U.S. patent application number 10/073625 was filed with the patent office on 2002-10-31 for radiative decay engineering.
Invention is credited to Lakowicz, Joseph R..
Application Number | 20020160400 10/073625 |
Document ID | / |
Family ID | 23022467 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160400 |
Kind Code |
A1 |
Lakowicz, Joseph R. |
October 31, 2002 |
Radiative decay engineering
Abstract
Compositions and methods for increasing the fluorescence
intensity of molecules are provided. In particular, compositions
and methods directed to increasing the intrinsic fluorescence of
biomolecules and low quantum yield fluorophores are described. The
intrinsic fluorescence of biomolecules is increased by positioning
a metal particle and a biomolecule at a distance apart sufficient
to increase the radiative decay rate of the biomolecule. Methods
for the identification of nucleic acids are also provided. The
compositions and methods can also be used to increase the emission
of any fluorophore, such as the extrinsic probes used to label
biomolecules.
Inventors: |
Lakowicz, Joseph R.;
(Ellicott City, MD) |
Correspondence
Address: |
TROUTMAN SANDERS LLP
BANK OF AMERICA PLAZA, SUITE 5200
600 PEACHTREE STREET , NE
ATLANTA
GA
30308-2216
US
|
Family ID: |
23022467 |
Appl. No.: |
10/073625 |
Filed: |
February 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60268326 |
Feb 14, 2001 |
|
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|
Current U.S.
Class: |
435/6.11 ;
435/174; 435/287.2; 435/6.12; 435/6.13; 435/7.9; 436/525;
436/94 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C12Q 1/682 20130101; C12Q 2563/107 20130101; C12Q 2563/143
20130101; C12Q 2563/149 20130101; C12Q 2563/149 20130101; C12Q
2563/143 20130101; C12Q 1/6818 20130101; C12Q 2563/107 20130101;
Y10T 436/143333 20150115; Y10S 436/805 20130101; C12Q 1/682
20130101 |
Class at
Publication: |
435/6 ; 435/7.9;
436/525; 436/94; 435/287.2; 435/174 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/542; G01N 033/553 |
Goverment Interests
[0002] The work leading to this invention was supported in part by
the U.S. Government under grant number RR-08119 awarded by the NIH
National Center for Research Resources. Therefore, the U.S.
Government may have certain rights in this invention.
Claims
What is claimed is:
1. A composition of matter comprising: a biomolecule in combination
with a metal particle, wherein said metal particle and said
biomolecule are positioned at a distance apart sufficient to adjust
intrinsic emission of electromagnetic radiation from the
biomolecule in response to an amount of exciting electromagnetic
radiation.
2. The composition of claim 1, wherein the biomolecule comprises a
nucleic acid.
3. The composition of claim 1, wherein the biomolecule comprises a
purine or pyrimidine.
4. The composition of claim 1, wherein the biomolecule comprises a
nucleoside or nucleotide.
5. The composition of claim 1, wherein the biomolecule comprises an
oligonucleotide.
6. The composition of claim 1, wherein the biomolecule comprises a
protein.
7. The composition of claim 1, wherein the biomolecule comprises an
amino acid.
8. The composition of claim 1, wherein the biomolecule comprises a
lipid.
9. The composition of claim 1, wherein the biomolecule comprises a
sugar moiety.
10. The composition of claim 1, wherein the metal particle is at a
distance of about 50 .ANG. to about 2000 .ANG. from the
biomolecule.
11. The composition of claim 1, wherein the metal particle
comprises a noble metal.
12. The composition of claim 11, wherein the noble metal is
selected from the group consisting of rhenium, ruthenium, rhodium,
palladium, silver, osmium, iridium, platinum, and gold.
13. The composition of claim 1, wherein the metal particle is
sub-wavelength in size.
14. The composition of claim 1, wherein the biomolecule is linked
to the metal particle.
15. A method for increasing the intrinsic fluorescence of a
biomolecule, said method comprising the step of: positioning a
metal particle and said biomolecule at a distance apart sufficient
to increase the electromagnetic emission from said biomolecule in
response to an amount of exciting radiation.
16. A method for detecting a biomolecule, said method comprising
the steps of: (a) positioning a metal particle and a biomolecule at
a distance apart sufficient to manipulate the electromagnetic
emission from said biomolecule; (b) exposing said biomolecule to an
amount of exciting radiation; and (c) detecting the electromagnetic
emission from said biomolecule.
17. A method for manipulating fluorescence intensity of a
biomolecule, said method comprising the steps of: (a) increasing
the rate of radiative decay of the biomolecule by positioning the
biomolecule adjacent to a metal particle; and (b) exposing the
biomolecule to an amount of exciting radiation.
18. A method for detecting the presence of a nucleic acid sequence
in a sample, said method comprising the steps of: (a) providing a
sample; (b) adding a nucleic acid sequence linked to a metal
particle; (c) exposing the sample to an amount of exciting
radiation; (d) detecting the fluorescence; and (e) determining the
presence of the nucleic acid sequence based on the detection of the
fluorescence.
19. A method for increasing the fluorescence intensity of a
fluorescently labeled biomolecule, said method comprising the steps
of: (a) labeling a biomolecule with a fluorophore; (b) positioning
the labeled biomolecule next to a metallic particle such that in
response to an amount of exciting radiation, the fluorophore emits
radiation.
20. A method for increasing fluorescence energy transfer on a
fluorescently labeled biomolecule, said method comprising the steps
of: (a) labeling a biomolecule with a donor fluorophore and an
acceptor fluorophore; (b) positioning the labeled biomolecule
adjacent to a metal particle such that in response to an amount of
exciting radiation, the donor fluorophore transfers energy to the
acceptor fluorophore causing the acceptor fluorophore to emit
electromagnetic radiation.
21. A method for increasing fluorescent intensity of a fluorophore,
said method comprising the steps of: (a) positioning a fluorophore
adjacent to a metal particle; and (b) exciting said fluorophore
with a plurality of photons.
22. A method for increasing fluorescent intensity of a biomolecule,
said method comprising the steps of: (a) positioning a biomolecule
adjacent to a metal particle; and (b) exciting said biomolecule
with a plurality of photons.
23. A method for selectively enhancing the region of
electromagnetic emission of a sample, said method comprising the
steps of: (a) directing a metal particle to a region of interest in
the sample; and (b) providing an amount of exciting radiation in
the region of interest.
24. A method for selectively enhancing the region of
electromagnetic emission of a sample, said method comprising the
steps of: (a) directing a metal particle to a region of interest in
the sample; (b) contacting the sample with a fluorophore; (c)
exposing the sample to an amount of exciting radiation.
25. A method for increasing fluorescence energy transfer on a
fluorescently labeled biomolecule, said method comprising the steps
of: (a) labeling a first biomolecule with a donor fluorophore (b)
labeling a second biomolecule with an acceptor fluorophore; (c)
positioning the labeled biomolecules adjacent to a metal particle
such that in response to an amount of exciting radiation, the donor
fluorophore transfers energy to the acceptor fluorophore increasing
the emission of electromagnetic radiation of the acceptor
fluorophore.
26. A microarray system comprising: a solid support, wherein the
solid support is coated with metal particles; and a matrix having
an array of biomolecules attached to the support such that when a
labeled probe hybridizes to the biomolecules, the fluorescence of
the labeled probe increases in response to an amount of exciting
radiation.
27. A composition of matter comprising: a biomolecule in
combination with a metal surface, wherein said metal surface and
said biomolecule are positioned at a distance apart sufficient to
adjust intrinsic emission of electromagnetic radiation from the
biomolecule in response to an amount of exciting electromagnetic
radiation.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S.
provisional application No. 60/268,326 entitled "RADIATIVE DECAY
ENGINEERING" filed on Feb. 14, 2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention is directed to compositions and
methods for increasing, and detecting the fluorescence of a
molecule, in particular, compositions and methods for increasing
the intrinsic fluorescence of a biomolecule. This invention also is
directed to methods and compositions for the detection of
biomolecules by increasing and detecting the fluorescence of
biomolecules.
[0005] 2. Description of Related Art
[0006] The use of fluorescence technology has greatly enhanced the
ability to detect specific molecules leading to rapid advancements
in diagnostics. For example, fluorescence detection is widely used
in medical testing and DNA analysis because of the high degree of
sensitivity obtained using fluorescent techniques. Small numbers of
molecules can be detected using fluorescence technology. Typically,
extrinsic fluorophores are added covalently or non-covalently to
allow molecules that do not ordinarily fluoresce or do not
fluoresce at previously commercially useful levels to be detected.
Biomolecules such as DNA ordinarily do not fluoresce at detectable
levels, and extrinsic fluorophores are added to DNA to facilitate
the detection of DNA on gels (Benson et al. (1993) Nucleic Acids
Res. 21, 5720-5726; Benson et al. (1995) Ananl. Biochem. 231,
247-255), in DNA sequencing (Smith et al. (1986) Nature 321,
674-679; Prober et al. (1987) Science 238, 336-343; Li et al.
(1999) Bioconjugate Chem. 10, 241-245), in fluorescence in-situ
hybridization (Denijn et al. (1992) APMIS 100, 669-681; Wiegant et
la. (2000) Genome Res. 10, 861-865), and for reading of DNA arrays
for gene expression (Lipshutz et al. (1999) Nat. Genet. SuppL. 1,
20-24; Ferea et al. (1999) Curr. Opin. Genet. Dev. 9, 715-722).
Extrinsic fluorophores are used with DNA because DNA absorbs in the
UV region near 260 nm. The short absorption wavelength is now less
of an obstacle because UV solid state lasers have become available.
Nonetheless, the intrinsic fluorescence from DNA is of little
practical usefulness because of the low quantum yields of 10.sup.-4
to 10.sup.-5 (Vigny et al. (1974) Photochem. Photobiol. 20,
345-349; Morgan et al. (1980) Photochem. Photobiol. 31, 101-113).
Because the intrinsic emission from DNA, nucleotides and nucleic
acid bases is very weak (Kneipp et al. (1999) Curr. Science 77,
915-924; Nie et al. (1997) Science 275, 1102-1106; Michaels et al.
(1999) J. Am. Chem. Soc. 121, 9932-9939), it is difficult to
observe the intrinsic fluorescence even with modem instrumentation
(Gersten et al. (1985) Surface Science 158, 165-189; Lakowicz
(2001) Anal. Biochem. 298, 1-24).
[0007] Some of the fluorescence techniques used to detect the
presence of molecules include Resonance Energy Transfer (RET),
immunofluorescent assays, and fluorescence in situ hybridization.
Detection of the molecule of interest is generally limited by the
properties of the fluorophore used. In some cases, labeling a
biomolecule with an extrinsic fluorophore can alter the biological
activity of the biomolecule potentially creating experimental
artifacts. Problems with current fluorescent techniques stem in
part from the low fluorescent intensities of commonly used
fluorophores. Additionally, background fluorescence can be
significant when using low wavelength excitation radiation required
by some fluorophores or when large quantities of fluorophore are
required.
[0008] DNA sequencing techniques using fluorescent dyes as markers
have their maximum emission spectra in the visible range, the DNA
is subject to irradiation in the visible spectra, and visible
spectra detectors and light sources are used. Generally
photomultipliers tubes are used for detection. As a result, these
DNA sequencing techniques have several disadvantages including high
costs resulting from the high cost of the lasers used to excite the
fluorescent markers which typically emit in the visible region of
light spectrum and the high noise to signal ratio due to the
background interferences by biomolecules.
[0009] Thus, there is a need for compositions and methods for
increasing the fluorescence intensity of biomolecules.
[0010] There is also a need for compositions and methods for
increasing the intrinsic fluorescence of biomolecules.
[0011] Another need exists for compositions and methods for
manipulating the fluorescence emission intensity of a biomolecule
in response to an amount of exciting radiation.
[0012] Yet another need exists for methods and compositions for
manipulating the radiative decay rate of biomolecules.
BRIEF SUMMARY OF THE INVENTION
[0013] One aspect of the present invention is directed to a
biomolecule in combination with a metal particle, wherein the metal
particle and the biomolecule are positioned at a distance apart
sufficient to enhance intrinsic emission of electromagnetic
radiation from the biomolecule in response to an amount of exciting
electromagentic radiation. Exemplary biomolecules include, but are
not limited to, purines, pyrimidines, nucleic acids,
oligonucleotides, peptide nucleic acids, RNA, DNA, amino acids,
proteins, peptides, vitamins, lipids, carbohydrates, steroids and
antibodies. Exemplary metals include, but are not limited to,
rhenium, ruthenium, rhodium, palladium, silver, copper, osmium,
iridium, platinum, and gold. The present invention is predicated on
the surprising discovery that the fluorescence intensity of a
biomolecule can be manipulated by varying the distance between the
biomolecule and a metal particle. It will be appreciated by one of
ordinary skill in the art that the scope of the present invention
includes increasing the intrinsic fluorescence of a biomolecule as
well as the fluorescence of a biomolecule labeled with extrinsic
probes. The extrinsic fluorescence of a biomolecule includes but is
not limited to the fluorescence of a fluorophore conjugated to the
biomolecule. Such extrinsic fluorophores can be covalently or
non-covalently attached to the biomolecule. Other aspects of the
present invention describe novel compositions and methods for the
detection of biomolecules. The present invention overcomes the
problems associated with background fluorescence because the signal
to noise ratio is increased when the fluorescence intensity of the
biomolecule is increased. Additionally, expensive lasers are not
required, thereby reducing costs. Extrinsic fluorophores may not be
required making fluorescence assays of biomolecules quicker and
less expensive.
[0014] Another aspect of the present invention provides a method
for increasing the intrinsic fluorescence of a biomolecule
including the step of positioning a metal particle and the
biomolecule at a distance apart sufficient to increase the
electromagnetic emission from the biomolecule in response to an
amount of exciting radiation. It will be appreciated that the
present invention includes positioning of a biomolecule adjacent to
a metal particle or positioning a metal particle adjacent to
biomolecule in any of the disclosed embodiments.
[0015] Still another aspect of the present invention provides a
method for detecting a biomolecule including the steps of
positioning a metal particle and a biomolecule at a distance apart
sufficient to manipulate the electromagnetic emission from the
biomolecule, exposing the biomolecule to an amount of exciting
radiation, and detecting the electromagnetic emission from the
biomolecule.
[0016] Yet another aspect provides a method for manipulating
fluorescence intensity of a biomolecule including the steps of
increasing the rate of radiative decay of the biomolecule by
positioning the biomolecule adjacent to a metal particle, and
exposing the biomolecule to an amount of exciting radiation. By
increasing the rate of radiative decay, the fluorescence intensity
of the biomolecule can be increased.
[0017] Another aspect of the present invention discloses a method
for detecting the presence of a nucleic acid sequence in a sample
including the steps of providing a sample, adding a nucleic
sequence linked to a metal particle, exposing the sample to an
amount of exciting radiation, detecting the fluorescence, and
determining the presence of a nucleic acid sequence based on the
detection of the fluorescence.
[0018] Still another aspect provides a method for increasing the
fluorescence intensity of a fluorescently labeled biomolecule
including the steps of labeling a biomolecule with a fluorophore,
positioning the labeled biomolecule next to a metallic particle
such that in response to an amount of exciting radiation, the
fluorophore emits radiation.
[0019] Yet another aspect provides a method for increasing
fluorescence energy transfer on a fluorescently labeled biomolecule
including the steps of labeling a first biomolecule with a donor
fluorophore and labeling a second biomolecule with an acceptor
fluorophore, positioning both the first labeled biomolecule and the
second labeled biomolecule adjacent to a metal particle such that
in response to an amount of exciting radiation, the donor
fluorophore transfers energy to the acceptor fluorophore causing
the acceptor fluorophore to emit electromagnetic radiation.
[0020] Another aspect provides a method for increasing fluorescence
energy transfer on a fluorescently labeled biomolecule including
the steps of labeling a biomolecule with a donor fluorophore and an
acceptor fluorophore, positioning the labeled adjacent to a metal
particle such that in response to an amount of exciting radiation,
the donor fluorophore transfers energy to the acceptor fluorophore
causing the acceptor fluorophore to emit electromagnetic
radiation.
[0021] Another aspect of the present invention provides a method
for increasing the fluorescent intensity of a fluorophore including
the steps of positioning a fluorophore adjacent to a metal
particle, and exciting the fluorophore with a plurality of photons
(this process is referred to as multi-photon excitation).
[0022] Another aspect of the present invention provides a method
for increasing the fluorescent intensity of a biomolecule including
the steps of positioning a biomolecule adjacent to a metal
particle, and exciting the biomolecule with a plurality of
photons.
[0023] Yet another aspect of the present invention provides a
method for selectively enhancing a region of electromagnetic
emission of a sample including the steps of directing a metal
particle to a region of interest in the sample, and providing an
amount of exciting radiation in the sample.
[0024] Another aspect of the invention provides a method for
selectively enhancing the region of electromagnetic emission of a
sample including the steps of directing a metal particle to a
region of interest in the sample, contacting the sample with a
fluorophore, and exposing the sample to an amount of exciting
radiation.
[0025] For a better understanding of the present invention,
together with other and further objects thereof, reference is made
to the following description, taken in conjunction with the
accompanying drawings, and its scope will be pointed out in the
appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A and 1B depict silver metal islands on a quartz
surface and its absorption spectrum, respectively.
[0027] FIGS. 2A-C are absorption spectra of rose bengal in a
cuvette, between quartz plates, and between quartz slides with
silver islands respectively.
[0028] FIGS. 3A, 3B are emission spectra of rhodamine B and rose
bengal between silver island films.
[0029] FIGS. 4A-4C are a graphs showing frequency-domain intensity
decays of rhodamine B under various conditions.
[0030] FIGS. 5A-5C are graphs showing frequency-domain intensity
decays of rose bengal under various conditions.
[0031] FIGS. 6A-6B are reconstructed time-domain intensity decays
of rhodamine B and rose bengal.
[0032] FIGS. 7A-7D are emission spectra of Erb, BF,
[Ru(bpy).sub.3].sup.2+, and [Ru(phen).sub.2dppz].sup.2+ between
silver island films (S) and between uncoated quartz plates (Q).
[0033] FIG. 8 is a graphical depiction of the enhancement of the
emission of fluorophores having different quantum yields when
placed between silver island films.
[0034] FIG. 9 is a graphical depiction of the fluorescence
intensity of [Ru(phen).sub.2dppz]2+ between silver island films
(I.sub.s) compared to the fluorescence intensity between quartz
plates (I.sub.Q) in solutions of DMF and water.
[Ru{phen}.sub.2dppx].sup.2+ decreases in fluorescence intensity in
the presence of water (inset). FIG. 9 shows that silver island
particles have a greater enhancement (I.sub.S/I.sub.Q) on
[Ru(phen).sub.2dppz].sup.2+ in solutions having more water than
DMF. Thus, silver island films have a greater enhancement of
fluorescence intensity on weak or quenched fluorophores than on
strong or non-quenched fluorophores.
[0035] FIGS. 10A and 10B are emission spectra of solvent-sensitive
fluorophores between silver island films (S) and between uncoated
quartz plates (Q).
[0036] FIGS. 11A and 11B are emission spectra of
.beta.-galactosidase and human glyoxalase between silver island
films (S) and between uncoated quartz plates (Q).
[0037] FIGS. 12A and 12B are emission spectra of nucleic acid bases
between silver island films (S) and between uncoated quartz plates
(Q).
[0038] FIGS. 13A and 13B are emission spectra of single stranded
nucleic acids between silver island films (S) and between uncoated
quartz plates (Q).
[0039] FIGS. 14A and 14B are absorption spectra of calf thymus DNA
in a cuvette (14A) and between silver island films or uncoated
quartz plates (14B).
[0040] FIGS. 15A and 15B are the emission spectra of DNA in a
cuvette (15A) and between silver island films or uncoated quartz
plates (15B).
[0041] FIGS. 16A and 16B are frequency-domain intensity decays of
calf thymus DNA in a cuvette (16A) and between silver island films
(16B).
[0042] FIG. 17 is time dependent intensity decays of calf thymus
DNA between silver island films and in a cuvette.
[0043] FIG. 18 is emission spectra of R6G as donor and SR101 as
acceptor between uncoated quartz plates and between silver island
films.
[0044] FIG. 19 is emission spectra of DNA labeled with DAPI and
acridine orange between silver island films and between uncoated
quartz plates.
[0045] FIG. 20 is emission spectra of DNA labeled with DAPI alone;
with propidium iodide (PI) alone; or with DAPI and PI between
uncoated quartz plates.
[0046] FIG. 21 is emission spectra of DAPI-labeled DNA and
PI-labeled DNA between silver island films and between uncoated
quartz plates.
[0047] FIG. 22 is emission spectra of DNA labeled with both DAPI
and PI between silver island films and between uncoated quartz
plates.
[0048] FIGS. 23A-23C are frequency-domain intensity decays of the
DAPI donor decay for calf thymus DNA labeled with both DAPI and PI
in a cuvette (23A), between uncoated quartz plates (23B), and
between silver islands (23C). The frequency-domain intensity decay
of DAPI alone is also shown.
[0049] FIGS. 24A-24C are the frequency-domain intensity decays of
DAPI in calf thymus DNA labeled with both DAPI and PI in a cuvette
(24A), between uncoated quartz plates (24B), and between silver
islands (24C).
[0050] FIG. 25 is an exemplary geometry for detecting fluorophores
between silver island films.
[0051] FIGS. 26A and 26B are emission spectra of rhodamine B
between silver island films and uncoated quartz plates using
one-photon excitation and two photon excitation.
[0052] FIG. 27 emission spectra of rhodamine B with two photon
excitation between uncoated quartz plates and between quartz plates
with silver islands on the outer surface of the quartz plates.
[0053] FIGS. 28A and 28B are frequency-domain intensity decays of
rhodamine B with one photon excitation between uncoated quartz
plates (28A) and between silver island films (28B).
[0054] FIGS. 29A and 29B are frequency-domain intensity decays of
rhodamine B with two photon excitation between uncoated quartz
plates (29A) and between silver island films (29B).
[0055] FIGS. 30A and 30B are graphs depicting the photostability of
rhodamine B between uncoated quartz plates (Q) and between silver
island films (S).
[0056] FIGS. 31A and 31B are emission spectra of eosin (31A) and
rose bengal (31B) between uncoated quartz plates (Q) and between
silver island films (S) using two photon excitation.
[0057] FIGS. 32A and 32B are emission spectra of coumarin (32A)
between uncoated quartz plates (Q) and between silver island films
(S) and ANS (32B) in a cuvette (C), between uncoated quartz plates
(Q), and between silver island films (S).
[0058] FIG. 33 is a depiction of selective multi-photon excitation
of fluorophores on metal colloids in the presence of free
fluorophore.
[0059] FIG. 34 is a depiction of localized multi-photon excitation
of intracellular autofluorescence by metal colloids.
[0060] FIG. 35 is an exemplary embodiment of a sandwich immunoassay
in conjunction with a silver island coated surface.
[0061] FIGS. 36A-C are an exemplary embodiment of voltage-activated
fluorescence assays.
[0062] FIGS. 37A-C are an exemplary embodiment of an energy
transfer immunoassay using donor and acceptor-labeled
antibodies.
[0063] FIG. 38A is an exemplary apparatus for surface plasmon
excitation and FIG. 38B is a graph of the angular distribution of
the fluorescence from rhodamine 6G.
DETAILED DESCRIPTION OF THE INVENTION
[0064]
1 Abbreviations: AO acridine orange BF Basic Fucsin bpy
2,2'-bipyridine CT calf thymus DAPI 4',6-diamidino-2-phenylindole
dppz dipyrido[3,2-a:2',3'-c]phenazine DMF Dimethylformamide ErB
Erythrosin B phen 9,10-phenanthroline prodan
6-Propionyl-2-(dimethylamino)naphthalene Py2 Pyridine 2 R6G
Rhodamine 6G RhB Rhodamine B RB Rose Bengal RET Resonance energy
transfer SERS Surface-enhanced Raman scattering SIF Silver island
films SR101 Sulforhodamine 101 A acceptor D donor DAPI
4',6-diamidino-2-phenylindole FD frequency-domain PI propidium
iodide RET resonance energy transfer
[0065] Definitions
[0066] The term "fluorophore" means any substance that emits
electromagnetic energy such as light at a certain wavelength
(emission wavelength) when the substance is illuminated by
radiation of a different wavelength (excitation wavelength).
Extrinsic fluorophores refers to fluorophores bound to another
substance. Intrinsic fluorophores refers to substances that are
fluorophores themselves. Exemplary fluorophores include but are not
limited to those listed in the Molecular Probes Catalogue which is
incorporated by reference herein. Representative fluorophores
include but are not limited to Alexa Fluor.RTM. 350, Dansyl
Chloride (DNS-Cl), 5-(iodoacetamida)fluoroscein (5-IAF);
fluoroscein 5-isothiocyanate (FITC), tetramethylrhodamine 5- (and
6-)isothiocyanate (TRITC), 6-acryloyl-2-dimethylaminonaphthalene
(acrylodan), 7-nitrobenzo-2-oxa-1,3,-diazol-4-yl chloride (NBD-Cl),
ethidium bromide, Lucifer Yellow, 5-carboxyrhodamine 6G
hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas
Red.TM. sulfonyl chloride, BODIPY.TM., naphthalamine sulfonic acids
including but not limited to 1-anilinonaphthalene-8-sulfonic acid
(ANS) and 6-(p-toluidinyl)naphthalen- e-2-sulfonic acid (TNS),
Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty
acid, Fluorescein-phosphatidylethanolamine, Texas
red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,
Fluorenyl-phosphotidylcholine, Merocyanine
540,1-(3-sulfonatopropyl)-4-[.-
beta.-[2[(di-n-butylamino)-6naphthyl]vinyl]pyridinium betaine
(Naphtyl Styryl), 3,3'dipropylthiadicarbocyanine (diS-C.sub.3-(5)),
4-(p-dipentyl aminostyryl)-l-methylpyridinium (di-5-ASP), Cy-3 lodo
Acetamide, Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate,
rhodamine 800, IR-125, Thiazole Orange, Azure B, Nile Blue, Al
Phthalocyanine, Oxaxine 1, 4', 6-diamidino-2-phenylindole (DAPI),
Hoechst 33342, TOTO, Acridine Orange, Ethidium Homodimer,
N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2,
Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofluors,
Coronene, and metal-ligand complexes. Representative intrinsic
fluorophores include but are not limited to organic compounds
having aromatic ring structures including but not limited to NADH,
FAD, tyrosine, tryptophan, purines, pyrimidines, lipids, fatty
acids, nucleic acids, nucleotides, nucleosides, amino acids,
proteins, peptides, DNA, RNA, sugars, and vitamins. Additional
suitable fluorophores include enzyme-cofactors; lanthanide, green
fluorescent protein, yellow fluorescent protein, red fluorescent
protein, or mutants and derivates thereof.
[0067] The term "biomolecule" means any carbon based molecule
occurring in nature or a derivative of such a molecule. The
biomolecule can be in active or inactive form. "Active form" means
the biomolecule is in a form that can perform a biological
function. "Inactive form" means the biomolecule must be processed
either naturally or synthetically before the biomolecule can
perform a biological function. Exemplary biomolecules include
nucleic acids, aromatic carbon ring structures, NADH, FAD, amino
acids, carbohydrates, steroids, flavins, proteins, DNA, RNA,
oligonucleotides, peptide nucleic acids, fatty acids, sugar groups
such as glucose etc., vitamins, cofactors, purines, pyrimidines,
formycin, lipids, phytochrome, phytofluor, and
phycobiliproptein.
[0068] The term "amount of exciting radiation" means an amount of
radiation that causes a molecule to emit radiation.
[0069] Exemplary Embodiments
[0070] One embodiment of the present invention is directed to a
biomolecule in combination with a metal particle, wherein said
metal particle and the biomolecule are positioned at a distance
apart sufficient to adjust, preferably enhance, intrinsic emission
of electromagnetic radiation from the biomolecule in response to an
amount of exciting electromagnetic radiation. Exemplary
biomolecules include but are not limited to purines, pyrimidines,
nucleic acids, oligonucleotides, peptide nucleic acids, RNA, DNA,
amino acids, flavins, proteins, peptides, vitamins, lipids,
antibodies, and aromatic carbon ring structures. Preferred
biomolecules and fluorophores of the present invention have quantum
yields of less than about 0.8, more preferably of less than about
0.5, and most preferably of less than about 0.2. Exemplary metals
include copper and noble metals such as rhenium, ruthenium,
rhodium, palladium, silver, osmium, iridium, platinum, and gold.
Similarly, another embodiment provides a composition of matter
including a biomolecule in combination with a metal surface wherein
said metal surface and the biomolecule are positioned at a distance
apart sufficient to adjust, preferably enhance, intrinsic emission
of electromagnetic radiation from the biomolecule in response to an
amount of exciting electromagnetic radiation. Still, another
embodiment provides that the metal surface can be a periodic metal
surface.
[0071] The present invention is predicated on the surprising
discovery that the fluorescence intensity of a biomolecule can be
manipulated by varying the distance between the biomolecule and a
metal particle. Indeed, it has been discovered that the intrinsic
fluorescence of a biomolecule can be increased by at least about 80
fold to about 140 fold when the biomolecule is positioned adjacent
to a metal particle. Preferably the metal particle and biomolecule
are separated by a distance of about 50 .ANG. to about 2000 .ANG.,
most preferably from about 50 .ANG. to about 200 .ANG.. In another
embodiment, the metal particle is sub-wavelength in size, typically
in the range of about 50 .ANG. to about 300 .ANG.. The metal
particles can be spheroid, ellipsoid, or of any other geometry. The
metal particles can be suspended in a colloid or combination of
colloids, alloys, or combinations of more than one metal. The metal
particles can be placed on surfaces as thin films, or deposited on
surfaces to form small islands. The surfaces can be metallic or
non-metallic. Additionally, the metal particles can be coated with
polymers, gels, adhesives, oxides, or biologic material. Exemplary
coatings include substances that increase the binding of the metal
particle to surfaces or other molecules. In one embodiment, the
metal particles can be modified on its surface to facilitate
binding to non-metallic molecules and biomolecules. In an exemplary
embodiment, metal particles, preferably noble metals, most
preferably silver, are chemically reduced on a surface. Chemical
reduction can be accomplished using known techniques. Exemplary
surfaces include but are not limited to glass or quartz.
[0072] In another embodiment, the biomolecule and the metal
particle can be attached to each other via an intermediate of a
length sufficient to have a desired effect on the intrinsic
fluorescence of the biomolecule. The attachment can be covalent or
non-covalent. Additionally, the metal and biomolecule can be stably
linked or can be linked such that the two can become separated as a
result of a chemical reaction, enzymatic reaction, or
photoreaction. For example, a biomolecule linked to a metal
particle can be internalized within a cell, cellular organelle, or
other compartment. Once internalized, the linked biomolecule can be
subjected to enzymatic or chemical reaction resulting in the
complete separation of the metal particle from the biomolecule.
Exemplary enzymatic reactions include, but are not limited to,
non-specific esterase reactions, and exemplary chemical reactions
include but are not limited to hydrolysis, oxidation, or
substitution. Once separated, the biomolecule can become
undetectable or less detectable because its intrinsic fluorescence
is no longer amplified by the metal particle. If the intrinsic
fluorescence of the biomolecule is desired to be quenched, the
biomolecule and the metal particle can be separated by a distance
of about 0 to less than 50 .ANG.. If the intrinsic fluorescence of
the biomolecule is to be increased, the biomolecule and the metal
particle can be separated by a distance of about 50 .ANG. to about
2000 .ANG., more preferably from about 50 .ANG. to about 200 .ANG..
Thus, the fluorescence intensity of a biomolecule can be
manipulated by varying the distance separating the metal particle
from the biomolecule.
[0073] It will be appreciated by one of ordinary skill in the art
that the scope of the present invention includes increasing the
extrinsic fluorescence of a biomolecule as well as the intrinsic
fluorescence of the biomolecule. The extrinsic fluorescence of a
biomolecule includes but is not limited to the fluorescence of a
fluorophore conjugated to the biomolecule. Such conjugated
fluorophores can be covalently or non-covalently attached to the
biomolecule. An increase or decrease in fluorescence intensity in
the present invention means an increase or decrease in intrinsic or
extrinsic fluorescence intensity when the biomolecule or
fluorophore is in combination with a metal particle compared to the
intrinsic or extrinsic fluorescence intensity of the biomolecule or
fluorophore in the absence of a metal particle.
[0074] Another embodiment of the present invention provides a
method for increasing the intrinsic fluorescence of a biomolecule
including the step of positioning the biomolecule and a metal
particle at a distance apart sufficient to increase the
electromagnetic emission from the biomolecule in response to an
amount of exciting radiation. It will be appreciated that the
present invention includes positioning of a biomolecule adjacent to
a metal particle or positioning a metal particle adjacent to the
biomolecule in any of the disclosed embodiments. In an exemplary
embodiment, the biomolecule and the metal particle are separated by
a distance of about 50 .ANG. to about 2000 .ANG., preferably from
about 50 .ANG. to about 200 .ANG., to increase the intrinsic
fluorescence of the biomolecule or separated by a distance of less
than about 50 .ANG. if the intrinsic fluorescence is to be
quenched. In other embodiments, the metal particles can be fixed on
a surface, and the biomolecule positioned adjacent to such a
surface. Such surfaces can form a part of cuvette or can be an
insert capable of being placed within a cuvette.
[0075] Positioning of the biomolecule or metal particle at a
desired distance can be achieved by using a linker that physically
links the two. Linkers can be one intervening atom or molecule,
preferably carbon chains of at least one carbon atom. Other linkers
include but are not limited to at least one amino acid.
Additionally, other chemical linkers known in the art can be used.
The linkers can be of any length, preferably of up to about 200
.ANG. depending the desired effect on fluorescence. In other
embodiments, the metal particle can be positioned adjacent to the
biomolecule using electromagnetic forces, charged fields, gravity
or other known methods. In one example, voltage can be regulated to
manipulate the position of the metal particle, or linked
biomolecule and metal particle. Alternatively, the biomolecule can
be positioned using electromagnetic fields, electric currents,
voltage, or gravity.
[0076] Yet another embodiment of the present invention provides a
method for detecting a biomolecule including the steps of
positioning said biomolecule and a metal particle at a distance
apart sufficient to manipulate the electromagnetic emission from
the biomolecule, exposing the biomolecule to an amount of exciting
radiation, and detecting the electromagnetic emission from the
biomolecule. Monitoring, detecting, and quantifying fluorescence is
known in the art. See for example Joseph R. Lakowicz. Principles in
Fluorescence Spectroscopy, Plenum Publishers 1999 which is
incorporated by reference herein in its entirety.
[0077] Briefly, fluorescence can be detected using devices
including, but not limited to, a spectrofluorometer having a light
source and detector. Light sources can include arc lamps and
lasers. Detectors can include photomultiplier tubes. Additionally,
it is advantageous for the device to have a monochromator so that
specific wavelengths of light may be used to excite a molecule or
to detect emissions at a specific wavelength. When a sample
containing a fluorophore is placed in the spectrofluorometer and
exposed to an amount of exciting radiation, the fluorophore emits
radiation that is detected by a photomultiplier tube. The
fluorescence intensity of a biomolecule can be increased in
response to an amount of exciting radiation when the distance
between the metal particle and the biomolecule is from about 50
.ANG. to about 2000 .ANG., preferably from about 50 .ANG. to about
200 .ANG.. Alternatively, the fluorescence intensity of the
biomolecule can be reduced when the distance between the
biomolecule and the metal particle is less than about 50 .ANG..
[0078] Yet another embodiment provides a method for manipulating
fluorescence intensity of a biomolecule including the steps of
increasing the rate of radiative decay of the biomolecule by
positioning the biomolecule adjacent to a metal particle, and
exposing the biomolecule to an amount of exciting radiation. By
increasing the rate of radiative decay, the fluorescence intensity
of the biomolecule can be increased. It has been discovered that by
manipulating the distance separating a biomolecule and a metal
particle, the radiative decay of the biomolecule can also be
manipulated.
[0079] Another embodiment of the present invention discloses a
method for detecting the presence of a nucleic acid sequence in a
sample including the steps of providing a sample, adding a nucleic
sequence linked to a metal particle, exposing the sample to an
amount of exciting radiation, detecting the fluorescence, and
determining the presence of the nucleic acid sequence based on the
detection of the fluorescence. In one embodiment, the nucleic acid
sequence linked to a metal particle is single stranded. In other
embodiments, the nucleic acid sequence linked to the metal particle
is double stranded. In a preferred embodiment, the nucleic acid
sequence linked to a metal particle is less than two hundred base
pairs in length, more preferably, less than 100 base pairs in
length, most preferably less than 50 base pairs in length, even
more preferably, about twenty or less nucleic acids in length. The
nucleic acids can be deoxyribonucleic acids, ribonucleic acids, or
chemically modified nucleic acids such as peptide nucleic acids and
the like.
[0080] Methods for the hybridization of nucleic acids are known in
the art. See for example Nonradioactive In Situ Hybridization
Application Manual at
biochem.roche.com/prod_inf/manuals/insitu/insi_toc.htm incorporated
by reference in its entirety. Historically, the detection of
hybridized nucleic acid used labeled nucleic acid probes to
hybridize to a nucleic acid sample. For example, in a Southern blot
technique, a nucleic acid sample is separated in an agarose gel
based on size and affixed to a membrane, denatured, and exposed to
the labeled nucleic acid probe under hybridizing conditions. If the
labeled nucleic acid probe forms a hybrid with the nucleic acid on
the blot, the label is bound to the membrane. Probes used in
Southern blots have been labeled with radioactivity, fluorescent
dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and
acridinium esters.
[0081] The present invention discloses a novel method for the
detection of nucleic acid sequences by increasing the intrinsic
fluorescence of the nucleic acids. This increase is accomplished by
linking the nucleic acid to a metal, preferably a noble metal. The
present invention does not require the use of an extrinsic probe.
Rather when the nucleic acid sequence linked to a metal particle is
added to a sample, this sequence can hybridize to complementary
nucleic acid sequences in the sample. In a preferred embodiment the
nucleic acid sequence to be detected can be affixed to a solid
support. Exemplary solid supports include films, membranes,
columns, nitrocellulose, plastic, quartz, glass, or metal. The
sample is irradiated with an amount of exciting radiation.
Fluorescence detected in the sample or, for example, on the solid
support would indicate that the nucleic acid linked to the metal
particle has hybridized to a complementary nucleic acid sequence
present in the sample. Therefore, the sample contains the nucleic
acid sequence of interest. Nucleic acids not linked to a metal
particle would not contribute significantly to fluorescence
emission because the quantum yield of nucleic acids is too low. It
will be appreciated that the nucleic hybridization detected by the
present invention can occur in situ. Additionally, it will be
appreciated that the detection of the nucleic acid can be based on
increasing the fluorescence intensity of an extrinsic fluorophore
attached to a nucleic acid by positioning the fluorophore adjacent
to a metal particle. Increasing the intrinsic fluorescence of
nucleic acids can be useful in DNA arrays or gene chips.
[0082] In another embodiment, the present invention provides a
method for identifying nucleic acids, the method including the
steps of positioning a nucleic acid adjacent to a metal particle,
irradiating the nucleic acid, detecting the fluorescence emission
from the nucleic acid, and identifying the nucleic acid based on
the fluorescence emission. The identification of nucleic acids
using the intrinsic fluorescence of the nucleic acid eliminates the
requirement for extrinsic probes. In one embodiment, the background
fluorescence is not problematic because the intrinsic fluorescence
can be increased by about 80 fold thereby reducing the noise to
signal ratio. In another embodiment, the nucleic acid can be
identified based on the emission spectra obtained from monitoring
the fluorescence of the sample. Thus, the sequence of nucleic acids
in a sample can be determined by sequentially removing a nucleic
acid, positioning the nucleic acid adjacent to metal particle,
irradiating the nucleic acid with an amount of exciting radiation,
detecting the emitted radiation, and correlating the emitted
radiation with the nucleic acid base. Methods for sequentially
removing a single nucleic acid form a nucleic acid sequence such as
an oligonucleotide are known in the art and include sequential
digestion, hydrolysis, and chemical cleavage. The nucleic acids can
be positioned adjacent to a metal particle by causing the stream of
a fluid sample containing a nucleic acid to pass near a surface
containing the metal particle. Such surfaces can be thin films or
islands of metal that form part of a sample chamber. The
irradiation of the nucleic acid can be timed to coincide with the
positioning of the nucleic acid adjacent to the metal. The nucleic
acids can be irradiated with one or more wavelengths. In a
preferred embodiment, the nucleic acids are excited at wavelengths
below 300 nm, preferably from 280 to about 295 nm. In another
embodiment, the excitation wavelength is near 520 nm for
multi-photon excitation.
[0083] It will be appreciated by those of ordinary skill in the
art, that the methods and compositions of the present invention can
be used in polymerase chain reaction techniques. Polymerase chain
reaction techniques are will known in the art. Nucleic acids
coupled to metal particles, preferably noble metal particles, can
be added to the polymerase chain reaction mixtures. The coupled
nucleic acids can be incorporated into the growing oligonucleotide
chain, and in response to an amount of exciting radiation, the
fluorescence of the coupled nucleic acid will be detectable
enabling the detection of the polymerase chain reaction
product.
[0084] Still another embodiment provides a method for increasing
the fluorescence intensity of a fluorescently labeled biomolecule
including the steps of labeling a biomolecule with a fluorophore,
positioning the labeled biomolecule adjacent to a metallic particle
such that in response to an amount of exciting radiation, the
fluorophore emits radiation, preferably detectable amounts of
radiation. In a preferred embodiment, the fluorophore has a quantum
yield of less than 0.8, preferably less than 0.5, more preferably
less than 0.2, and most preferably less than 0.1. In this
embodiment, the fluorescence intensity of an extrinsic fluorophore
can be used to detect the biomolecule.
[0085] Yet another embodiment provides a method for increasing
fluorescence energy transfer on a fluorescently labeled biomolecule
including the steps of labeling a first biomolecule with a donor
fluorophore and labeling a second biomolecule with an acceptor
fluorophore, positioning the labeled biomolecules adjacent to a
metal particle such that in response to an amount of exciting
radiation, the donor fluorophore transfers energy to the acceptor
fluorophore causing the acceptor fluorophore to emit
electromagnetic radiation.
[0086] Another embodiment provides a method for increasing
fluorescence energy transfer on a fluorescently labeled biomolecule
including the steps of labeling a biomolecule with a donor
fluorophore and an acceptor fluorophore, positioning the labeled
adjacent to a metal particle such that in response to an amount of
exciting radiation, the donor fluorophore transfers energy to the
acceptor fluorophore causing the acceptor fluorophore to emit
electromagnetic radiation.
[0087] Increases in energy transfer due to metallic particles can
be used in immunoassays. Thus, in one embodiment, the compositions
of the present invention can use fluorescence energy transfer to
measure an affinity reaction, preferably an antibody-antigen
reaction or a protein-carbohydrate interaction. Additionally, assay
chambers coated with or containing metallic particles can be used
to increase the efficiency or RET even between donor and acceptors
to span distances over 100 .ANG. apart. Metal enhanced energy
transfer is also useful with DNA arrays or gene chips. In another
embodiment, the compositions and methods of the present invention
can utilize fluorescence energy transfer to measure DNA
hybridization or the amount of double helical DNA. At present the
arrays are read by measuring the amount of two fluorophores
hybridized to the target DNA (Ferea et al. (1999) Curr. Opin.
Genetics Dev., 9, 715-722; Lipshutz et al. (1999). Nature Gen.
SuppL. 1, 20-24; Hacia et al. (1998) Molec. Psychiatry 3, 483-492).
Even though the two dyes are probably a good donor-acceptor pair,
energy transfer does not normally occur. The use of DNA arrays on
metallic surfaces provides a new type of DNA array analysis based
on RET between donors and acceptors positioned at long
distances.
[0088] Methods and procedures for producing biochips, gene chips.
or microarrays are known in the art. For example, U.S. Pat. No.
6,174,683 discloses methods and compositions relating to "biochips'
and the formation of "biochips" and is incorporated herein in its
entirety. Nucleic acid probes are affixed to a microarray surface.
In the present invention, the microarray surface is a metal
surface, preferably a noble metal, most preferably a silver
surface. The surface can be coated with metal islands as describe
above. Generally, total RNA is prepared from a sample or samples to
determine the pattern of gene expression. These samples can be
different cell lines, tumor samples, normal vs. disease, control or
drug-treated, etc. In most cases, a minimum of about 1 .mu.g of
polyA+RNA or about 5 .mu.g of total RNA is required. However, as
little as 0.2 .mu.g of "good quality" polyA+RNA can be used. As a
general rule, 1.times.10.sup.6 tissue culture cells should yield
10-15 .mu.g of total RNA.
[0089] Using Reverse Transcriptase, the RNA is converted into cDNA.
At this point the cDNA can be labeled directly by incorporation of
fluorescently-tagged dNTPs. More commonly, the cDNA is prepared
using an oligo-dT primer that incorporates a T7 RNA polymerase
promoter. The cDNA is then used in a subsequent step to make
fluorescently-tagged copy RNA, using T7 RNA polymerase. In general,
at least about 5 .mu.g of labeled cRNA or cDNA is required for
hybridizing to each microarray. However, the probes can be reused.
For example, one labeled probe can be used sequentially to
hybridize to five separate arrays.
[0090] The fluorescently-labeled probes are hybridized to the
microarrays, much as radioactive probes are hybridized to
conventional dot-blots. The fluorescence of the labeled probe will
increase in response to an amount of exciting radiation when the
probe hybridizes to a complementary sequence because it is
positioned near the metal microarray surface, preferably from about
50 .ANG. to about 2000 .ANG., more preferably from about 50 .ANG.
to about 200 .ANG., from the metal surface. In another embodiment,
the probe can be labeled with a donor and an acceptor fluorophore.
When the labeled probe hybridizes to its complement on the
microarray surface, it is positioned near the metal microarray
surface, preferably from about 50 .ANG. to about 2000 .ANG., more
preferably from about 50 .ANG. to about 200 .ANG., from the metal
surface. In this position, the fluorescence energy transfer from
the donor to the acceptor molecule is increased in response to an
amount of exciting radiation enabling detection of the labeled
probe. Detection of the labeled probe on the microarray indicates
that the probe has hybridized to a complementary sequence further
indicating expression of the corresponding gene. In still another
embodiment, the nucleic acid sequence affixed to the metal coated
microarray surface can be labeled with a donor or acceptor
molecule, and the nucleic acid probe can be labeled with acceptor
or donor molecule respectively such that when the nucleic acid
probe hybridizes with a labeled nucleic acid sequence on the
microarray surface, the fluorescence energy transfer from the donor
molecule to the acceptor molecule is increased in response to an
amount of exciting radiation.
[0091] After washing, the microarrays are analyzed using a
fluorescent scanner: a cross between a typical flat-bed scanner and
a confocal microscope. The data is an image of the fluorescent
spots on the microarray. The image can be analyzed using software
that identifies the spots and calculates the intensity of the
fluorescence in each one. By comparing the intensities obtained
with two different probes (e.g. control vs. drug-treated), one can
determine how the expression of each gene in the array changes.
[0092] In other embodiment, proteins can be arrayed on metal
surfaces, preferably noble metal surfaces, most preferably silver
coated surfaces. Generally, to array proteins on a surface, a GMS
417 Arrayer (Affymetrix, Santa Clara, Calif.) or other suitable
device can be used. The arrayer picks up about a microliter of
sample from four wells of a 96- or 384-well plate and deposits
about 1 nanoliter of each sample at defined locations on a series
of glass microscope slides. The arrayer can use a pin and ring
system: the samples are picked up in small rings that each hold
about 1 microliter and a solid pin (150 .mu.m diameter) then
punches repeatedly through the ring to deposit the proteins on the
slides. To prevent evaporation of the nanodroplets, 40% glycerol is
included in the protein samples. Nanoliter droplets of 40% glycerol
remain hydrated, even when left exposed to the atmosphere
overnight.
[0093] To study protein function, it is necessary to immobilize the
proteins in a way that preserves their folded conformations. In
addition, it is preferred to minimize nonspecific binding of other
proteins to the surface in subsequent steps. To accomplish these
goals, chemically derivatized slides can be used. For most
applications, slides that have been treated with an
aldehyde-containing silane reagent are used. These slides can also
be purchased from TeleChem International under the trade name
SuperAldehyde Substrates. The aldehydes react readily with primary
amines on the proteins to form a Schiffs base linkage. Because
typical proteins display many lysines on their surface as well as
the generally more reactive alpha-amine at their amino termini,
they can attach to the slide in a variety of orientations,
permitting different sides of the protein to interact with other
proteins or small molecules in solution. Following attachment of
the proteins to these slides, the unreacted aldehydes are quenched
and nonspecific binding minimized by immersing the slides in a
buffer containing bovine serum albumin (BSA).
[0094] Although appropriate for most applications, aldehyde slides
cannot be used when peptides or very small proteins are printed,
presumably because the BSA obscures the molecules of interest. For
such applications, BSA-NHS slides that are fabricated by first
attaching a molecular layer of BSA to the surface of glass slides
and then activating the BSA with N,N'-disuccinimidyl carbonate are
used. The activated lysine, aspartate, and glutamate residues on
the BSA react readily with surface amines on the proteins to form
covalent urea or amide linkages. The slides are then quenched with
glycine.
[0095] Aldehyde slides can be purchased from TeleChem International
(Cupertino, Calif.). BSA-NHS slides, displaying activated amino and
carboxyl groups on the surface of an immobilized layer of bovine
serum albumin (BSA), can be fabricated as follows. 10.24 g
N,N'-disuccinimidyl carbonate (100 mM) and 6.96 ml
N,N-diisopropylethylamine (100 mM) were dissolved in 400 ml
anhydrous N,N-dimethylformamide (DMF). 30 CMT-GAP slides (Corning
Incorporated, Coming, N.Y.), displaying amino groups on their
surface, were immersed in this solution for 3 hours at room
temperature. The slides are rinsed twice with 95% ethanol and then
immersed in 400 ml of phosphate buffered saline (PBS), pH 7.5
containing 1% BSA (w/v) for 12 hour at room temperature. The slides
are rinsed twice with ddH.sub.2O, twice with 95% ethanol, and
centrifuged at 200 g for 1 min to remove excess solvent. The slides
are then immersed in 400 ml DMF containing 100 mM
N,N'-disuccinimidyl carbonate and 100 MM N,N-diisopropylethylamine
for 3 hour at room temperature. The slides are rinsed four times
with 95% ethanol and centrifuged as above to yield BSA-NHS slides.
The slides are stored in a desiccator under vacuum at room
temperature for up to two months without noticeable loss of
activity.
[0096] Proteins are dissolved in 40% glycerol, 60% PBS, pH 7.5 at a
concentration of 100 .mu.g/ml unless indicated otherwise. The
proteins are spotted on aldehyde slides using a GMS 417 Arrayer
(Affymetrix, Santa Clara, Calif.). Following a 3 hours incubation
in a humid chamber at room temperature, the slides are inverted and
dropped onto a solution of PBS, pH 7.5 containing 1% BSA (w/v).
After 1 minute, the slides were turned right side up and immersed
in the BSA solution for 1 hour at room temperature with gentle
agitation. Following a brief rinse in PBS, the slides are ready for
further processing.
[0097] Proteins of interest can be labeled with a fluorophore and
hybridized to the array. The fluorescence of the fluorophore will
increase in response to an amount of exciting radiation when the
labeled protein is near the metal surface, preferably form about 50
.ANG. to about 2000 .ANG., more preferably from about 50 .ANG. to
about 200 .ANG., from the metal surface. In another embodiment, the
protein probe of interest can be labeled with an acceptor and donor
fluorophore such that when the protein probe binds to a protein on
the microarray, the protein probe is positioned near the metal
surface such that fluorescence energy transfer is increased from
the donor to the acceptor molecule. In still another embodiment,
the protein affixed to the metal coated microarray surface can be
labeled with a donor or acceptor molecule, and the protein probe
can be labeled with acceptor or donor molecule respectively such
that when the protein probe hybridizes with a labeled protein on
the microarray surface, the fluorescence energy transfer from the
donor molecule to the acceptor molecule is increased in response to
an amount of exciting radiation.
[0098] Thus, another embodiment of the present invention provides a
microarray system including a solid support, wherein the solid
support is coated with metal particles, preferably noble metal
particles, most preferably with silver particles; and a matrix
having an array of biomolecules, preferably nucleic acids or amino
acids, at desired lengths attached to the support such that when a
labeled probe hybridizes to a sequence of the biomolecules,
preferably such that the label is positioned about 50 .ANG. to
about 2000 .ANG., preferably from about 50 .ANG. to about 200 .ANG.
from a metal particle, the fluorescence of the labeled probe is
increased in response to an amount of exciting radiation. The term
"probe" includes proteins, nucleic acids, amino acids,
oligonucleotide, peptide nucleic acids, peptides, or other
molecules that hybridize, bind, or are complementary to the
molecules of the matrix attached to the metal coated surface of the
microarray.
[0099] The efficiency of light harvesting assemblies based on RET
(Adronov et al. (2000) J. Am. Chem. Soc. 122, 1175-1185; Swallen et
al. (1999) J. Molec. Structure 485-486:585-597) can be increased or
the extent of RET between donors and acceptors within cells
(Gonzalez et al. (1995). Biophys. J. 69, 1272-1280; (Ng et al.
(1999) Science 283, 2085-2089) but close to metallic particles can
be increased. The phenomenon of metal-enhanced RET provides a
unique opportunity of using the proximity of donor-acceptor pairs
to metallic particles to modify the rates of transfer. Such effects
are unique because the metal particles or surfaces, rather than the
solution composition, can be used to modify the spectral properties
of the probes.
[0100] Another embodiment of the present invention provides a
method for increasing the fluorescent intensity of a fluorophore
including the steps of positioning a fluorophore adjacent to a
metal particle, and exciting the fluorophore with a plurality of
photons, commonly referred to as multi-photon excitation.
Typically, the fluorophore is excited with short picosecond or
fentosecond laser pulses with a wavelength approximately twice the
longest single photon absorption maximum. Multi-photon excitation
instrumentation and methodology are known in the art, and can be
found, for example, in Topics in Fluorescence Spectroscopy, Volume
5, Nonlinear and Two-Photon-Induced Fluorescence, Edited by Joseph
R. Lakowicz. Plenum Press, New York, 1997, which is incorporated by
reference herein in its entirety. Generally, multi-photon
excitation is typically performed with a strongly focused laser
light source, such as, a mode-locked titanium sapphire laser,
providing pulses approximately 100 fentoseconds long, repetition
rate near 80 MHz, with a wavelength range from 700 to 900 nm.
Multi-photon excitation can also be accomplished with picosecond
dye lasers.
[0101] Another embodiment of the present invention provides a
method for increasing multi-photonic fluorescent intensity of a
biomolecule including the steps of positioning a biomolecule
adjacent to a metal particle, and exciting the biomolecule with a
plurality of photons.
[0102] Yet another embodiment of the present invention provides a
method for selectively enhancing the region of electromagnetic
emission of a sample including the steps of directing a metal
particle to a region of interest in the sample, and providing an
amount of exciting radiation in the sample.
[0103] Another embodiment of the invention provides a method for
selectively enhancing the region of electromagnetic emission of a
sample including the steps of directing a metal particle to a
region of interest in the sample, contacting the sample with a
fluorophore, exposing the sample to an amount of exciting
radiation. Exemplary metals are noble metals. The metal particle
can be positioned using electric potential, magnetisim, or
gravity.
[0104] Still another embodiment provides a method for selectively
enhancing the region of electromagnetic emission of a sample, the
method including the steps of directing a metal particle to a
region of interest in the sample, providing an amount of exciting
radiation in the region of interest. Samples can be living cells,
tissues, organs, or fluid samples in containers. The metal particle
can be positioned using electromagnetic fields, or the metal
particle can be linked to a protein, antibody, nucleic acid or the
like. An antibody linked to a metal particle can be used to bring
the metal adjacent to molecules recognized by the antibody. Thus,
if the antibody recognizes a particular biomolecule, the metal can
be positioned next to such biomolecule such that the fluorescence
intensity of the biomolecule will increase in response to an amount
of exciting radiation.
[0105] Another embodiment discloses a method for selectively
enhancing the region of electromagnetic emission of a sample, said
method including the steps of directing a metal particle to a
region of interest in the sample, contacting the sample with a
fluorophore, and exposing the sample to an amount of exciting
radiation. In a preferred embodiment, the fluorophore has a quantum
yield of less than 0.8, preferably less than 0.5, more preferably
less than 0.2, and most preferably less than 0.1. Fluorophores with
low quantum yields will not fluoresce detectably unless they are
adjacent to a metal particle. Thus, when the sample is exposed to
an amount of exciting radiation, only the fluorophores adjacent to
a metal particle will fluoresce enough to be detectable. Still
another embodiment discloses a kit for detecting the presence of an
analyte comprising an antibody capable of binding to said analyte,
the antibody linked to a fluorophore having a quantum yield of less
than about 0.5, preferably less than about 0.2, most preferably
less than about 0.1, and at least one quartz surface having metal
islands deposited thereon. Exemplary antibodies are directed to
proteins, peptides, and other biomolecules.
[0106] Another embodiment discloses a method for detecting an
analyte, the method including the steps of labeling a first
antibody with a donor, labeling a second antibody with an acceptor,
contacting a sample with the first and second antibodies to form a
complex with the analyte, positioning the analyte about 50 to about
2000 .ANG., preferably from about 50 .ANG. to about 200 .ANG., from
a metal particle, preferably a noble metal, providing an amount of
exciting radiation, and detecting the analyte based on the increase
in energy transfer from donor to acceptor. Exemplary donors are
fluorophores with wavelengths which overlap the absorption spectra
of the acceptor.
[0107] Exemplary acceptors have absorption spectra which overlap
with the emission spectra of the donors. Acceptors may also be
fluorescent. Exemplary donor-acceptor pairs include
fluorescein-rhodimine, DAPI-propidium iodide, and Cy3-Cy5. The
antibodies can be labeled using standard techniques. In one
embodiment, the complex can be positioned using gravity, electric
potential, or other known force.
EXAMPLE 1
Procedure for Making Metal Nanoparticle Films
[0108] Metal particles or metal particle films are known and can be
produced using known methods. The following example uses silver but
it will be appreciated that any metal can be used, preferably noble
metals. Chemicals used to generate silver particles, silver nitrate
(99+%), sodium hydroxide (pellets, 97%), ammonium hydroxide
(NH.sub.3 content 28-39%), and D-glucose (99.5%) were purchased
from commercial suppliers and used without further purification.
All procedures were performed using distilled water which was
further purified by Millipore filtration. Silver islands were
formed on quartz microscope slides. Quartz provided UV transmission
and less autofluorescence. The quartz slides used to deposit silver
particles were soaked in a 10:1 (v/v) mixture of H.sub.2SO.sub.4
(95-98%) and H.sub.2O.sub.2 (30%) overnight before the deposition.
They were washed with water and air-dried prior to use.
[0109] Silver deposition was carried out in a clean 30-ml beaker
equipped with a Teflon-coated stir bar. To a fast stirring silver
nitrate solution (0.22 g in 26 ml of Millipore filtered water),
eight drops of fresh 5% NaOH solution was added. Dark-brownish
precipitates were formed immediately. Less than 1 ml of ammonium
hydroxide was then added drop by drop to redissolve the
precipitates. The clear solution was cooled to 5.degree. C. by
placing the beaker in an ice bath, followed by soaking the cleaned
and dried quartz slides in the solution. At 5.degree. C., a fresh
solution of glucose (0.35 g in 4 ml of water) was added. The
mixture was stirred for 2 min at that temperature. Subsequently,
the beaker was removed from the ice bath. The temperature of the
mixture was allowed to warm up to 30.degree. C. As the color of the
mixture turning from yellow-greenish to yellow, the color of the
slides become greenish, the slides were removed and washed with
water and bath sonicated for 1 min at room temperature. After being
rinsed with water several times, the slides were stored in water
for several hours prior to the experiments.
[0110] Emission spectra were obtained using a SLM 8000
spectrofluorometer. Intensity decays were measured in the
frequency-domain using instrumentation described previously
(Lakowicz et al. (1985) Biophys. Chem. 21, 61-78; Laczko et al.
(1990) Rev. Sci. Instrum. 61, 2331-2337). For rhodamine 6G (R6G)
and rose bengal (RB) the excitation was at 514 nm from the
approximate 78 MHz output of a mode-locked argon ion laser. For the
frequency-domain measurements the emission was observed through a
580 interference filter. For all steady state and frequency-domain
measurements the excitation was vertically polarized and the
emission observed through a horizontally oriented polarizer to
minimize scattered light. The FD intensity decay data were analyzed
in terms of the multi-exponential model 1 I ( t ) = i i exp ( - t /
i ) ( 1 )
[0111] where .tau..sub.i are the lifetimes with amplitudes
.alpha..sub.i and .SIGMA..alpha..sub.i =1.0. Fitting to the
multi-exponential model was performed as described previously
(Lakowicz et al. (1994) Biophys. J. 46, 463-477). The contribution
of each component to the steady state intensity is given by 2 f i =
i i i j j ( 2 )
[0112] FIG. 1A shows the experimental geometry of the silver
islands on quartz slides, and FIG. 1B shows the absorption spectra
of silver island films. This spectrum indicates that the particles
are sub-wavelength in size. In the small-particle limit the
absorption maximum due to this plasmon resonance is expected to be
near 380 DM (Kerker, M. (1985) J. Colloid Interface Sci. 105,
297-314; Mulvaney, P. (1996) Langmuir, 12, 788-800). The absorption
maximum above 400 nm can be due to an asymmetric effective shape of
the particles with an axial ratio near 1.5 to 1.0 (Kerker, M.
(1985) J. Colloid Interface Sci. 105, 297-314) and is also
consistent with silver particles with spherical dimensions near
40-50 nm (Rivas et a l. (2001) Langmuir 17, 574-577; Jensen, et al.
(2000) J. Phys. Chem. B 104, 10549-10556; Singer et al. (1995) J.
Opt. Soc. Am. B, 12, 220-228). The shape and size distribution of
the particles is almost certainly heterogeneous, but it is clear
the particles are sub-wavelength in size.
[0113] To determine the effects of silver islands on fluorescence,
samples containing fluorophores were placed between two such silver
island plates. From the absorption spectra of rose bengal, between
two quartz plates, or two silver island coated plates (FIGS.
2A-2C). The distance between the plates is about 1 to 1.5
.mu.m.
EXAMPLE 2
Effects of Silver Island Films on Emission Spectra and
Lifetimes
[0114] As an initial experiment, the emission spectra of rhodamine
B (RhB) and rose bengal (RB) between uncoated quartz plates (Q) or
silver island films (S) were examined. These two fluorophores were
selected because of their similar absorption and emission spectra
but different quantum yields of 0.48 and 0.02 for RhB and RB,
respectively. In the case of RhB the intensities are similar in the
absence and presence of these silver islands (FIG. 3A). There may
be a small decrease in the RhB intensity due to the silver islands,
which may be due to the quenching effects of metals at short
distances.
[0115] Contrasting results were obtained for rose bengal (FIG. 3B).
In this case the intensity increased about 5-fold in the presence
of silver islands. It is important to recognize that the increased
intensity observed for RB represents an underestimation of the
quantum yield of RB near the silver islands. This is because only a
small fraction of the RB molecules are within the distance over
which metallic surfaces can exert effects. The region of enhanced
fluorescence extends about 200 .ANG. to about 2000 .ANG. into the
solution. Hence only about 4% of the liquid volume between the
plates is within the active volume. The low percentage of active
volume suggests that the quantum yield of RB within 200 .ANG. of
the islands is increased 125-fold. This increase is larger than
possible if the quantum yield of 0.02 is correct and reflects an
increased incident field because of the metal particles.
Nonetheless, the spectra for RB in FIG. 3 indicates a substantial
increased in quantum yield for the molecule within 200 .ANG. of the
silver islands.
[0116] The effect of a concentrated electric field is not the
dominant cause of the intensity increase for rose bengal in FIG.
3B. The emission occurs for RhB molecules both near to and distant
from the silver islands. The field concentration effects could be
masked by a dominant emission from the RhB molecules distant from
the silver islands.
[0117] The effects of an increased radiative rate and concentrated
electric field can be distinguished by lifetime measurements. An
increase in the radiative rate will decrease the lifetime; whereas,
an increased rate of excitation will not change the lifetime. The
intensity decays of RhB and rose bengal in the absence and presence
of silver islands were measured (FIGS. 4A-4C and FIGS. 5A-5C). In a
standard cuvette the intensity decay of RhB was found to be a
single exponential with a lifetime .tau.=1.56 ns (FIG. 4A). In the
presence of silver islands the intensity decay becomes strongly
heterogeneous (FIG. 4C).
[0118] The data could fit two decay times with the long lifetime of
1.81 ns being comparable to that found in a cuvette. Between silver
islands, a short lifetime of 0.14 ns appeared which is attributed
to RhB molecules in close proximity to the silver islands. The
fractional steady state intensity of this short component is about
10%. Control measurements showed that this component was not due to
scattered light. Measurements were also performed for RhB between
quartz plates without silver islands. In this case the decay was
also double exponential, but less heterogeneous than in the
presence of islands. Nonetheless, it is clear that a short lifetime
component appears for RhB between silver islands (FIG. 4B). Control
measurements showed there was no significant intensity for the
quartz slides alone without RhB. This result suggests that
scattered light is not the origin of the short component seen for
RhB between uncoated slides.
[0119] Frequency-domain (FD) intensity decays for rose bengal are
shown in FIGS. 5A-5C. In a cuvette the decay in a single
exponential with .tau.=94 ps. The decay become slightly
heterogeneous for rose bengal between uncoated quartz plates.
However, the intensity decay of rose bengal changed dramatically
when between silver islands. In this case the dominant lifetime
became a 6 ps component, which corresponds to rose bengal molecules
adjacent to the silver islands.
[0120] The effects of silver islands on the intensity decays of RhB
and rose bengal can be seen in the time-resolved decays
reconstructed from the frequency-domain data (FIGS. 6A and 6B). For
both fluorophores the intensity decay shows long decay time
components essentially identical to the values observed in
cuvettes. Silver islands result in the appearance of short decay
times. The larger contribution of the short decay times for rose
bengal can be understood from its lower quantum yield in bulk
solution. RhB has a higher quantum yield so that its emission is
detected from molecules throughout the 1 .mu.m thick sample. Rose
bengal has a low quantum yield in solution so the observed emission
is mostly due to rose bengal molecules near the silver islands. The
results from RhB and rose bengal were consistent with
expectations.
[0121] A number of additional fluorophores between uncoated quartz
plates and between silver island films were examined to account for
the contributions of artifacts. Emission spectra of four
fluorophores (Erb, BF, [Ru(bpy).sub.3].sup.2+, and
[Ru(phen).sub.2dppz].sup.2+) are shown in FIGS. 7A-7D. In all cases
the emission was more intense for the solutions between the silver
islands. For example, [Ru(bpy).sub.3].sup.2+ and [Ru
(phen).sub.2dppz].sup.2+ have quantum yields near zero,
respectively (Van Houten et al. (1975) J. Am. Chem. Soc. 97,
3843-3844; Harriman, A. (1977) J. Chem. Soc., Chem. Commun.
777-778; Nair et al. (1997) Inorg.Chem. 36, 962-965; Turro et al.
(1995) J. Am. Chem. Soc. 117, :9026-9032). A larger enhancement was
found for [Ru(phen).sub.2 dppz].sup.2+ than for
[RU(bpy).sub.3].sup.2+.
[0122] The enhancements for 10 different fluorophore solutions are
shown in FIG. 8. In all cases lower bulk-phase quantum yields
result in larger enhancements for samples between silver island
films. Additionally, [Ru(phen).sub.2dppz].sup.2+ in
water-dimethylformamide (DMF) mixtures were examined (FIG. 9). This
compound is quenched by water and the largest enhancements were
observed for the most-quenched solution (FIG. 9). The results in
FIGS. 7-9 provide strong support for the assertion that proximity
of the fluorophore to the metal islands resulted in increased
quantum yields. It is unlikely that these diverse fluorophores
would all bind to the silver islands or display other unknown
effect results which resulted in enhancements with increased
monotonically with decreased quantum yields.
EXAMPLE 3
Spectral Shifts in the Presence of Silver Islands
[0123] FIGS. 10A and 10B show the emission spectra of two solvent
sensitive fluorophores between quartz plates (Q) and silver islands
(S). In both cases blue shifts in the emission, which are
consistent with a decreased lifetime of fluorophores near the
islands were observed. Because fluorophores within 50 .ANG. of the
metal are likely to be quenched, it is unlikely that the blue
shifts seen in FIG. 10A and FIG. 10B are due to fluorophores bound
to the silver islands. Binding of fluorophores to the uncoated
quartz surface is also unlikely because uncoated quartz is present
for both emission spectra.
EXAMPLE 4
Effects of Silver Islands on Intrinsic Protein Fluorescence
[0124] The protein E. coli .beta.-galactosidase and human
glycoxidase were obtained from commercial suppliers. The proteins
were dissolved in 10 mM phosphate buffer, pH 6.5. The
concentrations of .beta.-galactosidase and human glyoxylase were
0.05 and 0.15 mg/ml, respectively. For studies of intrinsic protein
fluorescence the excitation wavelength was 295 nm. The emission
spectra of two proteins in the presence and absence of silver
islands were examined (FIGS. 11A and 11B). The proteins
.beta.-galactosidase and human glyoxylase were selected for their
modest and low quantum yields. .beta.-galactosidase has a quantum
yield approximately equal to that of N-acetyl-L-tryptophamide
(NATA) (D'Auria et al. (2001) J. Biochem., in press) which is
reported to be 0.13 (Demchenko, A. P. (1981). Ultraviolet
Spectroscopy of Proteins, Springer-Verlag, New York.). The quantum
yield of human glyoxylase was found to be about 10-fold less, and
thus near 0.013. For the higher quantum yield .beta.-galactosidase
there was no significant effect of the silver islands on the
emission spectra. For the lower quantum yield human glyoxylase are
observed both a blue shift and an increase in emission intensity.
.beta.-galactosidase is a tetrameric protein, 480,000 molecular
weight, which contains 26 tryptophan residues in each 120,000
dalton subunit (Jacobson et al. (1994) Nature 369, 761-766). Human
glyoxylase is a 66,000 dalton monomer which contains two tryptophan
residues (D'Auria, S., unpublished results). The spectra changes in
FIG. 11B are due to increased emission from a highly quenched
tryptophan residue in glyoxyalase which is shielded from the
solvent. The absence of a spectral shift or enhancement in
.beta.-galactosidase is understandable given its large number of
tryptophan residues and it being unlikely that a significant
fraction was highly quenched. Thus, silver islands can result in
increased emission from quenched aromatic amino acid residues in
proteins.
EXAMPLE 5
Effects of Silver Islands on Nucleic Acid Bases and DNA
[0125] Adenine, thymine and calf thymus DNA were obtained from
commercial suppliers. Poly T and poly C, each 15 bases long, were
obtained from the Biopolymer Core facility at the University of
Maryland, Baltimore School of Medicine. Emission spectra of the
bases adenine and thymine are shown in FIGS. 12A and 12B, showing
increased emission in the presence of the silver islands. Similar
results were obtained for the single stranded nucleotides poly T
and poly C (FIGS. 13A and 13B). The long wavelength emission maxima
of poly C is in agreement with that reported previously (Plessow et
al. (2000) J. Phys. Chem. B 104, 3695-3704). 1119] Calf-thymus DNA
was dissolved in 50 mM Tris, pH =7. The DNA concentration was 5 mM
as base pairs. Emission spectra were measured on a SLM 8000
spectrofluorometer with 287 nm excitation. Frequency-domain
lifetime measurements were obtained on a 10 GHz instrument (Laczko
et al. (1990) Rev. Sci. Instrum. 61, 2331-2337; Lakowicz et al.
(1994) Biophys. J. 46, 463-477). The excitation source was a
cavity-dumped rhodamine 6G dye laser providing approximately 100 ps
pulses which were frequency-doubled at 287 nm. Intensity decays
were measured through a combination 344 nm interference filter plus
a WG 335 long pass filter, which provided transmission from about
330 to 355 nm. Emission spectra and lifetimes were measured with
vertically polarized excitation and horizontally polarized
emission. This optical configuration reduced scattered light of the
excitation wavelength without significant distortion of the spectra
or lifetimes. The frequency-domain data were fit to the
multi-exponential model where the intensity decay is given by
equation (1) above where .alpha..sub.i are amplitude factors
associated with each decay time .tau..sub.i. The sum of the
.alpha..sub.i values are normalized to unity,
.SIGMA..alpha..sub.i=1.0.
[0126] If the mass thickness is restricted to near 40 .ANG., one
obtains particles on the surface with sub-wavelength dimensions, as
can be seen from the characteristic surface plasmon absorption
spectrum which are close to the small wavelength limit (FIG. 1B).
The DNA samples were placed between two such silver islands plates
with a separation near 1-1.5 .mu.m. The absorption spectrum for DNA
between the plates is approximately the sum of the DNA and silver
island absorption (FIGS. 14A and 14B), which suggests that the
islands did not significantly change the extinction coefficient of
the DNA.
[0127] The emission spectrum of DNA was examined in a thin 0.1 mm
cuvette and between the two island films (FIGS. 15A and 15B).
Excitation at 287 nm probably resulted in partially selective
excitation of the adenine and guanine residues (Wilson et al.
(1980) Photochem. & Photobiol. 31, 323-327; Georghiou et al.
(1996) Biophys. J. 70, 1909-1922). Surprisingly, the emission is
about 80-fold more intense near the metal islands. It is important
to note that this 80-fold increase is a considerable underestimate
of the increase displayed by DNA near the particles. The region of
enhancement is expected to extend about 200 .ANG. to about 2000
.ANG. into the solution. Taking into account the two island film
surfaces, only about {fraction (1/25)} of the DNA is near the
silver. This suggests that the emission of DNA near the silver is
enhanced 2000-fold. This is near the maximum enhancement predicted
for a molecule at the optimal distance from an ellipsoid of
appropriate size and shape. Amplified field effect can result in a
maximum of 140-fold enhancement (Gersten et al. (1981) J. Chem.
Phys. 75, 1139-1152) suggesting a minimum of a 15-fold increase in
the quantum yield of the DNA near the island films. It is unlikely
that the field enhancement is maximal. The actual increased quantum
yield of DNA is between about 15-fold and less than about
2000-fold.
[0128] One explanation of the increased intensity seen in FIG. 15B
could be a decrease in the non-radiative decay rate k.sub.nr, which
would result in a longer lifetime. Another reason for the increased
emission could be an amplified incident light field. This effect
would result in increased intensity, but is the lifetime would be
unchanged. Frequency-domain intensity decays are shown in FIGS. 16A
and 16B. These measurements were used to reconstruct the more
intuitive time-domain decays (FIG. 17). The decays are
multi-exponential in the absence or presence of metal islands
(Table 1). The intensity decays were strongly heterogeneous or
multi-exponential, which can be seen from the range of decay time
from 60 ps to 4.56 ns. The individual lifetimes of DNA are
uncertain because of its weak intrinsic fluorescence, but the mean
lifetimes ({overscore (.tau.)}) are reliable. Such a wide range of
lifetimes are in agreement with other published reports (Ballini et
al. (1983) Biophys. Chem. 18, 61-65; Georghiou et al. (1985)
Photochem. & Photobiol. 41, 209-212; Plessow et al. (2000) J.
Phys. Chem. B 104, 3695-3704). The important conclusion from these
experiments is that the mean lifetime ({overscore (.tau.)}) of DNA
decreased under the same conditions which we observed on 80-fold
increase in intensity (FIG. 15B). Such a decreased lifetime cannot
be explained by a decrease in k.sub.nr or increased rate of
excitation. However, the decreased lifetime can be explained by an
increase in the radiative decay rate. Let .GAMMA..sub.m represent
the rate of the radiative decay due to presence of the metal
particles. This new rate changes the quantum in the presence of
metal (m) to 3 Q m = + m + m + k nr ( 3 )
[0129] which will be larger than in the presence of the metal. The
lifetime in the presence of the metal (.tau..sub.m) will be
decreased to 4 m = 1 + m + k nr ( 4 )
[0130] The quantum yields and lifetimes in the absence of metals
are given by the equations 1 and 2 with .GAMMA..sup.m=0.0. Hence an
increase in the radiative decay rate of DNA by the metal can
explain both the increased intensity and decreased lifetime in the
presence of the silver islands. There is no quantitation agreement
between the 80-fold increase in intensity and the 3-fold decrease
in lifetime. There are numerous possible reasons, including
different spatial averaging across the sample by the intensity and
lifetime measurements. Nonetheless, the intrinsic DNA lifetime
decreased while the intensity increased, demonstrating an increase
in the rate of radiative decay.
2TABLE 1 Fluorescence intensity decay parameters of calf thymus DNA
in 50 mM TRIS, pH 7.0, 20.degree. C. CONDITIONS {overscore
(.tau.)}(ps) .sup.a) .alpha..sub.1 .tau..sub.1 (ps) .alpha..sub.2
.tau..sub.2 (ns) .alpha..sub.3 .tau..sub.3 (ns) X.sub.R.sup.2
.sup.b) 0.1 mm cuvette 60 0.974 12 0.021 1.17 0.005 4.56 2.7 on
silver islands 19 0.989 5 0.007 0.59 0.004 2.38 3.2 .sup.a)
{overscore (.tau.)} = .SIGMA..sub.i .alpha..sub.i .tau..sub.i
.sup.b) X.sub.R.sup.2 is the goodness-of-fit parameter calculated
with estimated uncertainties in the phase angle and modulation
values of 0.3.degree. and 0.007, respectively.
EXAMPLE 6
Effects of Silver Islands on Resonance Energy Transfer
[0131] Resonance energy transfer (RET) is widely used in
biochemical and biomedical research (Morrison et al. (1993)
Biochemistry 32, 3095-3104; Ju et al. (1996). Nat. Med, 292,
246-249). RET occurs whenever fluorophores with suitable spectral
properties come within the Forster distance R.sub.0. Forster
distances range from 20-40.ANG., and are rarely larger than 50
.ANG.. The effects of silver island films on RET between rhodamine
6G (R6G) and sulforhodamine 101 (SR101) when dissolved in
homogeneous solution were examined. Emission spectra of this
mixture are shown in FIG. 18, normalized to the donor emission.
Silver islands result in an increase in the acceptor emission near
590 nm. While this increase may appear modest, it is substantial
for a mixture of fluorophores in which the acceptors are present at
a concentration of 0.2 mM resulting in significant direct
excitation of the acceptor.
[0132] Additionally, RET of DAPI to acridine orange (AO) when bound
to double helical calf thymus DNA was examined (FIG. 19). In this
case the bulk concentration of the donor and acceptor are lower
because they are held in close proximity by the DNA. There is a
dramatic increase in the acceptor emission near 520 nm which we
believe is due to a metal-enhanced increase in the extent of energy
transfer.
[0133] Calf thymus DNA was obtained from commercial suppliers and
dissolved in 50 mM tris buffer, pH 7 to a concentration of 2 mM in
base pairs using 13,300 M.sup.-1 cm.sup.-1 per base pair. DAPI and
propidium iodide (PI) were obtained commercially. For the energy
transfer measurements the DAPI and PI concentrations were
1.5.times.10.sup.-5 M and 1.5.times.10.sup.-4 M, respectively.
These concentrations result in 133 base pairs per DAPI molecule and
13 base pairs per PI molecule.
[0134] Emission spectra were obtained using a SLM 8000
spectrofluorometer using 360 nm excitation. Intensity decays were
measured in the frequency-domain using instrumentation described
previously (Laczko et al. (1990) Rev. Sci. Instrum. 61, 2331-2337;
Lakowicz et al. (1994) Biophys. J. 46, 463-477). The excitation
wavelength of 360 nm was obtained from the frequency-doubled output
of a 3.80 MHz cavity dumped Pyridine 2 dye laser with a 10 ps or
less pulse width.
[0135] For the frequency-domain measurements the emission was
observed through a 460 nm interference filter. For steady state and
frequency-domain measurements the excitation was vertically
polarized and the emission observed through a horizontally oriented
polarizer to minimize scattered light. The FD intensity decay were
analyzed in terms of the multi-exponential model using equation (1)
above where .tau..sub.i are the lifetimes with amplitudes
.alpha..sub.i .sup.and .SIGMA..alpha..sub.i=1.0. Fitting to the
multi-exponential model was performed as described previously
(Lakowicz et al. (1994) Biophys. J 46, 463-477). The contribution
of each component to the steady state intensity is given by
equation (2) above.
[0136] The mean decay time is given by 5 _ = i f i i ( 5 )
[0137] The amplitude-weighted lifetime is given by 6 = i i i ( 6
)
[0138] The base pair length 3.4 .ANG. and r.sub.min=12 .ANG. were
fixed parameters.
[0139] Silver particles were obtained by chemical reduction of
silver onto quartz slides as above. If the mass thickness of the
deposition silver is kept near 40 .ANG. the silver particles have
sub-wavelengths dimensions and display the characteristics of
surface plasmon absorption (FIG. 1). From studies of the absorption
spectra of dyes between two such silver island films the sample
thickness was found to be near 1 to 1.5 .mu.m.
[0140] To examine the effect of silver islands on resonance energy
transfer double helical calf thymus DNA which was labeled with DAPI
as the donor and/or PI as the acceptor was used. Emission spectra
of DAPI-DNA and DNA labeled with both DAPI and PI in a cuvette are
shown in FIG. 20. The extent of energy transfer is about 20%, as
can be seen from the decrease in the DAPI donor intensity near 460
nm. The PI acceptor makes only a small contribution to the emission
seen at 610 nm. The extent of energy transfer near 20% is
consistent with the R.sub.0 value of 35.7 .ANG. for this D-A pair
(Murata et al. (2000) Biopolymers(Biospectrosc.) 57, 306-315).
Based on the extent of acceptor labeling of one per 13 base pairs,
and 3.4 .ANG. per base pair in the DNA helix, the acceptor
molecules are on average 45 .ANG. apart.
[0141] Next, the effects of the silver island films on DNA labeled
with only the DAPI donor or only the PI acceptor were examined
(FIG. 21). In the case of DAPI-DNA the intensity is essentially
unchanged when placed between quartz plates or between silver
island film. In the case of PI-DNA there is an approximate 2-fold
increase in the PI intensity. The larger effect of the silver
island film on PI-DNA is consistent with its lower quantum yield
near 0.15 as compared to 0.53 for DAPI-DNA (Murata et al. (2000)
Biopolymers (Biospectrosc.) 57, 306-315).
[0142] The emission spectra of donor and acceptor-labeled DNA are
shown in FIG. 22. A remarkable increase in the PI acceptor emission
was found for the DNA sample between the two silver island films as
compared to between two unsilvered quartz plates. The silver island
film had only a modest effect on acceptor-only DNA (PI-DNA). These
results show an increase in the efficiency of RET from DAPI to PI
due to proximity to the silver islands.
[0143] An increase in energy transfer from DAPI to PI is expected
to result in a decrease in the DAPI decay time. Frequency-domain
intensity decay of DAPI are shown in FIG. 23. The dashed lines in
each panel show the DAPI decays in the absence of the PI acceptor.
In all cases the mean DAPI lifetimes decreased in the presence of
PI (Table 2). The mean DAPI decay time ({overscore (.tau.)}=2.93 ns
without acceptor) was reduced about 20% to .tau.=2.39 ns in the
presence of the silver island, while the steady state intensity was
essentially unchanged (FIG. 21). This result suggests an increase
in the rate of radiative decay due to the silver islands. Control
measurements showed the absence of scattered light in all these
measurements.
3TABLE 2 Multi-exponential analysis of DAPI donor intensity decay
in the presence and absence of acceptor and silver islands
Sample.sup.1 {overscore (.tau.)} (ns).sup.2 .tau. (ns).sup.3
.alpha..sub.1 .tau..sub.1 (ns) .alpha..sub.2 .tau..sub.2 (ns)
.alpha..sub.3 .tau..sub.3 (ns) X.sub.R.sup.2 DAPI-DNA, C 2.93 2.42
0.278 0.63 0.722 3.11 -- -- 2.1 DAPI-DNA, Q 2.80 1.58 0.311 0.16
0.391 1.15 0.298 3.62 2.2 DAPI-DNA, S 2.39 1.10 0.447 0.09 0.414
1.29 0.139 3.70 2.1 DAPI-PI-DNA, C 2.16 1.04 0.427 0.11 0.308 0.84
0.265 2.77 1.0 DAPI-PI-DNA, Q 2.26 0.80 0.467 0.08 0.367 0.66 0.166
3.15 1.7 DAPI-PI-DNA, S 1.67 0.24 0.769 0.04 0.172 0.40 0.059 2.44
3.2 .sup.1C- in 0.5 mm cuvette, Q-between quartz plates without
silver, S-between quartz plates with silver .sup.2{overscore
(.tau.)} = .SIGMA. f.sub.i .tau..sub.i .sup.3<.tau.> =
.SIGMA. .alpha..sub.i .tau..sub.i
[0144] DAPI donor decay was examined when the donor and
acceptor-labeled DNA is in a 0.5 mm cuvette, between plain quartz
plates, and between silver island films (Table 2). The mean DAPI
lifetime was not changed going from the cuvette to the unsilvered
quartz plates, {overscore (.tau.)}=2.16 and 2.26 ns, respectively.
A dramatic decrease in the DAPI decay time to {overscore
(.tau.)}=1.67 ns was found for DAPI-DNA between the silver island
films (FIG. 23A C). This decrease in lifetime is attributable to
the increased in RET seen in the emission spectra (FIG. 22).
[0145] The frequency-domain donor decays in terms of the apparent
Forster distance were analyzed. This was accomplished by analyzing
the donor decay. The acceptor concentration was held constant at
0.075 acceptors per base pair and the values of R.sub.0 were
allowed to vary to obtain the best fit to the data (FIG. 24A-C).
The value of R.sub.0=37.4 .ANG. is close to that calculated for the
D-A pair, R.sub.0=35.7 A. The apparent value of Ro decreased to
33.5 A between the quartz plates. Importantly, the apparent value
of R.sub.0 increased 2-fold to 75.6 .ANG. for the sample between
the silver island films. This is an apparent R.sub.0 value.
Examination of this fit (FIG. 24C) reveals that the
frequency-domain intensity decay could not be fit to a single
R.sub.0 value. This lack of fit suggests the presence of at least
two populations of D-A pairs, with the pairs close to the silver
islands displaying a larger R.sub.0 value.
[0146] It is important to recognize that the 2-fold increase in the
apparent value of R.sub.0 represents a minimum estimate of the
effect of the silver islands on RET. The active space near the
silver islands extend approximately 200 .ANG. to about 2000 .ANG.
into the solution. Assuming a sample thickness of 1 .mu.m only
about {fraction (1/25)} of the sample is within the active value.
This suggests that the actual effect on RET is greater than a
2-fold increase in R.sub.0.
EXAMPLE 7
Localized Enhancement of Fluorescence Near Metallic Particles With
Multi-Photon Excitation
[0147] RhB, Eosin sodium salt, rose bengal and coumarin 152 were
obtained from commercial suppliers. The experimental sample
geometry is shown in FIG. 25. Two-photon excitation of RhB, eosin
and rose bengal was accomplished with the 852 nm output of a
Tsunami mode-locked Ti:Sapphire laser, 80 MHz repetition rate, 90
fs pulse, about 0.5 W average power. For C152 and ANS the
multi-photon excitation wavelength was near 800 nm. The excitation
was focused on the sample with a 15 cm radius concave mirror. The
solution was placed between two high quality quartz plates,
.lambda./4 flatness. The plates were half uncoated and half coated
with silver islands as described above. From absorption
measurements the thickness of the samples between the plates was
about 1 .mu.m. This sandwich sample was mounted on a x-y
positioner. The focused spot of the laser was about 4 mm in length
and 30 .mu.m in diameter. The x-y positioner was used to move the
sample so the laser illuminated regions with or without silver
islands. This position change was accomplished without any change
in the experimental geometry. Scattered excitation was eliminated
with a combination of a heat filter and BG-38 glass filter for the
emission spectra and a BG-38 and a 580 nm interference filter for
time-resolved measurements.
[0148] Intensity decays were measured in the frequency-domain using
instrumentation described previously (Lakowicz et al. (1985)
Biophys. Chem. 21, 61-78; Laczko et al. (1990) Rev. Sci. Instrum.
61, 2331-2337). For the frequency-domain measurements the emission
was observed through a 580 interference filter. For all steady
state and frequency-domain measurements the excitation was
vertically polarized and the emission observed through a
horizontally oriented polarizer to minimize scattered light. The FD
intensity decay were analyzed in terms of the multi-exponential
model using equation (1) above where .tau..sub.i are the lifetimes
with amplitudes .alpha..sub.i and .SIGMA..alpha..sub.i=1.0. Fitting
to the multi-exponential model was performed as described
previously (Lakowicz et al. (1994) Biophys. J. 46, 463-477). The
contribution of each component to the steady state intensity is
given by equation (2) above. The mean decay time is given by
equation (5) above.
[0149] The emission spectra of RhB between silver island films with
two-photon excitation at 852 nm was examined (FIG. 26A). The
emission intensity for RhB between the metal particles (indicated
as--in FIG. 26A) was increased about 4-fold relative to RhB between
uncoated quartz plates (indicated as - - - in FIG. 26A). When the
sample was first exposed to the focused 852 nm light, white light
from the illuminated region was visibly detected. This "spark"
decayed in less than one second, but some white light background
remained. This white light was also seen from the silver islands
alone without RhB (indicated as . . . in FIG. 26A). Such a white
continuum emission for illuminated metal probes in near-field
microscopy has been reported previously (Sanchez et al. (1999)
Phys. Rev. Letts. 82, 4014-4017). Importantly, the RhB signal
remained stable following the initial white light transient. RhB
was also examined with one-photon excitation of 490 nm (FIG. 26,
bottom). In this case there was almost no effect of the silver
islands as compared to the uncoated quartz plates.
[0150] As a control experiment, RhB with two-photon excitation in
the presence of silver islands was examined, but with the plates
rotated so that the islands were on the outer surface not in
contact with RhB (FIG. 27). In this case no difference between the
silvered and unsilvered plates were found. The white continuum
emission was still observed from the silver islands. This result
demonstrated that the enhanced emission of RhB seen by FIG. 26 is
due to two-photon excitation of RhB, and not due to second harmonic
generation by the metal particles which in turn excites RhB.
[0151] The results in FIG. 26 can be understood by considering the
nature of our layered sample (see FIG. 1). The fluorophore is
uniformly distributed in the 1 .mu. thick sample. The region
affected by the metal islands is expected to extend about 250 .ANG.
in the solution. Recalling that there were two silver island
surfaces, we estimated that only about 5% of the solution is within
the active area. In fact, even this percentage is probably too high
because the fluorophores within 50 .ANG. of the metal surface are
typically quenched. Assuming 5% of the sample is affected by the
metal, the 4-fold enhancement for RhB (FIG. 26, top) suggests an
80-fold enhancement of two-photon excitation due to the metal
particles. The small fraction of fluorophores near the metal
particles explains the absence of a significant effect with
one-photon (FIG. 26, bottom) because the majority of the emission
occurs from RhB molecules distant from the silver islands.
[0152] The frequency-domain intensity decays of RhB between coated
and uncoated quartz plates with one and two-photon excitation were
examined. The excitation wavelength for FIG. 29A was 852 nm, and
the observation wavelength was 580 nm. For one-photon excitation
the mean lifetime was essentially unchanged between the coated or
uncoated plates (FIGS. 28A-B). This result is consisted with FIG.
26B, which showed that most of the one-photon individual emission
of RhB occurred from the bulk sample. Contrasting results were
found for the intensity decay of RhB with two-photon excitation
(FIGS. 29A-B). In this case the mean lifetime is dramatically
shortened by the silver island films. The reduced RhB lifetime with
852 nm excitation is the result of localized two-photon excitation
of RhB molecules near the metal particles. The reduced RhB lifetime
also demonstrated that the excitation is not due to second harmonic
generation by the metal islands. The lifetime of RhB resulting from
excitation by the harmonic would be the same as that found in the
bulk solution.
[0153] In many applications of fluorescence, photostability of the
fluorophore is a primary consideration. This is particularly true
in single molecule detection where it has been estimated that
approximately 1,000 photons can be observed from a highly stable
fluorophore like rhodamine prior to photodestruction (Ambrose et
al. (1999) Chem. Rev. 99, 2929-2956). Since photochemistry occurs
in the excited state, a reduction in the fluorescence lifetime is
expected to result in increased photostability. The photostability
of Rhodamine B between coated and uncoated quartz slides with one-
and two-photon excitation was examined (FIGS. 30A-B). For
one-photon excitation, the photostability was unaffected by the
presence or absence of silver islands (FIG. 30B). For two-photon
excitation, photostability in the presence of silver islands was
enhanced (FIG. 30A). These results are consistent with the shorter
lifetime observed for Rhodamine B between silver islands and with
the assertion that two-photon excitation is occurring
preferentially near the silver island films.
[0154] In the proceeding experiments, Rhodamine B was used which
displays a quantum yield of 0.48 (Q=0.48) in bulk solution. As a
result, much of the emission occurred from the bulk solution in
regions unaffected by the silver islands. Multi-photon excitations
occurring near the silver islands are increasing the quantum yield
of the nearby fluorophores. FIG. 31A shows emission spectra for
eosin (Q=0.24), and FIG. 31B shows the emission spectra for rose
bengal (Q=0.02) between quartz plates and between silver island
films. Excitation wavelength was 852 nm. In these spectra, the
white light continuum resulting from the silver island films is
more evident because of the lower overall signal. Importantly, with
two-photon excitation, there is essentially no emission from eosin
or rose bengal under conditions where there is substantial emission
from the fluorophores between the silver islands. This result
suggested selective and localized two-photon excitation near metal
particles.
[0155] The concept of selective excitation was pursued further
using biochemically relevant fluorophores such as coumarin 152 and
ANS (FIG. 32A-B). In this case a remarkable enhancement of the
two-photon induced emission for these fluorophores between silver
island films was observed. In the case of ANS with a very low
quantum yield in water (Q<0.01) there was essentially no signal
seen for ANS between the uncoated slides, and even the signal
observed from a bulk solution in a cuvette was insignificant
compared to the two-photon induced emission in the presence of
silver particles. The results shown in FIGS. 31A-B and 32A-B
suggest that multi-photon excitation near silver particles is a
general phenomenon which can result in highly localized excitation
in regions near the metal particles.
[0156] FIG. 33 is an illustration of how fluorophores on metal
colloids can be selectively detected by multi-photon excitation in
the presence of free fluorophore. Only the fluorophores within
about 50 to about 2000 .ANG., preferably from about 50 .ANG. to
about 200 .ANG., of a metal particle will have increased
fluorescence when exposed to an amount of exciting radiation. The
free fluorophores will not fluoresce at detectable levels.
[0157] FIG. 34 is an illustration of localized multi-photon
excitation of intracellular autofluorescence by metal colloids. In
one embodiment, a metal particle, preferably a noble metal, is
attached to an antibody that binds to a desired target. When the
antibody binds to the desired target, the metal particle is
positioned near the target at a distance sufficient to increase the
fluorescence of the target, typically about 50 to about 2000 .ANG.,
preferably from about 50 .ANG. to about 200 .ANG., in response to
an exciting amount of fluorescence, preferably multi-photonic
excitation. The antibody can be from any host animal capable of
producing antibodies. Exemplary host animals include mammals,
preferably rabbits, goats, horses, and humans. The antibody can
also be conjugated with an extrinsic fluorophore.
EXAMPLE 8
Assays
[0158] FIG. 35 depicts a schematic for an immunoassay assay. A
capture antibody is covalently bound to the surface near the metal
particles. The presence of the analyte (An) results in surface
binding of a second antibody which is labeled with a
non-fluorescent chromophore. Exemplary non-fluorescent or weakly
fluorescent chromophores fluorophore include, but are not limited
to, rose bengal, eosin, malachite green, and organic molecules used
as dyes or stains in optical microscopy. Suitable organic molecules
used as dyes or stains in optical microscopy are well known in the
art and include, but are not limited to, acid fuchsin, alcian blue,
alizarin red, congo red, crystal violet, eosin, evans blue, light
green SF, luxol fast blue, methyl green, neutral red, nigrosin, oil
red o, orange g, picric acid, pyronin y, safranine o, sirius red,
sudan black b, and toluidine blue o. Upon binding to the antigen,
the previously non-fluorescent species emits in response to an
amount of exciting radiation due to the increased radiative rate.
The unbound species more distant from the metal site will not
interfere with the fluorescent signal because they do not
fluoresce. The non-fluorescent species becomes a "molecular beacon"
emitting only when close to the metal particles. It will be
appreciated that antibodies or antibody fragments from any host
capable of producing antibodies can be used. Exemplary hosts
include mammals such as primates, goats, horses, rabbits, and
rodents. Additionally, recombinant or chimeric antibodies can also
be used. This assay can be used to detect the presence of an
analyte in biologic fluids including, but not limited to, saliva,
urine, mucus, blood, plasma, and lymphatic fluid. Exemplary
analytes include steroids, small molecules, proteins, peptides,
bacteria, and fungi.
[0159] FIG. 36 depicts an assay of the present invention which uses
electrical potential to gate the fluorescence on and off. A
fluorophore is positioned at the end of a flexible polymer chain
which is attached to a surface coated with metal particles In one
embodiment, the entire chain and fluorophore are negatively
charged. When the voltage or the metal is positive, the fluorophore
is in the quenched zone. When the voltage is negative, the
fluorophore is displaced into the enhancement zone. Alternatively,
the fluorophore is moved in and out of the shorter range quenching
zone of the metal. Thus, the emission is gated by the voltage. The
electric potential can be generated using known techniques.
Suitable sources of electric potential include devices capable of
producing electricity including, but not limited to, batteries,
fuel cells, and transformers. In another embodiment, a method to
access array sensors using electric potential is provided using
methods known for linking DNA or proteins in desired patterns on
surfaces. For example, biomolecules can be linked to surfaces using
adhesives, polymers, lysine, or biotin-avidin.
[0160] Another embodiment of the present invention discloses an
immunoassay in wherein a first antibody is labeled with a donor
molecule and a second antibody is labeled with an acceptor molecule
(FIG. 37). The labeled antibodies will bind to their respective
antigens to form a complex. When the complex is near a metal
particle, the resonance energy transfer from donor to acceptor is
enhanced such that the emission from the acceptor is detectable.
The complexes can be positioned near a silver island surface using
electrical potential or other attractive forces. The metal-induced
increased in the transfer rate results in transfer over larger
distances, and the antigen is detectable by an increase in the
transfer efficiency.
[0161] FIG. 38 depicts another embodiment of the present invention,
an apparatus for surface plasmon excitation. For the control
surface without silver (FIG. 38B) the emission increases at the
critical angle for the fluorescence. When excitation is at the
plasmon resonance angle the emission is sharply distributed at the
plasmon angle for the emission wavelength. A typical metallic
surface for this purpose would be a continuous, semi-transparent
silver coating. This coating may be further modified by binding of
metallic colloids or particles to provide both enhanced and
directional emission.
[0162] Various modifications may be made to the invention as
described without departing from the spirit of the invention or the
scope of the appended claims.
* * * * *