Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
1999-03-15
2002-08-13
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C435S007100, C435S007200, C436S084000, C436S172000, C436S517000, C436S536000, C436S537000, C436S544000, C436S546000, C436S547000, C436S548000, C436S800000, C436S538000, C436S541000
Reexamination Certificate
active
06432722
ABSTRACT:
The invention concerns the stabilization and amplification of electrochemiluminescence signals in detection methods.
Luminescent metal complexes are known from the prior art. EP-A-0 178 450 discloses ruthenium complexes which are coupled to an immunologically active material where the ruthenium complexes contain three identical or different bicyclic or polycyclic ligands containing at least two nitrogen-containing heterocycles and at least one of these ligands is substituted with at least one group which renders it water-soluble such as —SO
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H or —COOH and at least one of these ligands is substituted directly or via a spacer group with at least one reactive group such as —COOH and the ligands are bound to the ruthenium via nitrogen atoms.
In addition the use of metal complexes as labelling reagents for an electrochemiluminescence detection method is also known (cf. e.g. EP-A-0 580 979, WO 87/06706, U.S. Pat. No. 5,238,108 or U.S. Pat. No. 5,310,687). Such an electrochemiluminescence detection method is based on transfer of the central atom of the metal complex e.g. ruthenium into an excited MLCT triplet state by electron transfer in a suitable measuring device. From this excited state it can relax into the basic state by means of a forbidden triplet-singlet transition with emission of a photon (cf. e.g. WO/90 05296, Leland and Powell, J.Electrochem.Soc. 137 (1990), 3127-313 1; Blackburn et al., Clin.Chem. 37 (1991), 1534-1539).
A disadvantage of this method is that the maximum obtainable level of the measurement signal is very limited due to a pronounced decrease of the signal intensity during the measuring phase. With the hitherto conventional procedure and the previous commercially used metal complexes, in particular ruthenium-(bipyridyl)
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complexes, this signal decrease already occurs after a measuring period of 100 ms and increases with the signal strength. This behaviour is not understandable on the basis of previous publications on the reaction mechanism. This decrease in the signal considerably limits the duration of the measurement and evaluation interval i.e. in practice to a maximum of 400 ms. In all previous cases this has led to a significantly reduced light yield which in turn results in a reduction of test sensitivity and test dynamics. Hence in practice it is only possible to use measurement and evaluation intervals of 400 ms at most without having problems with unspecific signals. Moreover imprecision and signal instabilities often occur in the decaying portion of the signal curve which lead to further inaccuracies.
New metal complexes with hydrophilic substituents or/and charge carriers on the linker are described in WO 96/03409 and WO 96/03410. Use of these complexes reduces an undesired adsorption which improves the stability and recovery in the detection method. Furthermore an increased quantum yield is described. However, no information is given about a possible extension of the maximum measurement interval in electrochemiluminescence measurements.
It was surprisingly found that the use of hydrophilic or/and charged metal complexes, for example according to EP-A-0 178 450, WO 96/03409 or WO 96/03410 results in considerable improvements in electrochemiluminescence detection since the signal decrease known for non-hydrophilic ruthenium-(bipyridine)
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complexes does not occur. Surprisingly the signal maintains its maximum value essentially over the entire duration of the measurement interval. This leads to a signal amplification or/and to an increase in the duration of the maximum possible measurement interval. This improvement is preferably achieved under method conditions in which a negative potential is applied before the measurement to the measuring electrode in the presence of the electrochemiluminescence cosubstrate.
Hence one subject matter of the invention is a method for the detection of an analyte in a sample by electrochemiluminescence measurement comprising the steps:
(a) providing an electrochemiluminescence device comprising a measuring electrode,
(b) bringing a conditioning liquid which contains an electrochemiluminescence cosubstrate into contact with the electrode
(c) adjusting conditions at the electrode which lead to the formation of an activated molecule of the layer containing the electrochemiluminescence cosubstrate on or/and in the boundary region of the electrode e.g. by applying a negative potential to the electrode,
(d) bringing the sample which contains a metal complex which contains at least one charge carrier or/and at least one hydrophilic group as an electrochemiluminescence marker group and an electrochemiluminescence cosubstrate into contact with the electrode,
(e) applying a potential to the electrode which enables an electrochemiluminescence reaction to proceed and measuring the electrochemiluminescence and
(f) correlating the measured luminescence with the presence or amount of the analyte in the sample.
The method according to the invention enables an at least two-fold to 5-fold higher measurement signal to be obtained due to the absence of a signal decrease compared to a conventional non-hydrophilic ruthenium-(bipyridyl)
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marker group. This higher signal strength enables the use of cheaper semiconductor detectors instead of the previously used photomultiplyer tubes. Furthermore under suitable measurement conditions, i.e. maintaining an adequate positive potential for the luminescence reaction and at the same time maintaining an adequate supply of electrochemiluminescence co-substrate, it is possible to generate a light quantity that remains constant per unit of time over any desired time interval. This enables a much larger quantity of light to be collected and achieves higher test sensitivities.
Additional advantages of the metal complexes used according to the invention are that less quenching by oxygen occurs and that there is less test interference for example by unspecific adsorption to test components or/and the electrode.
The electrochemiluminescence measuring device provided in step (a) of the method according to the invention can be a known device of the prior art (cf. for example N.R. Hoyle: The Application of electrochemiluminescence to Immunoassay-based Analyte Measurement, in: Bioluminescence and Chemiluminescence; Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge, September 1994, A.K. Campbell et al. (publ.) John Wiley & Sons; WO 89/10551; WO 90/11511). The device preferably comprises a measuring chamber which holds the measuring electrode, means for supplying and removing liquids to and from the measuring chamber and means for detecting the electro-chemiluminescence generated in the measuring chamber. In addition the device preferably contains magnetic means for immobilizing magnetic particles in the sample liquid on the measuring electrode.
Step (b) of the method comprises contacting the electrode with a conditioning liquid which contains an electrochemiluminescence cosubstrate which is effective as an oxidizing or reducing agent for the metal complex e.g. an amine or a persulfate. Tertiary amines such as trialkylamines are preferably used in which the alkyl residues each independently contain 1-4 C atoms. Tripropylamine is particularly preferred. The concentration of the cosubstrate in the conditioning liquid can be varied over wide ranges, it is preferably at least 1 mM, particularly preferably 10 to 500 mM and most preferably 100 to 300 mM. The conditioning liquid can additionally contain a suitable electrochemically inert buffer e.g. a phosphate buffer etc. and a detergent e.g. Thesit.
According to step (c) conditions are set on the electrode under which an attachment of activated and, in particular, reduced molecules of the cosubstrate occurs. The attachment can occur as an adsorption and also by formation of a boundary layer containing the cosubstrate molecules in the immediate vicinity of the electrode surface. For this purpose a negative potential is preferably applied to the electrode, preferably in the presence of the conditioning liquid. Th
Egger Martin
Josel Hans-Peter
Punzmann Gabriele
Amick Marilyn
Chin Christopher L.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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