Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1998-12-23
2003-06-10
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C556S015000, C556S016000, C556S136000, C556S138000, C556S143000, C585S025000, C435S007100, C435S006120, C530S391300
Reexamination Certificate
active
06576475
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an assay system and novel compounds for use therewith.
More particularly, it relates to an assay system which utilises the detection of light to qualitively or quantitively determine the presence of an analyte in a sample.
BACKGROUND OF THE INVENTION
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
The presence of such materials can often be determined by utilising an assay system which exploits the high degree of specificity which characterizes many biochemical and biological systems. Frequently used assay systems are based on, for example, antigen-antibody reactions, nucleic acid hybridization techniques, and protein-ligand reactions. In these assay systems, the presence or absence of an analyte is determined using an observable “label”—which has been attached directly or indirectly to one of the members of a binding pair.
Labels that can be detected luminometrically due to chemiluminescence or fluorimetrically as a result of the excitation of a fluorophore are particularly useful because of the sensitivity. In chemiluminescence light is emitted by a luminescent compound as a result of a chemical reaction. In fluorometry a fluorophore is excited with light of an appropriate wavelength and light that it emits is detected.
Electrochemiluminescence (ECL) is a specialised form of chemiluminescence in which the chemiluminescent reaction is preceded by an electrochemical redox reaction. Electrochemifluorescence is where a fluorophore is formed as the product of a chemical reaction which is preceded by an electrochemical redox reaction.
The present invention is concerned with assays that utilize electrochemiluminescence and electrochemifluorescence. It is also concerned with the enhancement of enzymatic reactions.
Amongst the prior art, the best known assay system utilising electrochemiluminescence (ECL) is a detection system developed by Igen Inc in which proteins and haptens are tagged with ruthenium II tris-bipyridine chelates which emit light when oxidised electrochemically in the present of tripropylamine which is also oxidised electrochemically.
Whilst this assay system has many benefits when compared to radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) it suffers a number of disadvantages compared with the invention described herein.
For example, sensitive detection systems for light (photomultipler tubes) are less sensitive to electrochemiluminescence at 620 nm (Igen) than to light at 425 nm (luminol), and each photon of light in the Igen system requires reoxidation of the label, whereas in the system described herein more than one photon of light is generated when the label is oxidised once.
DESCRIPTION OF THE INVENTION
It is one object of the present invention to provide an assay system which improves upon the current state of the art.
According to one aspect of the present invention there is provided a Bis (cyclopentadienyl) metal complex or a derivative thereof, for use as a label in an assay utilising electrochemiluminescence of fluorescence detection means.
Preferably, the Bis (cyclopentadienyl) metal complex is ferrocene, or a derivative thereof which gives rise to a ferricinium ion on electrochemical oxidation.
However, the use of other Bis (cyclopentadienyl) metal complexes or derivatives thereof are envisaged, such as, for example those of osmium or ruthenium.
The derivatives will include compounds which have the component of a binding pair linked to one of the cyclopentadienyl rings of the ferrocene, osmoscene or rutenocene. These derivatives are commonly referred to as ferrocenes, osmoscenes or ruthenocenes.
Thus, for example, a hapten or antigenic determinant might be linked to one or both of the cyclopentadienyl rings by reacting the amino group of a peptide with, for example, a carboxylic acid derivative of the ferrocene in a reaction as exemplified below:
Depending on the component of the binding pair, a number of well known chemical reactions can be utilised to covalently bond the first component of a binding pair to one or both of the cyclopentadienyl rings of, for example, ferrocene.
According to a further aspect of the present invention there is provided a Bis (cyclopentadienyl) metal complex derivative of the formula I:
Of course, depending on the reacting groups, the link could be a bond directly linking C
1
to the cyclopentadienyl ring.
Where M is a metal selected from the group consisting of iron, osmium and ruthenium;
L
1
is a link, and
C
1
is a first component of a binding pair.
Most preferably M is iron.
The link is most likely to be formed from reacting a carboxylic acid, aldehyde, thiol or hydroxy derivative of, for example, ferrocene with a reactant group from the first component of the binding pair.
For example, where ferrocene monocarboxylic acid is reacted with, for example, the amino group of an antigen, the link will be:
Of course, depending on the reacting groups, the link could be a bond directly linking C
1
to the cyclopentadienyl ring.
The link L
1
will usually be determined by the nature of C
1
. If C
1
is, for example, a protein, such as an antibody, L
1
is most likely to be a peptide bond (based on a carboxylic acid derivative of ferrocene and hydroxysuccinimide ester chemistry) and would be represented thus:
and gives rise to a ferrocene labelled protein which schematically looks like this:
If C
1
was DNA then L
1
could be a peptide bond (once again using hydroxysuccinimide ester chemistry). If C
1
is not a protein or DNA, then the L
1
may be an appropriate link. For example, where C
1
is the herbicide atrazine and the ferrocene is merceptomethylferrocene which has the structural formula:
The first component of the binding pair C
1
is preferably selected from:
i) Antibodies
ii) Antigens, and
iii) DNA
The derivative of formula I may include further modifications as shown in formula II
where
M, L
1
and C
1
are as defined in formula I,
L
2
is a link, and
F
1
is a fluorophore.
Preferably, the fluorophore is fluorescein, or another fluorophore, which can act as an acceptor of energy from the chemiluminescence reaction by resonance energy transfer.
The aim of attaching fluorescent compounds to the ferrocene is to utilise the phenomenon known as resonance energy transfer in which energy is transferred over short (effectively less than 10 nanometers) distances from an excited molecule (in this instance the product of a chemiluminescent reaction) to an fluorescent acceptor molecule.
The compounds of formula I and II may be further modified by the inclusion of substituents on the cyclopentadienyl ring which do not affect the ability of the compounds to bind a second component of the binding pair but which modulate the electrochemistry of the ferrocene.
Additional groups could be used to module the electrochemistry (for example ferrocene acetic acid is oxidised at a lower redox potential than ferrocene monocarboxylic acid) and so modulating substituents may be used.
According to yet a further aspect of the present invention there is provided a method for determining the presence of an analyte in a sample which is one of first and second components of a binding pair, the method comprising:
initiating, at an electrode, a reaction between the reactants of a chemiluminescent or a fluorescent detection system with a Bis (cyclopentadienyl) metal complex or derivative thereof, e.g. a complex or derivative of the formula I or II, and
identifying the presence of the analyte from a change in light emission due to the binding of the second component to the first component such that the Bis (cyclopentad
Schiffrin David
Wilson Robert
Chin Christopher L.
Merchant & Gould P.C.
University of Liverpool
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