U.S. patent application number 10/988781 was filed with the patent office on 2005-06-16 for small organometallic probes.
Invention is credited to Furuya, Frederic R., Hainfeld, James F., Powell, Richard D..
Application Number | 20050130207 10/988781 |
Document ID | / |
Family ID | 33425460 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130207 |
Kind Code |
A1 |
Hainfeld, James F. ; et
al. |
June 16, 2005 |
Small organometallic probes
Abstract
Small organometallic probes comprise a core of metal atoms
bonded to organic moieties. The metal atoms are gold, silver,
platinum, palladium, or combinations thereof. In one embodiment, a
multifunctional organometallic probe comprises a core of metal
atoms surrounded by a shell of organic moieties covalently attached
to the metal atoms, a fluorescent molecule, e.g., fluorescein,
covalently attached to one of the organic moieties, and a targeting
molecule, e.g., an antibody, covalently attached to another of the
organic moieties.
Inventors: |
Hainfeld, James F.;
(Shoreham, NY) ; Furuya, Frederic R.; (Williston
Park, NY) ; Powell, Richard D.; (Raleigh,
NC) |
Correspondence
Address: |
ALIX YALE & RISTAS LLP
750 MAIN STREET
SUITE 1400
HARTFORD
CT
06103
US
|
Family ID: |
33425460 |
Appl. No.: |
10/988781 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10988781 |
Nov 15, 2004 |
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10093770 |
Mar 8, 2002 |
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6818199 |
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10093770 |
Mar 8, 2002 |
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09619343 |
Jul 19, 2000 |
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6369206 |
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09619343 |
Jul 19, 2000 |
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09039601 |
Mar 16, 1998 |
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6121425 |
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09039601 |
Mar 16, 1998 |
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08652007 |
May 23, 1996 |
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5728590 |
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08652007 |
May 23, 1996 |
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08282929 |
Jul 29, 1994 |
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5521289 |
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Current U.S.
Class: |
435/6.11 ;
436/523 |
Current CPC
Class: |
B82Y 5/00 20130101; A61K
49/049 20130101; A61K 47/544 20170801; B82Y 30/00 20130101; C12Q
1/6816 20130101; G01N 33/533 20130101; C07F 9/5022 20130101; C12Q
1/6816 20130101; A61B 6/481 20130101; A61K 47/6923 20170801; C12Q
2563/149 20130101; C12Q 2563/137 20130101; C12Q 2563/107 20130101;
C07F 1/005 20130101 |
Class at
Publication: |
435/006 ;
436/523 |
International
Class: |
C12Q 001/68; B05D
007/00; G01N 033/543 |
Claims
What is claimed is:
1. A cluster complex or colloid comprising: a plurality of core
particles, each of the core particles being composed of a solid
metal; and a plurality of probe molecules covalently attached to
the core particles, the probe molecules functionalizing the cluster
complex or colloid to target at least one other substance or to
endow the particles with specified properties.
2. The cluster complex or colloid of claim 1 wherein the core
particles are less than 2 microns in size.
3. The cluster complex or colloid of claim 1 wherein the metal of
the core particles is gold, platinum, silver, palladium, or
combinations thereof.
4. The cluster complex or colloid of claim 1 wherein the probe
molecules are covalently bonded to the core particles by a specific
reaction of the probe molecules with a functional group
incorporated into an outer surface of the core particle by means of
chemical synthesis or modification, the functional group being
selected from the group comprising maleimide, N-hydroxysuccinimide,
Sulfo-N-hydroxysuccinimide, aliphatic amine, 1,2-dihydroxy,
aldehyde, nitrilotriaceitic acid (NTA) or its chelate with nickel,
copper, iron, cobalt, or other transition metal.
5. The cluster complex or colloid of claim 4 further comprising
nucleic acids, DNA, or RNA covalently bonded to the core particles
by the functional group.
6. The cluster complex or colloid of claim 1 wherein the covalently
attached probe molecules are selected from the group comprising
antibodies, antibody fragments, avidin or streptavidin, peptides,
drugs, antigens, hormones, DNA, and RNA.
7. The clusters or colloids of claim 1 further comprising a
plurality of fluorescently labeled molecules covalently attached to
the core particles, wherein the chemistry for covalently attaching
the probe molecules and the fluorescently labeled molecules to the
core particles controls the fluorophore of the fluorescently
labeled molecules.
8. The cluster complex or colloid of claim 1 further comprising a
plurality of lipid molecules attached to the core particles.
9. The cluster complex or colloid of claim 1 further comprising
nucleic acids, DNA, or RNA.
10. The cluster complex or colloid of claim 1 further comprising a
plurality of antibody fragments.
11. The cluster complex or colloid of claim 1 further comprising a
polymer coating the core particles, the polymer being chosen from a
group containing linear or branched polymer with functional groups,
polyamino acids, polyethylene derivatives, and mixtures
thereof.
12. The cluster complex or colloid of claim 1 wherein the core
particles are composed of gold and the probe molecules are
fluorescent dyes.
13. The cluster complex or colloid of claim 12 wherein the
fluorescent dye is Hoescht-33258.
14. The cluster complex or colloid of claim 1 wherein the probe
molecule is streptavidin.
15. A method for detecting a substance comprising mixing a sample
suspected of containing the substance with a cluster complex or a
colloid comprising a plurality of metal core particles and a
plurality of probe molecules covalently attached to the core
particles, the probe molecules functionalizing the cluster complex
or colloid to target the substance.
16. The method of 15 further comprising the step of detecting the
substance with a piezoelectric crystal mass measuring device.
17. The method of claim 15 further comprising the step of detecting
the substance by using changes in reflected light from a
surface.
18. The method of claim 15 further comprising the step of detecting
the substance using light microscopy.
19. The method of claim 15 further comprising the step of
visualizing the localization or distribution of the substance
within cells, tissue sections, organelles or organs using light
microscopy.
20. The method of claim 15 further comprising the step of detecting
the substance using fluorescent microscopy.
21. The method of claim 15 further comprising the step of
visualizing the localization or distribution of the substance
within cells, tissue sections, organelles or organs using
fluorescent microscopy.
22. The method of claim 15 further comprising the step of detecting
the substance using confocal microscopy.
23. The method of claim 15 further comprising the step of
visualizing the localization or distribution of the substance
within cells, tissue sections, organelles or organs using confocal
microscopy.
24. The method of claim 15 further comprising the step of detecting
the substance using electron microscopy.
25. The method of claim 15 further comprising the step of
visualizing the localization or distribution of the substance
within cells, tissue sections, organelles or organs using electron
microscopy.
26. The method of claim 15 wherein the cluster complex or colloid
further comprises a plurality of fluorescently labeled molecules
covalently attached to the core particles, the chemistry for
covalently attaching the probe molecules and the fluorescently
labeled molecules to the core particles controls the fluorophore of
the fluorescently labeled molecules, and wherein the method further
comprises the step of detecting the substance using both light,
fluorescence or confocal microscopy and electron microscopy.
27. The method of claim 15 wherein the cluster complex or colloid
further comprises a plurality of fluorescently labeled molecules
covalently attached to the core particles, the chemistry for
covalently attaching the probe molecules and the fluorescently
labeled molecules to the core particles controls the fluorophore of
the fluorescently labeled molecules, and wherein the method further
comprises the step of visualizing the localization or distribution
of the substance within cells, tissue sections, organelles or
organs using both light, fluorescence or confocal microscopy and
electron microscopy.
28. The method of claim 15 further comprising the step of detecting
the substance on blots, immunochromatographic lateral flow assay
devices, or test strips.
29. A metal cluster comprising: a plurality of metal core
particles; and a plurality of organic thiols covalently bound to
the core particles through a thiol moiety, the organic thiols being
selected from the group comprising alkyl thiols, aryl thiols,
proteins containing thiol, peptides or nucleic acids with thiol,
glutathione, cysteine, thioglucose, thiol benzoic acid, antibodies,
lipids, and carbohydrates.
30. A metal colloid comprising: a plurality of metal colloid
particles; and a plurality of organic thiols covalently bound to
the core particles through a thiol moiety, the organic thiols being
selected from the group comprising alkyl thiols, aryl thiols,
proteins containing thiol, peptides or nucleic acids with thiol,
glutathione, cysteine, thioglucose, thiol benzoic acid, antibodies,
lipids, and carbohydrates.
31. A metal colloid comprising: a plurality of metal colloid
particles; and a polymer coating the metal colloid particles.
32. The metal colloid of claim 31 further comprising a plurality of
covalently bonded molecules selected from the group including
proteins, peptides, antibodies, lipids, carbohydrates, nucleic
acids, drugs, and hormones.
33. A process for coating metal colloid particles comprising
synthesizing the metal colloid in the presence of a polymer.
34. The process of claim 33 further comprising the step of
stabilizing the polymer coating on the metal colloid particle by
warming the coated particle to 60.degree.-100.degree. C., microwave
heating, chemical crosslinking, continued polymerization, or
photocrosslinking.
35. A process for coating metal colloid particles comprising mixing
preformed metal colloid particles with a polymer.
36. The process of claim 35 further comprising the step of
stabilizing the polymer coating on the metal colloid particle by
warming the coated particle to 60.degree.-100.degree. C., microwave
heating, chemical crosslinking, continued polymerization, or
photocrosslinking.
37. A process for covalently incorporating organometallic particles
into nucleic acids comprising using an organometallic particle
containing a nucleic acid base or nucleic acid analog as a
substrate in automated nucleic acid synthesis.
38. A process for covalently incorporating organometallic particles
into nucleic acids comprising using an organometallic particle
attached to a nucleic acid base or analog in enzymatic nucleic acid
synthesis.
39. A process for covalently incorporating organometallic particles
into nucleic acids comprising reacting an organometallic particle
with functional groups incorporated into nucleic acids.
40. A process for covalently incorporating organometallic particles
into nucleic acids comprising reacting organometallic particles
containing photoactive groups with nucleic acids.
41. A process of exchanging thiol-containing molecules on metal
particles by incubating preformed metal particles containing bound
thiol molecules with a new thiol-containing molecule, whereby the
new thiol-containing molecule attaches to the metal particle.
42. The process of claim 41 where the new thiol-containing molecule
is chosen from whole IgG and Fab' antibody fragments.
43. A method for imparting specific chemical, spectroscopic, or
electrochemical properties or functions to a larger macromolecular
array or construct by covalently bonding a plurality of probe
molecules onto a plurality of metal core particles, the probe
molecules being covalently bonded to the core particles by a
specific reaction of the probe molecules with a functional group
incorporated into an outer surface of the core particle by means of
chemical synthesis or modification, the functional group being
selected from the group comprising maleimide, N-hydroxysuccinimide,
Sulfo-N-hydroxysuccinimide, aliphatic amine, 1,2-dihydroxy,
aldehyde, nitrilotriaceitic acid (NTA) or its chelate with nickel,
copper, iron, cobalt, or other transition metal.
44. A method for detecting changes in the conformation or binding
of probe molecules attached to specific targets, the probe
molecules being covalently attached to metal core particles, the
probe molecules functionalizing the cluster complex or colloid to
the target, the method comprising measuring changes in the
fluorescence properties of conjugated molecules labeled with
fluorescent tags.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending U.S. patent
application Ser. No. 10/093,770, filed Mar. 8, 2002; which is a
continuation-in-part of application Ser. No. 09/619,343, filed Jul.
19, 2000 and issued as U.S. Pat. No. 6,369,206; which is a
divisional of application Ser. No. 09/039,601, filed Mar. 16, 1998
and issued as U.S. Pat. No. 6,121,425; which is a
continuation-in-part of application Ser. No. 08/652,007, filed May
23, 1996 and issued as U.S. Pat. No. 5,728,590; which is a
continuation-in-part of application Ser. No. 08/282,929, filed Jul.
29, 1994 and issued as U.S. Pat. No. 5,521,289.
BACKGROUND OF THE INVENTION
[0002] The present invention is directed to small organometallic
probes, processes for making the small organometallic probes, and
applications of the small organometallic probes. In particular, the
small organometallic probes of the present invention are generally
less than two microns (2 .mu.m) across, and comprise a metal
cluster compound having a solid metal core, with organic groups
attached to the metal core. Alternatively, the organometallic
probes may comprise a metal colloid having organic groups attached
to the outer surface of the metal colloid. The metal clusters or
colloids may also be functionalized with other molecules attached
that can be used for targeting and detecting another substance,
generally, a biologically significant substance, such as an
antibody. The metal in the metal clusters or colloids is gold,
platinum, silver, palladium or combinations thereof.
[0003] Previous work by others has described the preparation of
gold and silver colloids. Such colloids do not have a fixed number
of metal atoms and vary considerably in size. For example, the
metal colloids could vary in size from 1 nm to 2 .mu.m in size and
could contain from about 10 metal atoms to thousands of metal
atoms, depending on size. It was found that a number of proteins,
such as IgG antibodies, could be adsorbed to these sol particles.
Gold colloids have been most commonly described. These conjugates
have been used in electron and light microscopy as well as on
immunodot blots for detection of target molecules. These conjugates
have many shortcomings. Since the molecules are only adsorbed onto
the colloids, they also desorb to varying extents. This leads to
free antibody which competes for antigen sites and lowers targeting
of gold. Furthermore, the shelf life of the conjugates is
compromised by this problem. The `sticky` colloids also tend to
aggregate. If fluorescence is used to detect the target molecules,
the gold particles quench most of it. Also, the gold colloids must
be stabilized against dramatic aggregation or `flocculation` when
salts are added by adsorbing bulky proteins, such as bovine serum
albumin. Due to the effects of aggregation and bulky additives, the
penetration of immunoprobes into tissues is generally <0.5
.mu.m. Accessibility of the probes to internal cell structures,
e.g., nuclear proteins, or to cells deeper in a tissue sample, is
impeded by these properties. Colloidal gold immunoprobes are also
used in diagnosis on blots. The sensitivity of these detection
schemes is again degraded by problems of aggregation, detachment of
antibodies from the gold, and problems with shelf life. The gold
prepared in standard ways also has low activity due to few adsorbed
antibodies and denaturation of some antibodies during
adsorption.
[0004] Various metal cluster containing organic shells have also
been previously described, such as Au.sub.11Ph.sub.7 (Ph=phenyl),
Au.sub.25R (R=organic), Pd.sub.561, and others. These metal
clusters have a fixed number of metal atoms in their metal cores
which range in size from ca. 0.7-2.2 nm. Most of these metal
clusters are based upon reduction of metal-triphenyl phosphines or
the use of 1,10-phenanthroline.
[0005] For example, Barlett, P. A. et al, in "Synthesis of
Water-Soluble Undecagold Cluster Compounds . . . ," J. Am. Chem.
Soc., 100, 5085 (1978), describe a metal cluster compound
(Au.sub.11) having a core of 11 gold atoms with a diameter of 0.8
nm. The metal core of 11 gold atoms in the undecagold metal cluster
compound is surrounded by an organic shell of PAr.sub.3 groups.
This metal cluster compound has been used to form gold
immunoprobes, for example, by conjugating Au.sub.11 to Fab'
antibody fragments as well as other biological compounds.
[0006] Another metal cluster compound which has been used as a
probe is Nanogold.TM. available from the assignee of the present
application. Nanogold.TM. has a metal core with 50-70 gold atoms
(the exact number not yet being known but believed to be 67 gold
atoms) surrounded by a similar shell of organic groups (PAr.sub.3)
as undecagold. The metal core of Nanogold.TM. is 1.4 nm in
diameter. The production of Nanogold is described in U.S. Pat. No.
5,360,895, of James F. Hainfeld and Frederic R. Furuya.
[0007] Although the preparation and properties vary for these metal
cluster compounds having organic shells, many of these can only be
synthesized in low yields, derivatization for use in coupling to
biomolecules is expensive in time and effort, and again in low
yields, and many of the cluster compounds are degraded rapidly by
heat or various chemical reagents.
SUMMARY OF THE INVENTION
[0008] Briefly stated, the invention in a preferred form is a
cluster complex or colloid including metal core particles and probe
molecules covalently attached to the core particles. The probe
molecules functionalize the cluster complex or colloid to target at
least one other substance or to endow the particles with specified
properties.
[0009] The core particles are less than 2 microns in size and may
be composed of gold, platinum, silver, palladium, or combinations
thereof.
[0010] The probe molecules may be covalently bonded to the core
particles by a specific reaction of the probe molecules with a
functional group incorporated into an outer surface of the core
particle by means of chemical synthesis or modification. The
functional group may be maleimide, N-hydroxysuccinimide,
Sulfo-N-hydroxysuccinimide, aliphatic amine, 1,2-dihydroxy,
aldehyde, nitrilotriaceitic acid (NTA) or its chelate with nickel,
copper, iron, cobalt, or other transition metal.
[0011] The probe molecules may be antibodies, antibody fragments,
avidin or streptavidin, peptides, drugs, antigens, hormones, DNA,
and RNA.
[0012] The clusters or colloids may also include fluorescently
labeled molecules covalently attached to the core particles, where
the chemistry for covalently attaching the probe molecules and the
fluorescently labeled molecules to the core particles controls the
fluorophore of the fluorescently labeled molecules.
[0013] The cluster complex or colloid may also include a polymer
coating the core particles, the polymer being chosen from a group
containing linear or branched polymer with functional groups,
polyamino acids, polyethylene derivatives, and mixtures
thereof.
[0014] The invention is also a method for detecting a substance,
where a sample suspected of containing the substance is mixed with
a cluster complex or a colloid having metal core particles and
probe molecules covalently attached to the core particles, the
probe molecules functionalizing the cluster complex or colloid to
target the substance.
[0015] The method may include detecting the substance with a
piezoelectric crystal mass measuring device, detecting the
substance by using changes in reflected light from a surface,
detecting the substance using light microscopy, detecting the
substance using fluorescent microscopy, detecting the substance
using confocal microscopy, or detecting the substance using
electron microscopy.
[0016] The method may include visualizing the localization or
distribution of the substance within cells, tissue sections,
organelles or organs using light microscopy, using fluorescent
microscopy, or using confocal microscopy, using electron
microscopy.
[0017] The method may include detecting the substance using both
light, fluorescence or confocal microscopy and electron microscopy,
the cluster complex or colloid also including fluorescently labeled
molecules covalently attached to the core particles, where the
chemistry for covalently attaching the probe molecules and the
fluorescently labeled molecules to the core particles controls the
fluorophore of the fluorescently labeled molecules.
[0018] The method may include visualizing the localization or
distribution of the substance within cells, tissue sections,
organelles or organs using both light, fluorescence or confocal
microscopy and electron microscopy, the cluster complex or colloid
also including fluorescently labeled molecules covalently attached
to the core particles, where the chemistry for covalently attaching
the probe molecules and the fluorescently labeled molecules to the
core particles controls the fluorophore of the fluorescently
labeled molecules.
[0019] Other objects and advantages of the invention will become
apparent from the drawings and specification.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0020] 1. "Thiol gold clusters" are novel gold clusters produced by
a novel synthesis. The procedure is: form an organic-gold complex
by reacting a compound containing a thiol with gold in solution. A
second equivalent is also added of the thiol compound. Finally the
gold organic is reduced with NaBH.sub.4 or other reducing agents
and organometallic particles are formed. These have the general
formula Au.sub.nR.sub.mR'.sub.i . . . , where n, m, and l are
integers, R and R' are organic thiols, (e.g., alkyl thiols, aryl
thiols, proteins containing thiol, peptides or nucleic acids with
thiol, glutathione, cysteine, thioglucose, thiol benzoic acid,
etc.) and the ellipsis indicates that one or more organic thiols
may be used. With two equivalents of organic thiol compound,
clusters with gold cores .about.1.4 nm are formed with many
organics. The organic moiety may then be reacted by usual reactions
to covalently link this particle to antibodies, lipids,
carbohydrates, nucleic acids, or other molecules to form probes.
Mixtures of organic thiols may be used to provide mixed
functionality to the clusters. These organo-gold clusters are
stable to heating at 100.degree. C.
[0021] 2. The thiol-gold preparation described in #1 above may be
altered such that a larger molar ratio of organic thiol to gold is
used. Ratios above approximately 2:1 or below 1:2 result in
organic-gold colloids whose size depends on this ratio. These are
useful when large gold particles are desired.
[0022] 3. The organic thiol-gold preparations described in #'s 1
and 2 above may be made using a similar process with alternatives
metals to gold, e.g., platinum, silver, palladium and other
metals.
[0023] 4. The organic thiol-metal particles described in #'s 1, 2,
and 3 above may be made using mixtures of metal ions, e.g., gold
and silver, resulting in mixed metal clusters.
[0024] 5. A novel process has been developed for coating colloidal
particles (of various types including gold, silver, and other
metals) with organic moieties having groups suitable for covalently
attaching additional molecules, such as antibodies, nucleic acids,
lipids, peptides, and other proteins. The process consists of
synthesizing the metal colloid in the presence of a suitable
polymer, e.g., HAuCl.sub.4 (0.01%) in 0.05M sodium hydrogen maleate
buffer (pH 6.0), with 0.004% tannic acid. The polymer may be chosen
from a linear or branched group with functional groups attached,
such as polyamino acids, polyethylene derivatives, other polymers,
or mixtures thereof. Optimal molecular weight of the polymer varies
with the specific ones chosen. A second method is to synthesize the
metal particle first, e.g., by combining 0.01% HAuCl.sub.4 with 1%
sodium citrate with heating. Once gold colloid is formed of the
desired size, it is coated with one of the above polymers by mixing
the two together and optionally warming to 60.degree.-100.degree.
C. for several minutes. The polymer coating may be further
stabilized by a) microwave heating, b) further chemical
crosslinking, e.g., by glutaraldehyde or other linkers, or by
continued polymerization adding substrate molecules for a brief
period. Use of N, N'-methylene bis acrylamide, for example, can
covalently further stabilize the polymer coating. Photocrosslinking
may also be used.
[0025] The functionalized polymer coating may now be used to
covalently attach proteins, peptides, antibodies, lipids,
carbohydrates, nucleic acids, drugs, hormones, or other substances.
This has the advantage that this step may be done mildly, in
physiological buffers if desired, using standard crosslinking
technology. This eliminates the usual restriction that conjugation
must be performed in very low ionic strength buffers, which
precludes attachment of certain molecules such as many IgM's which
cannot withstand the low ionic strength requirement.
[0026] 6. Combined (bifunctional) fluorescent and metal particle
probes have been synthesized that have the formula:
M.sub.n(OrF).sub.m(Or'T).sub- .l(Or").sub.p, where M is a metal
core consisting of multiple metal atoms (Au, Pt, Ag, Pd) that can
be mixed, covalently bonded to a shell of organic groups (Or, Or',
and Or"). Or and Or" are organic coupling moieties, (e.g.,
triphenyl phosphines 1,10 phenanthrolines, triphenyl phosphines or
phenanthrolines containing linkable groups such as amines or
carboxyls, triphenyl phosphines or 1,10 phenanthrolines containing
reactive groups such as maleimides or N-hydroxysuccinimide esters,
P(C.sub.6H.sub.4--CO--NH--(CH.sub.2).sub.3--NH.sub.2).sub.3,
P(C.sub.6H.sub.4--CO--NH--CH.sub.3).sub.2(C.sub.6H.sub.4--CO--NH--(CH.sub-
.2).sub.3--NH.sub.2), P(C.sub.6H.sub.4--CO--N
H--(CH.sub.2).sub.3--NC.sub.- 4O.sub.2H.sub.2).sub.3,
P(C.sub.6H.sub.4--CO--NH--(CH.sub.2).sub.3--NH--(C-
H.sub.2).sub.6--CO.sub.2--NC.sub.4O.sub.2H.sub.4).sub.3, etc.), Or"
is an organic group (e.g., triphenyl phosphines, 1,10
phenanthrolines, P(C.sub.6H.sub.4--CHOH--CH.sub.2OH).sub.3,
P(C.sub.6H.sub.4--CO--NH--CH.s- ub.3).sub.3,
P(C.sub.6H.sub.5).sub.3, P(C.sub.6H.sub.4SO.sub.3).sub.3, etc.),
part of the metal cluster, F is a fluorescent molecule, (e.g.,
fluorescein, rhodamine, aminomethyl coumarin, Texas Red, etc.) and
T is an optional targeting molecule such as antibody, peptide,
drug, etc. Or, Or', and Or" may be the same or different. The
subscripts 1, n, m and p indicate that multiple copies of each
moiety are possible and include mixtures of different metals,
fluorescent groups, organic groups and targeting molecules for
multifunctional conjugates. A specific example of this is:
Au.sub.11[Pph(CONHCH.sub.3)].sub.6 phCONH(CH.sub.2).sub.3 NH--F,
where ph is a phenyl group, and F is a fluorescent molecule.
[0027] A process to produce such multifunctional metal particles is
as follows: a metal cluster or metal colloid is synthesized having
one or more reactive groups such as an amine or carboxyl. These are
then reacted to covalently link a fluorescent molecule molecule
such as fluorescein, Texas Red, rhodamine, or aminomethyl coumarin,
and optionally a targeting or other molecule is covalently attached
such as an antibody or antibody fragment, a peptide, streptavidin,
or other proteins, a nucleic acid (RNA or DNA), drugs, hormones, or
other molecules. Alternatively, the fluorescent groups may be
incorporated into ligands which are then used to prepare the
cluster.
[0028] 7. Lipid molecules (fatty acids, phospholipids, or others)
are covalently attached to metal particles (clusters or coated
reactive colloids). This process uses a reactive lipid derivative,
e.g., a sulfonyl chloride or anhydride, which is reacted with an
amino group, for example, on the metal particle. Alternatively,
organic groups on the metal particle or lipid may be reacted with
bifunctional crosslinkers which are then reacted with the other
species. Another process is to presynthesize the organic components
of the metal particles (e.g., phosphines or polymers) with lipid
molecules attached and then to use these organo-lipids in
constructing the functionalized metal particle.
[0029] The general formula for the product is:
M-Or-L
[0030] where M is the metal particle (either cluster or colloid of
Au, Pt, Ag, Pd, and combinations), Or is an organic group of the
organometallic particle (such as, phosphines containing linkable
groups, polymers containing linkable groups,
P(C.sub.6H.sub.4--CO--NH--(CH.sub.2).sub.3--N- H.sub.2).sub.3,
polyethyleneimine, polyacrylamide hydrazide, polylysine, etc.), and
L is the lipid moiety.
[0031] 8. A novel gel stain product is a metal (preferably gold)
cluster of the form described in #1 above using appropriate organic
thiols. The thiols are preferably o-thiol benzoic acid,
glutathione, and thioglucose, although others may be used. The
general formula describing the product is:
M.sub.k(SOr).sub.m(SOr').sub.n . . .
[0032] where M is the metal, S is sulfur, Or and Or' are organic
groups (such as proteins or nucleic acids containing thiols, most
other organic thiols, glucose, benzoic acid, glutathione,
cholesterol, etc.), and k, m and n are integers. The ellipsis
indicates that one or more different (SOr) groups (organic thiols)
may be attached per metal core.
[0033] The process for a protein polyacrylamide gel is as follows:
mix the organometallic particle with the protein sample with or
without SDS (sodium dodecyl sulfate), for native or denaturing
gels; if a reducing gel is desired, the protein is first reduced
with a reductant, preferably .beta.-mercaptoethanol, followed by a
thiol blocking agent, preferably iodoacetamide, before adding the
metal stain. The sample is heated briefly (e.g., one minute),
loaded onto the gel and run normally. Stain development is effected
by soaking the gel in a silver enhancement medium (e.g., AgNO.sub.3
with hydroquinone) for several minutes followed by water or fixing
washes.
[0034] 9. The organometallic particles described above may be
covalently incorporated into nucleic acids by several techniques.
One is via synthesis of a metal particle attached to a nucleic acid
base or analogue that has its other functional groups protected so
that it is compatible with automated nucleic acids synthesis, such
as use of phosphoramadite chemistry. A second approach is to
incorporate appropriate organomettalic base analogs into nucleic
acids enzymatically. A third method is to react activated
organometallic clusters or colloids with functional groups
incorporated into nucleic acids (such as primary amines). A fourth
approach is to use organometallic particles that contain
photoactive group(s) that then covalently attach to the nucleic
acids when light activated.
[0035] The invention will further be described by reference to
specific examples.
EXAMPLES
[0036] Preparation of Fluorescent and Gold Immunoprobes
[0037] 1. Preparation of Fluorescein-Conjugated Nanoqold Using
Fluorescein-Pbosphine
[0038] A tris (aryl) phosphine ligand bearing a single fluorescein
substitutent, and a second tris (aryl) phosphine ligand bearing a
single primary amine, were mixed with tris
(p-N-methylcarboxamidophenyl) phosphine in the ratio 2:1:5. Ninety
mg of this ligand mixture in 25 mL of methanol (an estimated
twelve-fold molar excess) was added to a solution of freshly
prepared Nanogold (product from 0.4 g of gold (I)
triphenylphosphine chloride) in dichloromethane (25 mL) and stirred
at room temperature overnight.
[0039] The reaction mixture was extracted with 0.02M ammonium
acetate with acetic acid, pH 5.8, in 20% isopropanol/water
(3.times.150 mL), then evaporated to dryness, redissolved in DMSO
(2 mL) and 0.6M triethylammonium bicarbonate in 20%
isopropanol/water. The fluorescein-substituted Nanogold was
isolated by gel filtration, using a coarse gel in a large column
(length=120 cm, internal diameter=2.5 cm, volume=590 mL), eluting
with 0.6M triethylammonium bicarbonate in 20% isopropanol/water.
The brownish-green product is the first species to be eluted. Yield
was 820 mmol. UV/visible data suggested that the product
incorporated 6 fluorescein groups per cluster.
[0040] 2. Preparation of Fluorescein-Conjugated Undecagold Using
Fluorescein-Phosphine
[0041] Gold (I) cyanide (31 mg, 0.14 mmol) was stirred with a 4:1:1
mixture of a tris (aryl) phosphine ligand bearing a single
fluorescein substitutent, a second tris (aryl) phosphine ligand
bearing a single primary amine, and tris
(p-N-methylcarboxamidophenyl) phosphine (105 mg, 0.14 mmol) in a
mixture of methanol (3 mL) and ethanol (5 mL) for 4 hours. Sodium
borohydride (3 mg, 0.1 mmol) in ethanol (2.0 mL) was added dropwise
over 30 minutes, then 8 drops of acetone were added to stop the
reduction. The orange-brown solution was added to 10 mL of aqueous
3.0M sodium chloride, stirred for 30 minutes, then evaporated to
dryness, stirred with methanol (20 mL) and filtered through a
medium porosity glass frit in order to exchange the coordinated
cyanide ligands for chlorides.
[0042] The cluster was separated from uncoordinated ligands and
other smaller molecules by chromatography over a coarse gel
filtration column (dimensions as described in the fluorescent
Nanogold preparation) eluting with 0.6M triethylammonium
bicarbonate buffer in 5% methanol/water. The pale greenish-orange
cluster (yield close to 50 nmol) is eluted first, followed by
uncoordinated ligands and smaller molecules. Calculations based on
the UV/visible absorption spectrum suggested incorporation of 5.5
fluorescein groups per undecagold.
[0043] 3. Preparation of Texas Red-Conjugated Nanogold
[0044] Nanogold was prepared as described for
fluorescein-conjugated analog, except that a different mixture of
phosphines was used to perform the ligand exchange on the freshly
made compound. A 2:1:8 mixture of a phosphine bearing a single
primary amine, a phosphine containing a single primary amine
protected with a t-Boc group, and tris
(p-N-methylcarboxamidophenyl) phosphine. The protected cluster was
isolated in the same manner as the fluorescein-conjugated
analog.
[0045] 400 nmol of protected Nanogold was evaporated to dryness
five times from methanol to remove triethylammonium bicarbonate,
then dissolved in isopropanol (0.2 mL) and 0.1M sodium borate
buffer, pH 9.0 (0.4 mL). This solution was added to a solution of
Texas Red sulfonyl chloride (8-fold excess, 2.1 mg) in isopropanol
(0.2 mL) and the mixture incubated at 4.degree. C. for 1 hour. The
products were separated by coarse gel filtration (GH25 gel, Amicon,
using column with length=50 cm, internal diameter=0.66 cm,
volume=16 mL) eluting with 0.6M triethylammonium bicarbonate in 50%
isopropanol/water. The dark blue product eluted in the void volume:
it was evaporated to dryness from methanol five times, then the
t-Boc protecting group was removed with 0.1M hydrochloric acid in
methanol (1 hour). The solution was neutralized with
triethylammonium buffer, evaporated to dryness and
rechromatographed (GH25 gel, buffer as above) to remove
triethylammonium chloride. Final yield was 170 nmol (43%). Labeling
calculated from the UV/visible spectrum was 1.17 Texas Red groups
per cluster.
[0046] 4. Conjugation of Fluorescein-Nanoqold or Undecaqold to
Antibody Fab' Fragments
[0047] F(ab').sub.2 antibody fragments (1.0 mg) were reduced with
40 mM mercaptoethylamine hydrochloride in 0.1M sodium phosphate at
pH 6.0, with 5 mM EDTA (1.4 mL) for 1 hour at room temperature.
Then the Fab' fragments were separated on a coarse gel filtration
column (GH25, Amicon: length=50 cm, internal diameter=1.0 cm,
volume=40 mL), and eluted with 0.02M sodium phosphate, pH 6.5, with
150 mM NaCl and 1 mM EDTA.
[0048] Fluorescein-conjugated Nanogold (250 nmol) was evaporated to
dryness five times from methanol solution to remove any
triethylammonium bicarbonate, then dissolved in DMSO (0.5 mL) and
0.1M sodium phosphate buffer, pH 7.5 (0.9 mL) and added to a
solution of a 100-fold excess of N-methoxycarbonylmaleimide (NMCM,
8 mg) in DMSO (0.1 mL), mixed and incubated at 0.degree. C. for 30
minutes. Maleimido-[Nanogold-fluorescein- ] was separated from
unreacted NMCM on a coarse gel filtration column (GH25, Amicon:
length=50 cm, internal diameter=1.0 cm, volume=40 mL), eluted with
0.02M sodium phosphate, pH 6.5, with 150 mM NaCl and 1 mM EDTA in
10% isopropanol/water. Maleimido-[Nanogold-fluorescein] was eluted
in the excluded volume; a 5-fold excess was added to the Fab'
fragments and the mixture mixed and incubated at 4.degree. C.
overnight. The product was concentrated to 0.5 mL using centrifuge
membrane concentrators with a 30,000 molecular weight cutoff
(Centricon-30, Amicon), then isolated by gel filtration
chromatography using a medium gel (Amicon GCL-90: Column length=50
cm, internal diameter=0.66 cm, volume=16 mL). 0.02M sodium
phosphate with 150 mM sodium chloride, pH 7.40, was used as the
eluent. The labeled Fab' fragments eluted first, followed by
unbound fluorescein-Nanogold. The process was repeated once for
highest purity of product.
[0049] [Au.sub.11-Fluorescein] labeling was conducted in the same
manner, except that the buffer used to separate the activated
fluorescein gold was prepared as a solution in 40% DMSO/water.
[0050] 5. Labeling of Streptavidin with Fluorescein-Conjugated
Nanoqold
[0051] Fluorescein-conjugated Nanogold (250 nmol) was evaporated to
dryness five times from methanol to remove any triethylammonium
bicarbonate, then dissolved in DMSO (0.4 mL) and 0.02M HEPES-NaOH
buffer, pH 7.5 (0.9 mL) and added to a solution of bis
(sulfo-N-hydroxysuccinimid- yl) suberate (BS.sup.3) (250-fold
excess: 38 mg) in DMSO (0.1 mL). The solution was mixed thoroughly,
incubated at room temperature for 1 hour and 25 minutes, then
sulfo-NHS-[Fluorescein-Nanogold] was separated from excess
(BS.sup.3) by chromatography on a coarse gel filtration column
(GH25, Amicon: length=50 cm, internal diameter=1.0 cm, volume=40
mL), eluting with 0.02M HEPES-NaOH, pH 7.5, in 20%
isopropanol/water. Activated fluorescein-Nanogold was the first
species to be eluted, and was mixed in 12-fold excess to a solution
of streptavidin in an aqueous solution of the same buffer. The
mixture was incubated at 4.degree. C. overnight, then reduced to
0.5 mL by membrane centrifugation (Centricon-30, Amicon) and
purified twice by gel filtration (Amicon GCL-90 gel: Column
length=50 cm, internal diameter=0.66 cm, volume=16 mL) eluted with
0.02M sodium phosphate buffer, pH 7.4 with 150 mM NaCl. Labeled
streptavidin is the first species to be eluted.
[0052] Labeling of Antibody Fab' Fragments with Texas
Red-Conjugated Nanogold
[0053] Antibody reduction and fluorescent gold label conjugation
were conducted as described for fluorescein-Nanogold. Texas
Red-Nanogold (200 nmol) was converted to the maleimide form by
reaction with a 100-fold excess of NMCM (3 mg) for 30 minutes at
0.degree. C. in DMSO (0.30 mL), and 0.1M sodium phosphate buffer at
pH 7.5 (0.45 mL); the activated label was isolated by
chromatography over a coarse gel filtration column (GH25, Amicon:
length=50 cm, internal diameter=0.66 cm, volume=16 mL), eluted with
0.02M sodium phosphate with 150 mM sodium chloride and 1 MM EDTA in
isopropanol/water; gold-containing fractions were added in fivefold
excess to Fab' fragments, prepared from F(ab') fragments as
described in Example 3, and incubated at 2.degree.-8.degree. C.
overnight. The product was isolated on a medium gel filtration
column (Amicon GCL-90: 50 cm, internal diameter=0.66 cm, volume=16
mL), eluting with 0.02 P 7.4+150 mm NaCl. The blue-grey product was
the first species to be eluted.
[0054] 7. Conjugation of Hoescht-33258 to Nanogold
[0055] Cross-linking was performed with 1,1'-carbonyldiimidazole
(CDI). Hoescht-33258 (3 mg) was dissolved in 0.5 mL DMSO with a
stoichiometric amount of CDI and incubated at room temperature for
30 minutes, then polyamino-1.4 nm gold cluster (150 nmol, giving
100-fold excess of dye) in DMSO (0.4 mL) was added and incubation
continued for a further 1 hour at room temperature. The reaction
mixture was then diluted with deionized water to 20% DMSO and
concentrated three times to minimal volume using membrane
centrifugation (10,000 MW cutoff). The solution was switched to a
30,000 MW cutoff membrane concentrator and centrifuged to minimal
volume a further 8 times; no 1.4 nm gold was observed to pass
through the membrane. The concentration of fluorescent dye in the
filtrate was determined spectrophotometrically, and by the end of
the eighth centrifugation had fallen to less than half the
concentration of 1.4 nm gold particles remaining in the retained
solution, diluted to the same volume. The ratio of dye molecules to
gold clusters in the final product was estimated
spectrophotometrically to be 1.2.
[0056] Preparation of Gold-Labeled Dipalmitoyl Phosphatidyl
Ethanolamine (DPPE)
[0057] 8. Labeling of DPPE with Freshly Activated 1.4 nm Gold
[0058] Activation of the gold particles was conducted in methanol
with a small amount of triethylamine added, until the reading of a
pH meter inserted into the solution was between 7.5 and 8.0, to
promote the reaction. 300 nmol of monoamino 1.4 nm gold, isolated
by ion exchange chromatography (in 0.6M triethylammonium
bicarbonate buffer in 20% isopropanol/water) was evaporated to
dryness five times from methanol to remove the volatile buffer,
then dissolved in 1.5 mL of triethylamine-treated methanol in which
a 500-fold excess of bis(sulfo-succinimidyl) suberate (86 mg) was
dissolved. This mixture was incubated at room temperature for 1
hour 30 minutes. The activated 1.4 nm gold was separated over a
coarse gel filtration column (Amicon, GH25: length=50 cm, internal
diameter=1.0 cm, volume=40 mL), eluted with methanol; the dark
brown activated Nanogold is the first species to elute. Fractions
containing uncontaminated activated Nanogold were combined to yield
180 nmol of activated gold, in 4 or 6 mL. A solution of DPPE
(100-fold excess: 12 mg) in one-half this volume of
trichloromethane was added and the mixture incubated at 4.degree.
C. overnight.
[0059] The reaction mixture was evaporated to dryness, and stirred
with 0.02M ammonium acetate, pH 5.80 (50 mL) to extract any
unreacted Nanogold; this suspension was extracted three times with
chloroform (15 mL). The combined chloroform extracts were
evaporated to dryness, dissolved in a 2:1 methanol/chloroform
mixture, and separated on a column identical to that used above for
the Nanogold activation, eluted with the same solvent mixture. The
dark brown Nanogold-DPPE conjugate is the first species to be
eluted; unconjugated DPPE is eluted later.
[0060] 9. Labeling of DPPE with Lyophilized Mono-Sulfo-NHS-1.4 nm
Gold (Nanogold)
[0061] 60 nmol of lyophilized monofunctional sulfo-NHS-Nanogold
(Nanoprobes, catalog number 2025: two vials) was redissolved in
methanol (2 mL) with a drop of triethylamine, and added to a
150-fold excess of DPPE in a 2:1 methanol/chloroform mixture with a
small amount of dichloromethane (4 mL), mixed thoroughly and
incubated overnight at 4.degree. C.
[0062] The reaction mixture was evaporated to dryness, then
redissolved in a 2:1 methanol/chloroform mixture (0.8 mL),
filtered, and chromatographed over a coarse gel filtration column
(Amicon GH25: length=50 cm, internal diameter=0.66 cm, volume=16
mL), eluting with 2:1 methanol:trichlorometha- ne. The brown DPPE
conjugate is the first species to be eluted; colorless unreacted
DPPE is eluted later.
[0063] 10. Preparation of Undecagold-DPPE
[0064] Both the activation reaction of the gold, and
chromatographic separation of activated gold, are conducted in a
basic methanolic buffer, prepared as follows: sufficient HEPES to
give 200 mL of a 0.02M solution (0.956 g) are suspended in methanol
(200 mL), and triethylamine added slowly, dropwise, until all the
HEPES is dissolved. A pH electrode was then inserted into the
solution, and dilute triethylamine in methanol was added until the
reading on the pH meter was between 7.6 and 8.0.
[0065] Ion exchange-isolated monoamino undecagold (150 nmol, by
UV/visible absorption spectroscopy, dissolved in aqueous 0.6M
triethylammonium bicarbonate buffer in 5% methanol) was evaporated
to dryness five times from ethanol to remove the volatile buffer.
It was then dissolved in 0.6 mL of the methanolic HEPES buffer and
added to a solution containing a 200-fold excess of
bis(sulfosuccinimidyl) suberate (17 mg) in methanolic HEPES buffer
(0.2 mL) to give a total volume of 0.8 mL. The solution was mixed
thoroughly, then incubated at room temperature in a small
polyethylene vial for 1 hour and 30 minutes. Activated undecagold
was separated from excess BS.sup.3 using a coarse gel filtration
column (Amicon GH25; length=50 cm, internal diameter=0.66 cm;
volume=16 mL), eluting with methanolic HEPES buffer prepared
earlier. The activated undecagold cluster is the first species to
be eluted. Fractions containing uncontaminated activated undecagold
were pooled, to yield about 80 nmol of activated undecagold in 2 or
3 mL. This was then added to a solution of a 100-fold excess of
DPPE (5.4 mg) in a volume of chloroform equal to one-half the
volume of the activated undecagold (1 or 1.5 mL), mixed thoroughly,
and incubated overnight at 4.degree. C.
[0066] The mixture was then evaporated to dryness, and shaken with
a water/chloroform mixture to remove unreacted undecagold; the
aqueous layer was removed and the process repeated twice. The
organic layer and solid material accumulated at the phase boundary
were then evaporated to dryness, dissolved in a 1:1
methanol/dichloromethane mixture (0.8 mL), and separated on a
column identical to that used above to separate activated
undecagold. The eluent was a 1:1 methanol/dichloromethane mixture.
The Undecagold-DPPE conjugate is the first species to be eluted,
and is orange in color; colorless unconjugated DPPE is eluted
later.
Golden Lipids
[0067] Preparation of Alkylamido-1.4 nm Gold Cluster
Derivatives
[0068] General:
[0069] Monoamino-1.4 nm gold cluster is reacted in excess with
alkyl acid chloride or alkyl acid anhydride in dichloromethane. The
unreacted gold cluster was extracted from the reaction mixture into
aqueous buffer and the product was purified from alkyl carboxylic
acids, produced from the hydrolysis of unreacted acide derivatives,
by size exclusion chromatography. The alkyl carboxylic acid
derivatives include nonanoyl chloride, decanoyl chloride, decanoic
anhydride, lauroyl chloride, lauric anhydride, palmitoyl chloride,
palmitic anhydride, heptadecanoyl chloride, stearoyl chloride, and
stearic anhydride.
[0070] 11. 300 nmol of monoamino 1.4 nm gold cluster, isolated by
ion exchange chromotography and removal of volatile buffer salts by
evaporation, was placed in 3 ml dichloromethane and treated with
200 nmol palmitoyl chloride. The mixture was stirred for 1 hour and
then washed three times with 0.1M sodium phosphate buffer pH 6.5.
The remaining reaction mixture was evaporated to dryness,
redissolved in a 2:1 methanol/chloroform, and separated on a gel
filtration column (Amicon GH25) eluting with 2:1
methanol/chloroform. The first to elute is the dark brown
palmitamido gold cluster.
[0071] Preparation of Large Platinum and Palladium Cluster
Immunoprobes
[0072] 12. Preparation of Functionalized 1-2 nm Platinum Cluster
1
[0073] From platinum (II) acetylacetonate and bathophenanthroline:
A mixture of Pt.sup.11(acac).sub.2 (0.20 g, 0.5 mmol),
bathophenanthroline (1:35 mg, 0.064 mmol) and 5-10:1 mixed ligand
(3:5 mg, 0,016 mmol) was stirred in glacial acetic acid (20 mL)
under a slow flow of nitrogen. Meanwhile sodium borohydride (2.3 g)
was dissolved in 2-methoxyethyl ether (diglyme: 30 mL) and a 1:1
mixture of ethanol/water (20 mL) was added dropwise over 30
minutes; the hydrogen generated from this solution was bubbled into
the reaction mixture. After 30 minutes no color change was
observed: therefore, a small amount of platinum (II) chloride
(.about.0.05 g) suspended in acetic acid (1 mL) was added and
stirring continued. H.sub.2 bubbling was continued for a further 4
hours, during which time the solution darkened from yellow to green
and a greenish-black precipitate was formed. The solution was then
aerated for 2 hours by blowing air into the flask.
[0074] The contents of the reaction vessel were evaporated to
dryness, and the greenish-black solid extracted with DMSO (0.5 mL)
and 0.6M triethylammonium bicarbonate in 20% isopropanol/water (1.5
mL), filtered, then separated over a coarse gel filtration column
(GH25, Amicon) eluting with 0.6M triethylammonium bicarbonate in
20% isopropanol/water. A dark greenish-brown compound was eluted in
the void volume, and pale yellow species later. Electron microscopy
of the greenish-brown compound showed metal particles 1-2 nm in
diameter.
[0075] From Platinum (II) chloride and substituted phenanthrolines:
A mixture of platinum (II) chloride (0.5 mmol, 126 mg), ligands 2
(14.8 mg) and 3 (5.1 mg) were stirred in glacial acetic acid (25
mL) for 6 hours and heated to 100.degree. C. to partially dissolve
the solids. After cooling to room temperature overnight, hydrogen
(generated as described above) was bubbled slowly through the
reaction mixture for 3 hours; the color darkened from brown to
greenish-black. The reaction mixture was then poured into a glass
beaker and stirred overnight while air was bubbled through the
solution. All the acetic acid was removed to leave a greenish black
solid. This was then extracted into DMSO (0.5 mL) and 0.6M
triethylammonium bicarbonate in 20% DMSO/water (1.5 mL), filtered,
and separated on a coarse gel filtration column (GH25, Amicon)
eluting with 0.6M triethylammonium bicarbonate in 20% DMSO/water. A
brownish-green species was eluted in the void volume.
[0076] 13. Conjugation of Functionalized Platinum Cluster to
Antibodies
[0077] F(ab').sub.2 fragments or IgG molecules were reduced with 40
or 50 mM respectively mercaptoethylamine hydrochloride in 0.1M
sodium phosphate buffer with 5 mM EDTA, pH 6.0, for 1 hour and 5
minutes at room temperature (in 1.5 mL volume). Reduced antibody
was separated from excess mercaptoethylamine hydrochloride on a
coarse gel filtration column (GH25, Amicon), eluting with 0.02 m
sodium phosphate with 150 mM sodium chloride and 1 mM EDTA, pH 6.5;
reduced antibody was eluted in the excluded volume.
[0078] Platinum cluster (as isolated above; approximately one-third
of the total isolated) was evaporated to dryness, then reevaporated
to dryness five times from methanol solution to remove
triethylammonium bicarbonate. The compound was then dissolved in
DMSO (0.25 mL) and 0.1 m sodium phosphate, pH 7.5 (0.48 mL), and
added to a solution of N-methoxycarbonylmaleimide (NMCM: 6 mg) in
DMSO (0.07 mL); the solution was mixed thoroughly and incubated at
0.degree. C. (ice bath/refrigerator) for 30 minutes. The activated
platinum compound was then separated from excess smaller molecules
on a coarse gel filtration column (GH25, Amicon), eluting with 0.02
m sodium phosphate with 150 mM NaCl and 1 mM EDTA in 20%
DMSO/water; platinum-containing fractions were then added to those
containing reduced antibody, mixed thoroughly and the mixture was
incubated at 4.degree. C. overnight.
[0079] Next day, the reaction mixture was concentrated to between
0.5 and 1 mL (Centricon-30) and the products separated on a size
fractionation column (Pharmacia Superose-12) eluted with 0.02 m
sodium phosphate with 150 mM sodium chloride, pH 7.4; the first
species to be eluted was greenish-brown in color and was in the
position expected for antibody molecules. In the Fab' preparations,
fractions comprising this peak were concentrated and
re-chromatographed on a Superdex-75 column in the same buffer. The
first species to emerge was the antibody conjugate; some resolution
of unlabeled from labeled Fab' fragments was observed.
[0080] 14. Preparation and Antibody Conjugation of Functionalized
2-3 nm Pd Cluster
[0081] Palladium (II) acetate (1.0 mmol, 227 mg), and a mixture of
ligands 2 (27 mg) and 3 (8.5 mg) were stirred in glacial acetic
acid (25 mL) with stirring under a slow stream of nitrogen. The
mixture was warmed to 80.degree. C. to dissolve all the components,
to give an orange-brown solution. After cooling to room temperature
hydrogen (generated as described above) was bubbled slowly through
the mixture for 3 hours to give a near-black solution. This was
stirred in a glass beaker under a stream of air until dry, then
dissolved in DMSO (1.0 mL) and 0.6M triethylammonium bicarbonate in
20% DMSO/water (3.0 mL), filtered, and separated on a coarse gel
filtration column (GH25, Amicon) eluting with 0.6M triethylammonium
bicarbonate in 20% DMSO/water. The cluster was eluted in the
exclusion volume.
[0082] Fab' conjugation is conducted using the same procedure used
with the platinum cluster: F(ab').sub.2 is first reduced and
separated from excess reducing agent in the same manner. A suitable
amount of the cluster is evaporated to dryness three times from
methanol, with heating to 55.degree.-60.degree. C. to remove DMSO.
The black solid is dissolved in DMSO (0.32 mL) and 0.1M sodium
phosphate, pH 7.50 (0.40 mL) and added to a solution of
N-methoxycarbonylmaleimide (NMCM: 6 mg) in DMSO (0.05 mL). The
mixture is vortexed, incubated at 0.degree. C. for 30 minutes (ice
bath/refrigerator) then separated on a GH25 column. The activated
cluster is eluted in the void volume: cluster-containing fractions
are pooled and added to the reduced antibody. The mixture is left
at room temperature for 1 hour, then stored at 4.degree. C.
overnight.
[0083] 15. Immunoblotting of Conjugates
[0084] Immunoblotting was conducted using a single-layer technique:
mouse IgG was spotted onto a hydrated nitrocellulose membrane in
serial dilutions, and detected using platinum or palladium labeled
goat IgG and Fab' anti-mouse IgG conjugates.
1 Buffers required: PBS: 0.01M sodium phosphate with 150 mm sodium
chloride, pH 7.4 WASH: 0.02M sodium phosphate with 150 mm sodium
chloride, pH 7.4 0.8% w/w bovine serum albumin, fraction V by heat
shock 0.1% w/w gelatin, type B from bovine skin, approximately 60
bloom 2 mM sodium azide. BLOCK- 0.02M sodium phosphate with 150 mm
sodium chloride, ING: pH 7.4 4.0% w/w bovine serum albumin,
fraction V by heat shock. 0.1% w/w gelatin, type B from bovine
skin, approximately 60 bloom 2 mM sodium azide. INCUBA- 0.02M
sodium phosphate with 150 mm sodium chloride, TION: pH 7.4 0.8% w/w
bovine serum albumin, fraction V by heat shock. 0.1% w/w gelatin,
type B from bovine skin, approx. 60 bloom 1% W/W normal goat serum
2 mM sodium azide.
[0085] Procedure:
[0086] 1. A nitrocellulose membrane, marked with pencilled
divisions for antigen concentration identification, was simmered in
gently boiling water for 15 minutes.
[0087] 2. 1 .mu.l dilutions of mouse IgG were spotted onto
membrane, from 10.sup.-9 to 10.sup.-18 g.
[0088] 3. Membrane was blocked with blocking buffer for 30 minutes
at 45.degree. C.
[0089] 4. Membrane washed 5 minutes with wash buffer.
[0090] 5. Membrane was incubated with 5 mL of a {fraction (1/200)}
dilution of the NANOGOLD.TM. reagent in incubation buffer for 2.5
hours at room temperature, with slow agitation.
[0091] 6. Membrane was rinsed with buffer 3 (3.times.5 mins), then
PBS (3.times.30 seconds).
[0092] 7. Membrane postfixed with glutaraldehyde, 1% in PBS (10
minutes).
[0093] 8. Rinsed with deionized water (2.times.5 minutes).
[0094] 9. Rinse with 0.05M EDTA at pH 4.5 (2 minutes).
[0095] 10. Develop with freshly mixed LI SILVER.TM. (Nanoprobes,
Inc.), 2.times.30 minutes. Rinse thoroughly with deionized water
between developments to remove all the silver enhancement
reagent.
[0096] 11. Rinse repeatedly with deionized water, then let
air-dry.
[0097] The last visible spot in a series of decreasing
concentration contained 10 pg of mouse IgG for platinum-labeled
goat Fab'- and IgG-anti-mouse IgG, and also for palladium-labeled
goat anti-mouse Fab'. For the IgG conjugates, this was the same
level of sensitivity as was obtained from a second blot performed
with the analogous Nanogold conjugate.
[0098] Thiol-Gold Cluster Preparation and Antibody Labelling
Procedure
[0099] The following solutions are used in the preparation of
thiol-gold clusters:
[0100] A. NaBH.sub.4 solution: 0.2% solution of NaBH.sub.4 in
ethanol.
[0101] B. TEAH: 0.6M triethylammonium bicarbonate in distilled
water. Note: Unless otherwise specified, all reactions are
performed at 25.degree. C.
[0102] 16. Synthesis of Thiol Gold Cluster from Aurothioglucose
(I):
[0103] 5 mg of aurothioglucose (C.sub.6H.sub.11O.sub.5SAu) is
dissolved in 0.5 ml of distilled H.sub.2O in a test tube, forming a
light yellow solution. Two 10 .mu.L aliquots of NaBH.sub.4 solution
are added to the aurothioglucose solution over 15 minutes. Upon
NaBH.sub.4 addition the solution turns dark brown in color. The
cluster is purified by size exclusion chromatography using Amicon
GH-25 material (MW cutoff=3000 Daltons), with TEAH as the elution
buffer. The cluster is recovered from the void volume of the
column. Cluster formation is verified by UV/VIS spectroscopy and
electron microscopy.
[0104] 17. Synthesis of Thiol Gold Cluster from KAuBr.sub.4 and
Glutathione
[0105] (II): 5 mg of KAuBr.sub.4 (8.418.times.10.sup.-3 mMol) is
dissolved in 0.3 ml of distilled water. 5.17 mg of glutathione
(1.684.times.10.sup.-2 mMol), dissolved in 0.5 ml of distilled
water is added to the KAuBr.sub.4 solution in two 0.25 ml aliquots
at 5 minute intervals. After the first addition the dark reddish
brown KAuBr.sub.4 solution turns to a clear, colorless to pale
yellow solution. The reaction mixture is left for 5 minutes after
the second addition of glutathione solution. The pH of the reaction
mixture is adjusted to 8 using 6N NaOH solution. Two 10 .mu.L
aliquots of NaBH.sub.4 solution are added to the reaction mixture
over 15 minutes. The pH is adjusted to neutral with 1N HCl
solution, and a third 10 .mu.L aliquot of NaBH.sub.4 solution is
added. Two 10 .mu.L aliquot of NaBH4 solution are added to the
reaction mixture over 15 minutes. The pH is adjusted to neutral
with 1N HCl solution, and a third 10 .mu.L aliquot of NaBH.sub.4
solution is added. Upon NaBH.sub.4 addition the solution turns dark
brown in color. The cluster is purified by size exclusion
chromatography using Amicon GH-25 material (MW cutoff=3000
daltons), with TEAH as the elution buffer. The cluster is recovered
from the void volume of the column. Cluster formation is verified
by UV/VIS spectroscopy and electron microscopy.
[0106] 18. Synthesis of a Mixed Thiol Gold cluster from KAuBr.sub.4
and a Mixture of Glutathione and 1-thio-.beta.-D-Glucose (III):
[0107] 5 mg of KAuBr.sub.4 (8.418.times.10.sup.-3 mMol) is
dissolved in 0.3 ml of distilled water. A mixed thiol solution of
3.62 mg of glutathione (1.18.times.10.sup.-2 mMol) and 1.10 mg of
1-thio-.beta.-D-Glucose (5.05.times.10.sup.-3 mMol), dissolved in
0.5 ml of distilled water is added to the KAuBr.sub.4 solution in
two 0.25 ml aliquots at 5 minute intervals. After the first
addition the dark reddish brown KAuBr.sub.4 solution turns to a
clear, colorless to pale yellow solution. The reaction mixture is
left for 5 minutes after the second addition of the mixed thiol
solution. The pH of the reaction mixture is adjusted to 8 using 6N
NaOH solution. Two 10 .mu.L aliquots of NaBH.sub.4 solution are
added to the reaction mixture over 15 minutes. The pH is adjusted
to neutral with 1N HCl solution, and a third 10 .mu.L aliquots of
NaBH.sub.4 solution is added. Upon NaBH.sub.4 addition, the
solution turns dark brown in color. The cluster is purified by size
exclusion chromatography using Amicon GH-25 material (MW
cutoff=3000 daltons), with TEAH as the elution buffer. The cluster
is recovered from the void volume of the column. Cluster formation
is verified by UV/VIS spectroscopy and electron microscopy.
[0108] 19. Synthesis of Antibodies (Whole Molecule) Labelled with
III:
[0109] 2 mg of IgG is combined in a siliconized microcentrifuge
tube with 10 mg of mercaptoethanolamine in 1.4 ml of 0.1M sodium
phosphate, pH 6.0 which contains 5 mM EDTA. The reaction is allowed
to proceed 1 hour at room temperature, then the reduced antibody is
separated from excess MEA on a GH-25 column (Amicon), eluting with
0.1M borate buffer, pH 9.2, with 5 mM EDTA. The reduced antibody is
collected in the void volume of the column. Fractions are combined
and reduced in volume to 0.1 ml with centricon-30 (Amicon). The
reduced antibody fraction is then incubated at 37.degree. C. for 1
hour with a 20-fold excess of compound III, which is added in 0.1M
borate buffer, pH 9.2, with 5 mM EDTA. The total volume of this
reaction mixture is made up to 1.4 ml by adding 0.1M borate buffer,
pH 9.2, with 5 mM EDTA. After incubation period, the reaction
mixture is reduced in volume to 0.1 ml with centricon-30 (Amicon)
and injected on a Superdex-75 molecular weight fractionation column
(Pharmacia) to separate out the gold cluster-IgG conjugate from
excess III. Antibody labelling is verified by UV/VIS spectroscopy
and electron microscopy.
[0110] 20. Synthesis of Fab' Antibody Fragments Labelled with
III:
[0111] 2 mg of F(ab').sub.2 is combined in a siliconized
microcentrifuge tube with 7 mg of mercaptoethanolamine in 1.4 ml of
0.1M sodium phosphate, pH 6.0 which contains 5 mM EDTA. The
reaction is allowed to proceed 1 hour at room temperature, then the
reduced antibody fragment is separated from excess MEA on a GH-25
column (Amicon), eluting with 0.1M borate buffer, pH 0.2, with 5 mM
EDTA. The Fab' fragment is collected in the void volume of the
column. Fractions are combined and reduced in volume to 0.1 ml with
centricon-30 (Amicon). The Fab' fraction is then incubated at
37.degree. C. for 1 hour with a 20-fold excess of III, which is
added in 0.1M borate buffer, pH 9.2, with 5 mM EDTA. The total
volume of this reaction mixture is made up to 1.4 ml by adding 0.1M
borate buffer, pH 9.2, with 5 mM EDTA. After the incubation period,
the reaction mixture is reduced in volume to 0.1 ml with
centricon-30 (Amicon) and injected on a Superdex-75 molecular
weight fractionation column (Pharmacia) to separate out the gold
cluster-Fab' conjugate from excess III. Antibody labelling is
verified by UV/VIS spectroscopy and electron microscopy.
[0112] Preparation of Thiol-Metal Clusters (Silver, Silver-Gold,
Platinum, Thallium)
[0113] 21. Preparation of Silver-Organic Thiol Clusters
[0114] 5.times.10.sup.-6 moles of silver acetate in 0.5 ml of water
was mixed with 1.times.10.sup.-5 moles of thioglucose and heated to
70.degree. C. for 5 minutes. Then 5.times.10.sup.-6 moles of sodium
borohydride was added. Later, 5.times.1 01 moles of additional
sodium borohydride was added. A dark brown solution resulted, and
contained organo-silver clusters .about.1-3 nm in diameter. These
were purified by gel filtration size exclusion chromatography using
an Amicon GH25 column (cutoff 3,000 MW) in an aqueous buffer of
0.6M triethylammonium bicarbonate and 5% methanol. The product was
rotary evaporated under vacuum and resuspended in water. The size
was assayed by electron microscopy.
[0115] 22. Preparation of Mixed Silver-Gold Organic Thiol
Clusters.
[0116] 2.5.times.10.sup.-6 moles of silver acetate and
2.5.times.10.sup.-6 moles of chloroauric acid in 0.5 ml of water
were mixed with 1.times.10.sup.-5 moles of thioglucose. Then,
5.times.10.sup.-6 moles of sodium borohydride was added. A dark
brown solution resulted, and contained organo-silver/gold clusters
.about.1-3 nm in diameter. These were purified by gel filtration
size exclusion chromatography using an Amicon GH25 column (cutoff
3,000 MW) in an aqueous buffer of 0.6M triethylammonium bicarbonate
and 5% methanol. The product was rotary evaporated under vacuum and
resuspended in water. The size was assayed by electron
microscopy.
[0117] 23. Preparation of Platinum-Organic Thiol Clusters
[0118] 5.times.10.sup.-6 moles of platinum chloride in 0.5 ml of
water was mixed with 5.times.10.sup.-6 moles of thioglucose and
heated to 60.degree. C. for 5 minutes. Then 2.5.times.10.sup.-6
moles of sodium borohydride was added. Later, 2.5.times.10.sup.6
moles of additional sodium borohydride was added. A dark brown
solution resulted, and contained organo-platinum clusters
.about.1-3 nm in diameter. These were purified by gel filtration
size exclusion chromatography using an Amicon GH25 column (cutoff
3,000 MW) in an aqueous buffer of 0.6M triethylammonium bicarbonate
and 5% methanol. The product was rotary evaporated under vacuum and
resuspended in water. The size was assayed by electron
microscopy.
[0119] 24. Preparation of Thallium-Organic Thiol Clusters
[0120] 5.times.10.sup.-6 moles of thallium chloride in 0.5 ml of
water was mixed with 1.times.10.sup.-5 moles of thioglucose. After
10 minutes, 5.times.10.sup.-6 moles of sodium borohydride was
added. Later, 2.5.times.10.sup.-6 moles of additional sodium
borohydride was added. A dark brown solution resulted, and
contained organo-thallium clusters .about.1-3 nm in diameter. These
were purified by gel filtration size exclusion chromatography using
an Amicon GH25 column (cutoff 3,000 MW) in an aqueous buffer of
0.6M triethylammonium bicarbonate and 5% methanol. The product was
rotary evaporated under vacuum and resuspended in water. The size
was assayed by electron microscopy.
[0121] Thiol-Gold Clusters for Protein Staining in Polyacrylamide
Gel Electrophoresis (PAGE): Preparation and Application
[0122] The following solutions are used in the preparation of
thiol-gold clusters:
[0123] A. NaBH.sub.4 solution: 0.2% solution of NaBH.sub.4 in
ethanol.
[0124] B. TEAH: 0.6M triethylammonium bicarbonate in distilled
water.
[0125] Note: Unless otherwise specified, all reactions are
performed at 25.degree. C.
[0126] 25. Synthesis of Thiol-Gold Cluster from KAuBr.sub.4 and
o-mercaptobenzoic Acid (1):
[0127] 5 mg of KAuBr.sub.4 (8.41.times.10.sup.-3 mMol) is dissolved
in 0.3 ml of distilled water. 2.60 mg of o-mercaptobenzoic acid
(1.68.times.1 0-2 mMol), dissolved in 0.5 ml of TEAH is added to
the KAuBr.sub.4 solution in two 0.25 ml aliquots at 5 minute
intervals. After the first addition, the dark reddish brown
KAuBr.sub.4 solution turns to clear, colorless to pale yellow
solution. The reaction mixture is left for 5 minutes after the
second addition of glutathione solution. Two 10 .mu.L aliquots of
NaBH.sub.4 solution are added to the reaction mixture over 15
minutes. Upon NaBH.sub.4 addition the solution turns dark brown in
color. The cluster is purified by size exclusion chromatography
using Amicon GH25 material (MW cutoff=3000 daltons), with TEAH as
the elution buffer. The cluster is recovered from the void volume
of the column. The cluster containing fractions are combined in a
50 ml round bottom flask and the solution is evaporated to dryness
with a rotoevaporator (Buchi). The cluster is redissolved in
methanol and re-evaporated with the rotoevaporator. This methanol
evaporation is done 5 times. The cluster is then dissolved in 0.1
sodium phosphate buffer, pH 8.0, with 1.0 mM EDTA for use in
subsequent protein staining experiments. Cluster formation is
verified by UV/VIS spectroscopy and electron microscopy. As a thiol
scavenger, an excess amount of N-ethylmaleimide is added to the
cluster solution.
[0128] 26. Synthesis of a Mixed Thiol Gold Cluster from KAuBr.sub.4
and Mixture of o-mercaptobenzoic Acid and Nonylmercaptan(II):
[0129] 5 mg of KAuBr.sub.4 (8.41.times.10.sup.-3 mMol) is dissolved
in 0.3 ml of 90% ethanol in distilled H.sub.2O. A mixed thiol
solution of 2.34 mg of o-mercaptobenzoic acid (1.52.times.10.sup.-2
mMol) and 0.27 mg of nonylmercaptan (1.68.times.10.sup.-3 mMol),
dissolved in 0.5 ml of 95% ethanol in 6N NaOH, is added to the
KAuBr.sub.4 solution in two 0.25 ml aliquots at 5 minute intervals.
After the first addition, the dark reddish brown KAuBr.sub.4
solution turns to a pale yellow precipitate. Upon the addition of
the second thiol mixture addition, the precipitate is solubilized
to give a clear, colorless to pale yellow solution. The reaction
mixture is left for 5 minutes after the second addition of the
mixed thiol solution. Two 10 .mu.L aliquots of NaBH.sub.4 solution
are added to the reaction mixture over 15 minutes. The pH is
adjusted to neutral with 1N HCl solution, and a third 10 .mu.L
aliquots of NaBH.sub.4 solution is added. Upon NaBH.sub.4 addition,
the solution turns dark brown in color. The cluster is purified by
size exclusion chromatography using Amicon GH-25 material (MW
cutoff=3000 Daltons), with TEAH as the elution buffer. The cluster
is recovered from the void volume of the column. The cluster
containing fractions are combined in a 50 ml round bottom flask and
the solution is evaporated to dryness with a rotoevaporator
(Buchi). The cluster is redissolved in methanol and re-evaporated
with the rotoevaporator. This methanol evaporation is done 5 times.
The cluster is then dissolved in 0.1 sodium phosphate buffer, pH
8.0, with 1.0 mM EDTA for use in subsequent protein staining
experiments. Cluster formation is verified by UV/VIS spectroscopy
and electron microscopy. As a thiol scavenger, an excess amount of
N-ethylmaleimide is added to the cluster solution.
[0130] 27. Application of I and II to SDS-PAGE in Non-Reducing
Conditions:
[0131] Protein sample (Pharmacia LMW calibration kit proteins) to
be analyzed is incubated with excess I or II in 10 mM Tris/HCl, 1
mM EDTA, pH 8.0. To the sample is added Sodium Dodecyl Sulphate
(SDS) to 2.5%. The sample is heated for 5 minutes at 100.degree. C.
Bromophenol Blue is added to 0.01%. Using an eight "well" sample
applicator, eight 1 .mu.L aliquots of the sample mixture are
applied to a Phastgel gradient gel (8-25) and run on the Pharmacia
Phastgel system using the following protocol:
2 Sample Applicator down at step 1.1 1 Vh Sample Applicator up at
step 1.1 10 Vh SEP 1.1 250 V 10.0 mA 3.0 W 15.degree. C. 65 Vh SEP
1.2 50 V 0.1 mA 0.5 W 15.degree. C. 0 Vh
[0132] After electrophoresis, the gel is developed for 5-15 minutes
(or until background development) with LI SILVER silver enhancement
kit (Nanoprobes, Inc.). After silver development, gels are rinsed
thoroughly with deionized water, and preserved by incubation in 20%
glycerol in deionized water at 45.degree. C. for 10 minutes.
[0133] 28. Application of I and II to SDS-PAGE in Reducing
Conditions:
[0134] Protein sample (Pharmacia LMW calibration kit proteins) to
be analyzed is incubated with a 2.5% mercaptosuccinic acid solution
in 10 mM Tris/HCl, 1 mM EDTA, pH 8.0. To the sample is added Sodium
Dodecyl Sulphate (SDS) to 2.5%. The sample is heated for 5 minutes
at 100.degree. C. Bromophenol Blue is added to 0.01% and
N-ethylmaleimide (prepared as a 20% solution in DMSO) is added to
5%. The sample is incubated at room temperature for 10 minutes.
Excess amounts of I and II are added to the sample mixture. Using
an eight "well" sample applicator, eight 1 .mu.L aliquots of the
sample mixture are applied to a Phastgel gradient gel (8-25) and
run on the Pharmacia Phastgel system using the following
protocol:
3 Sample Applicator down at step 1.1 1 Vh Sample Applicator up at
step 1.1 10 Vh SEP 1.1 250 V 10.0 mA 3.0 W 15.degree. C. 65 Vh SEP
1.2 50 V 0.1 mA 0.5 W 15.degree. C. 0 Vh
[0135] After electrophoresis, the gel is developed for 5-15 minutes
(or until background development) with LI SILVER silver enhancement
kit (Nanoprobes, Inc.). After silver development, gels are rinsed
thoroughly with deionized water, and preserved by incubation in 20%
glycerol in deionized water at 45.degree. C. for 10 minutes.
Nucleic Acid
[0136] Detection of Oligonucleotides
[0137] General:
[0138] Oligonucleotides modified to contain biotin can be detected
through the use of either gold cluster labeled anti-biotin
antibodies, antibody fragments, gold cluster-labeled streptavidin,
or gold cluster-labeled avidin. Incorporation of the biotin into an
oligonucleotide strand can occur via a commercially available
biotin-NHS reagent and a primary amine that was introduced onto the
oligonucleotide via a modified nucleotide or nucleotide substitute
(phosphoramidite) by a DNA synthesizer. The biotin labeled
oligonucleotide can then be reacted with gold cluster labeled
reagent designed to react with the biotin moiety and observed via
silver development.
[0139] 29. An oligonucleotide having a primary amine attached to
the 5' end was reacted in sightly alkaine sodium phosphate buffer
with a large molar excess of biotin-LC-N-hydroxysuceinimide ester
(biotin-LC-NHS II; Pierce) dissolved in DMSO. After 1 hour at room
temperature the reaction mixture was purified on a GH25 (nominal
exclusion limit 3000 D) column and fractions measured for nucleic
acid content by monitoring at 260 nm. By comparing the ratio of the
260 and 240 nm peaks to gauge biotin incorporation. Recovered
nucleic acid yield 49%.
[0140] The biotin labeled oligonucleotides were reacted with
streptavidin gold cluster conjugates in phosphate buffered saline
(PBS), pH 7.4 for four hours at room temperature and separated by
size exclusion chromatography (Superdex 75, nominal exclusion limit
70 kD) using the same buffer as eluant. On this column, the major
peak coincided with the retention time of the cluster conjugate and
showed enhanced absorption at 260 nm indicating reaction had
occurred.
[0141] 30. Detection of Biotinylated DNA: Dot Blots
[0142] Blot tests were conducted to test the incorporation of gold
in the isolated product. In a typical run, biotin labeled
oligonucleotides was serially diluted in water by factors of ten
starting with a 2.5.times.10.sup.-7 M solution and ending at
2.5.times.10.sup.-166 M. 1 .mu.L spots were applied to a
nitrocellulose membrane by Drummond capillary pipettes. The
membrane was allowed to dry and then subjected to a 302 nm light
placed 18 cm from the membrane for ten minutes. The membrane was
blocked with 4% bovine serum albumin (BSA) for 30 minutes at
37.degree. C. The membrane was then incubated with the streptavidin
gold cluster conjugate diluted to about 2 .mu.g/ml in 0.8% BSA for
1.5 hours. After rinsing with buffer and then water, the membrane
was treated with silver developer. The membrane was again rinsed
with water and examined for spot generation after drying. The
silver developed gold particle is seen as a dark spot. The spots
appearing unambiguously above background provide a limit of
detection on the order of 10 to 100 attomole (1 attomol=10.sup.-188
mol) detected with more concentrated applications appearing darker
than dilute ones.
[0143] 31. Preparation and Detection of Biotinylated M13mp18
[0144] Another model system was the preparation of labeled
complementary fragments to the single stranded phage M13mp18. Using
the random primer method, biotin was introduced as modified dUTP
and incorporated with Klenow. After purification using GeneClean,
the biotinylated solution was applied to a nitrocellulose membrane,
immobilized in a vacuum oven and detected by streptavidin gold
cluster conjugate. In this procedure 10.sup.-15 mol M13mp18 was
detected. The detection limit was independent of the concentration
of the gold conjugate.
[0145] Preparation of Gold Cluster Labeled DNA Hybridization
Probes
[0146] 32. Random Primer Extension Method
[0147] Gold cluster labelled nucleotide triphosphate was prepared
by reaction of NHS-gold cluster with an amino modified nucleotide
triphosphate. Example: Amino-7-dUTP, available from Clontech, is a
dUTP analog with a primary amine covalently attached to the
pyrimidine ring through a seven atom spacer arm. 50 nmol
Amino-7-dUTP was reacted with 5 nmol
mono-N-hydroxy-sulfosuccinimide-1.4 nm gold cluster in 20 mM
HEPES-NaOH buffer pH 7.5 at 4.degree. C. overnight. The reaction
mixture was separated on a GH25 column to remove unreacted
nucleotide from the conjugate. The product was further purified by
ion exchange chromatography over TSK DEAE with elution at 0.3M
triethylammonium hydrogencarbonate.
[0148] The modified nucleotide was incorporated into an
oligonucleotide by enzymatic extension of random primers.
[0149] While the invention has been described by reference to
specific embodiments, this was for purposes of illustration only.
Numerous alternative embodiments will be apparent to those skilled
in the art and are considered to be within the scope of the
invention.
* * * * *