Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Reexamination Certificate
1998-06-26
2003-09-02
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
C435S006120, C436S500000, C436S519000, C436S806000, C436S815000, C436S816000, C436S817000, C530S391300, C530S391500, C530S402000
Reexamination Certificate
active
06613583
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of electrogenerated chemiluminescence (ECL). More particularly, it concerns a multimetallic assembly with ligands that bridge independent chromophores for use in ECL devices and ECL methods of improved sensitivity.
2. Description of Related Art
Electrogenerated chemiluminescence, alternatively referred to as electrochemiluminescence, involves the formation of excited state species as a result of highly energetic electron-transfer reactions of reactants formed electrochemically. ECL systems and devices that make use of molecules that luminesce upon electrical excitation have been widely studied and are used for such purposes as display devices and instruments for chemical analysis. Several reviews have appeared on both the theory and application of ECL (Faulkner and Bard, 1977; Faulkner and Glass, 1982; Yang et al., 1994; Knight and Greenway, 1994).
The first report of ECL in a metal chelate appeared in 1972, in which the excited state of Ru(bpy)
3
2+
was generated in nonaqueous media by electrochemical formation and subsequent annihilation of the reduced Ru(bpy)
3
+
and oxidized Ru(bpy)
3
3+
species (Tokel and Bard, 1972).
Ru(bpy)
3
2+
+e
−
→Ru(bpy)
3
+
(1)
Ru(bpy)
3
2+
−e
−
→Ru(bpy)
3
3+
(2)
Ru(bpy)
3
3+
+Ru(bpy)
3
+
→Ru(bpy)
3
2+
+Ru(bpy)
3
2+
* (3)
Ru(bpy)
3
2+
*→Ru(bpy)
3
2+
+hv (4)
The potential range (window of stability) in nonaqueous solvents (e.g., +2.5 to −2.5 V vs. NHE in MeCN) allows formation of the energetic precursors necessary in the annihilation sequence. However, given the limited potential window of water, alternative means must be used to produce the excited state (e.g., Ru(bpy)
3
2+
*) for aqueous ECL. For example, in the presence of a luminophore such as Ru(bpy)
3
2+
, oxidation of species like oxalate or tripropylamine (TPrA) or reduction of a species like peroxydisulfate (S
2
O
8
2−
) have been shown to generate the necessary energetic precursors for excited state formation (Yang et al., 1994; Knight and Greenway, 1994; Rubinstein and Bard, 1981; Rubinstein et al., 1983; Ege et al., 1984; White and Bard, 1982; McCord and Bard, 1991; Leland and Powell, 1990). The presumed mechanism involves formation of strong reductants (CO
2
−
.or TPrA.) or strong oxidants (SO
4
−
.) that can interact with Ru(bpy)
3
3+
or Ru(bpy)
3
+
respectively, to produce the excited state:
Ru(bpy)
3
3+
+TPrA.→Ru(bpy)
3
2+
*+products (5)
or
Ru(bpy)
3
+
+SO
4
−
.→Ru(bpy)
3
2+
*+SO
4
2−
(6)
Ru(bpy)
3
2+
is used as an ECL-active label in DNA and immunoassay probes and for clinical analyses (U.S. Pat. Nos. 5,221,605; 5,238,808; 5,310,687; 5,453,356; 5,714,089; 5,731,147; Ege et al., 1984; Blackburn et al., 1991). ECL has several advantages over other detection techniques like fluorescence because no excitation source is required, and thus, ECL is immune to interference from luminescent impurities and scattered light. However, as with fluorescence labeling techniques, the sensitivity of the analysis depends on the ECL efficiency of the label.
With the goal of increasing the magnitude of ECL emission, this earlier work was extended to the use of multimetallic systems. Several reports on ECL with multimetallic systems have appeared, including Mo and W clusters (Mussel and Nocera, 1990; Ouyang et al., 1986) and a bimetallic Pt complex, Pt
2
(&mgr;−P
2
O
5
H
2
)
4
4−
(Vogler and Kunkeley, 1984; Kim et al., 1985). However, the ECL efficiency (taken as the number of photons emitted per redox event) in these systems was much weaker than Ru(bpy)
3
2+
under the same experimental conditions. Moreover, these earlier studies precluded the use of water because of the insolubility and instability of these complexes in an aqueous environment (Mussel and Nocera, 1990: Ouyang et al., 1986; Vogler and Kunkeley, 1984; Kim et al., 1985).
There have been no reports of ECL in multimetallic ruthenium systems. Often, coordination of a second metal center through a bridging-ligand (BL) framework (e.g., L
2
M(BL)ML
2
) leads to decreased photoluminescence quantum efficiencies and excited-state lifetimes. For example, Ru(bpy)
3
2+
has an excited-state lifetime of emission (&tgr;em) of about 600 ns (Bock et al., 1974; Bock et al., 1979; Navon and Sutin, 1974; Sutin and Creutz, 1978; Meyer, 1978; Hage et al., 1990; Barigelletti et al., 1991; and references therein; Demas and Crosby, 1971) and an emission quantum efficiency (&phgr;em) in MeCN of 0.086 (Kawanishi et al., 1984). Replacement of one bipyridine with a ligand capable of bridging two independent metal centers such as 2,3-bis(2′-pyrifyl)pyrazine (dpp) results in a decrease of &phgr;em to 0.064 for Ru(bpy)
2
(dpp)
2+
and &tgr;em~200 ns. [In Brauenstein et al., 1984, Brauenstein reported the relative quantum efficiencies of Ru(bpy)
2
(dpp)
2+
and [(bpy)
2
Ru]
2
(dpp)
4+
compared to Os(bpy)
3
2+
(0.0348±0020) (Demas and Crosby, 1971). The values shown are scaled to Ru(bpy)
3
2+
(&tgr;em=0.086) (Kawanishi et al., 1984) to make comparisons more valid.] Addition of a second Ru(bpy)
2
2+
moiety to form [(bpy)
2
Ru]
2
(dpp)
4+
gives &phgr;em=0.0007 and &tgr;em<50 ns (Brauenstein et al., 1984). This appears to be the general behavior. Other studies on Ru(II) diimine systems have shown that the monometallic parent complex might be luminescent in fluid solution at room temperature, but the bimetallic system is usually not (Dose and Wilson, 1978; Hunziker and Ludi, 1977; Goldsby and Meyer, 1984; Richardson et al., 1982; Richter and Brewer, 1993). A number of these systems were prepared in mixed oxidation states (i.e., L
2
M
III
(BL)M
II
L
2
) with the goal of defining the intervalence charge-transfer transition that is often present in the mixed-valence state (Creutz and Taube, 1969; Creuiz and Taube, 1972; Elias and Drago, 1972; Callahan et al., 1974; Callahan et al., 1975; Tom and Taube, 1975; Krentzien and Taube, 1976; Powers et al., 1976). In such studies, luminescence is not necessary to probe the photophysical and charge-transfer behavior. However, luminescence is a necessary prerequisite for efficient ECL.
The emission displayed by [(bpy)
2
Ru]
2
(dpp)
4+
and its monometallic analogue in fluid solution at room temperature has been traced to the weak metal-metal interaction present in the bimetallic system and the bipyridine-like environment conferred by the bridging dpp ligand (Brauenstein et al., 1984). However, even in this case, luminescence in the bimetallic system is much weaker than that observed in the parent compound. Many photophysical studies on ruthenium and osmium multimetallic complexes have centered on systems where the degree of electronic coupling between metal centers, as mediated by the BL-based orbitals, varies over orders of magnitude (i.e., Robin and Day Class II and III systems) (Dose and Wilson, 1978; Hunziker and Ludi, 1977; Goldsby and Meyer, 1984; Richardson et al., 1982; Richter and Brewer, 1993; Creutz and Taube, 1969; Creutz and Taube, 1972; Elias and Drago, 1972; Callahan et al., 1974; Callahan et al., 1975; Tom and Taube, 1975; Krentzien and Taube, 1976; Powers et al., 1976; Robin and Day, 1967; Creutz, 1983). In such systems, increased electronic coupling between metal centers is directly influenced by the energy and density of states of the BL. Increasing electronic density on the lowest-unoccupied &pgr;* molecular orbitals and the acceptor orbitals active in the metal-to-ligand charge transfer (MLCT) transitions that produce the excited state leads to enhanced communication. However, such systems rarely display high photoluminescence efficiencies. In fact, these systems rarel
Bard Allen J.
Richter Mark M.
Schmehl Russel H.
Ceperley Mary E.
Evans, Esq. Barry
IGEN International Inc.
Kramer Levin Naftalis & Frankel LLP
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