Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-20
2002-11-12
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C436S149000, C436S518000, C530S300000, C530S388100, C536S026600
Reexamination Certificate
active
06479233
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to electrogenerated chemiluminescent methods and compositions of modified hydrocarbons and aromatic heterocyclic compounds that occur in aqueous solutions with coreactants such as tri-n-propylamine (TPrA) and peroxydisulfate (S
2
O
8
2−
) and in particular, those that provide simultaneous analyses and internal referencing. More specifically, the present invention provides substituted polycyclic organic luminescing compounds that have ring carbons optionally replaced with at least one hetero atom selected from N and S, such as substituted diphenylanthracene, thianthrene and promazine, for electrogenerated chemiluminescent applications.
BACKGROUND OF THE INVENTION
Luminescing labels have been used to tag a variety of bioanalytes, such as enzymes, antibodies, antigens, peptides, nucleotides, saccharides or cells compliments of the analyte of interest, in immunoassays, DNA probe assays and fluorescent and separation assays. Of particular interest are labels which can be made to luminesce through electrochemical reaction schemes. Labels that luminesce as a result of electrochemical excitation are beneficial because of their sensitive and nonhazardous properties.
One analytical technique that exploits the benefits of these labels is electrogenerated chemiluminescence (ECL). ECL arises from an energetic electron transfer reaction between electrogenerated redox species represented by A
−
and D
+
, typically radical ions, to form an excited state (A* or D*) that emits in the visible region:
A+e*→A
−
E
A
(1)
D−e*→D
+
E
D
(2)
A
−
+D
+
→A+D*(or A*+D) (3)
D*→D+h&ngr; (4)
Faulkner and Bard in
Electroanalytical Chemistry
, Vol. 10, p. 1, 1977 provide several examples of nonaqueous systems of this type.
A. W. Knight and G. M. Greenway in the
Analyst
, 1994, 119, 879 discuss ECL precursors that can be generated in aqueous solution even with the limited potential range imposed by the oxidation and reduction of water. ECL can be achieved by the simultaneous oxidation of tris (2,2-bipyridine)ruthenium(II) (Ru(bpy)
3
2+
) and a coreactant (XY
Z
) capable of generating a suitable reductant upon oxidation via an oxidative-reduction mechanism such as:
Ru(bpy)
3
2+
−e*→Ru(bpy)
3
3+
(5)
XY
Z
−e*→XY
Z+1
(6)
Ru(bpy)
3
3+
+XY
Z
→Ru(bpy)
3
2+
+XY
Z+1
(6a)
XY
Z+1
→X
x
+Y
y
(x+y=z+1) (7)
Ru(bpy)
3
3+
+X
x
→Ru(bpy)
3
2+*
+X
x+1
(8)
Ru(bpy)
3
2+*
→Ru(bpy)
3
2+
+h&ngr; (9)
Ru(bpy)
3
2+
is a typical precursor (D) for these schemes and has been used in chemiluminescence (CL) reactions with amines as well as ECL investigations utilizing a number of different coreactants including oxalate (where XY
z
is C
2
O
4
2−
and X
x
is CO
2
−*
) and aliphatic amines such as tri-n-propylamine (TPrA; where XY
z
is Pr
3
N and X
x
is the radical that results from deprotonation of Pr
3
N
+
). In these ECL schemes, the coreactant can be oxidized either at the electrode via equation (6) or in solution by the emitter via equation (6a). As an alternative to the reaction scheme of equation (8), the excited state can also be generated by the sequence of equations (10) and (11), below, as previously discussed for oxalate and TPrA.
Ru(bpy)
3
2+
+X
x
→Ru(bpy)
3
+
+X
x+1
(10)
Ru(bpy)
3
+
+Ru(bpy)
3
3+
→Ru(bpy)
3
2+*
+Ru(bpy)
3
2+
(11)
For the oxalate coreactant, the reaction scheme of equation (7) involves cleavage of the carbon—carbon bond while for the TPrA coreactant it is believed that this step involves the loss of a proton from the &agr;-carbon. These ECL reactions have been used for the determination of both Ru(bpy)
3
2+
and oxalate.
To complement these oxidative-reduction examples, peroxydisulfate and Ru(bpy)
3
2+
undergo an analogous inversion of this scheme (a reductive-oxidation mechanism, equations (12)-(14) followed by (9)) when the initial reactants are reduced, rather than oxidized, in acetonitrile (MeCN)-water solutions (1:1 volume ratio). In this case, the reduction of S
2
O
8
2−
results in the formation of the strong oxidizing agent SO
4
−*
:
Ru(bpy)
3
2+
+e*→Ru(bpy)
3
+
(12)
S
2
O
8
2−
+e*→SO
4
−*
+SO
4
2−
(13)
Ru(bpy)
3
+
+S
2
O
8
2−
→Ru(bpy)
3
2+
+SO
4
−*
+SO
4
−2−
(13a)
Ru(bpy)
3
+
+SO
4
−
→Ru(bpy)
3
2+*
+SO
4
2−
(14)
By analogy to equations (10) and (11) for oxalate and TPrA, an alternative to equation (14) for generating the excited state with peroxydisulfate is:
Ru(bpy)
3
2+
+SO
4
−*
→Ru(bpy)
3
3+
+SO
4
2−
(15)
followed by equation (11) above.
The sensitivity and selectivity of these coreactant analyses has led to the recent commercial application of the Ru(bpy)
3
2+
/TPrA system. For example, electrochemiluminescent ruthenium- and osmium-containing labels have been used in methods for detecting and quantifying analytes of interest in liquid media, U.S. Pat. Nos. 5,310,687; 5,238,808; and 5,221,605, incorporated herein by reference. In addition, the application of electrogenerated chemi-luminescence (ECL) measurements to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M. T. et al. (1990)
Bioconjupate Chem
2:257-263). Although such applications provide an acceptable analysis technique, it is often necessary to provide a system that allows simultaneous analyses and internal referencing. The present invention provides additional electrochemiluminescent systems that may be used in place of or along side of existing systems.
SUMMARY OF THE INVENTION
The present invention relates to new labels where: 1) the emitter must be soluble in aqueous solution; 2) the emission wavelength must be distinct from that of Ru(bpy)
3
2+*
; 3) the oxidative or reductive electrochemistry must proceed within the relatively narrow potential range imposed by the oxidation and reduction of water; and 4) the oxidized or reduced intermediate must react with the electrogenerated coreactant intermediate allowing formation of the excited state.
An object of the present invention is to provide substituted polycyclic organic luminescing compounds, e.g., aromatic polycyclics, that may have ring carbon substitutions selected from at least one hetero N or S atom having these four (4) properties. Substituted diphenylanthracene, thianthrene and promazine exemplify such reagent systems for electrogenerated chemiluminescent applications.
Another object of the present invention is to provide substituted polycyclic organic luminescing compounds, e.g., aromatic polycyclics, that may have ring carbon substitutions, that offer a complementary label to Ru(bpy)
3
2+
in bioanalytical applications.
It is an object of the present invention to provide label-coreactant compositions that allow for simultaneous analyses.
It is a still further object of the present invention to provide a method of detecting a plurality of analytes by (1) providing at least a first biomolecule with a luminescent label that has an emission wavelength distinct from that of Ru(bpy)
3
2+*
; (2) providing a second biomolecule analyte with a ruthenium or osmium containing label; (3) adding at least one coreactant; and (4) exposing the labelled biomolecule analytes and coreactants to electrochemical reaction or excitation and measuring the resulting luminescence to detect the various biomolecule analytes present.
A further object of the present invention is to provide a composition involving the anodic oxidation of aqueous sodium
Bard Allen J.
Leland Jonathan K.
Richards Thomas
Evans, Esq. Barry
IGEN International Inc.
Kramer Levin Naftalis & Frankel LLP
Riley Jezia
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