U.S. patent application number 12/354698 was filed with the patent office on 2009-08-06 for electrochemiluminescence of rare earth metal chelates.
This patent application is currently assigned to BioVeris Corporation. Invention is credited to Nicholas Cairns, Hongjun Yang.
Application Number | 20090197349 12/354698 |
Document ID | / |
Family ID | 23678750 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090197349 |
Kind Code |
A1 |
Yang; Hongjun ; et
al. |
August 6, 2009 |
ELECTROCHEMILUMINESCENCE OF RARE EARTH METAL CHELATES
Abstract
Luminescent chemical reagents that include complexes of rare
earth metals with ligands such as aromatic heterocyclic
nitrogen-containing compounds and semi-aromatic oxygen-containing
compounds are used to detect small quantities of complex substances
such as pharmaceuticals, metabolites, and microorganisms in complex
sample mixtures.
Inventors: |
Yang; Hongjun; (Rockville,
MD) ; Cairns; Nicholas; (Gaithersburg, MD) |
Correspondence
Address: |
FOLEY & LARDNER LLP
777 EAST WISCONSIN AVENUE
MILWAUKEE
WI
53202-5306
US
|
Assignee: |
BioVeris Corporation
|
Family ID: |
23678750 |
Appl. No.: |
12/354698 |
Filed: |
January 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11077076 |
Mar 11, 2005 |
7494820 |
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12354698 |
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09222443 |
Dec 29, 1998 |
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11077076 |
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08891337 |
Jul 10, 1997 |
5858676 |
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09222443 |
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08423394 |
Apr 18, 1995 |
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08891337 |
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Current U.S.
Class: |
436/501 ; 436/82;
534/15 |
Current CPC
Class: |
G01N 33/582 20130101;
Y10T 436/13 20150115; C07F 5/003 20130101; Y10S 436/806
20130101 |
Class at
Publication: |
436/501 ; 436/82;
534/15 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/20 20060101 G01N033/20; C07F 19/00 20060101
C07F019/00 |
Claims
1. A method of determining the presence of a chemical moiety, said
chemical moiety having the formula
[MPL.sup.1L.sup.2-(-link-)-].sub.tB wherein M is a lanthanide; P is
a polydentate ligand of M; L.sup.1 and L.sup.2 are ligands of M,
each of which may be a substance covalently bound to one or more of
P, L.sup.1 or L.sup.2 through one or more covalent bond linkages,
said linkages designated as (-link-) and being covalent bonds
linking B with at least one of P, L.sup.1 or L.sup.2; t is an
integer equal to or greater than 1; B is a biological substance or
a synthetic substance which is capable of competing with the
biological substance; P, L.sup.1 or L.sup.2, and B are of such
number that the total number of bonds to M provided by the ligands
of M equals the coordination number of M; and P, L.sup.1 or
L.sup.2, and B are of such composition that the chemical moiety can
be induced to repeatedly emit electromagnetic radiation; and
wherein the method comprises: (a) forming a reagent mixture
containing the chemical moiety; (b) exposing the reagent mixture to
electrochemical energy, the potential of which oscillates between a
potential sufficiently positive and sufficiently negative to induce
the chemical moiety to oscillate between at least two oxidization
states, thereby inducing the chemical moiety to repeatedly
electrochemiluminesce; and (c) detecting emitted luminescence
thereby to determine the presence of the chemical moiety.
2. A method of determining the presence of a chemical moiety, said
chemical moiety having the formula
[MPL.sup.1L.sup.2-(-link-)-].sub.tB wherein M is a lanthanide; P is
a polydentate ligand of M; L.sup.1 and L.sup.2 are ligands of M,
each of which may be a substance covalently bound to one or more of
P, L.sup.1 or L.sup.2 through one or more covalent bond linkages,
said linkages designated as (-link-) and being covalent bonds
linking B with at least one of P L.sup.1 or L.sup.2; t is an
integer equal to or greater than 1; B is a biological substance or
a synthetic substance which is capable of competing with the
biological substance; P, L.sup.1 or L.sup.2, and B are of such
number that the total number of bonds to M provided by the ligands
of M equals the coordination number of M; and P, L.sup.1 or
L.sup.1, and B are of such composition that the chemical moiety can
be induced to repeatedly emit electromagnetic radiation; and
wherein the method comprises: (a) forming a reagent mixture
containing the chemical moiety and an agent which upon exposure of
the reagent mixture to electrochemical energy forms a reductant;
(b) exposing the reagent mixture to electrochemical energy such
that the agent forms a reductant, thereby inducing the chemical
moiety to repeatedly electrochemiluminesce; and (c) detecting
emitted luminescence thereby to determine the presence of the
chemical moiety.
3. A competitive binding method of determining the presence of an
analyte of interest wherein the analyte and a chemical moiety bind
competitively to a complementary material, which comprises (a)
forming a reagent mixture comprising a sample that may contain the
analyte of interest, the complementary material, and the chemical
moiety such that the chemical moiety and the analyte of interest
bind competitively to the complementary material; said chemical
moiety having the formula [MPL.sup.1L.sup.2-(-link-)-].sub.tB
wherein M is a lanthanide; P is a polydentate ligand of M; L.sup.1
and L.sup.2 are ligands of M, each of which may be a substance
covalently bound to one or more of P, L.sup.1 or L.sup.2 through
one or more covalent bond linkages, said linkages designated as
(-link-) and being covalent bonds linking B with at least one of P,
L.sup.1 or L.sup.2; t is an integer equal to or greater than 1; B
is a biological substance or a synthetic substance which is capable
of competing with the analyte; P, L.sup.1, L.sup.2, and B are of
such number that the total number of bonds to M provided by the
ligands of M equals the coordination number of M; and P, L.sup.1,
L.sup.2, and B are of such composition that the chemical moiety can
be induced to repeatedly emit electromagnetic radiation; (b)
exposing the reagent mixture to electrochemical energy, the
potential of which oscillates between a potential sufficiently
positive and sufficiently negative to induce the chemical moiety to
oscillate between at least two oxidization states, thereby inducing
the chemical moiety to repeatedly electrochemiluminesce; and (c)
detecting emitted luminescence thereby to determine the presence of
the analyte of interest.
4. A competitive binding method of determining the presence of an
analyte of interest wherein the analyte and a chemical moiety bind
competitively to a complementary material, which comprises (a)
forming a reagent mixture comprising a sample that may contain the
analyte of interest, the complementary material, the chemical
moiety, and an agent which upon exposure of the reagent mixture to
electrochemical energy forms a reductant, such that the chemical
moiety and the analyte of interest bind competitively to the
complementary material; said chemical moiety having the formula
[MPL.sup.1L.sup.2-(-link-)-].sub.tB wherein M is a lanthanide; P is
a polydentate ligand of M; L.sup.1 and L.sup.1 are ligands of M,
each of which may be a substance covalently bound to one or more of
P, L.sup.1 or L.sup.2 through one or more covalent bond linkages,
said linkages designated as (-link-) and being covalent bonds
linking B with at least one of P, L.sup.1 or L.sup.2; t is an
integer equal to or greater than 1; B is a biological substance or
a synthetic substance which is capable of competing with the
analyte; P, L.sup.1, L.sup.2, and B are of such number that the
total number of bonds to M provided by the ligands of M equals the
coordination number of M; and P, L.sup.1, L.sup.2, and B are of
such composition that the chemical moiety can be induced to
repeatedly emit electromagnetic radiation; (b) exposing the reagent
mixture to electrochemical energy such that the agent forms a
reductant, thereby inducing the chemical moiety to repeatedly
electrochemiluminesce; and (c) detecting emitted luminescence
thereby to determine the presence of the analyte of interest.
5. A chemical moiety having the formula:
[MPL.sup.1L.sup.2-(-link-)-].sub.tB wherein M is a lanthanide
element; L.sup.1 and L.sup.2 are ligands of M; P is a bypyridyl or
diketone-type polydentate ligand of M, wherein P has at least one
linking group (-link-) attached thereto, the linking group (-link-)
being covalently bonded to B; and t is an integer equal to or
greater than 1; B is a biological substance, a synthetic substance
which is capable of competing with a biological substance in a
competitive binding reaction with a complementary material, or a
non-biological polymer; P, L.sup.1, and L.sup.2 are such that the
total number of bonds to M provided by the ligands of M equals the
coordination number of M; and P, L.sup.1, and L.sup.2, and B are of
such composition that the chemical moiety can be induced to emit
electromagnetic radiation.
6. A luminescent chemical reagent having the formula
MPL.sup.1L.sup.2-(-link-) wherein M is a lanthanide element;
L.sup.1 and L.sup.2 are ligands of M; P is a bypyridyl or
diketone-type polydentate ligand of M, wherein P has at least one
linking group (-link-) attached thereto, the linking group (-link-)
including an activator capable of reacting with a substance B to
form a covalent bond linkage therewith; wherein B is a biological
substance, a synthetic substance which is capable of competing with
a biological substance in a competitive binding reaction with a
complementary material, or a non-biological polymer; P, L.sup.1,
and L.sup.2 are of such number that the total number of bonds to M
provided by the ligands of M equals the coordination number of M;
and P, L.sup.1, and L.sup.2 are of such composition that the
luminescent chemical reagent can be induced to emit electromagnetic
radiation.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to luminescent metal chelate
labels for use in qualitative and quantitative chemical analysis.
More specifically, the present invention relates to the use of
chemical reagents that include complexes of rare earth metals with
ligands such as aromatic heterocyclic nitrogen-containing compounds
and semi-aromatic oxygen-containing compounds to detect small
quantities of complex substances such as pharmaceuticals,
metabolites, and microorganisms.
BACKGROUND OF THE INVENTION
[0002] There is a continuous and expanding need for rapid, highly
specific methods of detecting and quantifying chemical,
biochemical, and biological substances. Of particular value are
methods for measuring small quantities of pharmaceuticals,
metabolites, microorganisms, and other materials of diagnostic
value. Examples of such materials include narcotics and poisons,
drugs administered for therapeutic purposes, hormones, pathogenic
microorganisms and viruses, antibodies, metabolites, enzymes and
nucleic acids.
[0003] The presence of these materials can often be determined by
binding methods which exploit the high degree of specificity which
characterizes many biochemical and biological systems. Frequently
used methods are based on, for example, antigen-antibody systems,
nucleic acid hybridization techniques, and protein-ligand systems.
In these methods, the existence of a complex of diagnostic value is
typically indicated by the presence or absence of an observable
"label" which has been attached to one or more of the complexing
materials.
[0004] The specific labeling method chosen often dictates the
usefulness and versatility of a particular system for detecting a
material of interest. A preferred label should be inexpensive,
safe, and capable of being attached efficiently to a wide variety
of chemical, biochemical, and biological materials without changing
the important binding characteristics of those materials. The label
should give a highly characteristic signal, and should be rarely,
and preferably never, found in nature. The label should be stable
and detectable in aqueous systems over periods of time ranging from
hours to months. Detection of the label should be rapid, sensitive,
and reproducible without the need for expensive, specialized
facilities or personnel. Quantification of the label should be
relatively independent of variables such as temperature and the
composition of the mixture to be assayed. Most advantageous are
labels which can be used in homogeneous systems, i.e., systems in
which separation of the complexed and uncomplexed labeled material
is not necessary. This is possible if the detectability of the
label is modulated when the labeled material is incorporated into a
specific complex.
[0005] A wide variety of labels have been developed, each with
particular advantages and disadvantages. For example, radioactive
labels are quite versatile, and can be detected at very low
concentrations. However, they are expensive, hazardous, and their
use requires sophisticated equipment and trained personnel.
Furthermore, the sensitivity of radioactive labels is limited by
the fact that the detectable event can, in its essential nature,
occur only once per radioactive atom in the labeled material.
Moreover. radioactive labels cannot be used in homogeneous
methods.
[0006] Thus, there is wide interest in non-radioactive labels.
These include molecules observable by spectrophotometric, spin
resonance, and luminescence techniques, as well as enzymes which
produce such molecules. Among the useful non-radioactive labeling
materials are organometallic compounds. Because of the rarity of
some metals in biological systems, methods which specifically assay
the metal component of the organometallic compounds can be
successfully exploited. For example, Cais, U.S. Pat. No. 4,205,952
(1980) discloses the use of immunochemically active materials
labeled with certain organometallic compounds for use in
quantitating specific antigens. Any general method of detecting the
chosen metals can be used with these labels, including emission,
absorption and fluorescence spectroscopy, atomic absorption, and
neutron activation. These methods often suffer from lack of
sensitivity, can seldom be adapted to a homogeneous system, and as
with atomic absorption; sometimes entail destruction of the
sample.
[0007] Of particular interest are labels which can be made to
luminesce through photochemical, chemical, and electrochemical
means. "Photoluminescence" is the process whereby a material is
induced to luminesce when it absorbs electromagnetic radiation.
Fluorescence and phosphorescence are types of photoluminescence.
"Chemiluminescent" processes entail the creation of the luminescent
species by a chemical transfer of energy.
"Electrochemiluminescence" entails the creation of the luminescent
species electrochemically.
[0008] These luminescent items are of increasing importance. For
example, Mandle, U.S. Pat. No. 4,372,745 (1983) discloses the use
of chemiluminescent labels in immunochemical applications. In the
disclosed systems, the labels are excited into a luminescent state
by chemical means such as by reaction of the label with
H.sub.2O.sub.2 and an oxalate. In these systems, H.sub.2O.sub.2
oxidatively converts the oxalate into a high energy derivative,
which then excites the label. This system will, in principle, work
with any luminescent material that is stable in the oxidizing
conditions of the assay and can be excited by the high energy
oxalate derivative. Unfortunately, this very versatility is the
source of a major limitation of the technique; typical biological
fluids containing the analyte of interest also contain a large
number of potentially luminescent substances that can cause high
background levels of luminescence.
[0009] Another example of the immunochemical use of
chemiluminescence which suffers from the same disadvantages is
Oberhardt et al., U.S. Pat. No. 4,290,815, (1981) who disclose the
in situ electrochemical generation of an oxidant (e.g.,
H.sub.2O.sub.2) in close proximity to an immunoreactant labeled
with a chemiluminescent species. The electrogenerated oxidant
diffuses to the chemiluminescent species and chemically oxidizes
it, resulting in the net transfer of one or more electrons to the
electrogenerated oxidant. Upon oxidation, the chemiluminescent
species emits a photon. In contrast, the subject invention requires
the direct transfer of electrons from a source of electrochemical
energy to a chemiluminescent species which is capable of repeatedly
emitting photons.
[0010] The present invention is concerned with
electrochemiluminescent labels. Suitable labels comprise
electrochemiluminescent compounds, including organic compounds and
organometallic compounds. Electrochemiluminescent methods of
determining the presence of labeled materials are preferred over
other methods for many reasons. They are highly diagnostic of the
presence of a particular label, sensitive, nonhazardous,
inexpensive, and can be used in a wide variety of applications.
[0011] Organic compounds which are suitable electrochemical labels
include, for example, rubrene and 9,10-diphenyl anthracene. Many
organometallic compounds are suitable electrochemical labels. For
instance, Bard et al., in U.S. Pat. No. 5,310,687 (1994), discloses
that a wide variety of analytes of interest and chemical moieties
that bind to analytes of interest may be conveniently attached to
ruthenium- or osmium-containing labels through amide or amine
linkages. The labeled materials may then be determined by any of a
wide variety of means, including photoluminescent,
chemiluminescent, and electrochemiluminescent means. It is also
disclosed therein that electrochemiluminescent labels, including
ruthenium- and osmium-containing labels and organic molecules such
as rubrene and 9,10-diphenyl anthracene, are particularly versatile
and advantageous.
[0012] The series of elements having Atomic Numbers in the range
57-71 are known as the lanthanides; they are: cerium, dysprosium,
erbium, europium, gadolinium, holmium, lanthanum, lutetium,
neodymium, praseodymium, promethium, terbium, thulium, and
ytterbium. The lanthanides are classically referred to as the `rare
earths`. They are also referred to as the inner-transition
elements, because outer electron structure is identical across the
group; their electron structures differ only at an inner level.
Since the electronic diversity between the atoms is at some depth,
the elements are very similar chemically. All of the lanthanides
form trivalent ions and complexes. Their absorption bands are
narrow compared with those of the normal transition ions.
Complexing agents, which alter the absorption spectra of normal
transition ions by modifying their outer electron structures, have
little effect on the lanthanide ions. In spite of a high charge,
lanthanide ions are too large to cause significant polarization, so
complex formation is not facile.
[0013] With the transition metal chelates, the center metal atoms
are unfulfilled d-orbitals. Under the ligand field, d-orbitals
interact with the ligand field leading to splitting the d-orbitals
into two different energy levels, which correspond to the ground
state and to an excited state. Unlike the case with transition
metals, rare earth metals have unfulfilled f-orbitals. The emission
of rare earth metal chelates is due to the intromolecular energy
transfer from the ligand to the metal ion. Therefore, the emission
is more characteristic of the metal than of the interaction between
the metal and its ligands.
[0014] The rare earth metal chelated usually have eight
coordination sites rather than six as for transition metal
chelates. The eight coordination sites are arranged as four in one
plane and the other four in another plane, with the metal located
between the planes. However, the two planes twist 45.degree. with
respect to each other, so this type of compound is not of an
octahedral configuration.
[0015] The Bard et al. chemical moieties as disclosed in U.S. Pat.
No. 5,310,687 do have one characteristic that constitutes a
drawback in certain circumstances. The emission spectra of their
chelates has a band width on the order of 100 nm. This can make
signal discrimination difficult in multiple wavelength
electrochemiluminescence measurements. Thus there remains a need
for improvement in this area.
SUMMARY OF THE INVENTION
[0016] It has now been discovered that rare earth metal chelates
may be prepared that have emission spectra band widths of less than
50 nm. The great advantages of the use of the novel labeled
materials based upon the rare earth metal chelates, and of the
methods of detecting them, follows.
[0017] According to the present invention there is provided a
chemical moiety having the formula
[MPL.sup.1L.sup.2-(-link-)-].sub.tB wherein M is a lanthanide
element; P is a polydentate ligand of M; L.sup.1 and L.sup.2 are
ligands of M, each of which may be a substance covalently bound to
one or more of P, L.sup.1, or L.sup.2 through one or more covalent
bond linkages such as amide and amine linkages, said linkages
designated as (-link-) and linking B with at least one of P,
L.sup.1, or L.sup.1; t is an integer equal to or greater than 1;
and B is a substance of interest.
[0018] The present invention provides compounds particularly
suitable as intermediates for attaching a luminescent
lanthanide-containing label to amino groups of chemical,
biochemical, and biological substances. These intermediates are
thus particularly suitable for creating chemical moieties according
to the present invention. The intermediates are the mono- and
di-N-hydroxysuccinimide esters of lanthanide bis(2,2'-bipyridine)
(2,2'-bipyridine-4,4'-dicarboxylic acid) and their salts and
ruthenium or osmium
bis(2,2'-bipyridine)(4,4'-di(chloromethyl)-2,2'-bipyridine). These
compounds are generally synthesized by procedures which are known
in the art.
[0019] The present invention provides methods for determining the
presence of the novel chemical moieties. The present invention also
provides methods of determining the presence of a chemical moiety
as described above. The methods comprise: [0020] a) forming a
reagent mixture under suitable conditions containing the chemical
moiety; [0021] b) inducing the moiety to emit electromagnetic
radiation by exposing the reagent mixture to chemical energy or
electrochemical energy; and [0022] c) detecting the emitted
electromagnetic radiation and thereby determining the presence of
the chemical moiety.
[0023] This invention further provides for the use of
lanthanide-containing labels in binding methods for determining the
presence of substances of interest. These methods may be used to
determine labeled moieties of interest, to employ labeled moieties
to determine analytes of interest, or to use labelled analogues of
analytes of interest to determine analytes of interest in
competitive binding assays. These binding methods may be
homogeneous or heterogeneous binding methods.
[0024] Still further, the present invention provides systems for
determining the presence of the lanthanide-containing chemical
moieties of this invention. These systems comprise a means for
inducing the chemical moiety to emit electromagnetic radiation.
[0025] The present invention also provides for use of
electrochemiluminescent labels in binding methods for determining
the presence of substances of interest. These methods can be used
to determine labeled moieties of interest, to employ labeled
moieties to determine analytes of interest, or to use labeled
analogues of analytes of interest to determine analytes of interest
in competitive binding assays. These binding methods can be
homogeneous or heterogeneous binding methods.
[0026] A specific embodiment of the invention provides for
compositions which contain two or more different chemical moieties.
Each of the moieties may be chemical species which can be induced
to emit electromagnetic radiation of different wavelength. In
another embodiment of the invention the chemical moieties may be
chemical species each of which is induced to emit electromagnetic
radiation by exposure to energy of a different value or from a
different source. A different substance or analyte of interest may
then be specifically attached to each of the different chemical
moieties. By using these compositions and methods it is possible to
determine two or more different substances or analytes of interest
that may be present in the sample under examination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1(a) shows a cyclic voltammogram for 2 mM europium
chelate in 0.1 M tetrabutylammonium hexafluorophosphate
(hereinafter "TBAPF.sub.6") acetonitrile solution.
[0028] FIG. 1(b) shows a cyclic voltammogram for 2 mM europium
chelate with 10 mM tripropylamine (hereinafter "TPA") added in 0.1
M TBAPF.sub.6 acetonitrile solution.
DETAILED DESCRIPTION OF THE INVENTION
The Chemical Moiety: M and its Ligands
[0029] According to the present invention, there is provided a
chemical moiety having the formula
[MPL.sup.1L.sup.2-(-link-)-].sub.tB
wherein M is a lanthanide; P. is a polydentate ligand of M; L.sup.1
and L.sup.2 are ligands of M, each of which may be a substance
covalently bound to one or more of P, L.sup.1, or L.sup.2 through
one or more amide or amine linkages; t is an integer equal to or
greater than 1; P, L.sup.1, L.sup.2, and B are of such number that
the total number of bonds to M provided by the ligands of M equals
the coordination number of M; (-link-) designates the bond or bonds
that connect B to P, L.sup.1, and L.sup.2; and P, L.sup.1, L.sup.2,
and B are of such composition that the chemical moiety can be
induced to emit electromagnetic radiation.
[0030] In accordance with the present invention, M may be cerium,
dysprosium, erbium, europium, gadolinium, holmium, lanthanum,
lutetium, neodymium, praseodymium, promethium, terbium, thulium, or
ytterbium. In the currently preferred embodiments of the present
invention, M is cerium, europium, terbium, or ytterbium, with
europium being most preferred.
[0031] The chemical moiety in accordance with the present invention
should have at least one polydentate ligand of M. When the moiety
has more than one polydentate ligand, the polydentate ligands may
be the same or different. Polydentate ligands include aromatic and
aliphatic ligands. Suitable aromatic polydentate ligands include
aromatic heterocyclic ligands such as, for example, bipyridyl,
bipyrazyl, terpyridyl, phenanthrolyl, and porphyrins, as well as
semi-aromatic oxygen-containing compounds such as, for example,
diketone-type compounds. The semi-aromatic oxygen-containing
compounds are currently preferred.
[0032] Suitable polydentate ligands may be unsubstituted, or
substituted by any of a large number of substituents known to the
art. Suitable substituents include for example, alkyl, substituted
alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,
carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy,
imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium,
ureido, maleimide, sulfur-containing groups, phosphorus containing
groups, and the carboxylate ester of N-hydroxysuccinimide.
[0033] Additionally, at least one of L.sup.1 and L.sup.2 may be a
polydentate aromatic heterocyclic ligand that contains nitrogen as
well as semi-aromatic oxygen-containing compounds such as, for
example, diketone-type compounds. The semi-aromatic
oxygen-containing compounds are currently preferred. Suitable
polydentate ligands include, but are not limited to, bipyridyl,
bipyrazyl, terpyridyl; phenanthroyl, a porphyrin, substituted
bipyridyl, substituted bipyrazyl, substituted terpyridyl,
substituted phenanthroyl, or a substituted porphyrin. These
nitrogen-containing aromatic ligands as well the semi-aromatic
oxygen-containing ligands may be substituted with an alkyl,
substituted alkyl, aryl, substituted aryl, aralkyl, substituted
aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino,
hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine,
guanidinium, ureido, maleimide, a sulfur-containing group, a
phosphorus-containing group, or the carboxylate ester of
N-hydroxysuccinimide.
[0034] In one embodiment of the invention the chemical moiety
contains two bidentate ligands, each of which is bipyridyl,
bipyrazyl, terpyridyl, phenanthrolyl, substituted bipyridyl,
substituted bipyrazyl, substituted terpyridyl, or substituted
phenanthrolyl. In another embodiment of the invention the chemical
moiety contains three bidentate ligands, each of which is
bipyridyl, bipyrazyl, terpyridyl, phenanthrolyl, substituted
bipyridyl, or substituted phenanthrolyl, for instance
##STR00001##
[0035] This chemical moiety may have one or more monodentate
ligands, a wide variety of which are known to the art. Suitable
monodentate ligands include, for example, carbon monoxide,
cyanides, isocyanides, halides, and aliphatic, aromatic and
heterocyclic phosphines, amines, stibines, and arsines.
[0036] In still another embodiment of the invention the chemical
moiety contains a tetradentate ligand such as a porphyrin or
substituted porphyrin, or mixed nitrogen-containing and
oxygen-containing ligands, for instance
##STR00002##
[0037] In a preferred embodiment of the invention, the chemical
moiety contains three semi-aromatic oxygen-containing diketone-type
ligands.
[0038] Preferred embodiments of this chemical moiety comprise
bis(2,2'-bipyridyl)europium(II), tris(2,2'-bipyridyl)-europium(II),
europium
tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate),
which has the structure
##STR00003##
and terbium tris(2,2,6,6-tetramethyl-3,5-heptanedionate), which has
the structure
##STR00004##
[0039] Some of these chelates are known. The others can be prepared
by procedures analogous to those used for preparing the known
species. U.S. Pat. No. 3,700,410 discloses the preparation of
certain preferred chelates that may be used in accordance with the
present invention. The entire disclosure of that patent is
expressly incorporated herein by reference.
[0040] It is within the scope of the invention for one or more of
the ligands of M to be attached to additional chemical labels, such
as, for example, radioactive isotopes, fluorescent components, or
additional luminescent europium-containing centers.
The Chemical Moiety: Biological and Nonbiological Substances B
[0041] Suitable substances (B) include many biological substances,
for example, whole cells, viruses, subcellular particles, proteins,
lipoproteins, glycoproteins, peptides, nucleic acids
polysaccharides, lipopolysaccharides, lipids, fatty acids, cellular
metabolites, hormones, pharmacological agents, tranquilizers,
barbiturates, alkaloids, steroids, vitamins, amino acids and
sugars. Whole cell may be animal, plant, or bacterial, and may be
viable or dead. Examples include plant pathogens such as fungi and
nematodes. Within this application the term "subcellular particles"
means subcellular organelles, membrane particles as from disrupted
cells, fragments of cell walls, ribosomes, multienzyme complexes,
and other particles which can be derived from living organisms.
Also, within this application, nucleic acids means chromosomal rNA,
plasmid rNA, viral rNA, and recombinant INA derived from multiple
sources. Nucleic acids also include RiAs, for example messenger
RiAs, ribosomal IRNAs and transfer RNAS. Polypeptides include, for
example, enzymes, transport proteins, receptor proteins and
structural proteins such as viral coat proteins. Preferred
polypeptides are enzymes and serum-derived antibodies. Particularly
preferred polypeptides are monoclonal antibodies. Thus, in one
embodiment of the invention, B is a nucleotide or a polynucleotide.
In another embodiment, B is a serum-derived antibody or a
monoclonal antibody. Hormones include, for example, insulin and T4
thyroid hormone. Pharmacological agents include, for example,
cardiac glycosides. It is also within the scope of this invention
of include synthetic nucleic acids, and synthetic membranes,
vesicles and liposomes. The foregoing is not intended to be a
comprehensive list of the biological substances suitable for use in
this invention, but is meant only to illustrate the wide scope of
the invention.
[0042] It is within the scope of this invention to include labeled
nonbiological substances, including polymeric materials. These
substances may be in the form of soluble polymeric molecules, or
any of the large variety of known macroscopic forms such as, for
example, beads, or containers such as test tubes, bottles, assay
wells or the like.
The Chemical Moiety: Linkages
[0043] Biological and nonbiological substances (B) are covalently
bound to a ligand of M through one or more covalent linkages. These
are designated as (-link-) in the formula set forth above and in
the claims. Any type of covalent attachment is intended to be
covered by the general term (-link-). Amide and amine linkages are
preferred. In the case of amide linkages, the linkages may be
oriented so that material (B) is bonded directly either to the
carbonyl or to the nitrogen of the amide linkage. These chemical
moieties may be ionized. If so, it is understood in the art that
many different counterions will serve to neutralize the charge of
preparations of the chemical moiety. Suitable cations include, for
example, H.sup.+, NH.sub.4.sup.+, guanidinium, Ag.sup.+, Li.sup.+,
N.sup.+, K.sup.+, Mg.sup.2+, and Mn.sup.2+. Suitable anions
include, for example, halides, OH.sup.-, carbonate,
SO.sub.4.sup.2-, hexafluorophosphate and tetrafluoroborate.
[0044] It is also within the scope of this invention for the
labeled substance (B) to be labeled by greater than one, e.g., two,
three, four or more electrochemiluminescent centers.
Intermediates
[0045] The present invention also provides compounds that are
particularly suitable as intermediates for attaching a luminescent
europium-containing label to amino groups of chemical, biochemical
and biological substances. These intermediates are thus
particularly suitable for synthesizing chemical moieties according
to the present invention. They include compounds having the
formula
MPL.sup.1L.sup.2-(-link-)
wherein M is a lanthanide element; P is a polydentate ligand of M;
L.sup.1 and L.sup.2 are ligands of M, each of which may be a
substance covalently bound to one or more of P, L.sup.1, or L.sup.2
through one or more covalent bond linkages, said linkages
designated as (-link-) and being covalent bonds; P, L.sup.1, and
L.sup.2 are of such number that the total number of bonds to M
provided by the ligands of M equals the coordination number of M;
and P, L.sup.1, and L.sup.2 are of such composition that the
chemical moiety can be induced to emit electromagnetic radiation.
In these compounds, (-link-) may contain activators such as
N-hydroxysuccinimidyl moieties, amino moieties, or thiol moieties.
Among the intermediates of the invention are the mono- and
di-N-hydroxysuccinimide esters of europium
4,4'(dicarboxy)-2,2'-bipyridyl, bis(2,2'-bipyridyl) and their
salts; and europium 4,4'(dichloromethyl)-2,2'-bipyridyl,
bis(2,2'bipyridyl) and their salts.
[0046] A preferred method of synthesizing the europium-containing
N-hydroxysuccinimide esters is to first react europium
dichlorobis(2,2'-bipyridine) with 2,2'-bipyridine-4,4'-dicarboxylic
acid in a hot aqueous methanol solution of sodium bicarbonate.
After acidification, an aqueous solution of NAPF.sub.6 is added to
the solution of carboxylated europium compound. The isolated
hexafluorophosphate salt of the europium complex is then esterified
by reaction with N-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide in dimethylformamide of course, many
variations on the structure of the N-hydroxysuccinimide component
are possible without substantially altering the usefulness of the
inventive intermediates.
[0047] These intermediates may be ionized. If so, it is understood
in the art that many different counterions will serve to neutralize
the charge of preparations of the intermediate and form a salt.
Suitable cations for forming these salts include for example
NH.sub.4.sup.+, guandinium, Ag.sup.+, Li.sup.+, Na.sup.+, K.sup.-,
Ca.sup.2+, Mg.sup.2+, Mn.sup.2+, and Cd.sup.2+. Suitable anions for
forming these salts include, for example, halides, carbonate,
SO.sub.4.sup.2- hexafluorophosphate, and tetrafluoroborate.
[0048] These intermediates are useful for labeling substances
containing a free amino group capable of attacking the carboxylate
ester, and thereby displacing N-hydroxysuccinimide, or of attacking
the chloromethyl group, and thereby displacing chloride. Use of
these intermediates to label analytes of interest is preferred over
isothiocyanates of the prior art (e.g. Weber, U.S. Pat. No.
4,293,310). Isothiocyanates are generally prepared by reaction of a
primary amine with carbon disulfide or thiophosgene, each of which
is volatile and highly toxic. Carbon disulfide is also an acute
fire and explosion hazard. The required precursor primary aromatic
amines are more difficult to obtain than the precursor aromatic
carboxylic acids used in the present invention. Also, the
intermediates of the present invention are less reactive and more
easily stored and handled than the isothiocyanate derivatives.
Determining the Presence of Chemical Moieties: in General
[0049] The present invention provides methods for determining the
presence of the chemical moieties of the invention by forming a
reagent mixture which comprises the chemical moiety and detecting
the presence of the moiety in such reagent mixture. As such
throughout this application, and as will be readily appreciated by
those skilled in the art to which this invention pertains, "reagent
mixture" includes any and all combinations of the chemical moieties
with other substances or reagents which may be employed in the
practice of this invention. The specific combination may be in the
form of an aqueous or nonaqueous solution, a suspension or
emulsion, a solid or semisolid, or a gas, limited only by such
limitations as may be imposed by the detection method used to
detect the presence of the chemical moiety. For instance,
electrochemiluminescence identifications may be performed with
europium
tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate) in
aqueous solution and with terbium
tris(2,2,6,6-tetramethyl-3,5-heptanedionate) in methyl
chloride.
[0050] The chemical moieties may be detected by methods well known
in the art including, for example, emission and absorption
spectroscopy, e.g. ultraviolet absorption, infrared absorption, and
fluorescence emissions; atomic absorption, electrochemical, e.g.
anodic stripping voltametry; neutron activation and chemical
methods. Of particular interest are photoluminescence,
chemiluminescence and electrochemiluminescence methods. In one
embodiment of the invention, the presence of the chemical moiety
may be determined by inducing the chemical moiety to emit
electromagnetic radiation and detecting the emitted radiation. In
another embodiment of the invention, the chemical moiety may be
induced to emit electromagnetic radiation by exposing the reagent
mixture to electromagnetic, chemical or electrochemical energy. In
yet another embodiment of the invention, the chemical moiety may be
induced to emit electromagnetic radiation by exposing the reagent
mixture to chemical or electrochemical energy.
[0051] Eu chelate may be determined at very low concentrations
using luminescence techniques. Applicants' experience with Eu
chelate labeled substances indicates the advantages of using
europium-containing compounds as chemical labels. They are stable
for long periods and may be attached efficiently to a wide variety
of chemical, biochemical and biological materials. The labels are
safe and relatively inexpensive. They give a highly characteristic
signal and do not occur in nature. Measurements based on
luminescence of the labels are sensitive, fast, reproducible and
utilize simple instrumentation. There is very little interference
with detection based on luminescence of these labels by such
components as phosphate buffered saline, Tween (a surfactant),
liver tissue extract or serum. Luminescence-based measurement of
these labels does not destroy the sample or labeled materials and
may be performed repetitively. The signal is generated repeatedly
by each molecule of label, thereby enhancing the sensitivity with
which these label may be detected. The presence of labeled
materials may be determined qualitatively or quantitatively
depending on the needs of the particular application. The word
"determined", as used in this patent application, refers to either
qualitative or quantitative determinations of the labeled
material.
[0052] Accordingly, this invention provides a method of determining
the presence of a chemical moiety having the formula:
[MPL.sup.1L.sup.2-(-link-)-].sub.tB
wherein M is europium; P is a polydentate ligand of M; L.sup.1 and
L.sup.2 are ligands of M, each of which may be the same as, or
different from each other ligand; B is a substance which is a
ligand of M or is attached to one or more of P, L.sup.1, or
L.sup.2; t is an integer equal to or greater than 1; (-link-)
designates a covalent bond or bonds which connect B to P, L.sup.1,
or L.sup.2; and P, L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5 and
L.sup.6 and B are of such composition and number that the chemical
moiety can be induced to emit electromagnetic radiation and the
total number of bonds to M provided by the ligands of M equals the
coordination number of M.
[0053] The method comprises: [0054] a) forming a reagent mixture
under suitable conditions containing the chemical moiety; [0055] b)
inducing the moiety to emit electromagnetic radiation by exposing
the reagent mixture to chemical energy or electrochemical energy;
and [0056] c) detecting the emitted electromagnetic radiation and
thereby determining the presence of the chemical moiety.
[0057] Suitable conditions for forming the reagent mixture will be
known to those skilled in the art and will depend on the type of
reagent mixture involved. For example, suitable conditions for an
aqueous reagent mixture may include appropriate concentrations of
chemical moiety and other reagents such as oxidants, pH, salt
concentration and the like. For a solid sample, suitable conditions
for forming a reagent mixture may include addition of a conducting
liquid.
Determining the Presence of Bound Chemical Moieties
[0058] The methods of this invention include a method of
determining the chemical moiety wherein the moiety is capable of
binding to a chemical agent, i.e. forming a specific complex with
the chemical agent.
[0059] Suitable chemical agents include, but are not limited to,
whole cells, viruses, subcellular particles, nucleic acids
polysaccharides, proteins, glycoproteins, lipoproteins,
lipopolysaccharides, lipids, fatty acids, peptides cellular
metabolites, hormones, pharmacological agents, tranquilizers,
barbiturates, alkaloids, steroids, vitamins, amino acids, sugars or
non-biological polymers.
[0060] In one embodiment of the invention, the chemical agent may
be immobilized on the surface of an assay vessel. In another
embodiment, the chemical agent may be a serum-derived antibody or a
monoclonal antibody.
[0061] Of particular interest are antibody-antigen pairs of
materials. This binding method may be used to determine the
presence of labeled antigens, such as, for example, digoxin or
digitoxin in complex mixtures by first exposing the mixture to
immobilized antibodies specific for the antigen of interest, and
then measuring the amount of labeled material bound to the
immobilized antibodies.
[0062] the invention further provides methods for determining the
presence of analytes of interest which bind to a chemical moiety,
said moiety having the formula:
[MPL.sup.1L.sup.2-(-link-)-].sub.tB
wherein M is europium; P is a polydentate ligand of M; L.sup.1 and
L.sup.2 are ligands of M, each of which may be the same as, or
different from each other ligand; B is a substance which is a
ligand of M or is attached to one or more of P, L.sup.1, or
L.sup.2; t is an integer equal to or greater than 1; (-link-)
designates a covalent bond or bonds which connect B to P, L.sup.1,
or L.sup.2, and P, L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5 and
L.sup.6 and B are of such composition and number that the chemical
moiety can be induced to emit electromagnetic radiation and the
total number of bonds to M provided by the ligands of M equals the
coordination number of M.
[0063] The method comprises: [0064] a) forming a reagent mixture
under suitable conditions containing the chemical moiety; [0065] b)
inducing the moiety to emit electromagnetic radiation by exposing
the reagent mixture to chemical energy or electrochemical energy;
and [0066] c) detecting the emitted electromagnetic radiation and
thereby determining the analyte of interest. Within this
application "complementary material" means any substance capable of
forming complexes with both an analyte of interest and a labeled
analyte of interest or a labeled analogue of an analyte of
interest.
Electromagnetic Radiation
[0067] The phrase "inducing to emit electromagnetic radiation"
refers to creating an excited states of said moiety which
luminesces at wavelengths between 200 nanometers and 900 nanometers
at ambient temperatures. The present invention envisions
lanthanide-containing moieties such as europium-containing moieties
and encompasses the wide variety of luminescent moieties which can
be made by varying the chemical structure of the ligands. Each of
these variations in the metal and the ligands can change the
precise value of the energy input required to create the
luminescent excited state. Similarly, the wavelength of the emitted
electromagnetic radiation will be dependent upon the nature and
environment of the europium-containing material. Generally,
photoluminescence excitation and emission will occur with
electromagnetic radiation of between about 200 nanometers and about
900 nanometers in wavelength. Chemiluminescent and
electrochemiluminescent emission will generally occur with the
emitted electromagnetic radiation being between about 100
nanometers and about 1500 nanometers in wavelength, and preferably
of wavelengths between 200 and 900 nanometers. The potential at
which the reduction or oxidation of the chemical moiety will occur
depends upon its exact chemical structure as well as factors such
as the pH of the solution and the nature of the electrode used.
Generally, it is well known in the art how to determine the optimal
emission and excitation wavelengths in a photoluminescent system,
and the optimal potential and emission wavelength of an
electrochemiluminescent and chemiluminescent system.
[0068] It should be clear that there are many methods for
quantifying the amount of luminescent species present. The rate of
energy input into the system can provide a measure of the
luminescent species. Suitable measurements include, for example,
measurements of electric current when the luminescent species is
generated electrochemically, the rate of reductant or oxidant
utilization when the luminescent species is generated chemically or
the absorption of electromagnetic energy in photoluminescent
techniques. In addition, of course, the luminescent species can be
detected by measuring the emitted electromagnetic radiation. All of
these measurements can be made either as continuous, rate-based
measurements, or as cumulative methods which accumulate the signal
over a long period of time. An example of rate-based measurements
is the use of photomultiplier tubes, photodiodes or
phototransistors to produce electric currents proportional in
magnitude to the incident light intensity. Examples of cumulative
methods are the integration of rate-based data, and the use of
photographic film to provide cumulative data directly.
[0069] All of these luminescence-based methods entail repeated
luminescence by the europium-containing compound. the repetitive
nature of the detectable event distinguishes these labels from
radioactive isotopes or bound chemiluminescent molecules such a
luminal. The latter labels produce a detectable event only once per
molecular (or atom) of label, thereby limiting their
detectability.
Binding Methods
[0070] In the chemical moieties useful in these methods, biological
and nonbiological substance (B) may be incorporated into the
moieties by coordination directly to M or by attachment to a ligand
of M. Attachment may be through covalent bonding, or by
electrostatic or hydrogen bonding. Many diverse means of effecting
covalent bonding of substances (B) to ligands of M are available.
The attaching linkage may be, for example, an amide or amine bond,
an ester or thioester, an ether or thioester, or any of many other
means known to the art. The type of linkage will be determined by
the substituents of the ligand and the suitable chemical groups
available for binding with the ligand on the substance that is to
be labeled.
[0071] The analyte of interest and the chemical moiety may be any
pair of substances which are capable of binding together in a
specific manner. Such substances include for example, whole cells,
viruses, subcellular particles, nucleic acids, polysaccharides,
proteins, glycoproteins, lipoproteins, lipopolysaccharides, lipids,
fatty acids, peptides, cellular metabolites, hormones,
pharmacological agents, tranquilizers, barbiturates, alkaloids,
steroids, vitamins, amino acids, sugars and non-biological
polymers. Of particular interest are antibody-antigen pairs. One
embodiment of the invention provides the use of labeled antibodies
to determine the presence of cell surface antigens, or to label
particular cells for detection by cell sorting methods. Antigens
immobilized by, for example, attachment to immobilized, unlabeled
antibodies can be detected by labeled antibodies in a method
commonly known as a "sandwich" method.
[0072] In competitive binding assays, B may be the same substance
as the analyte of interest or an analogue of the analyte, and
capable of participating in the formation of a specific complex
with a complementary material. Such analytes and complementary
materials include, whole cells, viruses, subcellular particles,
nucleic acids, polysaccharides, proteins, glycoproteins,
lipoproteins, lipopolysaccharides, lipids, fatty acids, peptides,
cellular metabolites, hormones, pharmacological agents,
tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino
acids, sugars and non-biological polymers. Examples of such
analytes and complementary materials include insulin, digoxin,
digitoxin, T4 thyroid hormone, a fungus or nematode, a
serum-derived antibody or a monoclonal antibody, a DNA fragment or
an RNA fragment of particular interest are antibody-antigen based
methods. These methods "are analogous to the well known
radioimmunoassay, wherein an analyte of interest is detected when
it displaces a radioactive analogue of the analyte from an
antibody. The marry variations on radioimmunoassay known to the art
can, in principle, be used to advantage by employing moieties
labeled according to the present invention in place of
radioactively labeled compounds.
[0073] The present invention further provides heterogeneous and
homogeneous binding methods which utilize the chemical moieties
provided herein. In heterogeneous binding methods, the bound
labeled substance must be physically separated from the unbound
labeled substance before measurement of the presence of label. This
is frequently accomplished in antibody-antigen systems by
immobilizing one component, the antibody for example, by attachment
to an insoluble matrix such as a filter or to the surface of beads
or reaction vessels such as test tubes. The antigen-containing
solution is poured through the filter or into the reaction vessel,
and then washed away from the filter or sides of the reaction
vessel. Only antigen specifically bound to antibody will remain to
be determined.
[0074] In homogeneous methods, by contract, the bound and unbound
labeled material are present in the same reaction mixture when the
presence of label is measured. This is possible when binding
modifies the properties of the signal detectable from the label.
There are many ways that luminescent labels can be used in
homogeneous systems. For example, binding of the analyte to the
chemical moiety can directly influence the signal detectable from
the label. Additionally, a luminescence quencher may be positioned
on an antibody so that binding of a labeled antigen to the antibody
could result in suppression of the luminescence of the label by the
luminescence quencher on the antibody. Many homogeneous methods for
luminescent labels are known to the art; and some of them are
reviewed in Boguslaski and Li (1982), "Homogeneous Immunoassays,"
Applied Biochemistry and Biotechnology, 7, pp. 401-414.
[0075] In one embodiment of the invention, the analyte is fixed to
an insoluble matrix. Such a method may be performed as a sandwich
assay i.e. the chemical moiety becomes bound to the immobilized
analyte and unbound moiety is washed away from the matrix.
[0076] Another embodiment comprises a chemical agent to which the
moiety is capable of binding being fixed to an insoluble matrix and
the chemical moiety being a component of a biological fluid or
synthetic reaction.
[0077] Additionally, the competitive binding methods of the present
invention may comprise the complementary material being fixed to an
insoluble matrix.
[0078] Both the heterogeneous and homogeneous competitive methods
of the present invention comprise the complementary material being
monoclonal antibody and the insoluble matrix being the surface of
an assay vessel.
Inducing the Emission of Electromagnetic Radiation
[0079] The methods of the present invention may be performed by
exposing the reagent mixture to electrochemical energy or to
chemical energy. Additionally, the reagent mixture may be exposed
to a combination of electromagnetic radiation, chemical energy, and
electrochemical energy.
[0080] The chemical moiety may be oxidized by exposure to an energy
source. Such an energy source may be a chemical oxidizing agent.
Examples of such oxidizing agents include CE(IV) salts or
PbO.sub.2. Furthermore, the chemical moiety may be reduced by
exposure to an energy source. Such an energy source may be a
chemical reducing agent. An example of a suitable reducing agent is
magnesium.
[0081] The methods of the present invention may comprise nducing
the chemical moieties to emit electromagnetic radiation more than
once.
[0082] The reagent mixture may comprise oxalate, pyruvate, lactate,
malonate, citrate, tartrate or peroxydisulfate. Furthermore, the
chemical moiety may be reduced by exposure to an energy source and
the reagent mixture may comprise peroxydisulfate. Moreover, the
chemical moiety may be oxidized by exposure to an energy source and
the reagent mixture may comprise oxalate, pyruvate, lactate,
malonate, citrate or tartrate.
[0083] Methods of detecting the chemical moiety are provided
wherein the reagent mixture is continuously exposed to electrode
whose potential oscillates between a potential sufficient to effect
the reduction of said chemical moiety and a potential sufficient to
effect the oxidation of the chemical moiety.
[0084] The chemical moiety may be oxidized by exposure to an
electrode whose potential oscillates above and below a potential
sufficient to oxidize the chemical moiety, the reagent mixture
comprising oxalate, pyruvate, lactate, malonate, tartrate or
citrate. Moreover, the chemical moiety may be oxidized by exposure
to an electrode whose potential is constant and sufficient to
oxidize it, the reagent mixture comprising oxalate, pyruvate,
lactate, malonate, tartrate or citrate.
[0085] The chemical moiety may also be reduced by exposure to an
electrode whose potential oscillates above and below a potential
sufficient to reduce it, the reagent mixture comprising
peroxydisulfate. Such reagent mixture may additionally comprise
acetonitrile. Furthermore, the chemical moiety may be reduced by
exposure to an electrode whose potential is constant and sufficient
to reduce it, the reagent mixture comprising peroxydisulfate. Such
reagent mixture may also comprise acetonitrile.
[0086] When the chemical moiety is exposed to electrochemical or
chemical energy, the emitted electromagnetic radiation may be
continuously detected. Such electromagnetic radiation may be
detected visually or with a photodiode. Furthermore, when the
chemical moiety is exposed to electrochemical or chemical energy,
the emitted radiation may be detected cumulatively, e.g. with a
photographic film.
[0087] U.S. Pat. No. 5,147,806 (Kamin et al.) discloses a method
and apparatus for conducting electrochemiluminescence measurements.
The method and apparatus control the initial conditions relating to
the surface state of a triggering working electrode by reproducibly
creating and maintaining a favorable surface condition. This
enhances the precision and detection limit of the measurements.
Assays are performed with electrochemiluminescence detection in a
flow-through cell environment. The precision and detection limit
are enhanced by alternating initialization and measurement steps.
The entire disclosure of the Kamin et al. patent is expressly
incorporated herein by reference.
Means and Systems
[0088] The present invention also provides a system for determining
the presence of a chemical moiety having the formula:
[MPL.sup.1L.sup.2-(-link-)-].sub.tB
wherein M is europium; P is a polydentate ligand of M; L.sup.1 and
L.sup.2 are ligands of M, each of which may be the same as, or
different from each other ligand; B is a substance which is a
ligand of M or is attached to one or more of P, L.sup.1, or
L.sup.2; t is an integer equal to or greater than 1; and P,
L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5 and L.sup.6 and B are
of such composition and number that the chemical moiety can be
induced to emit electromagnetic radiation and the total number of
bonds to M provided by the ligands of M equals the coordination
number of M.
[0089] The system comprises: [0090] a) a reagent mixture comprising
the chemical moiety; [0091] b) means for inducing the chemical
moiety to emit electromagnetic radiation; and [0092] c) means for
detecting the emitted electromagnetic radiation.
[0093] A system for determining the presence of an analyte of
interest which binds to a chemical moiety is also provided, wherein
the moiety having the structural formula:
[MPL.sup.1L.sup.2-(-link-)-].sub.tB
is used, with the components of the structure being the same as
those described above. This system comprises: [0094] a) the
chemical moiety; [0095] b) a means for contact the chemical moiety
with the analyte of interest to form a reagent mixture; [0096] c) a
means for inducing the chemical moiety to emit electromagnetic
radiation; and [0097] d) a means for detecting the emitted
electromagnetic radiation.
Electromagnetic Radiation of Differing Wavelengths
[0098] This invention also concerns compositions which comprise the
europium-containing chemical moieties of this invention and one or
more different chemical moieties each of which can be induced to
emit electromagnetic radiation of a different distinct wavelength.
These compositions are useful in methods and systems of detecting
two or more different substances or analytes of interest contained
in a mixture of the same and other substances.
[0099] In one embodiment of the inventions the chemical moieties
are each attached to different analytes of interest.
[0100] The different chemical moiety or moieties may be any
suitable chemical moiety such as inorganic, organic or
organometallic compounds which can be induced to emit
electromagnetic radiation, e.g. rubrene or 9,10-diphenylanthracene.
These moieties may be such moieties that are induced to emit
electromagnetic radiation when exposed to energy of different
values or sources than the energy used to induce electromagnetic
radiation from the europium-containing chemical moieties. In a
specific embodiment of the invention, each other chemical moiety
emits electromagnetic radiation of a different distinct wavelength
when induced to emit electromagnetic radiation by energy of the
same source and value that induces the europium-containing chemical
moiety to emit electromagnetic radiation.
[0101] Methods for determining these chemical moieties comprise
forming a reagent mixture under suitable conditions containing the
chemical moieties and then inducing the chemical moieties to emit
electromagnetic radiation by exposing the reagent mixture to
chemical energy or electrochemical energy. The presence of each of
the moieties is determined by detecting the electromagnetic
radiation of different wavelengths emitted by each of the
moieties.
[0102] The invention also concerns a method of determining the
presence of one or more analytes of interest which bind selectively
to different chemical moieties present in the same mixture. The
method comprises contacting the analytes with the chemical moieties
under suitable conditions so as to form a reagent mixture. The
moieties are induced to emit electromagnetic radiation by exposing
the reagent mixture to chemical energy or electrochemical energy
and the emitted electromagnetic radiation of different wavelengths
is detected to determine the presence of each of the analytes of
interest.
[0103] These methods in which the presence of two or more chemical
moieties is determined in a mixture are applicable to all instances
described previously for determining the europium-containing
luminescent labels. This embodiment, however, allows for the
determination of two or more different substances present in the
same sample simultaneously.
[0104] Also provided are systems for determining the presence of
one or more different chemical moieties or analytes of interest
which bind to the chemical moieties, each of which may be induced
to emit electromagnetic radiation of a different wavelength. In one
embodiment of the invention, each moiety is attached to a different
analyte of interest.
EXAMPLES
[0105] This invention is illustrated in the examples which follow.
The examples are set forth to aid in an understanding of the
invention but are not intended to, and should not be construed to,
limit in any way the invention as set forth in the claims which
follow thereafter.
Example 1
Tris(6,6,7,7,8,8,8-heptofluoro-2,2-dimethyl-3,5-octanedionato)europium
[0106] The sodium salt of
2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione was prepared
as a white precipitate by the addition of 0.1M aqueous NaOH (0.7
ml, 0.7 mmol) to
2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione (208 mg, 0.7
mmol) to form a solution containing the ligand. Absolute ethanol
(0.7 ml) was then added with stirring and the solution was warmed
to 40.degree. C. for 15 minutes to effect complete dissolution of
the salt. Eu(NO.sub.3), (100 mg, 0.23 mmol) dissolved in a 1:1
water/ethanol mixture (1.0 ml) was added to the ligand solution in
one portion. A yellow oil formed immediately. The mixture was
stirred at room temperature for one hour. After this time, water
(15 ml) was added and the product was extracted with methylene
chloride (20 ml) and dried over anhydrous sodium sulfate. The
solvent was then removed under vacuum to afford 220 mg of
tris(6,6,7,7,8,8,8-heptofluoro-2,2-dimethyl-3,5-octanedionato)europium
as an oil (92% of theoretical yield). FAB mass spectrometry results
were as follows: m/e, 1077/1075 (M.sup.++K), 1061/1059
(M.sup.++Na), 1039/1037 (M.sup.++1). The product showed identical
properties (mass spectrometry, tlc) to the commercial compound
available from Aldrich (Cat. No. 16, 039-8, CAS No.
17631-68-4).
##STR00005##
Example 2
Effect of TPA on Electrochemiluminescence of Eu*
[0107] Cyclic voltammograms were established for 2 mM europium
chelate in 0.1 M TBAPF.sub.6 acetonitrile solution and for 2 mM
europium chelate with 10 mM TPA added in 0.1 M TBAPF.sub.6
acetonitrile solution. Both voltammograms were established with a
glassy carbon electrode (.theta.=3 mm) at a scan rate of 0.1
Volts/second. The results are depicted, respectively, in FIGS. 1(a)
and 1(b). The results confirm that the electrochemiluminescence of
Eu* is directly caused by the oxidation of TPA.
Example 3
N-Hydroxysuccinimide Ester Activator
[0108] An electrochemiluminescent rare earth chelate linker
containing an N-hydroxysuccinimide ("NHS") ester can be made by
forming europium
bis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate) and
separately synthesizing the key linking ligand,
N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate.
Reacting these two products together under basic conditions affords
the desired europium
bis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)(N-hydroxy-
succinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate). This
reaction, which is depicted below, is possible since the rate of
formation of the europium complexes is much faster than the rate of
NHS ester hydrolysis at pH 8-9.
[0109] NHS ester complexes are used to effect linkages to
melecules, biomolecules, and biopolymers of intereset that either
naturally contain primary or secondary amine functions or that have
been modified to contain such amines.
##STR00006## ##STR00007##
Example 4
N-Hydroxysuccinimide Ester Activator
[0110] Alternatively, an electrochemiluminescent rare earth chelate
linker containing an NHS ester can be made by an alternate route,
depicted below, in which the NHS ester is formed after the third
ligand has been complexed to the europium.
##STR00008##
Example 5
Dicyclohexylcarbodiimide Activator
[0111] An electrochemiluminescent rare earth chelate linker
containing a dicyclohexylcarbodiimide in place of the NHS ester can
be made by both the procedure of Example 3 and the alternative
procedure of Example 4.
[0112] Dicyclohexylcarbodiimide complexes are used to effect
linkages to melecules, biomolecules, and biopolymers of intereset
that either naturally contain primary or secondary amine functions
or that have been modified to contain such amines.
Example 6
Amine Activator
[0113] An example of an amine ligand, for linking to electrophilic
acyl centers such as NHS esters to form amides or to Michael-type
acceptors such as maleimides, is depicted below.
##STR00009##
Example 7
Thiol Activator
[0114] An example of a thiol linker, for linking to other thiols,
forming thioethers with electrophilic alkyl species such as alkyl
bromides, or reacting with Michael-type acceptors such as
maleimides, is depicted below.
##STR00010##
Example 8
Labelling of Bovine Serum Albumin with Activated Europium
Complex
[0115] Europium
bis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)
(N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate)
in dimethylformamide solution is added to a stirred solution of
bovine serum albumin (BSA) in aqueous Physiologic Buffered Saline
(PBS, 5 ml; 25 mg/ml BSA). The mixture is stirred overnight, and
precipitate is removed by centrifugation. The supernatant contains
europium-labelled BSA.
Example 9
Labelling of Human Immuoglobulin G with Activated Europium
Complex
[0116] Europium bis
(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)(N-hydroxysuc-
cinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate) in
dimethylformamide solution is added to a stirred solution of human
immunoglobulin G (IgG) in aqueous buffer. The europium-labelled IgG
solution fluoresces brightly after extensive dialysis, indicating
that the europium complex is bound to the high molecular weight
affinity-purified human IgG.
[0117] Although the present invention has been described and
illustrated with reference to certain preferred and other specific
embodiments thereof, those skilled in the art will be apprised by
the teachings herein of principles of wide applicability. The
invention patented, therefore, is determined only by the scope and
spirit of the appended claims.
* * * * *