Electrochemical assays

Details Electrochemical assays Electrochemical assays

2000-05-19

2003-04-22

Warden, Sr., Robert J. (Department: 1744)

Electrolysis: processes, compositions used therein, and methods

Electrolytic analysis or testing

Involving enzyme or micro-organism

C204S403010, C204S403120, C435S007100, C436S544000, C436S806000

Reexamination Certificate

active

06551495

ABSTRACT:

FIELD OF THE INVENTION
This invention relates, inter alia, to an assay method and to an assay device.
BACKGROUND OF THE INVENTION
This invention relates in general to electrochemical assays. In such assays, the presence of an analyte of interest in a sample causes a measurable change in an electrochemical property of a sensor device. Typically, electrochemical assays are classified as “potentiometric” or “amperometric”, which measure changes in either potential or current respectively.
A number of electrochemical assays have been described. For example, enzyme electrodes have been used for the direct measurement of biomolecules such as glucose, urea, amino acids, and others in physiological samples. These enzyme electrodes include a selective enzyme layer immobilized at the surface of a potentiometric or amperometric device that senses the steady state concentration of a product formed in the immobilized layer as the substrate for the enzyme diffuses into this reactive film.
Other assays involve the use of Nafion™ (E.I. Du Pont de Nemours and Co. Inc.) film. Nafion™ is a polyanionic perfluorosulphonate ionomer with permselective properties large hydrophobic cations rather than small, hydrophilic cations tend to accumulate in the polymer via cationic exchange.
Limoges & Degrand (1993 Analytical Chemistry 65, 1054-1060) described a model system is which a Nafion™-coated electrode was used to detect the presence of amphetamine. The assay took the form of a competitive immunoassay, in which amphetamine in a sample competed with known amounts of labelled amphetamine for binding to amphetamine—specific antibodies. The competitor amphetamine was labelled with cobalticenium, which is a redox label.
The basis for the assay is that once bound by antibody, the labelled amphetamine is excluded from the Nafion™ film because of the large size of the resulting amphetamine/antibody complex. Thus, in the presence of high concentrations of amphetamine in the sample, there is more free labelled amphetamine available for ion exchange and incorporation into the Nafion™ film. Accordingly, the current corresponding to the oxidation or reduction of the cobalticenium-label in the film is proportional to the concentration of amphetamine in the introduced sample.
This sort of assay has several disadvantages and accordingly has not been widely adopted. In particular it requires the performance of a number of reaction steps before detection can effected by the Nafion™-coated electrode. The assay apparatus disclosed by Limoges & Degrand comprised a sensor which was not disposable or re-usable.
A different sort of electrochemical assay involves the use of an “Antibody responsive membrane electrode”, as described by Solsky & Rechnitz (1979 Science 204, 1308; 1981 Anal. Chim. Acta 123, 135). The antibody responsive membrane comprised ionophores (crown ethers) within a polyvinyl chloride matrix, the ionophores being conjugated to a hapten in such a way that the haptens projected from the surface of the membrane. The membrane was mounted in the tip of a conventional potentiometric membrane electrode. The addition of a sample containing antibodies specific for the hapten would allow antibodies to bind to the hapten which, in some unknown way, altered the electrochemical properties of the ionophores, changing the potential across the membrane.
The manner in which the device works is not understood, making it impossible rationally to design improvements thereon. Also, the associated detection system is large and cumbersome and not readily re-usable.
WO 89/11649 discloses a device for use in an electrochemical assay, the device comprising an electroactive polymer layer, within which layer are entrapped antibody molecules having binding specificity for an analyte of interest. Binding of the analyte of interest to the antibody inhibits the flow of counter ions from the environment surrounding the device into the space around the electroactive polymer, hence inhibiting electron flow to or from the polymer during a redox reaction. There is no disclosure of the electrochemical properties of the polymer being affected by the binding of a binding partner directly to the polymer.
WO 95/29199 discloses an electrode having a similar arrangement, wherein binding of a binding partner to a chemical moiety attached to an electroactive polymer can indirectly affect the electrochemical properties of the polymer. There is no disclosure of a binding partner having binding specificity for the electroactive polymer itself. A similar arrangement is disclosed in EP 0239969.
WO 93/25907 discloses a competition assay system involving an antigen of interest, and a derivatised antigen carrying a redox label, competing for binding to limiting amounts of antibody. Excess redox-labelled antigen is bound electrostatically to a polymer-coated film, so as to alter the redox potential across the film, which is measured in a conventional manner. The polymeric layer is not electroactive and essentially non-conducting.
EP 0402917 discloses a biosensor operating on a very similar principle to that disclosed in WO 89/11649: a conducting surface with an electroactive surfactant coating is modified by inclusion of one member of a specific binding pair. The analyte of interest is the other member of the specific binding pair. Binding of the reciprocal members of the specific binding pair blocks the movement of counter ions. There is no binding event involving binding directly to the electroactive surfactant.
WO 97/27474 discloses a method of determining the presence of an analyte of interest, whereby an electrode is coated with a member of a specific binding pair. In the absence of analyte of interest (which is the reciprocal member of the specific binding pair), an electroactive redox molecule can come into proximity with the electrode and donate electrons to, or accept electrons from, the electrode. This process is inhibited by the analyte of interest, which blocks the redox molecule from coming into proximity with the electrode. Thus there is no disclosure of the direct binding of a binding partner to an electroactive molecule so as to modify the electrochemical properties thereof. (All documents cited in the present specification are incorporated herein by reference).
The present invention aims to provide an improved type of electrochemical assay, particularly one which will be suitable for forming the basis of disposable, easy-to-use assay devices.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of: providing an electrically conducting solid support having immobilised thereon a chemical moiety having an electroactive portion with an electrochemical property capable of being modulated in a detectable manner directly by the binding thereto of a binding partner having specific binding activity for the electroactive portion; and causing the binding partner to contact the electroactive portion of the chemical moiety as a result of the presence in the sample of the analyte of interest.
In a second aspect, the invention provides a component for a device for detecting the presence of an analyte of interest in a sample, the component comprising an electrically conducting solid support having immobilised thereon a chemical moiety, said chemical moiety comprising an electroactive portion with an electrochemical property capable of being directly modulated in a detectable manner by the binding thereto of a binding partner having specific binding activity for the electroactive portion.
The method of the invention may be used qualitatively, so as to indicate the presence or absence of the analyte of interest. Alternatively, and preferably, the method may be used quantitatively so as to indicate the amount (in relative or absolute units) of the analyte present. It will be apparent that all sorts of substances may be analytes of interest, although biological molecules (i.e. molecules present in or produced by living organisms) will typically b

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