Redox reversible bipyridyl-osmium complex conjugates

Details Redox reversible bipyridyl-osmium complex conjugates Redox reversible bipyridyl-osmium complex conjugates

1999-05-28

2002-03-05

Le, Long V. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007200, C435S005000, C435S006120, C435S007100, C436S544000, C436S547000, C436S548000, C436S805000, C436S806000, C436S084000, C436S172000, C436S501000, C436S517000, C436S518000, C436S536000

Reexamination Certificate

active

06352824

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to novel redox-reversible conjugates. More particularly the invention is directed bipyridyl complexed-osmium conjugates useful for detection and quantification of biologically significant analytes in a liquid sample.
BACKGROUND AND SUMMARY OF THE INVENTION
Therapeutic protocols used today by medical practitioners in treatment of their patient population requires accurate and convenient methods of monitoring patient disease states. Much effort has been directed to research and development of methods for measuring the presence and/or concentration of biologically significant substances indicative of a clinical condition or disease state, particularly in body fluids such as blood, urine or saliva. Such methods have been developed to detect the existence or severity of a wide variety of disease states such as diabetes, metabolic disorders, hormonal disorders, and for monitoring the presence and/or concentration of ethical or illegal drugs. More recently there have been significant advancements in the use of affinity-based electrochemical detection/measurement techniques which rely, at least in part, on the formation of a complex between the chemical species being assayed (the “analyte”) and another species to which it will bind specifically (a “specific binding partner”). Such methods typically employ a labeled ligand analog of the target analyte, the ligand analog selected so that it binds competitively with the analyte to the specific binding partner. The ligand analog is labeled so that the extent of binding of the labeled ligand analog with the specific binding partner can be measured and correlated with the presence and/or concentration of the target analyte in the biological sample.
Numerous labels have been employed in such affinity based sample analysis techniques, including enzyme labeling, radioisotopic labeling, fluorescent labeling, and labeling with chemical species subject to electrochemical oxidation and/or reduction. The use of redox reversible species, sometimes referred to as electron transfer agents or electron mediators as labels for ligand analogs, have proven to provide a practical and dependable results in affinity-based electrochemical assays. However, the use of electrochemical techniques in detecting and quantifying concentrations of such redox reversible species (correlating with analyte concentrations) is not without problem. Electrochemical measurements are subject to many influences that affect the accuracy of the measurements, including not only those relating to variations in the electrode structure itself and/or matrix effects deriving from variability in liquid samples, but as well those deriving from interference between multiple electroactive species, especially when assay protocols require detection or quantification of multiple electroactive species.
The present invention relates to novel diffusible, redox-reversible osmium-bipyridyl conjugates useful in immunosensors based on either indirect amplified electrochemical detection techniques or on direct electrochemical measurement of detectable species with microarray electrodes under bipotentiostatic control. An Os-bipyridyl complex can, for example be covalently attached to a peptide which has amino acid sequence of the binding epitope for an antibody. When Os complex/peptide conjugate is bound to antibody, the conjugate does not function electrochemically; it is said to be “inhibited”. Typically an analyte present in sample will compete with Os-bipyridyl complex/peptide conjugate for the limited number of binding sites on the antibody. When more analyte is present, more free Os-bipyridyl complex/peptide conjugate will be left in an unbound diffusible state producing higher current at a sensor electrode, i.e. one of the working electrodes where measured events (oxidation or reduction) are taking place. In the opposite case, when less analyte is present, more indicator/peptide conjugate will be bound to antibody resulting less free conjugates and producing lower current levels at the working electrodes. Therefore the current detected at either one of the working electrodes will be a function of analyte concentration.
It is frequently desired to measure more than one analyte species in a liquid sample. Measurement of multiple species in a mixture has been achieved with photometry and fluorescence, via selection of the appropriate wavelengths. Electrochemical measurements of a single species in a complex mixture are routinely made by selecting a potential at which only the desired species is oxidized or reduced (amperometry) or by stepping or varying the potential over a range in which only the desired species changes its electrochemical properties (AC and pulse methods). These methods suffer from disadvantages including lack of sensitivity and lack of specificity, interference by charging and matrix polarization currents (pulse methods) and electrode fouling due to the inability to apply an adequate over potential. Moreover, electrochemical measurements are complicated by interference between the multiplicity of electroactive species commonly extant in biological samples.
Electrode structures which generate steady state current via diffusional feedback, including interdigitated array electrodes (IDAs) (
FIGS. 1 and 2
) and parallel plate arrangements with bipotentiostatic control are known. They have been used to measure reversible species based on the steady state current achieved by cycling of the reversible species. A reversible mediator (redox reversible species) is alternately oxidized and reduced on the interdigitated electrode fingers. The steady state current is proportionate to mediator concentration (
FIG. 3
) and limited by mediator diffusion. A steady state current is achieved within seconds of applying the predetermined anodic (more positive) and cathodic (less positive or negative) potentials (
FIG. 6
) to the microelectrode array. The slope of a plot of the IDA current vs. mediator concentration is dependent on IDA dimensions, and the slope increases with narrower electrode spacings (FIG.
7
).
The present invention provides novel osmium-bipyridyl complex conjugates useful in a method for measuring multiple analyte species in the same sample, and optimally on the same electrode structure, thus improving the accuracy of the relative measurements. The present conjugates can be used with other electroactive conjugate species having unique redox potentials to provide an electrochemical biosensor with capacity to provide improved accuracy. Analyte concentration can be measured/calculated from electrometric data obtained on the same liquid sample with the same electrode structure, thereby minimizing perturbations due to variability in sample or electrode structure.
The diffusible osmium conjugates of this invention find use in assay based on the principle of diffusional recycling, where a diffusible redox reversible species is alternately oxidized and reduced at nearby electrodes (the working electrodes), thereby generating a measurable current. As alternate oxidation and reduction is required for measurement, only electroactive species which are electrochemically reversible at the predetermined redox potential are measured thereby eliminating, or at least reducing, the impact or interference from non-reversible electroactive species in the sample or other reversible-redox species having unique (at least 50 millivolts different) redox potential. Redox reversible species having different oxidation potentials can be independently measured in a mixture by selecting and bipotentiostatically controlling the oxidizing and reducing potentials for neighboring electrode pairs so that only the species of interest is oxidized at the anode and reduced at the cathode. When the working electrodes are dimensioned to allow diffusional recycling of the redox-reversible-species at the selected oxidizing and reducing potentials appropriate for that species, a steady state current is quickly established through the sample and the electrode structure. The magni

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