Hemophilus influenzae type b, Corynebacterium diphtheriae,
Corynebacterium minutissimum, Bordetella pertussis, Streptococcus
pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Shigella
dysenteriae, Pseudomonas aeruginosa, Bacteroides fragilis,
Prevotella melaninogenica, Fusobacterium, Erysipelothrix
rhusiopathiae, Listeria monocytogenes, Bacillus anthracis,
Hemophilus ducreyi, Francisella tularensis, Yersinia pestis,
Bartonella henselae, Klebsiella, Enterobacter, Serratia, Proteus,
and Shigella.
[0152] Exemplary spirochetes which may be encoded include Treponema
pallidum, T. pertenue, T. carateum, Borrelia recurrentis, B.
vincentii, B. burgdorferi, and Leptospira icterohaemorrhagiae.
[0153] Exemplary fungi which may be encoded include Actinomyces
bovis, Aspergillus fumigatus, Blastomyces dermatitidis, Candida
albicans, Coccidioides immitis, Cryptococcus enoformans,
Histoplasma capsulatum, Sporotrichum schenckii, Actinomyces
israelii, Actinomyces bovis, Aspergillus fumigatus, Blastomyces
dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus
neoformans, Histoplasma capsulatum, Nocardia asteroides,
Pneumocystis carinii, Sporothrix schenckii, Pichia pastoris,
Saccharomyces cerevisiae, and Schizosaccharomyces pombe.
[0154] Exemplary protozoa and parasites which may be encoded
include Plasmodium falciparum, Entamoeba histolytica, trypansomes,
Leishmania, Toxpolasma gondii, Giardia lamblia, Chlamydia
trachomatis.
[0155] Spectrally Encoded Cells
[0156] Semiconductor nanocrystals, other fluorescent species, or
otherwise detectable species and combinations thereof can be used
to spectrally encode cells either by allowing SCNCs producing a
single color or mixtures of colors to associate specifically or
non-specifically to the surface of the cells or to be incorporated
into the cells. Populations of cells thus encoded can then be mixed
with other populations of cells with different mixtures of colors
encoding them. The mixed samples of encoded cells can then be
decoded.
[0157] There are several methods whereby SCNCs can be used to
spectrally encode cells. The SCNCs can be coated with a substance,
e.g., a carboxyl or amine group-containing ligand that allows the
SCNCs to be linked to the proteinaceous lipid bilayer of cells or
to the surface of prokaryotic cells. This is done by mixing cells
(e.g., from 1 cell to .about.10.sup.11 cells) for an appropriate
period of time (e.g., about 1 minute to about 24 hours) with an
appropriate concentration of SCNCs (e.g., 1 pM to 1 M). The excess
SCNCs can be separated by filtering out SCNCs or centrifuging the
cells at a speed slow enough to sediment the cells but not the
SCNCs.
[0158] Alternatively, SCNCs can be conjugated with a specific
molecule, e.g., a cell surface marker-specific antibody, that has a
known affinity for a molecule on the surface of the cell and by
this means the SCNCs could encode the cells by incubating cells and
SCNCs. The binding partner on the surface of the cell can be an
endogenous protein or a protein which is not normally endogenous to
the cell, but which the cell is induced to express.
[0159] Cells can also be encoded by introducing SCNCs to the
interior of the cell either by coating the SCNC with a molecule
recognized by a molecule on the surface of the cell and allowing an
active uptake procedure to occur (e.g., receptor mediated
endocytosis), by forcing the SCNCs into the cell (by transient
permeabilization or via lipid vesicles or by high speed injection),
or by using a cationic polymer to facilitate transport across the
cell membrane.
[0160] The encoded cells can be subjected to an assay which
introduces a specific label to interact with the cells; for
example, the label can be an SCNC or another fluorescent or
non-fluorescent label. The specific interaction can be via
receptor-ligand interactions, an adhesion molecule and its binding
partner, a drug and the cellular protein to which it binds, or any
other specific interaction between an entity on the cell and an
introduced, labeled analyte. The labeled or unlabeled encoded cells
can then be interrogated using a detection system or systems which
can decode the cells and identify which cells are labeled, for
example flow cytometry or another detection system described
herein.
[0161] The initial mixed samples of encoded cells can then be grown
in the presence or absence of a selective force (e.g., heat,
ultraviolet light, osmotic stress, shear stress, selective media, a
cytostatic or cytotoxic agent, and the like). After a certain
growth period (for example, from 1 minute to 1 week depending on
the cell type and type of assay being performed) the number of
cells bearing the diluted code can be determined.
[0162] Through the use of the techniques described herein, a number
of assays may be performed simultaneously in a single tube for a
number of different analytes. This may be accomplished using a
number of differently encoded cells in the same tube. The cells may
then be categorized and detected by exposure to a 488 nm laser. The
relative emission intensities of the different fluorescence
channels are used to detect and classify which assay (which cell)
is being measured.
[0163] The use of SCNCs greatly reduces the difficulty encountered
with coding schemes using dye molecules because it allows simple
and efficient classification and detection simultaneously with a
single light source. The usually narrow dye molecule excitation
spectra demand multiple excitation sources in order to successfully
classify the dyes and their relative abundances.
[0164] Cells can be spectrally encoded through incorporation of
nanoparticles, semiconductive, e.g., SCNCs, or metallic
nanoparticles, or other fluorophores. The desired fluorescence
characteristics of the cells may be obtained by mixing SCNCs of
different sizes and/or compositions in a fixed amount and/or ratio
to obtain the desired spectrum, which can be determined prior to
association with the cells. Subsequent treatment of the cells
(through for example covalent attachment, or passive absorption or
adsorption) with the staining solution results in a material having
the designed fluorescence characteristics.
[0165] A number of cell encoding or staining solutions can be
prepared, each having a distinct distribution of sizes and
compositions, to achieve the desired fluorescence characteristics.
These solutions may be mixed in fixed proportions to arrive at a
spectrum having the predetermined ratios and intensities of
emission from the distinct SCNCs suspended in that solution. Upon
exposure of this solution to a light source, the emission spectrum
can be measured by techniques that are well established in the art.
If the spectrum is not the desired spectrum, then more of the SCNC
solution needed to achieve the desired spectrum can be added and
the solution "titrated" to have the correct emission spectrum.
These solutions may be colloidal solutions of SCNCs dispersed in a
solvent, or they may be pre-polymeric colloidal solutions, which
can be polymerized to form a matrix with SCNCs contained
within.
[0166] The composition of the staining solution can be adjusted to
have the desired fluorescence characteristics, preferably under the
exact excitation source that will be used for the decoding. A
multichannel auto-pipetter connected to a feedback circuit can be
used to prepare an SCNC solution having the desired spectral
characteristics, as described above. If the several channels of the
titrater/pipetter are charged with several unique solutions of
SCNCs, each having a unique excitation and emission spectrum, then
these can be combined stepwise through addition of stock
solutions.
[0167] Once the staining solution has been prepared, it can be used
to incorporate a unique spectral code into a given cell or a cell
population. The staining procedure can also be carried out in
sequential steps.
[0168] In another method, the cell or the population of cells can
be spectrally encoded through incorporation of microspheres or
beads that make up a beadset, usually referred to as fluorospheres
or fluospheres. Fluorospheres suitable for use in accordance with
the invention are generally known in the art and may be obtained
from manufacturers such as Spherotech and Molecular Probes.
Examples of fluorospheres include blue fluorescent fluorospheres,
with excitation/emission maxima of 350/440 nm, yellow-green
fluorescent fluorospheres having excitation/emission maxima of
505/515 nm, red fluorescent fluorospheres having
excitation/emission maxima of 580/605 nm, infrared fluorescent
fluorospheres having excitation/emission maxima of 715/755 nm.
Alternatively, fluorospheres having different surface functional
groups for conjugation can be used in the present invention. The
surface functional groups can include, for example, carboxylate,
sulfate, aldyhyde-sulfate, amine, and the like. In addition,
fluorospheres labeled with biotin, streptavidin, avidin,
neutravidin, protein A, or the like can also be used for encoding
the cells for use in the invention.
[0169] In another method, the cell or the population of cells can
be spectrally encoded through incorporation of colloidal rod
particles, also referred to as nanoparticles, nanorods, or
nanobars. Typically, nanobar codes have a plurality of segments
with the entire width of the nanobar particle being about 30 nm to
about 1,000 nanometers, and length being about 1 to 15 microns.
Nanobar codes are usually composed of two or more different
materials, such as metal, metal chalcogenide, metal oxide, metal
alloy, a semiconductor, or an organic or inorganic material. The
method of manufacture of colloidal rod particles as nanobar codes
is described in PCT publications WO 01/25002 and WO 01/25510. In
general, the nanobar code particles are manufactured by
electrochemical deposition in an alumina or polycarbonate template,
followed by template dissolution, or by alternating electrochemical
reduction of metal ions. The cell or the population of cells can
then be spectrally encoded with nanobars using the methods
described in detail above.
[0170] In another method of spectrally encoding the cells or
population of cells, light scattering metallic particles of
nanometer size onto which Raman-active moieties are adsorbed or
attached are used, and the cells thus encoded are then detected by
surface-enhanced Raman scattering (SERS). The metal particles can
be made from a SERS active metal such as silver, gold, copper,
lithium, aluminum, platinum, palladium, or the like. In addition,
the particles can be in a core-shell configuration, e.g., a gold
"core" encased in a silver "shell" (see, e.g., Freeman et al.
(1996) J. Phys. Chem. 100:718-724). Furthermore, the particles can
be composites of two or more metals. Preferably, the metal particle
is a silver particle, a gold particle or a gold core-silver shell
particle.
[0171] The colloids can be prepared from a reduction of a soluble
precursor, for example, a metal salt in aqueous or solvent
environment, by controlled addition of a colloid-generating agent
such as citrate or borohydride, or by other conventional
comminution techniques. See, e.g., Lee et al. (1982) J. Phys. Chem.
86:3391. The size of the colloids can be between about 2 and 150
nm, preferably between about 5 and 100 nm, more preferably between
about 20 and 100 nm. The reduction can be carried out over a
temperature range from 0.degree. C. to 100.degree. C. The SERS or
SERRS active structures can also be an aggregate of the
aforementioned particles. Both single particle and aggregates of
particles that exhibit SERS or SERRS activity will be referred to
as SERS colloids.
[0172] A Raman-active tag is adsorbed to the surface of the SERS
colloid. The Raman-active tag can be any chemical molecule or
portion thereof that exhibits a characteristic Raman spectrum and
is capable of adsorbing or binding to a SERS colloid. The tag can
be a dye or pigment and/or can be, for example, a nitrile, a
pyridine, an imidazole, a pyrrole, an isonitrile, a thiocyanate, a
urea, an isourea, a carbamate, a thiocarbamate, an imide, a thiol,
an amine, an amide, a carbonate, a carbonyl or a carboxylate. See,
also, Rahman et al. (1998) J. Org. Chem. 63:6196-6199, for
additional Raman-active moieties. The tag can have a Raman-active
mode relative to the excitation light source in the range of
between about 100 to 5000 cm.sup.-1, preferably between about
1000-5000 cm.sup.-1 and, more preferably, between about 1000-2500
cm.sup.-1. SERRS-active particles can be used that have a suitable
electronic transition such that the excitation light source is
chosen to emit light having a frequency close to that of the
electronic transition and/or the frequency of the SERS plasmon
resonance of the SERS particle.
[0173] Methods by which Raman-active moieties can be adsorbed or
bound to the surface of the particle are well known in the art.
See, e.g., EP 0806460(A1). Thus, for example, the Raman-active tag
may be added to the medium containing the SERS colloid as a solid
or as a solution. It can be added before, during or after the
reduction of the soluble metal precursor. The amount of
Raman-active tag can be added to provide between about 1 and
1,000,000, preferably between about 10 and 10,000, more preferably
between about 10 and 100 Raman-active tags on each particle. The
cell or the population of cells can then be spectrally encoded with
SERS and/or SERRS particle using the methods described in detail
herein.
[0174] Spectrally encoding cells can be effected by any combination
of the above described methods and detectable species.
[0175] Attaching SCNCs to Cells
[0176] The SCNCs can be attached to the cells by covalent
attachment as well as by entrapment, or can be coupled to one
member of a binding pair the other member of which is attached to
the cells. For instance, SCNCs are prepared by a number of
techniques that result in reactive groups on the surface of the
SCNC. See, e.g., Bruchez et al. (1998) Science 281:2013-2016, Chan
et al. (1998) Science 281:2016-2018, Colvin et al. (1992) J. Am.
Chem. Soc. 114:5221-5230, Katari et al. (1994) J. Phys. Chem.
98:4109-4117, Steigerwald et al. (1987) J. Am. Chem. Soc. 110:3046.
The reactive groups present on the surface of the SCNCs can be
coupled to reactive groups present on the cell. For example, SCNCs
which have carboxylate groups present on their surface can be
coupled to cells with amine groups using a carbodiimide activation
step.
[0177] Any cross-linking method that links a SCNC to a cell and
does not adversely affect the properties of the SCNC or the cell
can be used. In a cross-linking approach, the relative amounts of
the different SCNCs can be used to control the relative
intensities, while the absolute intensities can be controlled by
adjusting the reaction time to control the number of reacted sites
in total. After the cells are crosslinked to the SCNCs, the cells
are optionally rinsed to wash away unreacted SCNCs.
[0178] A sufficient amount of fluorophore must be used to encode
the cells so that the intensity of the emission from the
fluorophores can be detected by the detection system used and the
different intensity levels must be distinguishable, where intensity
is used in the coding scheme but the fluorescence emission from the
SCNCs or other fluorophores used to encode the cells must not be so
intense to as to saturate the detector used in the decoding
scheme.
[0179] Where intact cellular structures are desired, the methods
used to encode the cells cause minimal disruption of the viability
of the cell and of the integrity of membranes. Alternatively, the
cells can be fixed and treated with routine histochemical or
cytochemical procedures. A fixative that does not affect the
encoding should be used.
[0180] Semiconductor nanocrystals of varying core sizes (10-150
angstroms), composition and/or size distribution can be conjugated
to a specific-binding molecule which bind specifically to an
molecule on a cell membrane or within a cell. Any specific
"anti-molecule" can be used, for example, an antibody, an
immunoreactive fragment of an antibody, and the like. Preferably,
the anti-molecule is an antibody. The semiconductor nanocrystal
conjugates are used to associate the SCNC with the cell or, once
within the cell, to identify intracellular components, organelles,
molecules or the like.
[0181] More specifically, the specific-binding molecule may be
derived from polyclonal or monoclonal antibody preparations, may be
a human antibody, or may be a hybrid or chimeric antibody, such as
a humanized antibody, an altered antibody, F(ab').sub.2 fragments,
F(ab) fragments, Fv fragments, a single-domain antibody, a dimeric
or trimeric antibody fragment construct, a minibody, or functional
fragments thereof which bind to the analyte of interest. Antibodies
are produced using techniques well known to those of skill in the
art and disclosed in, for example, U.S. Pat. Nos. 4,011,308;
4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745.
[0182] For example, polyclonal antibodies are generated by
immunizing a suitable animal, such as a mouse, rat, rabbit, sheep
or goat, with an antigen of interest. In order to enhance
immunogenicity, the antigen can be linked to a carrier prior to
immunization. Such carriers are well known to those of ordinary
skill in the art.
[0183] Immunization is generally performed by mixing or emulsifying
the antigen in saline, preferably in an adjuvant such as Freund's
complete adjuvant, and injecting the mixture or emulsion
parenterally (generally subcutaneously or intramuscularly). The
animal is generally boosted 2-6 weeks later with one or more
injections of the antigen in saline, preferably using Freund's
incomplete adjuvant. Antibodies may also be generated by in vitro
immunization, using methods known in the art. Polyclonal antiserum
is then obtained from the immunized animal.
[0184] Monoclonal antibodies are generally prepared using the
method of Kohler and Milstein (1975) Nature 256:495-497, or a
modification thereof. Typically, a mouse or rat is immunized as
described above. However, rather than bleeding the animal to
extract serum, the spleen (and optionally several large lymph
nodes) is removed and dissociated into single cells. If desired,
the spleen cells may be screened (after removal of nonspecifically
adherent cells) by applying a cell suspension to a plate or well
coated with the antigen. B-cells, expressing membrane-bound
immunoglobulin specific for the antigen, will bind to the plate,
and are not rinsed away with the rest of the suspension. Resulting
B-cells, or all dissociated spleen cells, are then induced to fuse
with myeloma cells to form hybridomas, and are cultured in a
selective medium (e.g., hypoxanthine, aminopterin, thymidine
medium, "HAT"). The resulting hybridomas are plated by limiting
dilution, and are assayed for the production of antibodies which
bind specifically to the immunizing antigen (and which do not bind
to unrelated antigens). The selected monoclonal antibody-secreting
hybridomas are then cultured either in vitro (e.g., in tissue
culture bottles or hollow fiber reactors), or in vivo (e.g., as
ascites in mice).
[0185] Human monoclonal antibodies are obtained by using human
rather than murine hybridomas. See, e.g., Cote, et al. Monclonal
Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77.
[0186] Monoclonal antibodies or portions thereof may be identified
by first screening a B-cell cDNA library for DNA molecules that
encode antibodies that specifically bind to p185, according to the
method generally set forth by Huse et al. (1989) Science
246:1275-1281. The DNA molecule may then be cloned and amplified to
obtain sequences that encode the antibody (or binding domain) of
the desired specificity.
[0187] As explained above, antibody fragments which retain the
ability to recognize the molecule of interest, will also find use
in the subject invention. A number of antibody fragments are known
in the art which comprise antigen-binding sites capable of
exhibiting immunological binding properties of an intact antibody
molecule. For example, functional antibody fragments can be
produced by cleaving a constant region, not responsible for antigen
binding, from the antibody molecule, using e.g., pepsin, to produce
F(ab').sub.2 fragments. These fragments will contain two antigen
binding sites, but lack a portion of the constant region from each
of the heavy chains. Similarly, if desired, Fab fragments,
comprising a single antigen binding site, can be produced, e.g., by
digestion of polyclonal or monoclonal antibodies with papain.
Functional fragments, including only the variable regions of the
heavy and light chains, can also be produced, using standard
techniques such as recombinant production or preferential
proteolytic cleavage of immunoglobulin molecules. These fragments
are known as F.sub.V. See, e.g., Inbar et al. (1972) Proc. Nat.
Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem
15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
[0188] A single-chain Fv ("sFv" or "scFv") polypeptide is a
covalently linked V.sub.H-V.sub.L heterodimer which is expressed
from a gene fusion including V.sub.H- and V.sub.L-encoding genes
linked by a peptide-encoding linker. Huston et al. (1988) Proc.
Nat. Acad. Sci. USA 85:5879-5883. A number of methods have been
described to discern and develop chemical structures (linkers) for
converting the naturally aggregated, but chemically separated,
light and heavy polypeptide chains from an antibody V region into
an sFv molecule which will fold into a three dimensional structure
substantially similar to the structure of an antigen-binding site.
See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The
sFv molecules may be produced using methods described in the art.
See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci. USA
85:5879-5883; U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778.
Design criteria include determining the appropriate length to span
the distance between the C-terminus of one chain and the N-terminus
of the other, wherein the linker is generally formed from small
hydrophilic amino acid residues that do not tend to coil or form
secondary structures. Such methods have been described in the art.
See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778.
Suitable linkers generally comprise polypeptide chains of
alternating sets of glycine and serine residues, and may include
glutamic acid and lysine residues inserted to enhance
solubility.
[0189] "Mini-antibodies" or "minibodies" will also find use with
the present invention. Minibodies are sFv polypeptide chains which
include oligomerization domains at their C-termini, separated from
the sFv by a hinge region. Pack et al. (1992) Biochem 31:1579-1584.
The oligomerization domain comprises self-associating
.alpha.-helices, e.g., leucine zippers, that can be further
stabilized by additional disulfide bonds. The oligomerization
domain is designed to be compatible with vectorial folding across a
membrane, a process thought to facilitate in vivo folding of the
polypeptide into a functional binding protein. Generally,
minibodies are produced using recombinant methods well known in the
art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et
al. (1992) J Immunology 149B:120-126.
[0190] Introduction of the SCNCs into the Cell
[0191] In general, transfer methods into cells can be divided into
three categories: physical (e.g., electroporation, direct transfer,
and particle bombardment), chemical (e.g., proteinoids,
microemulsions, and liposomes), and biological (e.g., virus-derived
vectors, receptor-mediated uptake, phagocytosis). Derivatizing a
ligand for a cellular receptor which is endocytosed with an agent
acts as a means to ferry that agent into the cell.
[0192] The procedure for attaching an agent such as an SCNC to a
ligand varies according to the chemical structure of the ligand.
Generally, the ligand contains a variety of functional groups which
are available for reaction with a suitable functional group on a
biologically active molecule to bind the agent thereto.
Alternatively, the ligand and/or agent may be derivatized to expose
or attach additional reactive functional groups. The derivatization
may involve attachment of any of a number of linker molecules such
as those available from Pierce Chemical Company, Rockford Ill.
[0193] A linker can be used to join, covalently or noncovalently,
the ligand and agent. Suitable linkers are well known to those of
skill in the art and include, but are not limited to, straight or
branched-chain carbon linkers, heterocyclic carbon linkers, or
peptide linkers. See, e.g., Birch and Lennox, Monoclonal
Antibodies: Principles and Applications, Chapter 4, Wiley-Liss, New
York, N.Y. (1995); U.S. Pat. Nos. 5,218,112 and 5,090,914;
Hermanson (1996) Bioconjugate Techniques, Academic Press, San
Diego, Calif.
[0194] A bifunctional linker having one functional group reactive
with a group on a particular agent, and another group reactive with
a ligand, may be used to form the desired conjugate. Alternatively,
derivatization may involve chemical treatment of the ligand and/or
agent; e.g., glycol cleavage of a sugar moiety with periodate to
generate free aldehyde groups. The free aldehyde groups may then be
reacted with free amine or hydrazine groups on an agent to bind the
agent thereto. See, U.S. Pat. No. 4,671,958. Procedures for
generation of free sulfhydryl groups on antibodies or antibody
fragments are also known. See, U.S. Pat. No. 4,659,839. Many
procedures and linker molecules for attachment of proteins to other
molecules are known. See, e.g., European Patent Application No.
188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;
4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)
Cancer Res. 47:4071-4075.
[0195] Conjugates comprising cleavable linkages may be used.
Cleaving of the linkage to release the agent from the ligand and/or
linker may be prompted by enzymatic activity or conditions to which
the conjugate is subjected. The cis-aconitic acid spacer can be
used to release the agent from the ligand in endosomes. Disulfide
linkages are also cleavable in the reducing environment of the
endosomes.
[0196] A number of different cleavable linkers are known to those
of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and
4,625,014. The mechanisms for release of an agent from these linker
groups include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 5,141,648 discloses
conjugates comprising linkers of specified chemical structure,
wherein the linkage is cleaved in vivo thereby releasing the
attached compound. The linker is susceptible to cleavage at a
mildly acidic pH, and is believed to be cleaved during transport
into the cytoplasm of a target cell, thereby releasing the agent
inside a target cell. U.S. Pat. No. 4,671,958 includes a
description of conjugates comprising linkers which are cleaved by
proteolytic enzymes of the complement system.
[0197] Alternatively, methods may be used to transport SCNCs out of
the endosome. A number of suitable methods are known in the art.
SCNC bound to a ligand which binds specifically to the polymeric
immunoglobulin receptor can be used for efficient introduction into
cells. Ferkol et al. (1993) J. Clin. Invest. 92:2394-2400; and
Ferkol et al. (1995) J. Clin. Invest. 95:493-502. As an example,
SCNC may be linked to ricin A, which is capable of penetrating the
endosomal membrane into the cytosol. Beaumell et al. (1993) J.
Biol. Chem. 268:23661-23669.
[0198] Nonlimiting examples of artificial means for transporting
SCNCs across cell membranes include action of chemical agents such
as detergents, enzymes or adenosine triphosphate; receptor- or
transport protein-mediated uptake; liposomes or alginate hydrogels;
phagocytosis; pore-forming proteins; microinjection;
electroporation; hypoosmotic shock; or minimal physical disruption
such as scrape loading, patch clamp methods, or bombardment with
solid particles coated with or in the presence of the SCNCs of the
invention.
[0199] These techniques include transfection, infection, biolistic
impact, electroporation, microinjection, scraping, or any other
method which introduces the gene of interest into the host cell
(see, U.S. Pat. No. 4,743,548, U.S. Pat. No. 4,795,855, U.S. Pat.
No. 5,068,193, U.S. Pat. No. 5,188,958, U.S. Pat. No. 5,463,174,
U.S. Pat. No. 5,565,346 and U.S. Pat. No. 5,565,347).
[0200] One method for introducing SCNCs into cells involves the use
of micelles and liposomes. Micelles can be formed in aqueous
solution by the use of micelle forming agents such as emulsifying
agents, cholic acid and derivatives thereof, phosphatides,
detergents, cationic lipids, and the like. Emulsifying agents
include, for example, those marketed under the tradenames
Cremophore EL, the Tweens, and the pluronics. Cholic acid and its
derivatives include the trihydroxycholic acids, such as glyocholic
acid, taurocholic acids, and their salts. Phosphatides for use in
the invention include especially those that contain at least one
saturated fatty acid residue that is branched, such as glycerol
where two of the hydroxyl groups are esterified with residues from
saturated fatty acids of C.sub.10-20 where at least one of the
carbon atoms has an alkyl group. Examples of such phosphatides
includes, for example,
1,2-di(8-methylheptadecanoyl)-sn-glycero-3-phosphocholine,
1,2-di(10-methylstearoyl)-sn-glycero-3-phosphocholine,
1,2-(10-methylnonadecanoyl)-sn-glycero-3-phosphocholine, and the
like. As will be evident to one of skill in the art, other
compounds may also be added to the micelle forming agents, such as
a lipoid component, bile acid salts, dihexanoyl lecithin, and the
like.
[0201] SCNCs can be introduced into cells using transfection by
micelle-based or liposome-based methods. Conjugated or unconjugated
SCNCs in solution (about 1 fm to about 10 mM) are mixed with a
micelle forming agent or any other species that can be used to form
effective micelles or liposomes, at various concentrations to form
SCNC trapped in the micelles. The solution containing the SCNCs
trapped in the micelles can then be added to mammalian or other
eukaryotic or prokaryotic cells, wherein the lipid and SCNC
compositions and concentrations are varied, the micelle forming
agent:SCNC ratio is varied, the cell density is varied and the time
of exposure to the SCNC trapped in the micelles or liposomes is
varied (e.g., from 1 minute to 48 hours) to determine the optimum
transfection conditions. The efficacy of introduction of such SCNCs
into cells can be assessed by standard epi-fluorescent microscopy
or by any other detection system utilizing the broadband excitation
and flexible emission spectra of SCNCs.
[0202] Useful liposomes include cationic phospholipids, neutral
phospholipids, lipids and mixtures thereof. Additional components
may be included, such as targeting peptides or proteins, fusion
peptides (e.g., from Sendai virus, influenza virus,
hemagluttinating virus of Japan (HVJ)), envelope proteins of
viruses, polycationic substances such as poly-L-lysine or
DEAE-dextran, molecules which bind to the surface of airway
epithelial cells including antibodies, adhesion molecules and
growth factors, and the like.
[0203] The SCNC can be formulated as an SCNC-liposome complex
formulation. Such complexes comprise a mixture of lipids which bind
to the SCNC or a ligand attached to the SCNC, providing a
hydrophobic coat which allows the agent to be delivered into cells.
Liposomes that can be used include DOPE (dioleoyl phosphatidyl
ethanol amine) and CUDMEDA (N-(5-cholestrum-3-ol
3-urethanyl)-N',N'-dimethylethylene diamine). Cationic liposomes
which may be used in the present invention include
3-[N--(N',N'-dimethyl-aminoethane)-carbamoyl]-cholesterol
(DC-Chol),
N,N,N-trimethyl-2,3-bis((1-oxo-9-octadecenyl)oxy)-(Z,Z)-1-propanaminium
methyl sulfate (DOTAP), lipopolyamines such as lipospermine (DOGS),
(+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium
bromide (DLRIE), DOTMA, DOSPA, DMRIE, GL-67, GL-89, Lipofectin, and
Lipofectamine (Thiery et al. (1997) Gene Ther. 4:226-237; Felgner
et al. (1995) Annals N.Y. Acad. Sci. 772:126-139; Eastman et al.
(1997) Hum. Gene Ther. 8:765-773). Also encompassed are the
cationic phospholipids described in U.S. Pat. Nos. 5,264,618,
5,223,263 and 5,459,127. Other suitable phospholipids which may be
used include phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, sphingomyelin, phosphatidylinositol, and
the like. Cholesterol or DC-cholesterol may also be included.
[0204] For preparing liposomes, the procedure described by Kato et
al. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids
and lumen composition containing the SCNC are combined in an
appropriate aqueous medium, conveniently a saline medium where the
total solids is in the range of about 1-10 weight percent. After
intense agitation for short periods of time, from about 5-60 sec.,
the tube is placed in a warm water bath, from about 25-40.degree.
C. and this cycle repeated from about 5-10 times. The composition
is then sonicated for a convenient period of time, generally from
about 1-10 sec. and may be further agitated by vortexing. The
volume is then expanded by adding aqueous medium, generally
increasing the volume by about from 1-2 fold, followed by shaking
and cooling. This method allows for the incorporation into the
lumen of high molecular weight molecules.
[0205] The process of receptor-mediated endocytosis results in the
contents of an endosome fusing with liposomes and their subsequent
degradation. Certain viruses (e.g., Semliki forest virus) avoid
being transported to liposomes by being released from the endosome
prior to endosome-lysosome fusion. These viruses behave in this
manner because proteins in the viral coat (e.g., hemagglutinin) are
induced to cause fusion and release of viral particles in the
acidic environment of the endosomes. Thus, SCNCs and proteins to
trigger receptor-mediated endocytosis can be enclosed in liposomes,
thereby permitting acid-induced fusogenic proteins to be introduced
into cells (FIG. 4). SCNC conjugates with a proteins or peptides of
interest (e.g., the signal transduction domains of a receptor) can
be entrapped within lipsomes using standard techniques. The
lipsomes bilayers have proteins incorporated therein or attracted
to their surface using methods already described. The proteins
associated with liposome membrane can include a ligand train to
induce receptor-mediated endocytosis (e.g., transferrin) and
proteins that induce fusion to the endosome under acidic conditions
e.g., hemagglutinin, or some portion of such a proteins that is
sufficient to generate its activity. The ligand used for
receptor-mediated endocytosis can also act as a specific
cell-targeting agent when introducing the lipsomes to mixed cell
cultures or in whole organisms or in mixed blood cell or tissue
cell populations. The SCNC-liposome can then be added to a
population of cells and will be taken up and deposited into the
cytoplasm of the target cells. Liposomes could be loaded with
multiple SCNC conjugates and the cells subsequently treated in the
appropriate manner and the localization of each type of SCNC
analyzed microscopically.
[0206] SCNCs can also be incorporated into cells using an
artificial viral envelope, either alone or in combination with
other materials. Artificial membranes can be prepared, for example,
by double detergent dialysis as described in U.S. Pat. No.
5,252,348 and published EP patent application 0 555 333 B 1. These
viral envelopes have a cholesterol:phospholipid ratio of about 0.8
to about 1.2, preferably 1.0, similar to natural viral envelopes.
The particles also have a homogenous size structure similar to that
of natural viral particles and a physically stable unilamellar
membrane structure.
[0207] In another method, SCNCs conjugated to specific biomolecules
or unconjugated SCNCs can be incorporated into cells by forming
pores in the cells. The pores can be formed by, for example,
electroporation, osmotic shock, or by the use of a porogen.
Electroporation is a common method for introducing foreign
material, such as DNA, into cells (see Hui, 1995, Methods in
Molecular Biology, Chapter 2, 48:29-40). The electroporation method
of the invention consists of delivering high voltage pulses to
cells thereby making pores in the cell membrane to facilitate the
transport of SCNCs into cells. The electroporation process consists
of two major steps: reversible breakdown of the cell membranes, and
recovery of permeablized cells. Thus, the electrical and incubation
parameters are optimized to facilitate the transfer of SCNCs across
the membrane. In general, cells in suspension (from 1 to 10.sup.10
cells) can be placed in an electroporation cuvette with an
appropriately sized SCNC (10 .ANG. to 150 .ANG.) at various
concentrations (approximately 1 fmol to approximately 10 mM). The
cuvette is then connected to an appropriate power supply and the
cells/SCNCs are subjected to a high voltage pulse of defined
magnitude and length. The voltage, capacitance and resistance can
be varied appropriately depending on the cells or efficiency of the
protocol. For example the voltage can be varied between about 1 V
to about 100 kV, preferably 1 to 5 kV), the capacitance can be
varied between about 0.1 Ff to about 100 f, preferably between
about 1 Ff to about 50 Ff, and the resistance can be varied from
about 0.1 S to about infinity. Cells should then be allowed to
recover in the appropriate medium and detection of successfully
transfected cells assessed using the appropriate detection systems
for the SCNC.
[0208] Alternatively, the porogen can be digitonin, saporin, or a
member of the complement cascade. Cells may be permeabilized with
digitonin as described in Hagstrom et al. (1997) J. Cell. Sci.
110:2323-31, and in Sterne-Marr et al. (1992) Meth. Enzymol.
219:97-111, to allow the SCNC to be incorporated into the cell.
[0209] There are many other ways in which SCNC can be introduced
into cells, e.g., microinjection, passive pinocytosis or uptake via
coating with viral fusogenic proteins. SCNCs can be used as
flexible markers for identifying microinjected cells. To this end,
SCNCs are microinjected either alone (as control) or together with
other molecules of interest to allow color-coded identification of
a particular microinjected cell. The method of microinjection uses
a syringe needle, usually a heat-drawn sharp-ended glass tube, to
puncture the cell membrane to deliver a solution containing SCNCs.
SCNCs are suspended in an appropriate microinjected buffer at the
required concentration (1 fm to 10 mM). The needle is aimed and
actuated by using a micro-manipulator and viewed under a
microscope. Once the cell is punctured, a controlled quantity of
SCNCs is injected by applying a controlled pressure to the syringe
plunger. After microinjection, the cells are left to recover. The
size and composition of the SCNC (10 .ANG. to 150 .ANG.) determines
the emission wavelength. This type of marker can be used, for
example, to differentially mark cells injected with a particular
molecule within a population of cells injected with multiple
molecule (FIG. 2). Because all SCNCs can be excited at a common
wavelength of light, or a wavelength may be selected to excite all
species that have been used to encode the population of cells, all
injected cells can be visualized concurrently and the effects of
the co-injected molecule observed. In addition, the use of SCNCs as
markers allows a flexible third, fourth, fifth, and greater, color
to be used in multicolor immunofluorescent staining experiments
(FIG. 3).
[0210] Transport of SCNCs Across Biological Membranes Using
Cationic Polymers
[0211] One method for transporting SCNCs and other semiconductor
nanoparticles across biological membranes, including across cell
membranes and intracellular organelle membranes, such as the
mitochondrial membrane, nuclear membranes, and the like, employs
the use of peptides and cationic polymers that encourage entry of
SCNCs into the cell.
[0212] One such peptide is an HIV-Tat peptide that facilitates
viral passage into cells. The Tat peptide has been used to
introduce magnetic nanoparticles into mammalian cells. Tat peptides
for use herein can include peptides from the protein transduction
domain of Tat. One particular Tat peptide for use herein is a
fragment of the tat protein containing the tat basic region
(residues 49-57 having the sequence RKKRRQRRR (SEQ ID NO: 1). SCNCs
can be coated with Tat peptide sequences alone or along with other
peptides, oligonucleotide or other affinity molecule to facilitate
SCNC uptake by the cells and delivered to their appropriate binding
partner or cellular compartment. Attachment can be achieved via any
standard bioconjugation process well known in the art. SCNCs of any
size and composition can be coated with both a peptide that
recognizes a specific binding motif on an intracellular protein and
also with a peptide that corresponds to the HIV-Tat sequence.
Incubation of such modified SCNCs with a mammalian cell allows the
SCNCs to enter the cell, probably via adsorptive endocytosis. Once
inside the cell, the modified SCNCs can interact with and bind to
the protein of oligonucleotide that contains the region recognized
by the other peptide or oligonucleotide, respectively, on the
surface of the SCNC. This will provide information as to the
localization, trafficking and abundance of that protein. If a
second color of SCNC that carries an affinity molecule for a second
intracellular protein or oligonucleotide is introduced via a
similar method then the relative positions of the two molecules can
be determined (see FIG. 1).
[0213] Other viral-based peptides for enhancing transport of
molecules into the cytoplasm and nucleus of cells are described in
e.g., Tkachendo et al., J. Am. Chem. Soc. (2003) 125:4700-4701, and
include peptides derived from the SV40 large T NLS, such as the
peptide with the sequence CGGGPKKKRKVG (SEQ ID NO:2), peptides from
the adenoviral NLS, such as the peptide with the sequence
CGGFSTSLRARKA (SEQ ID NO:3), peptides from the adenoviral RME, such
as a peptide with the sequence CKKKKKKSEDEYPYVPN (SEQ ID NO:4) and
peptides from the adenoviral fiber protein such as a peptide having
the sequence CKKKKKKKSEDEYPYVPNFSTSLRARKA (SEQ ID NO:5).
[0214] Similarly, other cationic polymers, i.e., polymers with a
series of positively charged monomers, can be used to facilitate
transport of SCNCs over biological membranes. Typically, although
not exclusively, the positively charged groups are primary,
secondary or tertiary amines which ionize at or around neutral pH.
Such amine groups can be present as amino groups in side chains as
in poly(amino acids); amino groups included in a polymer backbone
as in poly(amines); or amino substituents added to an uncharged
polymer, such as result in dextran substituted with
diethylaminoethyl groups. Polymers containing other positively
charged groups, such as quaternary amines, the sulfur group in
S-methyl methionine, etc., would also be suitable.
[0215] Specific poly(amino acids) which are suitable include, but
are not limited to, poly-L-lysine, poly-L-ornithine,
poly-L-arginine, poly-L-homoarginine, poly-L-diaminobutyric acid,
poly-L-histidine, the D-optical isomers thereof and copolymers
thereof. Copolymers may include non-cationic amino acid residues.
Cationic poly(amino acids) are preferred. Additionally, it may be
desirable to employ cationic polymers which are digested by
proteolytic enzymes present in mammalian cells, such as, for
example, poly-L-lysine and poly-L-arginine.
[0216] Other cationic polymers suitable for use with the present
methods include polymers with neutral or anionic backbones to which
cationic groups have been bonded, such as substituted
polysaccharides (e.g., diethylaminoethyl dextran), substituted
cellulose, substituted copolymers of ethylene and maleic anhydride,
substituted lactic or glycolic acid polymers, and the like.
Polyamines, such as for instance, poly(vinyl amine), or other
cationic synthetic polymers, will also find use herein.
[0217] Additionally, other positively charged, naturally occurring
macromolecules will also serve as suitable cationic polymers.
Specific examples include protamines and histones, such as those
found to increase cellular uptake of albumin by their simple
presence. See, Ryser et al., Science (1965) 150:501-503.
[0218] Generally, the multiple positive charges present in the
cationic polymer will give the molecule a net positive charge. In
other cases, however, the multiple charges may form an adequate
sequence in the primary structure, or an adequate spacial
arrangement in the tertiary structure, or both, to cause enhanced
transport, even though the molecule does not itself have an overall
net positive charge. For example, a molecule containing a limited
number of positive charges at various intervals in its primary
structure may fold in a manner such that a cluster of positive
charges will be positioned in the same spatial area of its tertiary
structure. Alternatively, a copolymer of poly(amino acid) with a
neutral or negative net charge may contain a functionally important
cluster of positive charges. Therefore, as used herein, the term
"cationic polymer" refers not only to a polymer which has an
overall positive net charge, but also includes polymers that
contain sequential portions or spatial arrangements of positive
charges sufficient to confer on them the transport properties of
cationic polymers having a net positive charge.
[0219] Preferably, the cationic polymer will include from about 5
to about 50 subunits, more preferably from about 5 to about 25
subunits, or any number within these ranges, such as 6, 7, 8, 9, 10
. . . 15 . . . 20 . . . 25, and so forth. In some cases, at least
half of the subunits will include a guanidino or amidino sidechain
moiety. Such polymers are described in e.g., U.S. Pat. No.
6,495,663, the disclosure of which is incorporated herein by
reference in its entirety. Particularly preferred, are polymers
containing about 4 to 25 contiguous Arg or Lys residues, preferably
from 7-15 such residues. However, the residues need not be
contiguous and it may sometimes be desirable to include, e.g.,
repeating units of two or three Arg or Lys residues, or the like,
separated by other amino acids. See, e.g., U.S. Patent Application
Publication No. 2003/0032593, incorporated herein by reference in
its entirety.
[0220] The polymers are constructed by any method known in the art.
For example, peptides can be conveniently synthesized chemically,
by any of several techniques that are known to those skilled in the
peptide art. In general, these methods employ the sequential
addition of one or more amino acids to a growing peptide chain.
Normally, either the amino or carboxyl group of the first amino
acid is protected by a suitable protecting group. The protected or
derivatized amino acid can then be either attached to an inert
solid support or utilized in solution by adding the next amino acid
in the sequence having the complementary (amino or carboxyl) group
suitably protected, under conditions that allow for the formation
of an amide linkage. The protecting group is then removed from the
newly added amino acid residue and the next amino acid (suitably
protected) is then added, and so forth. After the desired amino
acids have been linked in the proper sequence, any remaining
protecting groups (and any solid support, if solid phase synthesis
techniques are used) are removed sequentially or concurrently, to
render the final peptide. By simple modification of this general
procedure, it is possible to add more than one amino acid at a time
to a growing chain, for example, by coupling (under conditions
which do not racemize chiral centers) a protected tripeptide with a
properly protected dipeptide to form, after deprotection, a
pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase
Peptide Synthesis (Pierce Chemical Co., Rockford, Ill. 1984) and G.
Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis,
Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic
Press, New York, 1980), pp. 3-254, for solid phase peptide
synthesis techniques; and M. Bodansky, Principles of Peptide
Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J.
Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol.
1, for classical solution synthesis.
[0221] Typical protecting groups include t-butyloxycarbonyl (Boc),
9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);
p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);
biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,
isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl,
isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
[0222] Typical solid supports are cross-linked polymeric supports.
These can include divinylbenzene cross-linked-styrene-based
polymers, for example, divinylbenzene-hydroxymethylstyrene
copolymers, divinylbenzene-chloromethylstyrene copolymers and
divinylbenzene-benzhydrylaminopolystyrene copolymers.
[0223] N-methyl and hydroxy-amino acids can be substituted for
conventional amino acids in solid phase peptide synthesis. However,
production of polymers with reduced peptide bonds requires
synthesis of the dimer of amino acids containing the reduced
peptide bond. Such dimers are incorporated into polymers using
standard solid phase synthesis procedures. Other synthesis
procedures are well known and can be found, for example, in
Fletcher et al., Chem. Rev. (1998) 98:763-795, Simon et al., Proc.
Natl. Acad. Sci. (1992) 89:9367-9371, and references cited
therein.
[0224] Alternatively, the peptides can be produced by recombinant
techniques, e.g., by synthesizing DNA encoding the desired peptide,
along with an ATG initiation codon. The nucleotide sequence can be
designed with the appropriate codons for the particular amino acid
sequence desired. In general, one selects preferred codons for the
intended host in which the sequence is expressed. The complete
sequence is generally assembled from overlapping oligonucleotides
prepared by standard methods and assembled into a complete coding
sequence. See, e.g., Edge Nature (1981) 292:756; Nambair et al.
Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.
Automated synthetic techniques such as phosphoramide solid-phase
synthesis, can be used to generate the nucleotide sequence. See,
e.g., Beaucage, S. L. et al. Tet. Lett. (1981) 22:1859-1862;
Matteucci, M. D. et al. J. Am. Chem. Soc. (1981) 103:3185-3191.
Next the DNA is cloned into an appropriate expression vector,
either procaryotic or eucaryotic, using conventional methods.
[0225] Transport polymers of the invention can be associated with
SCNCs by simply mixing the particular polymer with the
nanoparticles thereby forming a non-covalent association. See,
e.g., Example 1 herein. Alternatively, the transport polymers can
be attached covalently to SCNCs by a number of chemical methods,
methods known in the art (see, for example, Wong, S. S., Ed.,
Chemistry of Protein Conjugation and Cross-Linking, (1991) CRC
Press, Inc., Boca Raton, Fla., either directly (e.g., with a
carbodiimide) or via a linking moiety. In particular, carbamate,
ester, thioether, disulfide, and hydrazone linkages are generally
easy to form and suitable for most applications. Ester and
disulfide linkages are preferred if the linkage is to be readily
degraded in the cytosol, after transport of the substance across
the cell membrane.
[0226] Various functional groups (hydroxyl, amino, halogen, etc.)
can be used to attach the SCNC to the transport polymer.
[0227] Moreover, the SCNC can be associated with the cationic
polymer using first and second members of a binding pair. In a
preferred embodiment, steptavidin, avidin or neutravidin is coupled
to the SCNC and biotin is coupled to the cationic polymer.
Alternatively, biotin is coupled to the SCNC and steptavidin,
avidin or neutravidin is coupled to the cationic polymer. Methods
for biotinylating peptides are well known in the art, and various
commercial sources, such as Pierce Chemical Co., Rockford Ill.,
sell kits for doing so
[0228] Cationic polymers are generally produced with an amino
terminal protecting group, such as FMOC. The FMOC may be cleaved
from the N-terminus of the completed resin-bound polypeptide so
that the SCNC can be linked to the free N-terminal amine. In such
cases, the SCNC is typically activated by methods well known in the
art to produce an active ester or active carbonate moiety effective
to form an amide or carbamate linkage, respectively, with the
polymer amino group. Of course, other linking chemistries can also
be used.
[0229] To help minimize side-reactions, guanidino and amidino
moieties can be blocked using conventional protecting groups, such
as carbobenzyloxy groups (CBZ), di-t-BOC, PMC, Pbf, N--NO.sub.2,
and the like.
[0230] Coupling reactions are performed by known coupling methods
in any of an array of solvents, such as N,N-dimethyl formamide
(DMF), N-methyl pyrrolidinone, dichloromethane, water, and the
like. Exemplary coupling reagents include O-benzotriazolyloxy
tetramethyluronium hexafluorophosphate (HATU), dicyclohexyl
carbodiimide, bromo-tris(pyrrolidino)phosphonium bromide (PyBroP),
etc. Other reagents can be included, such as N,N-dimethylamino
pyridine (DMAP), 4-pyrrolidino pyridine, N-hydroxy succinimide,
N-hydroxy benzotriazole, and the like.
[0231] The linker may be a readily cleavable linker, meaning that
it is susceptible to enzymatic or solvent-mediated cleavage in
vivo. For this purpose, linkers containing carboxylic acid esters
and disulfide bonds are preferred, where the former groups are
hydrolyzed enzymatically or chemically, and the latter are severed
by disulfide exchange, e.g., in the presence of glutathione.
[0232] In one embodiment, the cleavable linker contains a first
cleavable group that is distal to the SCNC, and a second cleavable
group that is proximal to the SCNC, such that cleavage of the first
cleavable group yields a linker-SCNC conjugate containing a
nucleophilic moiety capable of reacting intramolecularly to cleave
the second cleavable group, thereby releasing the SCNC from the
linker and polymer.
[0233] Methods for conjugating cationic polymers to molecules to be
transported are known in the art and described in e.g., U.S. Pat.
Nos. 4,847,240 and 6,495,663; and U.S. Patent Application
Publication No. 2003/0032593, the disclosures of which are
incorporated herein by reference in their entireties.
[0234] The Coding Scheme
[0235] The cells are encoded to allow rapid analysis of cell,
identity, as well as allowing multiplexing. The coding scheme
preferably employs one or more different SCNCs, although a variety
of additional agents, including chromophores, fluorophores and
dyes, and combinations thereof can be used alternatively or in
combination with SCNCs. For organic dyes, different dyes that have
distinguishable fluorescence characteristics can be used. Different
SCNC populations having the same peak emission wavelength but
different peak widths can be used to create different codes if
sufficient spectral data can be gathered to allow the populations
to be distinguished. Such different populations can also be mixed
to create intermediate linewidths and hence more unique codes. In
addition, the coding scheme can be based on differences in
excitation wavelength, emission wavelength, emission intensity,
FWHM (full width at half maximum peak height), fluorescence
lifetime, or combinations thereof.
[0236] The number of SCNCs used to encode a single cell locale can
be selected based on the particular application. Single SCNCs can
be detected (see, e.g., U.S. application Ser. No. 09/784,866, filed
Feb. 15, 2001 and entitled "Single Target Counting Assays Using
Semiconductor Nanocrystals: Empedocles et al. inventors); however,
a plurality of SCNCs from a given population is preferably
incorporated in a single cell to provide a stronger, more
continuous emission signal from each cell and thus allow shorter
analysis time.
[0237] Different SCNC populations can be prepared with peak
wavelengths separated by approximately 1 nm, and the peak
wavelength of an individual SCNC can be readily determined with 1
nm accuracy. In the case of a single-peak spectral code, each
wavelength is a different code. For example, CdSe SCNCs have a
range of emission wavelengths of approximately 490-640 nm and thus
can be used to generate about 150 single-peak codes at 1 nm
resolution.
[0238] A spectral coding system that uses only highly separated
spectral peaks having minimal spectral overlap and does not require
stringent intensity regulation within the peaks allows for
approximately 100,000 to 10,000,000 or more unique codes in
different schemes.
[0239] A binary coding scheme combining a first SCNC population
having an emission wavelength within a 490-565 nm channel and a
second SCNC population within a 575-650 nm channel produces 3000
valid codes using 1-nm resolved SCNC populations if a minimum peak
separation of 75 nm is used. The system can be expanded to include
many peaks, the only requirement being that the minimum separation
between peak wavelengths in valid codes is sufficient to allow
their resolution by the detection methods used in that
application.
[0240] A binary code using a spectral bandwidth of 300 nm, a
coding-peak resolution, i.e., the minimum step size for a peak
within a single channel, of 4 nm, a minimum interpeak spacing of 50
nm, and a maximum of 6 peaks in each code results in approximately
200,000 different codes. This assumes a purely binary code, in
which the peak within each channel is either "on" or "off." By
adding a second "on" intensity, i.e., wherein intensity is 0, 1 or
2, the number of potential codes increases to approximately 5
million. If the coding-peak resolution is reduced to 1 nm, the
number of codes increases to approximately 1.times.10.sup.10.
[0241] Valid codes within a given coding scheme can be identified
using an algorithm. Potential codes are represented as a binary
code, with the number of digits in the code corresponding to the
total number of different SCNC populations having different peak
wavelengths used for the coding scheme. For example, a 16-bit code
could represent 16 different SCNC populations having peak emission
wavelengths from 500 nm through 575 nm, at 5 nm spacing. A binary
code 1000 0000 0000 0001 in this scheme represents the presence of
the 500 nm and 575 nm peaks. Each of these 16-bit numbers can be
evaluated for validity, depending on the spacing that is required
between adjacent peaks; for example, 0010 0100 0000 0000 is a valid
code if peaks spaced by 15 nm or greater can be resolved, but is
not valid if the minimum spacing between adjacent peaks must be 20
nm. Using a 16-bit code with 500 to 575 nm range and 5 nm spacing
between peaks, the different number of possible valid codes for
different minimum spectral spacings between adjacent peaks is shown
in Table 2.
TABLE-US-00002 TABLE 2 The number of unique codes with a binary
16-bit system. Spectral Separation 5 nm 10 nm 15 nm 20 nm 25 nm 30
nm Number of 65535 2583 594 249 139 91 unique codes
[0242] If different distinguishable intensities are used, then the
number of valid codes dramatically increases. For example, using
the 16-bit code above, with 15 nm minimum spacing between adjacent
peaks in a code, 7,372 different valid codes are possible if two
intensities, i.e., a ternary system, are used for each peak, and
38,154 different valid codes are possible for a quaternary system,
i.e., wherein three "on" intensities can be distinguished.
[0243] Codes utilizing intensities require either precise matching
of excitation sources or incorporation of an internal intensity
standard into the cells due to the variation in extinction
coefficient exhibited by individual SCNCs when excited by different
wavelengths.
[0244] It is preferred that the light source used for the encoding
procedure be as similar as possible (preferably of the same
wavelength and intensity) to the light source that will be used for
decoding. The light source may be related in a quantitative manner,
so that the emission spectrum of the final material may be deduced
from the spectrum of the staining solution.
[0245] Codes can optionally be created by using substantially
non-overlapping colors of SCNCs, and then combining the SCNCs in
unique ratios, or according to absolute levels. Alternative codes
might be created by relying on overlapping signal
deconvolution.
[0246] The code creation methods optionally use a computer program
to combine or mix together, in silico (that is, using computer
modeling), emission signals from SCNCs. These individual marker
signal spectra can be real spectra from SCNCs that have already
been manufactured, or simulated spectra for SCNC batches that can
be manufactured. Candidate code spectra are then compared against
one another, with acceptable codes added to the library in order to
create an optimal set of codes that are sufficiently different from
each other to allow robust code assignment given constraints such
as code-number requirements and instrument resolution. A further
method uses stored patterns of known code spectra against which to
evaluate an unknown spectrum, in order to assign a code to the
unknown spectrum, or to declare it as "no match." To do this,
several steps are performed, some optional: (1) creation of a code;
(2) creation of a template for the code; (3) comparison of a sample
spectrum against all possible templates; and (4) assignment of
"match" or "no match" to the sample based upon its degree of
similarity to one of the templates and/or dissimilarity to the
remainder.
[0247] Coded objects can be created by attaching one or more SCNC
batches to an object or to many objects simultaneously. One
criterion for creating useful codes is that, when a code is
analyzed, it can be uniquely identified within the statistical
confines of the experiment or actual code reading equipment.
Generally, all codes to be used in a given application should be
spectrally resolvable, i.e., sufficiently spectrally dissimilar
within manufacturing tolerances and/or reading error, such that the
rate of incorrect decoding is very low. The acceptable error rate
depends on the application. Codes may be created randomly or
systematically. Using the random approach, mixtures of SCNCs are
created and then used as codes. Using the systematic approach, SCNC
batches are chosen, and mixed together in the appropriate ratios to
generate the codes. In both approaches, the composite emission
spectrum of each new code is compared to the emission spectrum of
all other codes that will be used in the application. This can be
done prior to the actual physical creation of the code, by using
predicted spectra, or can be done by reading the spectrum of the
new code prior to, or after, attaching the code to the object(s).
If the code is non-overlapping, i.e., will not be misclassified
when noise, aging, reader differences, or other factors are taken
into account, then the code is valid to be used. The emission
spectrum of the new code is stored digitally so that putative new
codes, and unknown codes during code reading, can be compared
against it. Preferably, reading accuracy will be incorporated into
the comparison of prior codes with new codes, the reading accuracy
generally being determined based on known properties of one or more
of the excitation energy source, the sensor, and the data
manipulation performed by the processor.
[0248] When many items are being coded with the same code, e.g.,
when attaching SCNCs to cells, microspheres or beads in a batch
mode, it is useful to analyze more than one of those items and
store an average, or representative, spectrum for the code. Once
this has been done, the actual spectrum for each sample item can be
compared with the average spectrum to ensure that they are
correctly identified. They may also be compared against the spectra
of other codes to ensure that they are not mis-identified.
Furthermore, statistical information regarding, for example,
reproducibility and confidence levels can be gleaned at this
stage.
[0249] The stored emission spectrum may herein be called the code's
"template" and can have been generated experimentally by analyzing
coded object(s) or SCNC mixtures, or can be generated in silico by
adding together emission spectra from the SCNCs that make up the
code, along with any required correction factors.
[0250] Template emission spectra may be generated by using the
instrument (or a similar instrument, or a computer model of the
instrument) that will be used for reading the code, optionally
correcting for any instrument-to-instrument variation. For example,
for SCNC-encoded cell assays it is desirable to analyze wells that
contain a single or a few different known coded cells that have
been processed through assay conditions. The template emission
spectra may be generated for each encoded cell reader instrument so
that during analysis, the templates for a given reader or assay are
used.
[0251] Many different systematic methods for creating codes can be
envisioned. For example, two colors of SCNCs may be used and the
ratio of color 1:color-2 varied to create different codes. Using
additional colors, the different ratios can be varied to create
codes that are more complex.
[0252] SCNC batches that have the same color, i.e., the same peak
wavelength, but have different peak widths, can be used to create
two different codes if sufficient spectral data is gathered to
allow these to be defined as being significantly different. These
batches can also be mixed to create intermediate linewidths and
hence more unique codes.
[0253] A computer-based method that uses all physically available
SCNC spectra, or that uses electronically generated spectra of all
manufacturable SCNC batches, can be used. In this case, the
computer is programmed to combine systematically or randomly
different amounts of these SCNC spectra, in silico, along with any
correction factors desired due to energy or electron transfer,
emission intensity variations, or wavelength changes that may
occur. The electronically created spectra are compared against
current codes and any that are sufficiently distinguishable are
candidates for manufacturing into real physical codes. This type of
approach can also be used to create code sets, i.e., manufacturable
emission spectra that are chosen to be maximally different from one
another according to predetermined comparison criteria such as the
residual value from a least squares fitting, or other methods known
in the art.
[0254] Data on the overall emission spectrum of a code can be
gathered by exciting the SCNCs with an appropriate source, e.g.,
laser, lamp, light-emitting diode, or the like, and reading the
emitted light with a device that provides spectral information for
the object, e.g., grating spectrometer, prism spectrometer, imaging
spectrometer, or the like, or use of interference (bandpass)
filters. Using a two-dimensional area imager such as a CCD camera,
many objects may be imaged simultaneously. Spectral information can
be generated by collecting more than one image via different
bandpass, longpass, or shortpass filters (interference filters,
colored glass filters, or electronically tunable filters are
appropriate). More than one imager may be used to gather data
simultaneously through dedicated filters, or the filter may be
changed in front of a single imager. Once this data has been
gathered, it can be processed to generate spectral information
about objects in the image, or for each pixel, or group of pixels
in the image, via straightforward image processing techniques.
[0255] The emission spectrum from the sample object is compared
against all the known templates. This can be done using many
techniques known in the art such as least squares fitting, Fourier
analysis, Kolmogorov-Smirnov Test, Pearson Rank Correlation test,
or the like (see Numerical Recipes in C, Press et al., Cambridge
University Press, 1996). In each case, a measure of the goodness of
fit of the unknown to each template is generated, (e.g., a residual
value for a least squares approach, or other fit measure dependent
on the fitting algorithm used such as one of the "robust" or
absolute magnitude methods described by Press et al., supra). If
this goodness of fit falls within the predetermined range for only
one of the codes then this is the identity of the unknown code,
otherwise the unknown is classified as "no match," or as matching
too many templates.
[0256] It might be desirable to make the matching process
insensitive to absolute intensity variations. This can be done by
including a linear or non-linear intensity normalization factor
during the matching process, which is varied to generate the lowest
residual value or other match parameter for each comparison. The
normalization factor can be allowed to vary without limits or can
be constrained to be within a given range to limit the amount of
correction for intensity variations.
[0257] The spectral data can also be normalized spectrally, i.e.,
shifting the data spectrally in a linear or nonlinear manner, to
correct for variations in the wavelength that may occur due to the
instrument or due to temperature changes, degradation, or other
effects that cause the SCNCs to emit at different wavelengths.
Again, the spectral shift factor may be constrained to be within a
given range.
[0258] When the emission spectrum also contains signal from a
reporter or reference SCNC, e.g., in the case of encoded cell
assays, this may be quantitated at the same time, and may also be
normalized according to the factors described above. Any spectral
overlap from the code into the assay signal may also be corrected
for in this way.
[0259] Spectral data will often be collected from more windows
and/or allowed discrete wavelengths than there are colors of SCNCs
present. This allows SCNCs of only slightly differing wavelengths
to be used to create the codes. Additional spectral data also makes
the classification process more robust than simple one-color,
one-data point approaches. An advantage of the pattern matching
approach for analysis is that, independent of the method of code
creation, any sufficiently different spectra can be used as unique
codes. Since unique fingerprints can be obtained for each code
based on individual raw spectra, concrete statistical estimates can
be used in determinations such as goodness of fit, confidence
intervals, and determination of uniqueness. In addition, this
method allows for empirical determination of codes following
chemical processing as blanks, removing much of the ambiguity
associated with pre-formatted idealized code sets.
[0260] Spectrally Encoded Microspheres.
[0261] Microspheres for use in the invention disclosed herein can
be spectrally encoded through incorporation of SCNCs See, e.g.,
U.S. Pat. No. 6,207,392 to Weiss et al., issued Mar. 27, 2001,
International Pat. Publ. No. WO 00/17103 (inventors Bawendi et
al.), published Mar. 30, 2000, and Han et al. (2001) Nature
Biotech. 19:632-635.
[0262] Preferably, microspheres or beads used to encode cells are
approximately less than about 1 micrometer, preferably 0.01 to
about 0.5 micrometer, more preferably 0.01 to about 0.1 micrometer,
and can be manipulated using normal solution techniques when
suspended in a solution. Each individual cell can be encoded with a
single microsphere having a unique code. Alternatively, each
individual cell can be encoded with more than one microsphere as
needed to provide a uniquely encoded cell. The beads can be
prepared to contain a population of SCNCs having a single peak
emission wavelength or the beads can be prepared to contain more
than a single population of SCNCs, each population having a peak
emission wavelength, or other fluorescence characteristic (for
example excitation wavelength, emission wavelength, emission
intensity, FWHM (full width at half maximum peak height), or
fluorescence lifetime) that is distinguishable from that of the
other populations, such that each bead has a unique spectral
signature.
[0263] Polymeric microspheres or beads can be prepared from a
variety of different polymers, including but not limited to
polystyrene, cross-linked polystyrene, polyacrylic, polylactic
acid, polyglycolic acid, poly(lactide coglycolide), polyanhydrides,
poly(methyl methacrylate), poly(ethylene-co-vinyl acetate),
polysiloxanes, polymeric silica, latexes, dextran polymers and
epoxies. The materials have a variety of different properties with
regard to swelling and porosity, which are well understood in the
art. The terms "bead," "sphere," "microbead" and "microsphere" are
used interchangeably herein.
[0264] The desired fluorescence characteristics of the microspheres
may be obtained by mixing SCNCs of different sizes and/or
compositions in a fixed amount and ratio to obtain the desired
spectrum, which can be determined prior to association with the
microspheres. Subsequent treatment of the micro spheres (through,
for example, covalent attachment, co-polymerization, or passive
absorption or adsorption) with the staining solution results in a
material having the designed fluorescence characteristics.
[0265] A number of SCNC solutions can be prepared, each having a
distinct distribution of sizes and compositions, to achieve the
desired fluorescence characteristics. These solutions may be mixed
in fixed proportions to arrive at a spectrum having the
predetermined ratios and intensities of emission from the distinct
SCNCs suspended in that solution. Upon exposure of this solution to
a light source, the emission spectrum can be measured by techniques
that are well established in the art. If the spectrum is not the
desired spectrum, then more of the SCNC solution needed to achieve
the desired spectrum can be added and the solution "titrated" to
have the correct emission spectrum. These solutions may be
colloidal solutions of SCNCs dispersed in a solvent, or they may be
pre-polymeric colloidal solutions, which can be polymerized to form
a matrix with SCNCs contained within.
[0266] The composition of the staining solution can be adjusted to
have the desired fluorescence characteristics, preferably under the
exact excitation source that will be used for the decoding. A
multichannel auto-pipettor connected to a feedback circuit can be
used to prepare an SCNC solution having the desired spectral
characteristics, as described above. If the several channels of the
titrator/pipettor are charged with several unique solutions of
SCNCs, each having a unique excitation and emission spectrum, then
these can be combined stepwise through addition of stock
solutions.
[0267] Once the staining solution has been prepared, it can be used
to incorporate a unique spectral code into a given bead population.
If the method of incorporation of the SCNCs into the beads is
absorption or adsorption, then the solvent that is used for the
staining solution should be one that is suitable for swelling the
microspheres, and can be selected based on the microsphere
composition. Typical solvents for swelling microspheres include
those in which the microsphere material is more soluble, for
example dichloromethane, chloroform, dimethylformamide,
tetrahydrofuran and the like. These can be mixed with a solvent in
which the microsphere material is less soluble, for example
methanol or ethanol, to control the degree and rate of
incorporation of the staining solution into the material.
[0268] The microspheres swell when added to the staining solution
and incorporate a plurality of SCNCs in the relative proportions
that are present in the staining solution. After removal of the
staining solution from the material, a nonswelling solvent is
added, the material shrinks, or unswells, thereby trapping the
SCNCs in the material. Alternatively, SCNCs can be trapped by
evaporation of the swelling solvent from the material. After
rinsing with a nonswelling solvent in which the SCNCs can be
suspended, the SCNCs are trapped in the material, and can be
retained by further use of a nonswelling solvent. Typical
nonswelling solvents include hexane and toluene. The thus-encoded
beads can be separated and exposed to a variety of solvents without
a change in the emission spectrum under the light source. When the
material used is a polymer bead, the material can be separated from
the rinsing solvent by any suitable technique, for example,
centrifugation, evaporation, fluidized bed drying, etc., or
combined procedures, and can be redispersed into aqueous solvents
and buffers through the use of detergents in the suspending
buffer.
[0269] The staining procedure can also be carried out in sequential
steps. A first staining solution can be used to stain the beads
with one population of SCNCs. The beads can then be separated from
the first staining solution and added to a second staining solution
to stain the beads with a second population of SCNCs. These steps
can be repeated until the desired spectral properties are obtained
from the material when excited by a light source.
[0270] The SCNCs can be attached to the beads by covalent
attachment as well as by entrapment in swelled beads, or can be
coupled to one member of a binding pair the other member of which
is attached to the beads. For instance, SCNCs are prepared by a
number of techniques that result in reactive groups on the surface
of the SCNC. See, e.g., Bruchez et al. (1998) Science
281:2013-2016, Chan et al. (1998) Science 281:2016-2018, Colvin et
al. (1992) J. Am. Chem. Soc. 114:5221-5230, Katari et al. (1994) J.
Phys. Chem. 98:4109-4117, Steigerwald et al. (1987) J. Am. Chem.
Soc. 110:3046. The reactive groups present on the surface of the
SCNCs can be coupled to reactive groups present on a surface of the
material. For example, SCNCs which have carboxylate groups present
on their surface can be coupled to beads with amine groups using a
carbodiimide activation step.
[0271] Any cross-linking method that links a SCNC to a bead and
does not adversely affect the properties of the SCNC or the bead
can be used. In a cross-linking approach, the relative amounts of
the different SCNCs can be used to control the relative
intensities, while the absolute intensities can be controlled by
adjusting the reaction time to control the number of reacted sites
in total. After the beads are crosslinked to the SCNCs, the beads
are optionally rinsed to wash away unreacted SCNCs.
[0272] A sufficient amount of fluorophore must be used to encode
the beads so that the intensity of the emission from the
fluorophores can be detected by the detection system used and the
different intensity levels must be distinguishable, where intensity
is used in the coding scheme but the fluorescence emission from the
SCNCs or other fluorophores used to encode the beads must not be so
intense to as to saturate the detector used in the decoding
scheme.
[0273] The beads can be encoded to allow rapid analysis thereof,
and thus of the cell encoded therewith, identity, as well as
allowing multiplexing. The coding scheme preferably employs one or
more different SCNCs, although a variety of additional agents,
including chromophores, fluorophores and dyes, and combinations
thereof can be used alternatively or in combination with SCNCs. For
organic dyes, different dyes that have distinguishable fluorescence
characteristics can be used. Different SCNC populations having the
same peak emission wavelength but different peak widths can be used
to create different codes if sufficient spectral data can be
gathered to allow the populations to be distinguished. Such
different populations can also be mixed to create intermediate
linewidths and hence more unique codes.
[0274] The number of SCNCs used to encode a single bead or
substrate locale can be selected based on the particular
application. Single SCNCs can be detected; however, a plurality of
SCNCs from a given population is preferably incorporated in a
single bead to provide a stronger, more continuous emission signal
from each bead and thus allow shorter analysis time.
[0275] The beads can be encoded using the coding scheme described
supra.
[0276] The Excitation Source
[0277] By exposing the encoded cells prepared and described as
above to light of an excitation source, the SCNCs disposed in or on
the cell will be excited to emit light. This excitation source is
of an energy capable of exciting at least one population of SCNCs
used in the experiment to emit light and preferably chosen to be of
higher energy than the shortest emission wavelength of the SCNCs
used. Further, the excitation source can be chosen such that it
excites a minimum number of SCNCs in the sample to produce
detectable light. Preferably the excitation source will excite a
sufficient number of different populations of SCNCs to allow unique
identification of all the encoded materials used in the experiment.
For example, using two different populations of cells having
different ratios of red to blue SCNCs, it would not be sufficient
to only excite the red emitting SCNCs, e.g., by using green or
yellow light, of the sample in order to decode the cells. It would
be necessary to use a light source comprising at least one
wavelength that is capable of exciting the blue emitting and the
red emitting SCNCs simultaneously, e.g., violet or ultraviolet.
There may be one or more light sources used to excite the different
populations of SCNCs simultaneously, or sequentially, but a given
light source will only excite subpopulations of SCNCs that emit at
lower energy than the light source, due to the absorbance spectra
of the SCNCs.
[0278] In addition, if a lamp source is used, degradation of the
lamp can result in changes in the excitation source, thereby
compromising the codes.
[0279] Detection of Emission
[0280] An example of an imaging system for automated detection for
use with the present methods comprises an excitation source, a
monochromator (or any device capable of spectrally resolving the
image, or a set of narrow band filters) and a detector array. The
excitation source can comprise blue or UV wavelengths shorter than
the emission wavelength(s) to be detected. This may be: a broadband
UV light source, such as a deuterium lamp with a filter in front;
the output of a white light source such as a xenon lamp or a
deuterium lamp after passing through a monochromator to extract out
the desired wavelengths; or any of a number of continuous wave (cw)
gas lasers, including but not limited to any of the Argon Ion laser
lines (457, 488, 514, etc. nm) or a HeCd laser; solid state diode
lasers in the blue such as GaN and GaAs (doubled) based lasers or
the doubled or tripled output of YAG or YLF based lasers; or any of
the pulsed lasers with output in the blue.
[0281] The emitted light can be detected with a device that
provides spectral information for the substrate, e.g., grating
spectrometer, prism spectrometer, imaging spectrometer, or the
like, or use of interference (bandpass) filters. Using a
two-dimensional area imager such as a CCD camera, many objects may
be imaged simultaneously. Spectral information can be generated by
collecting more than one image via different bandpass, longpass, or
shortpass filters (interference filters, or electronically tunable
filters are appropriate). More than one imager may be used to
gather data simultaneously through dedicated filters, or the filter
may be changed in front of a single imager. Imaging based systems,
like the Biometric Imaging system, scan a surface to find
fluorescent signals.
[0282] A scanning system can be used in which the sample to be
analyzed is scanned with respect to a microscope objective. The
luminescence is put through a single monochromator or a grating or
prism to spectrally resolve the colors. The detector is a diode
array that then records the colors that are emitted at a particular
spatial position. The software then recreates the scanned
image.
[0283] In the embodiment where cell or the population of cells is
encoded with light-scattering SERS or SERRS particle, the Raman
signal is detected using an epifluorescence laser confocal
microscope comprising a visible or infra red excitation laser, a
dichroic beam-splitter, a microscope objective, an excitation
cutoff filter, spectrometer and high efficiency detector such as a
CCD camera. Alternatively, the detection system can use line
illumination and collection by adding a cylindrical lens to the
excitation pathway and using a 2D detector. Alternatively, the
detection system can use area illumination and detection, and
generate spectral data by using a tunable bandpass filter or a
number of fixed bandpass filters placed in the detection
pathway.
[0284] Decoding Multiple Fluorescence Emissions
[0285] When imaging samples labeled with multiple fluorophores, it
is desirable to resolve spectrally the fluorescence from each
discrete region within the sample. Such samples can arise, for
example, from multiple types of SCNCs (and/or other fluorophores)
being used to encode cells, from a single type of SCNC being used
to encode cells but bound to a molecule labeled with a different
fluorophore, or from multiple cells labeled with different types of
fluorophores which overlap. Decoding the spectral code of an
encoded substrate can take place prior to, simultaneously with, or
subsequent to obtaining information from a functional assay
performed on the cells.
[0286] Many techniques have been developed to solve this problem,
including Fourier transform spectral imaging (Malik et al. (1996)
J. Microsc. 182:133; Brenan et al. (1994) Appl. Opt. 33:7520) and
Hadamard transform spectral imaging (Treado et al. (1989) Anal.
Chem. 61:732A; Treado et al. (1990) Appl. Spectrosc. 44:1-4; Treado
et al. (1990) Appl. Spectrosc. 44:1270; Hammaker et al. (1995) J.
Mol. Struct. 348:135; Mei et al. (1996) J. Anal. Chem. 354:250;
Flateley et al. (1993) Appl. Spectrosc. 47:1464), imaging through
variable interference (Youvan (1994) Nature 369:79; Goldman et al.
(1992) Biotechnology 10:1557), acousto-optical (Mortensen et al.
(1996) IEEE Trans. Inst. Meas. 45:394; Turner et al. (1996) Appl.
Spectrosc. 50:277) or liquid crystal filters (Morris et al. (1994)
Appl. Spectrosc. 48:857) or simply scanning a slit or point across
the sample surface (Colarusso et al. (1998) Appl. Spectrosc.
52:106A), all of which are capable of generating spectral and
spatial information across a two-dimensional region of a
sample.
[0287] One-dimensional spectral imaging can easily be achieved by
projecting a fluorescent image onto the entrance slit of a linear
spectrometer. In this configuration, spatial information is
retained along the y-axis, while spectral information is dispersed
along the x-axis (Empedocles et al. (1996) Phys. Rev. Lett.
77(18):3873). The entrance slit restricts the spatial position of
the light entering the spectrometer, defining the calibration for
each spectrum. The width of the entrance slit, in part, defines the
spectral resolution of the system.
[0288] Two-dimensional images can be obtained by eliminating the
entrance slit and allowing the discrete images from individual
points to define the spatial position of the light entering the
spectrometer. In this case, the spectral resolution of the system
is defined, in part, by the size of the discrete images. Since the
spatial position of the light from each point varies across the
x-axis, however, the calibration for each spectrum will be
different, resulting in an error in the absolute energy values.
Splitting the original image and passing one half through a
dispersive grating to create a separate image and spectra can
eliminate this calibration error. With appropriate alignment, a
correlation can be made between the spatial position and the
absolute spectral energy.
[0289] To avoid ambiguity between images that fall along the same
horizontal line, a second beam-splitter can be added, with a second
dispersive element oriented at 90 degrees to the original. By
dispersing the image along two orthogonal directions, it is
possible to unambiguously distinguish the spectra from each
discrete point within the image. The spectral dispersion can be
performed using gratings, for example holographic transmission
gratings or standard reflection gratings. For example, using
holographic transmission gratings, the original image is split into
2 (or 3) images at ratios that provide more light to the spectrally
dispersed images, which have several sources of light loss, than
the direct image. This method can be used to spectrally image a
sample containing discrete point signals, for example in high
throughput screening of discrete spectral images such as single
cells or ensembles of cells immobilized on a substrate, and for
highly parallel reading of spectrally encoded cells. The images are
then projected onto a detector and the signals are recombined to
produce an image that contains information about the amount of
light within each band-pass.
[0290] Alternatively, techniques for calibrating point spectra
within a two-dimensional image are unnecessary if an internal
wavelength reference (the "reference channel") is included within
the spectrally encoded cell. The reference channel is preferably
either the longest or shortest wavelength emitting fluorophore in
the code. The known emission wavelength of the reference channel
allows determination of the emission wavelengths of the
fluorophores in the dispersed spectral code image. In addition to
wavelength calibration, the reference channel can serve as an
intensity calibration where coding schemes with multiple
intensities at single emission wavelengths are used. Additionally,
a fixed intensity of the reference channel can also be used as an
internal calibration standard for the quantity of label bound to
the surface of each bead.
[0291] In one system for reading spectrally encoded cells, a
confocal excitation source is scanned across the surface of a
sample. When the source passes over an encoded cell, the
fluorescence spectrum is acquired. By raster-scanning the
point-excitation source over the sample, all of the cells within a
sample can be read sequentially.
[0292] Encoded Cells Immobilized on Chips
[0293] Qcell.TM. encoding technology may be used to study membrane
receptor proteins. Membrane receptor proteins constitute an
important target class for drug development, yet are difficult to
purify and immobilize on protein chips. Native expression of
membrane proteins in SCNC-encoded cells greatly facilitates the
correct folding and identification of these proteins for use in a
variety of proteomics and diagnostics applications. Encoded cells
are randomly deposited on the chip surface, and there is no need to
spatially arrange each receptor for encoding. This assay platform
is compatible with a variety of detection technologies that measure
binding of fluorescent-tagged ligands to proteins on a chip
surface.
[0294] Encoded cells may be utilized in conjunction with a
substrate, and may be grown on, attached to, or placed upon the
substrate. The substrate can comprise a wide range of material,
either biological, nonbiological, organic, inorganic, or a
combination of any of these. For example, the substrate may be a
polymerized Langmuir Blodgett film, functionalized glass, Si, Ge,
GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a
wide variety of gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene, cross-linked polystyrene,
polyacrylic, polylactic acid, polyglycolic acid, poly(lactide
coglycolide), polyanhydrides, poly(methyl methacrylate),
poly(ethylene-co-vinyl acetate), polysiloxanes, polymeric silica,
latexes, dextran polymers, epoxies, polycarbonate, or combinations
thereof.
[0295] Substrates can be planar crystalline substrates such as
silica-based substrates (e.g. glass, quartz, or the like), or
crystalline substrates used in, e.g., the semiconductor and
microprocessor industries, such as silicon, gallium arsenide and
the like.
[0296] Silica aerogels can also be used as substrates, and can be
prepared by methods known in the art. Aerogel substrates may be
used as freestanding substrates or as a surface coating for another
substrate material.
[0297] The substrate can take any form and typically is a plate,
slide, bead, pellet, disk, particle, strand, precipitate, membrane,
optionally porous gel, sheets, tube, sphere, container, capillary,
pad, slice, film, chip, multiwell plate or dish, optical fiber, and
the like. The substrate may contain raised or depressed regions on
which an encoded cell is located. The surface of the substrate can
be etched using well known techniques to provide for desired
surface features, for example trenches, v-grooves, mesa structures,
or the like.
[0298] Surfaces on the substrate can be composed of the same
material as the substrate or can be made from a different material,
and can be coupled to the substrate by chemical or physical means.
Such coupled surfaces may be composed of any of a wide variety of
materials, for example, polymers, plastics, resins,
polysaccharides, silica or silica-based materials, carbon, metals,
inorganic glasses, membranes, or any of the above-listed substrate
materials. In one embodiment, the surface will be optically
transparent and will have surface Si--OH functionalities, such as
those found on silica surfaces.
[0299] The substrate and/or its optional surface are chosen to
provide appropriate optical characteristics for the synthetic
and/or detection methods used. The substrate and/or surface can be
transparent to allow the exposure of the substrate by light applied
from multiple directions. The substrate and/or surface may be
provided with reflective "minor" structures to increase the
recovery of light emitted by the semiconductor nanocrystal or other
label. The substrate and/or its surface may also be coated to
decrease the amount of spurious incident light.
[0300] The substrate and/or its surface is generally resistant to,
or is treated to resist, the conditions to which it is to be
exposed in use, and can be optionally treated to remove any
resistant material after exposure to such conditions.
[0301] SCNCs as Labels to Study Intracellular Protein/Protein
Interactions.
[0302] To measure intracellular protein/protein interactions, a
cell line expressing, for example, a gene fusion of renilla
luciferase and a protein of interest (protein A) can be used. An
SCNC, conjugated to a potential interacting protein (protein B), is
delivered into cells using the Chariot reagent, a peptide reagent
based on the HIV-tat sequence (see Example 1). The binding of
protein A and protein B is measured by bioluminescence resonance
energy transfer (BRET). See FIG. 5. The light emitted by renilla
luciferase is transferred to the SCNC only if protein A is bound to
protein B, and the distance between luciferase and the SCNC is less
than 100 angstroms. Alternatively, several different SCNC/protein
conjugates are delivered into cells and their interactions with
protein A are studied in real time by measuring the SCNC emission
properties. This approach can be used to study the assembly of
complex structures such as transcription factor complexes or the
splicesome inside living cells.
[0303] Encoding DNA Transfections to Screen, in a Combinatorial
Fashion, the Functions of Genes Identified in Gene Expression
Microarray Experiments.
[0304] As explained above, there are a number of distinct methods
for delivering SCNCs into cells. One of these methods relies on a
cationic lipid that is similar to commercial reagents for
transfecting DNA molecules into cells which can be used to
co-deliver DNA and SCNC codes into cells. The encoding of DNA
transfections greatly facilitates the functional analysis of genes
identified in microarray experiments. For example, a microarray
experiment can identify hundreds of genes that are specifically
turned on in response to a compound that induces cell apoptosis. To
identify single genes or gene combinations responsible for the
apoptotic phenotype, for example, many separate DNA transfections
and assays are required using conventional methods. Multiplexing
the assays with encoded DNA greatly facilitates these assays
because the genotype is linked to phenotype via an easily read
optical SCNC code. The encoded transfectants are mixed and added to
wells, and the effects of a specific compound or incubation
condition can be screened simultaneously against the phenotypes of
many gene combinations within a single well.
[0305] Encoded Cells for Multiplex Screening of Different Drug
Targets Expressed in a Common Host Cell Line
[0306] Encoded cell technology can be used to screen multiple
targets simultaneously in the same assay well. Each target is
expressed in a common host cell line, and the identity of the
receptor is encoded in the semiconductor nanocrystal code. Examples
of high-value drug targets and corresponding cell-based assays
include the following:
[0307] G protein coupled receptors: competition binding assays,
reporter gene assays, calcium assays;
[0308] Ion channels: competition binding assays, reporter gene
assays, calcium assays, membrane potential assays;
[0309] Nuclear receptors: reporter gene assays, calcium assays;
and
[0310] Cytokine receptors: competition binding assays, reporter
gene assays, calcium assays.
[0311] Encoded Cells for Comparing Complex Phenotypes Among
Different Cell Types
[0312] Encoded cell technology is not limited to target-specific
cell based assays. A complex phenotype, such as apoptosis or cell
migration, can be compared between different cell types in the same
assay well because each cell type is encoded with a unique SCNC
code. Multiplexing complex phenotypic assays in the same assay well
may be valuable for kinetic assays, or for measuring the effects of
a single compound on cell-type specific responses. Examples of such
phenotypic assays include the following: apoptosis, cell migration,
cytoplasm to nucleus translocation, retrograde transport, neurite
outgrowth, and receptor internalization.
[0313] SCNCs as Labels for Imaging Intracellular Organelles or
Studying Protein Trafficking in Live Cells
[0314] Delivery of SCNCs conjugated to specific peptides, proteins
or antibodies into cells may provide a new and powerful method for
live cell imaging (FIG. 6, in which X is, for example, a peptide
ligand, protein, localization sequence or antibody). A peptide
sequence may act as an affinity handle for binding the SCNC to a
specific intracellular target, or it can target the SCNC to a
specific intracellular organelle. Mulitiplex analysis of several
proteins in a live cell is invaluable in screening and target
validation applications.
[0315] Encoding Fixed Cells for his to Chemical Applications
[0316] Encoded cell technology can also be used to multiplex any
histochemical staining assay. For example, kits are commercially
available for measuring the cytoplasm to nucleus translocation of
several transcription factors (Cellomics). The kit is comprised of
an Alexa-488-conjugated antibody that recognizes a specific
transcription factor, and buffers to fix and mount the cells. Cells
are incubated and fixed at various times after adding a compound,
and the cytoplasm to nucleus translocation of the transcription
factor is measured by fluorescence microscopy. Different encoded
cell types can be used to study the translocation of a single
protein among different cell types. Thus, the effect of a single
compound can be screened for its ability to block or activate the
translocation of a protein among different cell types.
[0317] Examples of Cellomics kits that can be encoded with SCNCs
include those for NF.epsilon.B, STAT1, STAT2, STAT3, STATS, c-Jun,
ATF-2, p38 MAPK, JNK/SAPK, ERK MAPK.
[0318] Other cell-based assay kits that can be used with encoded
cells include cell viability, neurite outgrowth, apoptosis, mitotic
index, cell motility, and receptor internalization.
[0319] Multiplex Screening of Cell Viability
[0320] A single compound can be screened for toxicity against
multiple cell types in a single well (FIG. 7).
[0321] Selectivity Profiling as a Tool for Predicting Compound
Toxicity.
[0322] Encoded cells can be used to measure the selectivity of
compounds against many drug targets and cell types. This
information can be used to predict toxicity, because many compounds
are toxic due to non-selective interactions.
[0323] Selectivity Profiling as a Tool to Increase the Efficiency
of Lead Optimization
[0324] Selectivity profiling can also aid lead optimization. A
thorough understanding of target selectivity at an early stage in
the drug discovery pipeline can lead to better choices for lead
optimization.
[0325] Combining Target Distribution and Compound Selectivity to
Predict Biodistribution of Compounds
[0326] Combining a compound's proteome-wide selectivity with the
proteome-wide tissue distribution of targets enables predictive in
silico biodistribution models (FIG. 8).
[0327] Transporter Assays
[0328] Transporter proteins are a high-value target class because
of their role in drug uptake. For example, selective serotinin
reuptake inhibitors (SSRIs) interact with the serotonin transporter
protein. SCNCs can be applied to transporter assays in several
ways. First, SCNCs can be conjugated to transporter ligands and
used in competition uptake assays to screen for compounds that
block uptake of the SCNC conjugate. Another use is to encode cell
lines expressing different transporters and to compare the uptake
efficiency of a fluorescent-labeled ligand among different
transporter types.
[0329] Thus, the applications of encoded cells are extremely
wide-ranging (FIG. 9).
[0330] GPCR Pathway Assays
[0331] The present invention provides a method of screening test
compounds and test conditions for the ability to modulate (activate
or inhibit, enhance or depress) a GPCR pathway, and provides
methods of assessing GPCR pathway function, such as the function of
an orphan GPCR, in a cell in general. In the present methods, SCNCs
are coupled with a candidate ligand or a library of candidate
ligands, as detailed above, and translocation of the ligand by the
GPCR pathway is followed by detecting the spatial location of
SCNCs, or the change in spatial location of SCNCs, in extracellular
fluid (natural or artificial, e.g., a growth or assay medium), in a
cell, the cell cytosol, a cell membrane, or an intracellular
compartment or membrane, e.g., an intracellular vesicle, the cell
nucleus or nuclear membrane, mitochondria or mitochondria membrane,
golgi apparatus, other organelle, or other intracellular
compartment or membrane. The relative extent of translocation or
change of spatial location of SCNCs under varied test conditions
may be compared, or a test condition may be compared, to a control
condition or to a predetermined standard. Depending on the assay
design, the determination of translocation of the ligand is an
indicator of modulation, e.g., agonist stimulation, of GPCR
activity or of the presence of a GPCR in a cell, in a cell membrane
or the like.
[0332] Translocation of the ligand is evidenced by an increase in
the intensity of the detectable signal located within the cell
cytosol, cell membrane, or an intracellular compartment and/or a
decrease in the intensity of the detectable signal located within
the cytosol, membrane, or intracellular compartment, wherein the
change occurs after exposure to the test compound. Translocation
may thus be detected by comparing changes in the detectable signal
in the same cell over time (i.e., pre- and post-exposure to the
test compound or to one or more members of the library of test
compounds). Alternatively, a test cell may be compared to a
pre-established standard. If a known modulator, e.g., an agonist or
antagonist ligand, is available, the present methods can be used to
screen a chemical compound library for and study candidate GPCR
agonists and antagonists.
[0333] The methods of the present invention provide easily
detectable results. For example, translocation of a ligand, such as
a GPCR ligand or beta-arrestin, coupled to an SCNC, in response to
GPCR activation or inhibition, results in a relative change in the
spatial location of the detectable signal within the cell cytosol,
membrane or intracellular compartment. In addition, the concomitant
decrease in detectable signal from the original location of the
signal in the cell cytosol, membrane or intracellular compartment
can be used to measure translocation of the ligand. In certain
cells, the activation of the GPCRs will result in essential
clearing of detectable signal from the original location of the
signal, and an concomitant increase in the detectable signal within
the cell cytosol, membrane or intracellular compartment. In the
present methods, it is preferred that the assay design results in
an increase in the detectable signal within the cell cytosol,
membrane or intracellular compartment after GPCR activation.
Preferably, the signal will increase at least two-fold, more
preferably at least three-fold, still more preferably at least
five-fold, and most preferably at least ten-fold.
[0334] In one embodiment, the present invention provides a method
for screening modulators of GPCR activity comprising: a) providing
a cell expressing a known or unknown GPCR, wherein the cell is
encoded with an SCNC, other detectable label as disclosed herein or
combination thereof; b) exposing the cell to a test compound; c)
detecting the signal from the SCNC; and (d) comparing the signal
produced in the presence of the test compound with the signal
produced in the absence, wherein changes in the spatial location of
the signal indicates that the compound is a modulator of a
GPCR.
[0335] In another embodiment, the present invention provides a
method for screening candidate GPCR modulator compounds comprising:
a) providing a cell expressing a known or unknown GPCR; b)
contacting the cell with a translocatable ligand that is conjugated
to a SCNC; c) exposing the cell to a predetermined concentration of
a test compound or each member of a library of test compounds; d)
detecting the translocation of the translocatable ligand into the
cell cytosol, cell membrane or intracellular compartment, and
comparing the translocation in the presence and absence of the
candidate modulator.
[0336] In yet another embodiment, the present invention provides
methods for screening a cell or a population of cells for the
presence of a GPCR, comprising (a) providing a cell or a population
of cells; (b) associating the cell or population of cells with an
SCNC; (c) exposing the cell or population of cells to a test
solution containing a known agonist to a GPCR; and either (d)
detecting in the cell translocation of a translocatable ligand
either (i) from the cellular membrane to the cytosol of the cell or
to an intracellular compartment or (ii) from the cytosol of the
cell to the membrane, and subsequently to an intracellular
compartment, (iii) from the cytosol to an intracellular
compartment, or (iv) from one intracellular compartment to another
intracellular compartment, or (e) detecting those cells in which
translocation of the translocatable ligand occurs, wherein the
translocation of the ligand indicates the presence of such a GPCR.
Translocation of the ligand can be detected as discussed above.
Populations of cells to be screened are discussed above, and can
additionally include a tissue, an organ, or an organism.
[0337] The present invention thus provides a convenient method of
identifying modulators for an orphan GPCR. Orphan GPCRs are novel
receptors and are typically identified by the sequence
comparison-based methods, but whose cognate ligands are not known.
It is estimated that from 400 to as many as 5000 orphan GPCRs may
be coded for in the human genome, representing a vast potential for
developing new drugs.
[0338] The present invention provides a convenient and efficient
method for identifying a natural or synthetic ligand that initiates
orphan GPCR activation, and for identifying ligands that inhibit
such activation, thereby characterizing the pharmacology of the
orphan GPCR. The method of the invention can be used to detect the
orphan GPCRs GPCR10, OX1R and OX2R, and GPCR 24 using the ligands
prolactin-releasing peptide, orexin-A/orexin-B, and melanin
concentrating hormone, respectively. Thus, the functions of orphan
GPCRs can be identified as controlling feeding behavior.
[0339] Preparation of Cells that Express GPCRs
[0340] Methods for preparing cells that express GPCRs have been
described. See, e.g., U.S. Pat. Nos. 6,051,386, 6,069,296,
6,111,076 and 6,280,934, the disclosures of which are incorporated
herein by reference. Generally, complementary DNA encoding GPCRs
can be obtained and can be expressed in an appropriate cell host
using techniques well known in the art. Typically, once a
full-length GPCR cDNA has been obtained, it can be expressed in a
mammalian cell line, yeast cell, amphibian cell or insect cell for
functional analysis. Preferably, the cell line is a mammalian cell
line that has been characterized for GPCR expression and that
optionally contains a wide repertoire of G-proteins to allow
functional coupling to downstream effectors. Examples of such cell
lines include Chinese Hamster Ovary (CHO) or Human Embryonic Kidney
293 (HEK293) lines. Cells in which the cDNA is expressed can be
encoded using the methods disclosed herein, thus allowing the
multiplex screening of ligands. The expressed receptor can then be
screened in a variety of functional assays to identify an
activating ligand as disclosed above and in U.S. Pat. Nos.
6,051,386, 6,069,296, 6,111,076 and 6,280,934. Preferably, the
functional assay methods use SCNCs, although other functional
responses can be monitored can also be used. Other functional
responses include changes in intracellular calcium or cAMP levels,
and metabolic activation, which can be measured using the
Cytosensor microphysiometer. In another embodiment, the receptor is
co-expressed with promiscuous G-proteins thereby aggregating signal
transduction through a common pathway involving phospholipase C and
calcium mobilization. Changes in calcium mobilization may be
detected using SCNCs, as discussed above, or via standard
fluorescence-based techniques using a high throughput imaging
system such as FLIPR.RTM. (Fluorescent Imaging Plate Reader).
Examples of high throughput microscopes include Discovery 1 from
Universal Imaging Corporation, CellPix from Axon Instruments,
LeadSeeker from Amersham/Pharmacia, and Explorer from Acumen. The
ability to screen in a high-throughput manner permits the screening
of orphan receptors against a wide range of candidate ligands, such
as those contained in a library. The library of candidate ligands
may contain known or suspected GPCR ligands, as well as molecules
for which the receptor is unknown. In addition, the methods of the
invention permit screening against biological extracts of tissues,
fluids, and cell supernatants, thereby identifying novel ligands
for GPCRs. Additionally, the methods of the invention can be used
to screen against peptide libraries or compound libraries. Once an
activating ligand is obtained, high-throughput screens of the
invention can be used to search for modulators of the receptor,
such as agonists and antagonists. The invention thus allows for the
identification of various agonists and antagonists of the known and
orphan GPCRs that can be used to evaluate the physiological role of
the receptor and its potential as a therapeutic target for drug
discovery.
[0341] Kits
[0342] Kits comprising reagents useful for performing the methods
of the invention are also provided. The components of the kit are
retained by a housing. Instructions for using the kit to perform a
method of the invention are provided with the housing, and may be
located inside the housing or outside the housing, and may be
printed on the interior or exterior of any surface forming the
housing which renders the instructions legible. In one embodiment,
a kit comprises an SCNC population, and a reagent useful for
encoding a cell using the SCNC population. Exemplary reagents
useful for encoding a cell are described above. These reagents may
be used alone or in combination. Additionally, the kit may be
designed for multiplex applications and contain a plurality of SCNC
populations useful for simultaneously encoding a plurality of
different cell populations.
EXAMPLES
[0343] The following examples are set forth so as to provide those
of ordinary skill in the art with a complete description of how to
make and use the present invention, and are not intended to limit
the scope of what is regarded as the invention. Efforts have been
made to ensure accuracy with respect to numbers used (e.g.,
amounts, temperature, etc.) but some experimental error and
deviation should be accounted for. Unless otherwise indicated,
parts are parts by weight, temperature is degree centigrade and
pressure is at or near atmospheric, and all materials are
commercially available.
[0344] Preparation of Polymer-Coated SCNCs
[0345] A. Synthesis of Hydrophobically Modified Hydrophilic
Polymers: A modified polyacrylic acid was prepared by diluting 100
g [0.48 mol COONa] of poly(acrylic acid, sodium salt) (obtained
from Aldrich, molecular weight 1200) was diluted two-fold in water
and acidified in a 1.0 L round bottom flask with 150 ml (1.9 mol)
of concentrated HCl. The acidified polymer solution was
concentrated to dryness on a rotary evaporator (100 mbar,
80.degree. C.). The dry polymer was evacuated for 12 hours at
<10 mbar to ensure water removal. A stirbar and 47.0 g (0.24
mol) of 1-[3-(dimethyl-amino)-propyl]-ethylcarbodiimide
hydrochloride (EDC-Aldrich 98%) were added to the flask, then the
flask was sealed and purged with N.sub.2, and fit with a balloon.
500 ml of anhydrous N--N,dimethylformamide (Aldrich) was
transferred under positive pressure through a cannula to this
mixture; and the flask was swirled gently to dissolve the solids.
32 ml (0.19 mol) of octylamine was transferred dropwise under
positive pressure through a cannula from a sealed oven-dried
graduated cylinder into the stirring polymer/EDC solution, and the
stifling continued for 12 hours. This solution was concentrated to
<100 ml on a rotary evaporator (30 mbar, 80.degree. C.), and the
polymer was precipitated by addition of 200 ml di-H.sub.2O to the
cooled concentrate, which produced a gummy white material. This
material was separated from the supernatant and triturated with 100
ml di-H.sub.2O three more times. The product was dissolved into 400
ml ethyl acetate (Aldrich) with gentle heating, and basified with
200 ml di-H.sub.2O and 100 g N--N--N--N-tetramethylammonium
hydroxide pentahydrate (0.55 mo) for 12 hours. The aqueous layer
was removed and precipitated to a gummy white product with 400 ml
of 1.27 M HCl. The product was decanted and triturated with 100 ml
of di-H.sub.2O twice more, after which the aqueous washings were
back-extracted into 6.times.100 ml portions of ethyl acetate. These
ethyl acetate solutions were added to the product flask, and
concentrated to dryness (100 mbar, 60.degree. C.). The crude
polymer was dissolved in 300 ml of methanol and purified in two
aliquots over LH-20 (Amersham-Pharmacia-5.5 cm.times.60 cm column)
at a 3 ml/minute flow rate. Fractions were tested by NMR for
purity, and the pure fractions were pooled, while the impure
fractions were re-purified on the LH-20 column. After pooling all
of the pure fractions, the polymer solution was concentrated by
rotary evaporation to dryness, and evacuated for 12 hours at <10
mbar. The product was a white powder (25.5 g, 45% of theoretical
yield), which showed broad NMR peaks in CD.sub.3OD [d=3.1 b (9.4),
2.3 b (9.7), 1.9 1.7 1.5 1.3 b (63.3) 0.9 bt (11.3)], and clear IR
signal for both carboxylic acid (1712 cm.sup.-1) and amide groups
(1626 cm.sup.-1, 1544 cm.sup.-1).
[0346] B. Preparation of Surface-Modified Nanocrystals: Twenty
milliliters of 3-5 mM (3-5 nmoles) of TOPO/TOP coated CdSe/ZnS
nanocrystals (see, Murray et al. (1993) J. Am. Chem. Soc. 115:8706)
were precipitated with 20 milliliters of methanol. The flocculate
was centrifuged at 3000.times.g for 3 minutes to form a pellet of
the nanocrystals. The supernatant was thereafter removed and 20
milliliters of methanol was again added to the particles. The
particles were vortexed to loosely disperse the flocculate
throughout the methanol. The flocculate was centrifuged an
additional time to form a pellet of the nanocrystals. This
precipitation/centrifugation step was repeated an additional time.
to remove any excess reactants remaining from the nanocrystal
synthesis. Twenty milliliters of chloroform were added to the
nanocrystal pellet to yield a freely dispersed sol.
[0347] 300 milligrams of hydrophobically modified poly(acrylic
acid) was dissolved in 20 ml of chloroform. Tetrabutylammonium
hydroxide (1.0 M in methanol) was added to the polymer solution to
raise the solution to pH 10 (pH was measured by spotting a small
aliquot of the chloroform solution on pH paper, evaporating the
solvent and thereafter wetting the pH paper with distilled water).
Thereafter the polymer solution was added to 20 ml of chloroform in
a 250 ml round bottom flask equipped with a stir bar. The solution
was stirred for 1 minute to ensure complete admixture of the
polymer solution. With continued stifling the washed nanocrystal
dispersion described above was added dropwise to the polymer
solution. The dispersion was then stirred for two minutes to ensure
complete mixing of the components and thereafter the chloroform was
removed in vacuo with low heat to yield a thin film of the
particle-polymer complex on the wall of the flask. Twenty
milliliters of distilled water were added to the flask and swirled
along the walls of the flask to aid in dispersing the particles in
the aqueous medium. The dispersion was then allowed to stir
overnight at room temperature. At this point the nanocrystals are
freely dispersed in the aqueous medium, possess pendant chemical
functionalities and may therefore be linked to affinity molecules
of interest using methods well known in the art for biolabeling
experiments. In addition, the fact that the nanocrystals now have a
highly charged surface means they can be readily utilized in
polyelectrolyte layering experiments for the formation of thin
films and composite materials.
[0348] C. Crosslinking of Polymer Stabilized Nanocrystals with a
Diamino Crosslinker: Ten milliliters of nanocrystals at 3.5 .mu.M,
stabilized as described supra, were purified by tangential flow
filtration using a 100 K polyethersulfone membrane against one
liter of distilled water and one liter of 50 mM
Morpholinoethanesulfonic acid buffer, pH 5.9. The nanocrystals were
concentrated to 10 milliliters and the pH of the aqueous dispersion
was decreased to pH 6.5 with 50 .mu.l additions of 0.1M HCl. 67
milligrams (315 .mu.moles) EDC were added to the stifling
nanocrystal dispersion. The reaction was allowed to proceed for 10
minutes before 1 milliliter of 0.5M borate buffer (pH 8.5)
containing 3.94 .PHI.moles of the crosslinking reagent lysine (a
diamino carboxylic acid) were added to the reaction mixture. The
reaction mixture was stirred for 2 hours at room temperature and
then transferred to a 50,000 molecular weight cut-off
polyethersulfone dialysis bag. Dialysis was performed for 24 hours
against 2 changes of 4 liters of water.
Example 1
Peptide-Mediated Uptake of SCNCs
[0349] Chariot (Active Motif, Carlsbad, Calif.) is a peptide
reagent based on the HIV-tat sequence (Schwarze et al. (1999)
Science 285:1569-1572), and has been used to deliver a variety of
macromolecules into cells. Chariot forms a non-covalent complex
with a molecule of interest (protein, peptide, antibody, or SCNC),
and acts as a carrier to deliver molecules into cells.
[0350] To deliver SCNCs into cells using Chariot, tissue culture
cells were seeded into six-well tissue culture plates (surface area
of 962 mm.sup.2 per well) at a cell density of 3.times.10.sup.5
cells per well and incubated overnight at 37.degree. C. in a 5%
CO.sub.2 atmosphere. The transfection efficiency was dependent on
the percent confluency of cells; the optimal percent confluency for
Chinese Hamster Ovary (CHO) cells was about 50-70%.
[0351] The transfection mixture was prepared by first diluting 616
nm emitting SCNCs into PBS in a final volume of 100 .mu.l. The
diluted SCNCs were combined with a mixture containing 94 .mu.l
sterile water and 6 .mu.l Chariot reagent, and the 200 .mu.l
transfection mix was incubated at room temperature for 30 min.
[0352] To transfect, cells were rinsed with PBS, and the 200 .mu.l
transfection mix was added directly to the cell monolayer, followed
by 400 .mu.l of serum-free growth medium. The final SCNC
concentration ranged from 10 to 120 nM, depending on the cell type
and SCNC material. The cells were incubated at 37.degree. C., 5%
CO.sub.2 for 1 hour, and 1 ml of serum containing growth medium was
added to each well. The cells were allowed to incubate for an
additional 2 hours. To visualize internalized SCNCs, the cells were
analyzed by fluorescence microscopy (FIG. 10A and FIG. 10B) or flow
cytometry using the appropriate filter sets.
Example 2
Nonspecific Uptake of SCNCs
[0353] SCNCs can be internalized by cells in the absence of a
specific carrier molecule. Non-crosslinked polymer-coated SCNCs
prepared as described above are sufficiently hydrophobic that they
bind to cells and are taken up by nonspecific endocytotic pathways.
Cells encoded with SCNCs were prepared as described in Example 1,
except the Chariot reagent was omitted from the transfection mix.
An example of nonspecific uptake of SCNCs is shown in FIG. 11.
Example 3
Cationic Lipid-Mediated and Micelle-Mediated Uptake of SCNCs
[0354] BioPORTER (BioPORTER, Gene Therapy Systems, San Diego,
Calif.) is a cationic lipid that is similar to other lipid-based
reagents for DNA transfections. It forms ionic interactions with
negatively charged groups of a molecule (protein, peptide,
antibody, or SCNC), and delivers the molecule into cells via fusion
with the cell membrane.
[0355] Cells were seeded at the same density as described in
Example 1. A transfection mix, comprised of carboxylated SCNCs and
PBS in a final volume of 100 .mu.l, was added to a tube containing
10 .mu.l of dried BioPORTER reagent. The solution was mixed gently
by pipetting, incubated at room temperature for 5 minutes, and
diluted by adding 900 .mu.l of serum free medium. Cells were washed
with PBS, and the diluted SCNC solution (1 ml) was added to the
cell monolayer. The final SCNC concentration was 2-60 nM, depending
on the cell line and SCNC material being tested. The cells were
incubated at 37.degree. C., 5% CO.sub.2 for 3 hr, and internalized
SCNCs were visualized by fluorescence microscopy (FIG. 12).
Alternatively, 3 ml of serum-containing medium was added to each
well and the cells were incubated overnight for analysis the next
day.
[0356] Micelle-Mediated Uptake of SCNCs
[0357] SCNCs were stabilized by entrapping phosphine/phosphine
oxide ligands onto the surface of SCNCs with specific polymers
through hydrophobic interaction. The most common ligands used in
the synthesis of SCNCs are TOP and TOPO. TOP and TOPO bind to the
surface Cd or Se through P-metal bond and their hydrophobic octyl
chains are pointing toward solvent making the surface of SCNCs
hydrophobic. Partially grafted poly(acrylic acid) (PAA), in which
octylamines were attached to about 40% carboxyl groups of PAA
through amide bond formation, were adsorbed onto the hydrophobic
surface of SCNCs through hydrophobic interaction, leading
water-soluble SCNCs. The remaining carboxyl groups can be used to
conjugate to biological molecules or to be crosslinked with each
other in order to make stable SCNCs.
[0358] In another method, the hydrophilic shell of micelles was
chemically crosslinked, where the surface of the micelles is made
up with carboxyl groups, which can then be used to form
bioconjugates for biological applications. The amphiphilic block
coploymer entraps or encapsulates SCNCs rendering the SCNCs
water-soluble. The polymers can be diblock, triblock or multiblock
copolymer, which contains at least one block of a hydrophobic
segment and at least one block of hydrophilic segment. The surface
of the micelles or functional groups in hydrophilic block of the
block copolymer can be carboxyl, aldehyde, alcohol, amine or any
reactive groups. The micelles encoded with one or more SCNCs were
further stabilized through crosslinking of the hydrophilic
shell.
[0359] For micelle-mediated uptake of SCNC, cells and aqueous
solutions of SCNCs trapped in micelles were prepared as described
above. To transfect, a mixture comprised of SCNCs/micelles and
serum free medium was prepared at a final volume of 500 ul and
incubated for 5 min at room temperature.
[0360] Cells were washed with PBS, and the transfection mixture was
added to the cell monolayer such that the final SCNC/micelle
concentration was approximately 20 nM. Cells were incubated at
37.degree. C., 5% CO.sub.2 for 1 hr, and 1 ml of serum containing
growth medium was added to each well. The cells were incubated for
an additional 2 hr and analyzed. To analyze cells the following
day, 2 ml of serum containing medium was added and the cells were
incubated overnight. The incorporation of SCNCs into the cells was
detected by fluorescence microscopy using a 535 nm emission filter
or a 625 nm emission filter.
Example 4
Co-Delivery of DNA and SCNCs
[0361] It is possible to co-deliver SCNCs and DNA using cationic
lipids for encoded DNA transfection applications. A transfection
solution comprised of 2 nM red (emitting at 630 nm) SCNC polymer
cross-linked prepared as described above, and 3 .mu.g of DNA
carrying an EGFP (enhanced green fluorescent protein)/rac kinase
fusion sequence was prepared and added to the BioPORTER reagent as
described in Example 3. Cells were cultured 2 days for EFGP
expression and analyzed by fluorescence microscopy and flow
cytometry. The microscopy results show that it was possible to find
cells that expressed EGFP and contained red SCNCs (FIGS. 13A, 13B
and 13C). Control experiments using the EGFP fusion DNA or red
SCNCs alone suggest that the DNA transfection efficiency decreased
in the presence of SCNCs. This may be caused by SCNC competing with
DNA for the BioPORTER reagent.
Example 5
Decoding of SCNC-Labeled Cells
[0362] SCNC codes can be detected inside cells using the green (530
nm) or red (630 nm) crosslinked polymer-coated SCNCs prepared as
described above were delivered into CHO cells as single colors or
mixtures of two colors using the Chariot reagent. Individual cells
were identified and analyzed over the range of 510 nm to 680 nm
using an 18-filter set. The results show that for individual cells,
absolute SCNC fluorescence intensity can vary more than 10-fold,
but that normalized spectral patterns are very similar for green or
red SCNCs (FIGS. 14A, 14B, 15A and 15B). Mixing green and red SCNCs
prior to adding them to the cell monolayer results in cells that
also have very similar spectral patterns after fluorescence
normalization (FIG. 16). Thus, these results suggest that pattern
recognition can be used as an encoding strategy for cells.
Example 6
Encoding Multiple Cell Lines with SCNCs
[0363] A first cell line expressing a G-protein coupled receptor,
e.g., a serotonin receptor, is taken into suspension and fixed in
an appropriate fixative (e.g., 3% paraformaldehyde). A specific
mixture of SCNCs having known fluorescence characteristics is used
to encode this population of cells. A second cell line expressing a
different G-protein coupled receptor, e.g., a beta adrenergic
receptor, is encoded with a second spectral code in a similar
manner. The first and second spectral codes have distinguishable
fluorescence characteristics.
[0364] The separately encoded cells are then mixed together in the
well of a microtiter plate and this mixed population is
interrogated with a labeled ligand (labeled with either a
fluorophore or a SCNC detectable different from the code) which may
or may not bind to the G-protein coupled receptors on the cell
lines. After an incubation period the encoded cells are allowed to
settle to the bottom of the well and each encoded population of
cells is measured to determine if label is associated with it using
a scanning spectrometer based detection system.
Example 7
Incorporation of SCNC into Yeast Mutant Cells
[0365] Populations of specific and distinct yeast mutants are
permeabilized to introduce a specific color set of SCNCs. Each of
the populations of yeast mutants are prepared with an SCNC code
that is distinguishable from the other of the populations of yeast
mutants (see FIG. 17). The encoded mutants are then used to
inoculate a common plate containing a suitable growth medium.
Several plates containing such mixed inocula of yeast mutants can
be prepared.
[0366] Sets of inoculated plates are incubated under a chosen
condition having altered temperature, light source, humidity or
nutrient availability as compared to standard growth conditions.
After an appropriate growth period (1 hour to 1 week) colonies
which have formed can be spectrally decoded to identify the
original mutant from which it derived.
Example 8
Immunostaining of SCNC-Encoded Cells
Herceptin Antibody Immunostaining of SKBR3 Cells
[0367] SKBR3 cells were seeded into an 8-well chamber slide at a
density of 80,000 cells per well and encoded with green (530 nm)
SCNCs using the cationic lipid BioPorter as described in Example 3.
Encoded or unencoded cells were incubated overnight at 37.degree.
C., 5% CO.sub.2. Cells were washed three times with PBS, and fixed
in the presence of 3.7% formaldehyde for 10 minutes. The cells were
washed 3 times with PBS, and incubated in the presence of PBS/1%
bovine serum albumin (BSA) at room temperature for 30 minutes to
minimize non-specific binding.
[0368] The cells in each well were incubated with 5 g/ml herceptin
antibody in PBS/1% BSA in a total volume of 150 l for 30 min at
room temperature. The cells were washed five times with PBS, and
incubated with a 1:500 dilution of biotinylated goat anti-human IgG
(Vector Laboratories, 1.5 mg/ml) for 30 minutes. The cells were
washed again with PBS and incubated with a 1:400 dilution of
streptavidin-conjugated Cy3 (Amersham, 1 mg/ml) for 30 minutes. The
cells were washed again with PBS and the slide was mounted using
50% glycerol in PBS. The cells were imaged using a Nikon
fluorescence microscope equipped with a Cy3 and green SCNC filter
set. Control experiments indicate that binding of herceptin is
unaffected by the SCNC code. The results indicate that the binding
of herceptin is unaffected by the SCNC encoding process or by the
presence of intracellular SCNC.
Example 9
Immunostaining of SCNC-Encoded Cells
Encoded Anti-Tubulin Immunostaining of CHO Cells
[0369] Chinese hamster ovary (CHO) cells were seeded into an 8-well
chamber slide at a density of 15,000 cells per chamber. The cells
were encoded with green (530 nm) SCNCs dots using Chariot reagent
as described in Example 1. The cells were incubated overnight in
complete medium (DMEM-F12, 10% fetal bovine serum (FBS), 2 mM
L-glutamine). Cells were washed 3 times with PBS, and fixed with
3.7% formaldehyde in PBS at room temperature for 10 minutes. The
cells were washed 4 times with PBS, and incubated for 30 minutes at
room temperature in the presence of PBS/1% bovine serum albumin
(BSA). Anti-tubulin antibody (rabbit IgG fraction, whole
de-lipidized antsera, Sigma) was diluted 1:200 in PBS/1% BSA and
incubated with cells at room temperature for 30 minutes. The cells
were washed 5 times with PBS, and incubated with biotinylated goat
anti rabbit IgG (Vector Laboratories, Burlingame, Calif. stock is
1.5 mg/ml) at a 1:500 dilution in PBS/1% BSA for 30 minutes. The
cells were washed 5 times with PBS and incubated with streptavidin
conjugated Cy3 (Amersham, 1 mg/ml stock solution) diluted 1:400 in
PBS for 30 minutes. The cells were washed 5 times with PBS and the
slide was mounted using 50% glycerol in PBS. Cells were imaged
using a Nikon fluorescence microscope equipped with a Cy3 and green
SCNC filter set. The results indicate that the binding of
anti-tubulin is unaffected by the SCNC encoding process or by the
presence of intracellular SCNC.
Example 10
A Reporter Gene Assay for the 2 Adrenergic Receptor Using
SCNC-Encoded CHO Cells
[0370] Chinese hamster ovary (CHO) cells expressing the 2
adrenergic receptor and renilla luciferase reporter gene were
encoded with green (530 nm) SCNCs using the Chariot reagent as
described in Example 1. Encoded cells or unencoded cells were
seeded into the wells of a white, clear-bottom, 96-well plate at a
seeding density of 100,000 cells per well. The cells were incubated
overnight in complete medium (DMEM-F12, 10% fetal bovine serum
(FBS), 2 mM L-glutamine, and 1 mg/ml G418 (Gibco BRL)).
[0371] The cells were washed and starved for 20-24 hrs by
incubating them in DMEM-F12 medium lacking serum and phenol red.
The beta receptor agonist isoproterenol (Sigma) was diluted at
various concentrations in DMEM-F12 medium and incubated with cells
for four hours. To measure expression of the reporter gene, cells
were washed with PBS and assayed for renilla luciferase activity
using the RenLuc kit (Promega). Luminescence was measured using a
Tecan SpectraFluor Plus plate reader. The dose response curves
shown in FIG. 18 indicate that the EC.sub.50 values for encoded or
unencoded cells are nearly identical, but that the encoded cells
have a smaller signal dynamic range.
Example 11
An Fluorescence Competition Binding Assay for the .beta.2
Adrenergic Receptor Using SCNC-Encoded CHO Cells
[0372] CHO cells expressing the 2 adrenergic receptor were encoded
with green (530 nm) SCNCs using Chariot reagent as described in
Example 1. Encoded or unencoded cells were seeded into 8-well
chamber slides at a density of 40,000 cells per chamber. The cells
were incubated overnight in complete medium.
[0373] The chamber slides were chilled at 4.degree. C. for
approximately 20 minutes, and the cells were washed once with cold
binding buffer (serum- and phenol red-free DMEM-F12 supplemented
with 0.1% BSA). The cells were incubated in binding buffer in the
presence or absence of 1 M unlabeled CGP12177 ligand (Sigma). The
slides were incubated at 4.degree. C. for 30 minutes. To bind the
fluorescent ligand, BODIPY.RTM. TMR (.+-.) CGP 12177 (Molecular
Probes) was added to a final concentration of 250 nM in binding
buffer, and the slides were wrapped in aluminum foil and incubated
at 4.degree. C. for 1 hour. Each well was washed 4 times with
binding buffer and the slides were mounted with 50% glycerol in
PBS. Cells were imaged using a Nikon fluorescence microscope
equipped with a Cy3 and green SCNC filter set (FIGS. 19A and 19B).
Control experiments indicate that competition binding of CGP12177
is essentially the same for either encoded or unencoded cells.
Example 12
A Calcium Assay for the M1 Muscarinic Receptor Using SCNC-Encoded
CHO Cells
[0374] CHO cells expressing the M1 muscarinic receptor M1WT3
(American Type Culture Collection, catalog number CRL-1985) were
encoded with green (530 nm) SCNCs using the Chariot reagent as
described in Example 1. Encoded or unencoded cells were seeded into
the wells of a 96-well assay plate at a density of 10,000 cells per
well and grown overnight in complete medium (Ham's F12K, 10% fetal
bovine serum (FBS), 2 mM L-glutamine). A calcium dye loading
solution using the FLEXstation calcium assay kit (Molecular
Devices) was prepared according the manufacturer's directions. The
loading buffer was supplemented with 2.5 mM probenecid to inhibit
anion-exchange proteins and prevent loss of internalized dye. To
load cells with the calcium indicator dye, 100 l of loading
solution is added to 100 l of medium per well, and the plate was
incubated at 37.degree. C., 5% CO.sub.2 for 1 hr.
[0375] The plate was removed and placed on the microscope-based
system for visualizing fluorescent images described above and in
commonly owned U.S. application Ser. No. 09/827,076, entitled
"Two-dimensional Spectral Imaging System" by Empedocles et al.,
filed Apr. 5, 2001, for imaging. Compounds were diluted in complete
medium and added to the wells. The plate was incubated at room
temperature for 5 minutes, and the cells were imaged as described
in Example 5. The results indicate that the agonist carbachol can
stimulate the calcium response of either unencoded or encoded
cells.
Example 13
A GPCR Internalization Assay for Multiplex Screening of Agonist or
Antagonist Ligands Using SCNC-Encoded Cells
[0376] A method is described for encoding and multiplexing a GPCR
internalization assay. Many, if not all, GPCRs undergo
agonist-dependent aggregation on the cell surface and subsequent
internalization via clathrin coated pits. The internalized GPCR is
contained within an endo some, which is either recycled back to the
membrane or targeted to the lysosome for degradation. An assay,
based on visualizing the movement of a fluorescent-tagged receptor
from the cell surface to an endosomal compartment, has been shown
for several GPCRs, including the parathyroid hormone receptor
(Conway et al. (1999) J. Biomol. Screening 4(2):75-86),
cholecystokinin receptor type A (Tarasova et al. (1997) J. Biol.
Chem. 272(23):14817-24) and 2 adrenergic receptor (Kallal et al.
(1998) J. Biol. Chem. 273(1):322-8). A receptor chimera, comprised
of green fluorescent protein (GFP) fused to the cytoplamic
C-terminal tail of the GPCR, can be used to visualize receptor
trafficking. It should also be possible, however, to tag the GPCR
with a short epitope sequence displayed on one of its extracellular
loops, and to label the receptor with an anti-epitope antibody
conjugated to a fluorescent dye molecule or SCNC. Examples of such
epitope sequences include the eight amino acid sequence FLAG
peptide (Chubet et al. (1996) Biotechniques 20(1):136-41), or the
nine amino acid sequence influenza virus hemagglutinin (HA) peptide
(Koller et al. (1997) Anal. Biochem. 250(1):51-60).
[0377] To multiplex an internalization assay using epitope-tagged
GPCRs, cell lines expressing various GPCRs are encoded as described
in, e.g., Example 1, 2 or 3, and mixed. The mixed cells are added
to the wells of a clear bottom assay plate. The fluorescent
dye-labeled antibody is added, followed by the compound. The cells
are incubated at 37.degree. C. for 30-60 minutes, and the assay
plate and cells are imaged using a fluorescence microscope.
Alternatively, the cells can be fixed with paraformaldehyde or some
other fixative agent, and the plates are stored at 4.degree. C. for
imaging at a later time. Binding of an agonist ligand to the GPCR
will cause internalization of the GPCR and its bound antibody,
which can be visualized under the microscope as a movement of
fluorescence from the cell surface to an intracellular compartment.
To screen for antagonists, the compounds are screened for their
ability to block the agonist-dependent internalization of the
receptor. This method can also be used to screen for agonist
ligands of orphan GPCRs.
Example 14
A Method for Encoding and Assaying Cells Grown in a Macroporous
Gelatin Microcarrier
[0378] A method is described for encoding and screening cells grown
on a microcarrier bead surface. An example of such a microcarrier
is CultiSpher.TM. from HyClone Laboratories, Inc. CultiSpher.TM. is
a macroporous gelatin microcarrier bead that provides a very large
interior surface for cell attachment. The large surface-to-area
ratio of the beads results in much higher cell yields compared to
conventional liquid cell cultures.
[0379] Microcarrier beads can be encoded using chemical methods
(see, e.g., U.S. Pat. No. 6,207,392, PCT Publication No. WO
00/17103, and Han et al. (2001) Nature Biotech. 19:632-635) and
then used as a substrate on which to grow cells. An advantage of
this method is that encoding is done on the bead scaffold used to
grow the cells, and not on individual cells. Methods for encoding
the beads include adsorbing a unique SCNC code to the bead surface,
or encapsulating the code within the interior of the bead.
[0380] To perform a multiplex assay using this method, the
microcarrier beads are encoded and stored until ready for use.
Cells expressing a receptor target of interest are added to the
encoded beads and incubated in culture medium to allow cell
attachment. The beads on which each cell line have been grown are
combined, and aliquots of the mixture are added to the wells of an
assay plate.
[0381] There are a variety of assays that can be adapted for use
with encoded microcarrier beads and cells. For example, binding of
a fluorescent ligand to a cell surface receptor can be measured by
flow cytometry of the microcarrier/cell complexes. Fluorescence
microscopy can be used to image calcium flux or expression of a
reporter gene using cells grown on microcarrier beads.
Example 15
A Method for Screening 600 GPCRs Using a 10-Plex SCNC-Encoded Cell
Assay
[0382] A method is described for screening 600 GPCRs against 96
compounds using a 10-plex encoded cell assay.
[0383] The compounds from a 96-well compound plate are replica
plated to 60 96-well daughter plates. Alternatively, a 20-plex
assay would require 30 compound daughter plates, and a 60-plex
assay would only require 10 daughter plates. For a 10-plex assay,
the 60 daughter plates are divided into 6 groups of 10 plates
each.
[0384] The 600 GPCR cell lines are stored as frozen cells, and are
thawed as needed. An important advantage of this method is that far
fewer cells are used for screening compared to conventional
screening methods. For example, fewer than 100 cells per GPCR are
screened in a well using encoded cell technology, compared to
50,000 cells per GPCR using a conventional calcium assay such as
the Fluorescent Imaging Plate Reader (FLIPR) from Molecular
Devices. Therefore, all of the GPCR cell lines required for the
encoded cell technology can be grown in 6-well culture plates
compared to the large flasks or bioreactors that are required for
conventional screening. Growing the cells in 6-well culture plates
is also more amenable to automation compared to conventional
screening.
[0385] To encode the 600 GPCR cell lines, the cells are transferred
to 100 6-well culture plates and grown to a cell density of about
0.5-1.0.times.10.sup.6 cells per well. The 600 wells are organized
according to the multiplexing capability of the assay. For example,
a 10-plex assay would require that 600 wells be organized into 60
groups, each group comprised of 10 wells, while a 60-plex assay
would be organized as 10 groups of 60 wells each. The cell codes
are stored as premixed color combinations of SCNCs, and are used as
previously described. The number of SCNC colors necessary for a
10-plex, 20-plex, and 60-plex assays using single emission
intensity levels are 4, 5, and 6, respectively.
[0386] For a 10-plex assay, the cell lines growing in 10 different
wells are encoded with one of the 10 SCNC codes as described, for
example, in Examples 1 and 3. The cells are encoded, lifted from
each of the 10 wells, counted, and pooled such that the number of
cells comprising each GPCR cell line is approximately equal. For
example, pooling 0.5.times.10.sup.6 cells from each well would
result in a mixture containing a total of 5.times.10.sup.6 cells.
Cells from the mix are distributed to all the wells of a 96-well
assay plate at a seeding density of 10,000 total cells per well
(equivalent to 1000 cells per GPCR cell line). This process is
repeated 60 times until all 600 GPCR cell lines are contained
within the wells of 60 assay plates.
[0387] This screening method can be adapted to a variety of assay
formats. One such assay is the GPCR internalization assay described
above using epitope-tagged GPCRs. The anti-epitope fluorescent
antibody is added to the wells of the 60 assay plates. Compounds
from the 60 compound replica plates are transferred to the assay
plates, and the plates are incubated at 37.degree. C., 5% CO.sub.2
for 30-60 minutes for receptor internalization to occur. The cells
are fixed with paraformaldehyde and stored at 4.degree. C. until
ready for imaging.
[0388] The cells are imaged using an automated high-throughput
fluorescence microscope. A nuclear stain such as Hoechst 33258 (350
nm excitation, 461 nm emission) is used to identify single cells
within a field of view. To screen for agonist compounds, the cells
are screened for the internalization of the fluorescent reporter
bound to the antibody. Positive cells are then scanned using
multiple filters to determine the SCNC code. This process is
repeated at either single or multiple fields of view per well until
a statistically significant number of data points are collected.
Image and data processing are used to store and analyze the
data.
Example 16
Peptide-Mediated Uptake of SCNCs Using Binding Pairs
[0389] A labeling solution was made by pre-mixing 1 .mu.L of 2
.mu.M streptavidin conjugated quantum dots with an emission maximum
of 655 nm (Quantum Dot Corporation, Hayward, Calif.) and 100 .mu.M
biotinylated D-Arg9 (nine contiguous D-Arg molecules) (CS Bio Co.,
Inc., San Carlos, Calif.) into a 1.5 mL microcentrifuge tube. 200
.mu.L of fresh full growth medium with 10% serum (Invitrogen,
Carlsbad, Calif.) was added and vortexed for 30 seconds. The medium
used was the ATTC recommended medium for HeLa cells. This medium
includes minimum essential medium (Eagle) with 2 mM L-glutamine and
Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
non-essential amino acids, and 1.0 mM sodium pyruvate, 10% fetal
bovine serum. The entire quantity of labeling solution was added to
a monolayer of HeLa cells subcultured overnight in one well of an
8-well Lab-Tek chambered coverglass system (Nunc, Rochester, N.Y.).
After incubation at 37.degree. C. in a humidified atmosphere of 95%
air, 5% CO.sub.2 for 45-60 minutes, the cells were washed twice
with fresh growth medium. The image shown in FIG. 20 was captured
using Leica SP-2 confocal microscope with 488 nm excitation.
[0390] Although the invention has been described in some detail
with reference to the preferred embodiments, those of skill in the
art will realize, in light of the teachings herein, that certain
changes and modifications can be made without departing from the
spirit and scope of the invention. Accordingly, the invention is
limited only by the claims.
Sequence CWU 1
1
519PRTArtificialpeptide fragment of HIV-TAT basic region 1Arg Lys
Lys Arg Arg Gln Arg Arg Arg 1 5 212PRTArtificialpeptide fragment
from SV40 large T NLS 2Cys Gly Gly Gly Pro Lys Lys Lys Arg Lys Val
Gly 1 5 10 313PRTArtificialpeptide fragment from adenoviral NLS
3Cys Gly Gly Phe Ser Thr Ser Leu Arg Ala Arg Lys Ala 1 5 10
417PRTArtificialpeptide fragment from adenoviral RME 4Cys Lys Lys
Lys Lys Lys Lys Ser Glu Asp Glu Tyr Pro Tyr Val Pro 1 5 10 15 Asn
528PRTArtificialpeptide fragment from adenoviral fiber protein 5Cys
Lys Lys Lys Lys Lys Lys Lys Ser Glu Asp Glu Tyr Pro Tyr Val 1 5 10
15 Pro Asn Phe Ser Thr Ser Leu Arg Ala Arg Lys Ala 20 25
* * * * *