CA1254616A - Electrochemical enzymic assay procedures - Google Patents
Electrochemical enzymic assay proceduresInfo
- Publication number
- CA1254616A CA1254616A CA000522577A CA522577A CA1254616A CA 1254616 A CA1254616 A CA 1254616A CA 000522577 A CA000522577 A CA 000522577A CA 522577 A CA522577 A CA 522577A CA 1254616 A CA1254616 A CA 1254616A
- Authority
- CA
- Canada
- Prior art keywords
- enzyme
- redox
- substrate
- assay
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/806—Electrical property or magnetic property
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/904—Oxidation - reduction indicators
Abstract
ABSTRACT
Electrochemical detection of mediator level is employed in a method of assay using a redox enzyme and redox substrate to detect conversion to an effective mediator by an assay enzyme label of a compound which is non-mediating under the assay conditions.
Electrochemical detection of mediator level is employed in a method of assay using a redox enzyme and redox substrate to detect conversion to an effective mediator by an assay enzyme label of a compound which is non-mediating under the assay conditions.
Description
gkr/m~c52471 page 1 M&C FOLIO: 230P52471 WANGDOC: 0020C
ELECTROCHEMICAL ENZYMIC ASSAY PROCEDURES
This invention relates to electrochemical enzymic assay procedures.
More particularly, but not exclusively, the present invention relates to techniques and equipment useful in immunoassays. The invention is especially concerned with immunoassays of use for instance in a wide range of diagnostic or investigative techniques for humans or animals, or for example in investigating and monitoring of food chemistry or process chemistry.
~n example of a known enzyme-labelled assay is the heterogeneous immunoassay known as the ELISA
(enzyme-labelled immunosorbent assay) technique. In the commonly adopted ELISA sandwich procedure, the technique will typically involve three steps, separated by washes and rinses:
(a) immobilisation of a suitable antibody on a support sur~ace, such as polymer beads or possibly the walls o~ a plastics vessel or recesses in a plastics dish;
(b) contacting the surface carrying immobilised antibody with the sample to be investigated, whereby gkr~m~cS2471 page 2 a speci~ic analyte in the sample can bind to the antibody in a proportion dependent on the analyte concentration; and (c) further contacting the surface with an antibody labelled with an enzyme, this enzyme-labelled antibody binding only to the antigen and thus providing at the surface the enzyme in a measurable concentration corresponding to the analyte concentration in the sample.
The sandwich technique has certain limitations, most especially in that it can only be used with analyte molecules having at least two epitopes, so as to react both with the immobilised antibody and with the enzyme-labelled antibody.
In the alternative ELISA competitive procedure for assay of a specific analyte, a known concentration of enzyme-labelled specific analyte is added to a sample which Is then contacted with a sllrface supporting an known concentration of antibody, whereby labelled and unlabelled analyte compete proportionally for binding sites, and a srlbsequent measurement of enzyme concentration will indicate the labelled/unlabelled ratio.
gkr/m~cs2471 page 3 Most of the existinq ~LISA and other enzymic assay procedures use a colour-forming substrate which at the end of the incubation releases a chromophore whose concentration can be determined spectrophotometrically. The use of colour-forming substrates may have their drawbacks in view of the length of time required to obtain a result and also the instability o~ some chcomophores. Thus there is a need for an improved assay procedure.
As an altecnative to colour detection, an instance of electrochemical detection is described in Anal. Chem.
(1984) 56, 2355, where the assay enzyme label converts an electroinactive compound to an electroactive compound which can be electrochemically detected. The electroactive compound, phenol, has a redox potential of +750 mV, and as such the method is not generally applicable because the system will encounter other components which will oxidise at the potential of 750 mV. In practice, this system therefore can not be adopted with .~ blood or serum sample.
The present invention especially relates to assay techniques llt ilising amplification of a response, particularly ~oc detecting the presence o~ or monitorin~
the level ot one or more substances in a mixture of components, where the analyte is present in particularly gkr/m~c52471 page 4 low concentra~ion and/or in admixture with potentialy interfering substances, and wherein the presence or absence of the response is linked to the extent of a specific binding reaction.
European Patent Specification 78636 describes the use of a mediator compound such as a ferrocene derivative to transfer electrons between an enzyme and an electrode when the enzyme catalyses a redox reaction on a substrate. Such a procedure is of major utility for electrochemical detection of glucose as substrate by adoption of a glucose oxidase as the redox enzyme.
In more recent European Patent Specifications such as EP125139, assays are described in which the concentration of substrate is fixed. and the concentration of available mediator compound represents the vaEiable factor. In this way, the procedure o~
EP~8636 is inverted, and the combination of the redox enzyme and substrate allows electrochemical detection of the mediator.
The present invention is concerned with a further development o~ assays based on electrochemical detection of mediator levels using a redox enzyme and substrate.
gkr/m~c52g71 paqe 5 Objects of the Invention A principal object of the present invention is the provision of improved enzyme-labelled assays. More specifically, a particular object of the present invention is an improved ELISA ~echnique. Other objects include reagents for novel enzyme-labelled assays.
SummarY of the Invention The ~resent invention provides for the use of electrochemical detection of mediator level using a redox enzyme and redox substrate to detect conversion in an assay of a non-mediating compound to an effective mediator by an assay enzyme label.
Thus, the present invention employs electrochemical procedures ~or detecting the effective mediator generated from a compound which is not a mediator under the assay conditions. The efective mediator is generated by an enzyme used as label in an assay. In turn, this allows the determination of an analyte in d qualitatative or quarltitative sense. A relatively sensltive and fast immunoassay in the nanomolar range can be achieved.
~2~
The conditions are selected so that a substrate for the assay enzyme label is not effective as a mediator, and the action of the assay enzyme label converts the substrate to an effective mediator. The non-mediating compound may itself be a potential mediator, except that under the selected conditions the compound is not effective as a mediator, and is thus ~non-mediating~, In this way, there is provided a method of assay in which an enzyme label is employed, characterized in that the label is electrochemically detected with a redox enzyme and a redox substrate as a result of conversion by the labelling enzyme of a non-mediating substrate from an ineffective mediator to an effective mediator for mediating electron transfer between the redox enzyme and an electrode.
The use of assay enzyme labels together with a media-tor/redox enzyme system facilitates the extension of known spe-cific bindin~ assays to finer levels of resolution than those previously accomplished, without the requirement for pretreatment of samples to remove interfering substances.
The present system is distinguished from that described in the Canadian Patent application No. 502,464 filed February 2, 1986 in that the redox substrate for the present invention is fr~ely gkr/m~c52471 pa~e 7 diffusing before conversion into the effective mediator, whereas in the prior system, the potentially active component is inactivated by linkage to a relatively large molecule of an immobiliser material.
Preferred E~bodiments of the Invention The assay of this invention is typically an immunoassay. The assay suitably involves a sandwich or competitive procedure. Thus, in a preferred aspect, the assay of this invention is a sandwich ELISA or a competitive ELISA.
For a sandwich assay, a mono- or polyclonal antibody is immobilized, the sample is contacted with the immobilized antibody, enzyme-labelled antibody is then contacted with the system, the substrate is added with the system conditions being such that the substrate is non-mediating, and then the electrochemical determination of the effective mediator is carried out with the redox enzyme and the redox substrate.
For a competitive assay, the sample and an enzyme-labelled analyte are contacted with immobilized antibody, the non-mediating substrate is added, and the electrochemical determination is effected.
~;:5~
gkr/m&c52471 page 8 Generally, in the electrochemical detection, the non-mediating substrate will preferably itself be a potential mediator rendered non-mediating because of the operating conditions. To this end, an electrode is poised at a preselected potential which is intermediate between the redox potential of the substrate for the redox enzyme and the product, such that only the product havinq a redox potential lower than that at which the electrode is poised can undergo a redox reaction at the electrode.
By employing the product as a mediator which exhibits electron transfer at a preselected potential, and which can be formed by an enzyme reaction from a substrate which exhibits no mediator activity at the preselected potential, it is possible to determine the extent of conversion of the substrate into the product and consequently the presence or activity of the enzyme.
It will be cea~ised that a variety of assay protocols may be envisaged, in which the conversion of the substrate into the product is accompanied by an increase of mediator a(tivity under the conditions of the assay.
The substrdte tor the labelling enzyme is a mediatoc derivative which is non-mediating under the assay conditions. The nature of the derivative will depend on the nature ot ~he labelling enzyme.
~z~
gkr/m~c52471 page 9 Examples of mediator compounds which can form the basis of the substrate include metallocenes; ruthenium compounds; carboranes; conductive salts of TCNQ;
haloanils and derivatives thereof; viologens; quinones;
alkyl substituted phenazine derivatives; bis-cyclo pentadienyl (Cp)2MXX complexes of transition metals;
and phenol derivatives including ferrocene-phenol and indophenol compounds. Such compounds are readily decivatised to provide suitable substrates.
By the use of water-soluble, air-insensitive derivatives it is possible that the assay may be performed in samples of biological fluids which have undergone a minimum of pretreatment.
Suitable derivatives which provide the substrate for a given enzyme can readily be synthesised. For example, phospha~e derivatives are recognised by acid or alkaline phosphatases.
Conveniently, the substrate for a phospha~ase labelling enzyme is a metallocene derivative, preferably a ferrocene derivative.
Examples of suitable phosphate derivatives of ferrocene gkr/m~c52471 page lO
are:
~Co~ l ~ O - P- ~ H
~ o~
,_ ~O~ o ~CH~,-O- P-o~
In a pacticularly preferred embodiment of the yresent invention, the redox substrate i5 the phosphate compound 5(I), otherwise termed, C5H5.Fe.C5H5.CO.NH-C6H4-OPo3 and herein called [N-ferrocenoyl]-4-aminophenyl phosphate. This compound is a novel compound and also forms part of the present invention.
The compound [N-ferrocenoyl]-4-aminophenyl phosphate has an El/2 of +390m~ against a standard calomel electrode ("SCE"). When the compound is treated with a phosphatase, such as acid or alkaline phosphatase, it is converted into the phenol derivative;
15(A) c5H5 Fe c5H5 co NH C6Hq which has an oxidation potential of +180mV against SCE. Hence, when an amperometric measurement is made at about +230m~ against SCE, only the catalytic curren~
4~
gkr/m~c52471 page 11 due to compound (A) is detected, and the compound (I) shows no electrode response at this potential.
In one alternative preferred embodiment, the substrate is a phenol derivative such as 2,6-dichloroindophenyl phosphate, which is of the formula (III) O ~ I (III).
Cl ~H
Although phosphatases are particularly exemplified, the the invention is applicable to any enzyme substrate coupled to any electroactive species. Therefore, this invention is not limited to such phosphate compounds for use with a phosphatase as labelling enzyme. Mediator derivatives can readily be synthesised so as to be coupled to other enzymic systems, and accordingly the nature of the reaction catalysed by the assay enzyme label is not critical. For example, the enzyme can be a protease, amidase or hydrolase.
According to another aspect of the present invention there is provided a system for detecting the activity of a first enzyme, comprising;
gkr/m~cS2471 page 12 a) a freely diffusinq reagent convertible by the first enzyme into a product exhibiting mediator activity thereby enabling detection of the product, and, b) detection means comprising a redo~ enzyme, a substrate for the redox enzyme, and an electrode, whereby, in the presence of an active form of the first enzyme, the reagent is converted into the product and a detectable change occurs in mediated electron transfer to the electrode. Typically, the characteristic mediator property of ~he product is electron transfer at a preselected electrode potential.
Such a system can be used to provide an assay for the first enzyme. whether or not the first enzyme is a label.
In another aspect of the present invention there is provided a bioelectrochemical cell (BEC) incorporating a substrate for an assay enzyme label whereby consumption of the substrate by said assay enzyme label produces a change in output from the BEC enabling said enzyme to be detected. ~he B~C comprises a second enzymic reaction system which provides the electrical output through conversion ot the substrate to a mediator.
For the second enzymic reaction used in the BEC, d ferrocene mediator is preferred for mediating electron transfer in the reaction between glucose and glucose gkr/m~cS2471 page 13 oxidase. Such a system results in amplification of the effect of the redox potential change in the ferrocene compound, owing to the mediatoc action of the ferrocene compound. The chemical change in the ferrocene compound or other substrate as it is consumed by the enzyme analyte can have a marked effect on the mediator characteristics, and hence on the output from the BEC.
According to a yet further aspect of the invention there is provided a method for detecting the activity of a non-redox enzyme, in the presence at least one electrode poised at a fixed potential, which method comprises:
a) treating a sample suspected of containing the non-redox enzyme with a reagent which has a redox potential higher than the poised potential of the lS electrode, the reagent being a substrate for the non-redox enzyme, and convertible by the activity of the non-redox enzyme into a product which acts as a mediator compound and has a redox potential lower than the poised potential of the electrode, and, b) treatinq the sample with a redox enzyme, and a substrate for the redox enzyme whereby in the presence of a mediatoc compound having a redox potential lowec than the poised potential of the electrode, a measurable transfer of charge to the electrode occurs.
The present Invention enables the detection of t~e 6~
gkr/m~c52471 page 14 activity of enzymes which do not themselves exhibit redox activity but which in one aspect of the invention can catalyse the transformation of a substrate in to a product which has mediator properties under the conditions of assay.
It should be noted that while the invention will be described heceaftec with refecence to a phosphatase enzyme, the invention extends to other assay systems and to other reagents. For example, it is envisaged that In other labelling enzymes may be employed, each being capable of reacting with a reagent to produce a product which has mediator properties. Moreover, while the invention is described with reference to the labeling of antigens, it is envisaged that enzyme labels in other types of specific binding reactions, such as those between complementary strands of nucleic acid, may be employed.
SUMMARY OF THE DRAWINGS
Figure 1 is a general scheme ~or an assay of the presen~
invention;
Figure 2 is a plot of peak current at 180mV against phosphatase concentration, obtained in Example 5;
gkr/m~cS2471 page lS
Figure 3 is a plot of corrected peak current at l~OmV
against oestriol concentration for a set of serum standards in the assay of Example 6; and Figure 4 comprises a set of cyclic voltammograms obtained in Example 7tb).
In Figure 1, mediator compound (Mi) is for example the compound (I). This compound is a water soluble reagent which with ar- electrode poised at a potential of ~230mV
against SCE exhibits no mediator activity between glucose oxidase (GOD~ and its substcate glucose.
In the presence of a phospha~ase enzyme (E) pre~ent as a label, the compound (I) is converted into the compound (A), which does exhibit mediator activity (Mred~oX) between glucose oxidase (GOD), its substrate glucose, and an electrode poised at ~230mV against SCE.
Thus, the system of Figure 1 enables the detection of the activity of the phosphatase enzyme. The detected current at the electrode is due only to the presence of compound (A).
~0 In Figure 1, the phosphatase enzyme is a label for the antibody (Ab), and consequently the concentration of the phosphatase enzyme is affected by specific binding 1;2~L6 gkr/m6c52471 page 16 reactions between the antibody and its corresponding antigen. The steps of exposing the antibody to the antigen and separating bound and free fractions may be carried out in accordance with known techniques of S immunoassay. In a variation, the phosphatase enzyme can be a label for the antigen.
SPECIFIC EXAMPLES OF THE INVENTION
In order that the invention may be better understood and carried into effect, various embodiments given by way of non-limiting example will now be described with reference, where appropriate, to the accompanying drawings.
Example 1 Preparation o~ a Substrate, Compound (I) (a) Preparation of Compound (A):
~ ~\~ 0~
F~ (A) Ferrocene monocarboxylic acid was converted to the gkr/m~cS2471 page 17 ferrocenoyl chloride by the method described in J. Org.
Chem., 24, ~80-281, (1959).
Ferrocenoyl chlo~ide (lmM) was then dissol~ed in 5ml of ice-cold dry pyridine. To this solution was added in S one portion a solution of p-aminophenol (1.1 mM) in Sml dry pyridine. The reaction was allowed to proceed foc
ELECTROCHEMICAL ENZYMIC ASSAY PROCEDURES
This invention relates to electrochemical enzymic assay procedures.
More particularly, but not exclusively, the present invention relates to techniques and equipment useful in immunoassays. The invention is especially concerned with immunoassays of use for instance in a wide range of diagnostic or investigative techniques for humans or animals, or for example in investigating and monitoring of food chemistry or process chemistry.
~n example of a known enzyme-labelled assay is the heterogeneous immunoassay known as the ELISA
(enzyme-labelled immunosorbent assay) technique. In the commonly adopted ELISA sandwich procedure, the technique will typically involve three steps, separated by washes and rinses:
(a) immobilisation of a suitable antibody on a support sur~ace, such as polymer beads or possibly the walls o~ a plastics vessel or recesses in a plastics dish;
(b) contacting the surface carrying immobilised antibody with the sample to be investigated, whereby gkr~m~cS2471 page 2 a speci~ic analyte in the sample can bind to the antibody in a proportion dependent on the analyte concentration; and (c) further contacting the surface with an antibody labelled with an enzyme, this enzyme-labelled antibody binding only to the antigen and thus providing at the surface the enzyme in a measurable concentration corresponding to the analyte concentration in the sample.
The sandwich technique has certain limitations, most especially in that it can only be used with analyte molecules having at least two epitopes, so as to react both with the immobilised antibody and with the enzyme-labelled antibody.
In the alternative ELISA competitive procedure for assay of a specific analyte, a known concentration of enzyme-labelled specific analyte is added to a sample which Is then contacted with a sllrface supporting an known concentration of antibody, whereby labelled and unlabelled analyte compete proportionally for binding sites, and a srlbsequent measurement of enzyme concentration will indicate the labelled/unlabelled ratio.
gkr/m~cs2471 page 3 Most of the existinq ~LISA and other enzymic assay procedures use a colour-forming substrate which at the end of the incubation releases a chromophore whose concentration can be determined spectrophotometrically. The use of colour-forming substrates may have their drawbacks in view of the length of time required to obtain a result and also the instability o~ some chcomophores. Thus there is a need for an improved assay procedure.
As an altecnative to colour detection, an instance of electrochemical detection is described in Anal. Chem.
(1984) 56, 2355, where the assay enzyme label converts an electroinactive compound to an electroactive compound which can be electrochemically detected. The electroactive compound, phenol, has a redox potential of +750 mV, and as such the method is not generally applicable because the system will encounter other components which will oxidise at the potential of 750 mV. In practice, this system therefore can not be adopted with .~ blood or serum sample.
The present invention especially relates to assay techniques llt ilising amplification of a response, particularly ~oc detecting the presence o~ or monitorin~
the level ot one or more substances in a mixture of components, where the analyte is present in particularly gkr/m~c52471 page 4 low concentra~ion and/or in admixture with potentialy interfering substances, and wherein the presence or absence of the response is linked to the extent of a specific binding reaction.
European Patent Specification 78636 describes the use of a mediator compound such as a ferrocene derivative to transfer electrons between an enzyme and an electrode when the enzyme catalyses a redox reaction on a substrate. Such a procedure is of major utility for electrochemical detection of glucose as substrate by adoption of a glucose oxidase as the redox enzyme.
In more recent European Patent Specifications such as EP125139, assays are described in which the concentration of substrate is fixed. and the concentration of available mediator compound represents the vaEiable factor. In this way, the procedure o~
EP~8636 is inverted, and the combination of the redox enzyme and substrate allows electrochemical detection of the mediator.
The present invention is concerned with a further development o~ assays based on electrochemical detection of mediator levels using a redox enzyme and substrate.
gkr/m~c52g71 paqe 5 Objects of the Invention A principal object of the present invention is the provision of improved enzyme-labelled assays. More specifically, a particular object of the present invention is an improved ELISA ~echnique. Other objects include reagents for novel enzyme-labelled assays.
SummarY of the Invention The ~resent invention provides for the use of electrochemical detection of mediator level using a redox enzyme and redox substrate to detect conversion in an assay of a non-mediating compound to an effective mediator by an assay enzyme label.
Thus, the present invention employs electrochemical procedures ~or detecting the effective mediator generated from a compound which is not a mediator under the assay conditions. The efective mediator is generated by an enzyme used as label in an assay. In turn, this allows the determination of an analyte in d qualitatative or quarltitative sense. A relatively sensltive and fast immunoassay in the nanomolar range can be achieved.
~2~
The conditions are selected so that a substrate for the assay enzyme label is not effective as a mediator, and the action of the assay enzyme label converts the substrate to an effective mediator. The non-mediating compound may itself be a potential mediator, except that under the selected conditions the compound is not effective as a mediator, and is thus ~non-mediating~, In this way, there is provided a method of assay in which an enzyme label is employed, characterized in that the label is electrochemically detected with a redox enzyme and a redox substrate as a result of conversion by the labelling enzyme of a non-mediating substrate from an ineffective mediator to an effective mediator for mediating electron transfer between the redox enzyme and an electrode.
The use of assay enzyme labels together with a media-tor/redox enzyme system facilitates the extension of known spe-cific bindin~ assays to finer levels of resolution than those previously accomplished, without the requirement for pretreatment of samples to remove interfering substances.
The present system is distinguished from that described in the Canadian Patent application No. 502,464 filed February 2, 1986 in that the redox substrate for the present invention is fr~ely gkr/m~c52471 pa~e 7 diffusing before conversion into the effective mediator, whereas in the prior system, the potentially active component is inactivated by linkage to a relatively large molecule of an immobiliser material.
Preferred E~bodiments of the Invention The assay of this invention is typically an immunoassay. The assay suitably involves a sandwich or competitive procedure. Thus, in a preferred aspect, the assay of this invention is a sandwich ELISA or a competitive ELISA.
For a sandwich assay, a mono- or polyclonal antibody is immobilized, the sample is contacted with the immobilized antibody, enzyme-labelled antibody is then contacted with the system, the substrate is added with the system conditions being such that the substrate is non-mediating, and then the electrochemical determination of the effective mediator is carried out with the redox enzyme and the redox substrate.
For a competitive assay, the sample and an enzyme-labelled analyte are contacted with immobilized antibody, the non-mediating substrate is added, and the electrochemical determination is effected.
~;:5~
gkr/m&c52471 page 8 Generally, in the electrochemical detection, the non-mediating substrate will preferably itself be a potential mediator rendered non-mediating because of the operating conditions. To this end, an electrode is poised at a preselected potential which is intermediate between the redox potential of the substrate for the redox enzyme and the product, such that only the product havinq a redox potential lower than that at which the electrode is poised can undergo a redox reaction at the electrode.
By employing the product as a mediator which exhibits electron transfer at a preselected potential, and which can be formed by an enzyme reaction from a substrate which exhibits no mediator activity at the preselected potential, it is possible to determine the extent of conversion of the substrate into the product and consequently the presence or activity of the enzyme.
It will be cea~ised that a variety of assay protocols may be envisaged, in which the conversion of the substrate into the product is accompanied by an increase of mediator a(tivity under the conditions of the assay.
The substrdte tor the labelling enzyme is a mediatoc derivative which is non-mediating under the assay conditions. The nature of the derivative will depend on the nature ot ~he labelling enzyme.
~z~
gkr/m~c52471 page 9 Examples of mediator compounds which can form the basis of the substrate include metallocenes; ruthenium compounds; carboranes; conductive salts of TCNQ;
haloanils and derivatives thereof; viologens; quinones;
alkyl substituted phenazine derivatives; bis-cyclo pentadienyl (Cp)2MXX complexes of transition metals;
and phenol derivatives including ferrocene-phenol and indophenol compounds. Such compounds are readily decivatised to provide suitable substrates.
By the use of water-soluble, air-insensitive derivatives it is possible that the assay may be performed in samples of biological fluids which have undergone a minimum of pretreatment.
Suitable derivatives which provide the substrate for a given enzyme can readily be synthesised. For example, phospha~e derivatives are recognised by acid or alkaline phosphatases.
Conveniently, the substrate for a phospha~ase labelling enzyme is a metallocene derivative, preferably a ferrocene derivative.
Examples of suitable phosphate derivatives of ferrocene gkr/m~c52471 page lO
are:
~Co~ l ~ O - P- ~ H
~ o~
,_ ~O~ o ~CH~,-O- P-o~
In a pacticularly preferred embodiment of the yresent invention, the redox substrate i5 the phosphate compound 5(I), otherwise termed, C5H5.Fe.C5H5.CO.NH-C6H4-OPo3 and herein called [N-ferrocenoyl]-4-aminophenyl phosphate. This compound is a novel compound and also forms part of the present invention.
The compound [N-ferrocenoyl]-4-aminophenyl phosphate has an El/2 of +390m~ against a standard calomel electrode ("SCE"). When the compound is treated with a phosphatase, such as acid or alkaline phosphatase, it is converted into the phenol derivative;
15(A) c5H5 Fe c5H5 co NH C6Hq which has an oxidation potential of +180mV against SCE. Hence, when an amperometric measurement is made at about +230m~ against SCE, only the catalytic curren~
4~
gkr/m~c52471 page 11 due to compound (A) is detected, and the compound (I) shows no electrode response at this potential.
In one alternative preferred embodiment, the substrate is a phenol derivative such as 2,6-dichloroindophenyl phosphate, which is of the formula (III) O ~ I (III).
Cl ~H
Although phosphatases are particularly exemplified, the the invention is applicable to any enzyme substrate coupled to any electroactive species. Therefore, this invention is not limited to such phosphate compounds for use with a phosphatase as labelling enzyme. Mediator derivatives can readily be synthesised so as to be coupled to other enzymic systems, and accordingly the nature of the reaction catalysed by the assay enzyme label is not critical. For example, the enzyme can be a protease, amidase or hydrolase.
According to another aspect of the present invention there is provided a system for detecting the activity of a first enzyme, comprising;
gkr/m~cS2471 page 12 a) a freely diffusinq reagent convertible by the first enzyme into a product exhibiting mediator activity thereby enabling detection of the product, and, b) detection means comprising a redo~ enzyme, a substrate for the redox enzyme, and an electrode, whereby, in the presence of an active form of the first enzyme, the reagent is converted into the product and a detectable change occurs in mediated electron transfer to the electrode. Typically, the characteristic mediator property of ~he product is electron transfer at a preselected electrode potential.
Such a system can be used to provide an assay for the first enzyme. whether or not the first enzyme is a label.
In another aspect of the present invention there is provided a bioelectrochemical cell (BEC) incorporating a substrate for an assay enzyme label whereby consumption of the substrate by said assay enzyme label produces a change in output from the BEC enabling said enzyme to be detected. ~he B~C comprises a second enzymic reaction system which provides the electrical output through conversion ot the substrate to a mediator.
For the second enzymic reaction used in the BEC, d ferrocene mediator is preferred for mediating electron transfer in the reaction between glucose and glucose gkr/m~cS2471 page 13 oxidase. Such a system results in amplification of the effect of the redox potential change in the ferrocene compound, owing to the mediatoc action of the ferrocene compound. The chemical change in the ferrocene compound or other substrate as it is consumed by the enzyme analyte can have a marked effect on the mediator characteristics, and hence on the output from the BEC.
According to a yet further aspect of the invention there is provided a method for detecting the activity of a non-redox enzyme, in the presence at least one electrode poised at a fixed potential, which method comprises:
a) treating a sample suspected of containing the non-redox enzyme with a reagent which has a redox potential higher than the poised potential of the lS electrode, the reagent being a substrate for the non-redox enzyme, and convertible by the activity of the non-redox enzyme into a product which acts as a mediator compound and has a redox potential lower than the poised potential of the electrode, and, b) treatinq the sample with a redox enzyme, and a substrate for the redox enzyme whereby in the presence of a mediatoc compound having a redox potential lowec than the poised potential of the electrode, a measurable transfer of charge to the electrode occurs.
The present Invention enables the detection of t~e 6~
gkr/m~c52471 page 14 activity of enzymes which do not themselves exhibit redox activity but which in one aspect of the invention can catalyse the transformation of a substrate in to a product which has mediator properties under the conditions of assay.
It should be noted that while the invention will be described heceaftec with refecence to a phosphatase enzyme, the invention extends to other assay systems and to other reagents. For example, it is envisaged that In other labelling enzymes may be employed, each being capable of reacting with a reagent to produce a product which has mediator properties. Moreover, while the invention is described with reference to the labeling of antigens, it is envisaged that enzyme labels in other types of specific binding reactions, such as those between complementary strands of nucleic acid, may be employed.
SUMMARY OF THE DRAWINGS
Figure 1 is a general scheme ~or an assay of the presen~
invention;
Figure 2 is a plot of peak current at 180mV against phosphatase concentration, obtained in Example 5;
gkr/m~cS2471 page lS
Figure 3 is a plot of corrected peak current at l~OmV
against oestriol concentration for a set of serum standards in the assay of Example 6; and Figure 4 comprises a set of cyclic voltammograms obtained in Example 7tb).
In Figure 1, mediator compound (Mi) is for example the compound (I). This compound is a water soluble reagent which with ar- electrode poised at a potential of ~230mV
against SCE exhibits no mediator activity between glucose oxidase (GOD~ and its substcate glucose.
In the presence of a phospha~ase enzyme (E) pre~ent as a label, the compound (I) is converted into the compound (A), which does exhibit mediator activity (Mred~oX) between glucose oxidase (GOD), its substrate glucose, and an electrode poised at ~230mV against SCE.
Thus, the system of Figure 1 enables the detection of the activity of the phosphatase enzyme. The detected current at the electrode is due only to the presence of compound (A).
~0 In Figure 1, the phosphatase enzyme is a label for the antibody (Ab), and consequently the concentration of the phosphatase enzyme is affected by specific binding 1;2~L6 gkr/m6c52471 page 16 reactions between the antibody and its corresponding antigen. The steps of exposing the antibody to the antigen and separating bound and free fractions may be carried out in accordance with known techniques of S immunoassay. In a variation, the phosphatase enzyme can be a label for the antigen.
SPECIFIC EXAMPLES OF THE INVENTION
In order that the invention may be better understood and carried into effect, various embodiments given by way of non-limiting example will now be described with reference, where appropriate, to the accompanying drawings.
Example 1 Preparation o~ a Substrate, Compound (I) (a) Preparation of Compound (A):
~ ~\~ 0~
F~ (A) Ferrocene monocarboxylic acid was converted to the gkr/m~cS2471 page 17 ferrocenoyl chloride by the method described in J. Org.
Chem., 24, ~80-281, (1959).
Ferrocenoyl chlo~ide (lmM) was then dissol~ed in 5ml of ice-cold dry pyridine. To this solution was added in S one portion a solution of p-aminophenol (1.1 mM) in Sml dry pyridine. The reaction was allowed to proceed foc
2 hours at 4C and then the solution was warmed to coom temperature and stirred for a further 2 hours. After this time the solution was filtered and the solvent removed in vacuo. The residue was then dissolved in the minimum amount of ethyl acetate and the compound purified by column chromatography on silica using ethyl acetate/hexane (3:1 v/v) as the eluent. The purified compound was crystallised as orange needles by slow diffusion of hexane into a solution of the compound in a minimum amount of ethyl acetate Mass spectroscopy gave a parent ion peak at m/e = 321, corresponding to the desired compound (A).
(b) Preparation of Compound I:
o P- Olt ~FL O~ ( I ) ,~
~f~:~'6~
gkr/m~c52471 page 18 The substrate for the alkaline phosphatase assay was peepared by modification of the methods in Biochem. J., 47, 93-95, (1950) and Biochem. J., 33, 1182-1184, (1939).
Compound (A) (100 mg, 0.31 mM) was dissolved in 5ml of dry pyridine, and the mixture cooled in an ice bath.
To this solution was added freshly distilled phosphorous oxychloride (30 ~1, 0.32 mM) and the resulting mixture stirred at ooc until the ceaction was complete. The reaction was followed by tlc using ethyl acetate as eluent.
Once the reaction was complete, a small amount of water (about 5 ml) was added and thereafter saturated barium hydroxide was added at 0C until the solution was alkaline. After the addition of barium hydroxide, an l~ equal volume of ice-cold ethanol was added to precipitate the barium salt of the phosphoric ester.
The barium salt was collected by filtration, washed with ethanol and dried ~n vacuo.
The crude bd~ium salt was only slightly soluhle in water. It WdS treated with a stoichiometric amount o~
H2SO4, The ;olution was filtered and subsequently lyophillised to yield the free acid.
gkr/m~c52471 page 19 ExamPle 2 Preparation of an alternative substrate, Compound (II~
o C41~,- 0 P ~ o~
O ~ (II) Hydroxymethyl ferrocene (216 mg) was dissolved in 5 ml of dry pyridine and thereafter lll ~l of fresh phosphorus oxychloride was added in one portion. The cleac yellow solution immediately turned dark red, and hydrogen chloride gas was released. The solution was stirred for approximately two hours. The reaction of hydroxymethyl ferrocene was confirmed by tlc on silica plates using ethyl acetate as eluent. After five minutes reaction, no hydroxymethyl ferrocene could be detected in the mixture.
After two hours, 1.0 ml of slightly basic distilled water (pH around ~) was added to the solution, which was then extracted ~our times with ethyl acetate to remove pyridine. The aqueous layer was further extracted four times with chloro~orm and then evaporated to dryness on a rotary evaporator. The residue was taken up four gkrtm6c52g71 page 20 times in slightly acidic distilled water (pH 5.5) and repeatedly evaporated to dryness in order to remove any residual pyridine.
The product was dissolved in ethanol in order to precipitate any inorganic impurities which were then filtered off.
Finally, in order to dry the product, it was dissolved in a minimum amount of distilled water and then freeze-dried for forty-eight hours.
A crystalline material, compound (II), was obtained upon standing at 4C for forty-eight hours.
Example 3 Determination of redox Potentials The redox potentials of compound (I) and compound tII) were determined.
An electrochemical cell was set up using a gold working electrode, a saturated calomel reference electtode (SC~) and a platinum counter electrode, connected to a potentiostat. The clean electrochemical cell was filled with a solution of the ferrocene derivative (2 mM) and glucose ( ~0 mM) in lOOmM Tris at pH 10.15. T~e gkr/m~cS2471 page 21 final volume was 400 ~1. Two different buffers were used to make up the solution, but did not affect the eesults.
The voltage was swept from 0 to 600 mV at a rate of 5 mVsec . From the resultant voltammograms, the tedox potentials of compounds (I) and (II) were calculated.
For compound (I), the redox potential is around +390mV.
For compound (II), the redox potential is around +370 mV.
The redox potentials of compounds (I) and (II) were pH-independent in the range studied.
Example 4 PreParation o~ an alternative substrate, Compound (III) ~ à~ (III) 5g of the sodium salt of 2,6-dichloroindophenol was dissolved in water and hydrogen chloride gas was added until a deep blue precipitate was deposited. The solid was filtered, dissolved in dichloromethane and dried gkr/m~c52471 page 22 over magnesium sulphate, The solution was filtered and the solvent removed to yield 2,6-dichloroindophenol.
The 2,6-dichloroindophenol was dissolved in dry pyridine and one equivalent of phosphorus oxychloride was added dropwise with stirring. After L5 minutes, ice was added and the solution neutralized with sodium carbonate. The solvent was removed and the residue extracted with boiling ethanol. The ethanol was filtered and evaporated. The residue was washed with ethanol to yield the desired compound (III).
_xamPle S
Determination o~ Alkaline PhosPhatase Activity A two compartment electrochemical cell was u6ed. In addition to a 4mm diameter graphite disc working electrode, the cell contained a lcm platinum gauze counter electrode and a saturated calomel electrode ("SCE") as a re~erence.
D.C. cyclic voltammetry experiments were carried out using a BAS-100 Electrochemical Analysec.
Calf intestine alkaline phosphatase (supplied by Calzyme) was dialysed into O.lM Tris buffer, pH 10.15 The ~inal protein concentration was 0.86 mq/ml.
6~
gkr/m~cS2471 page 23 0.6ml of a 2mM substrate solution in O.lM Tris pH 10.15, containing lOmM MgC12 and SOmM NaCl, was placed in the sample compartment of the electrochemical cell and the D.C. cyclic voltammogram recorded. The peak current, if any, at 180mV was measured and thereafter, lOo~l of a suitably diluted stock solution of alkaline phosphatase was added and the cell contents incubated at room temperature for 15 minutes. After incubation the cyclic voltammogram was again recorded and the current at 180mV measured. This procedure was repeated for a range of alkaline phosphatase concentrations.
The peak current at L80mV due to the oxidation of ferrocene to the ferricinium ion in the phenolic product was used to estimate alkaline phosphatase activity.
The peak currents at 180mV as a function of alkaline phosphatase concentration are shown graphically in Figure 2.
An in-cell concentration of 1.23 x 10 mol alkaline phosphatase is equivalent to 8.6 x 10 moles of ~ yme in the electrochemical cell.
Accordingly, d very low amount of alkaline phosphatase can be determined using cyclic voltammetry under relatively miId conditions, with lS minutes incubation at room temperature (as opposed to the more common conditions IJse(i in immunoassays, with at least 30 ~z~
gkr/m~cS2471 page 24 minutes incubation at 37C).
~xamPle 6 AmPerometric END~B EnzYme Immunoassav Having demonstrated the inherent sensitivity of the electrochemical alkaline phosphatase assay, a detection system for enzyme immunoassay was tried. An ENDAB
enzyme immunoassay kit for unconjugated oestriol in serum was purchased from CMD (UK) Ltd.
The assay procedure supplied with the oestriol kit was followed until the second antibody precipitation step, and then modified to give an assay of this invention.
Thus, 50 ~l of each oestriol standard t0, l, 3, ~0, 20 and 40 ng ml ) were pipetted into the appropriate glass tubes. In order to allow for non-specific binding (~'NSB~), 50 ~l of the 0 ng ml standard was placed in the NSB tube. Thereafter, 25 ~l of the oestriol-enzyme conjugate was added to all the tubes, followed by 100 l~l of oestriol anti-serurn. Oestriol anti-serum was not added to the NSB tube, and instead 100 ~l of Background Reagent was added.
The reagents were mixed and incubated for 20 minutes at room temperature. l ml of the second antibody-separating reagent was then added to all gkr/m~cS2471 page 25 tubes. The tubes were centrifuged at 3000 tpm for 10 minutes and the supernatant discarded. The tubes were then blotted on absorbent paper to remove excess liquid.
At this point, instead of following the procedure specified with the kit for spectrophotometric determination of oestriol, the centrifugal pellet, which contained the antibody-bound oestriol-alkaline phosphatase, was resuspended in a solution containing the ferrocene linked substrate (0.6ml of a 2mM solution in O.lM Tris, pH 10.15 containing lO~M MgC12 and 50mM
NaCl). The solution was incubated at room temperature for 15 minutes. ~fter 15 minutes incubation, the solution was transferred to the sample compartment of the electrochemical cell.
The cyclic voltammogram of the solution was recorded over the range 0 to +650mV versus SCE, and the peak current at 180mV measured.
This procedure was carried out for each seru~ oestriol standard, that is, 0, 1, 3, 20 and 40 ng/ml.
The background current at 180mV for the substrate in the absence of any alkaline phosphatase, that is, the non-specific binding current, was 1.15~A. This value was subtracted from the results obtained using the seru~
gkr~m~c52471 page 26 standards.
The corrected peak current at 180mV for each serum standard was plotted on the standard--curve paper supplied with the kit to give Figure 3.
Plotting - ln i~io against oestriol concentration gave a straight line (y = 0.04 x + 0.43, r = 0.98).
This ~NDAB immunoassay with amperometric detection was achieved using shorter incubation times and lower temperatures than the standacd kit method.
xamPle 7 (a) Use of compounds (I) and (II) The same apparatus and voltage sweep was used as in Example 3.
In this experiment 1 ~1 of the enzyme glucose oxidase (GOD), 175 mg/ml, was added to the solutions as in Example 3, giving a einal volume o~ 0.35 ml.
The voltage sweep was carried out as before and the results, cuerent against voltage, were recorded.
It was found ~hat both compounds (I) and (II) will act l~S~
gkr/m~c52471 page 27 as mediators for the glucose oxidase enzymic reaction and that the resulting current produced at oxidising potentials had been amplified. This amplification is due to the catalytic reduction of the ferricinium ion by the enzyme and the subsequent reoxidation of ferrocene at the electrode.
Measurements against time of current at a constant potentia} of 450 mV were performed in the three-electrode system, with detection of hydrolysis of the phosphate group of the ferrocene derivative in the presence of a crude preparation of an acid phosphatase. 8 ~1 compound (II) (50 ~M in ethanol) and 50 ~1 glucose (lM) in 100 mM citrate buffer, pH
4.66, were used, giving a final volume of 400 ~1.
The samples were all stirred throughout the experiment.
Four sets of measurements were recorded:
(a) Compound (II) plus glucose, no acid phosphatase or glucose oxidase.
(b) As (a) but with the addition of 25 ~1 of 2 mM
glucose oxidase after ten minutes incubation of compound (II) and glucose at 37C. The incubation was used to show that any decrease in current occurring afte~
addition of acid phosphatase was not due to breakdown in ~,4~
gkr/m~cS2g71 page 28 solution of compound (II).
Thece was a clear difference in current between (a) and (b), due to the oxidase enzymic reaction acting through compound (II) as a media~or.
(c) As (b) but with 10 ~1 of crude acid phosphatase solution incubated with compound (II) and glucose for ten minutes at 37C prior to addition of glucose oxidase.
A decrease in current compared with (b) was observed, presumably due to reaction of acid phosphatase on compound (Il). which alters its mediator characteristics.
(d) As (c), but 30 ~1 of acid phosphatase solution was added.
A further decrease in current was observed, showing an 1~ inverse relationship between the acid phosphatase concentration and the catalytic current.
(b) Use of compound (III) In a similar manner to Example 7(a), the cyclic voltammogram o~ compound (II) with O.lM glucose was determined at pH ~.8 using a scan rate of 20mV/s, givin~
gkr/m~c52471 page 29 curves (a), (b) and (c) of Figure 4 respectively for the compound (IrI), the compound (III) after 5 minutes with alkaline phosphatase, and the compound (III) with alkaline phosphatase and glucose oxidase. Again the amplification was marked.
(b) Preparation of Compound I:
o P- Olt ~FL O~ ( I ) ,~
~f~:~'6~
gkr/m~c52471 page 18 The substrate for the alkaline phosphatase assay was peepared by modification of the methods in Biochem. J., 47, 93-95, (1950) and Biochem. J., 33, 1182-1184, (1939).
Compound (A) (100 mg, 0.31 mM) was dissolved in 5ml of dry pyridine, and the mixture cooled in an ice bath.
To this solution was added freshly distilled phosphorous oxychloride (30 ~1, 0.32 mM) and the resulting mixture stirred at ooc until the ceaction was complete. The reaction was followed by tlc using ethyl acetate as eluent.
Once the reaction was complete, a small amount of water (about 5 ml) was added and thereafter saturated barium hydroxide was added at 0C until the solution was alkaline. After the addition of barium hydroxide, an l~ equal volume of ice-cold ethanol was added to precipitate the barium salt of the phosphoric ester.
The barium salt was collected by filtration, washed with ethanol and dried ~n vacuo.
The crude bd~ium salt was only slightly soluhle in water. It WdS treated with a stoichiometric amount o~
H2SO4, The ;olution was filtered and subsequently lyophillised to yield the free acid.
gkr/m~c52471 page 19 ExamPle 2 Preparation of an alternative substrate, Compound (II~
o C41~,- 0 P ~ o~
O ~ (II) Hydroxymethyl ferrocene (216 mg) was dissolved in 5 ml of dry pyridine and thereafter lll ~l of fresh phosphorus oxychloride was added in one portion. The cleac yellow solution immediately turned dark red, and hydrogen chloride gas was released. The solution was stirred for approximately two hours. The reaction of hydroxymethyl ferrocene was confirmed by tlc on silica plates using ethyl acetate as eluent. After five minutes reaction, no hydroxymethyl ferrocene could be detected in the mixture.
After two hours, 1.0 ml of slightly basic distilled water (pH around ~) was added to the solution, which was then extracted ~our times with ethyl acetate to remove pyridine. The aqueous layer was further extracted four times with chloro~orm and then evaporated to dryness on a rotary evaporator. The residue was taken up four gkrtm6c52g71 page 20 times in slightly acidic distilled water (pH 5.5) and repeatedly evaporated to dryness in order to remove any residual pyridine.
The product was dissolved in ethanol in order to precipitate any inorganic impurities which were then filtered off.
Finally, in order to dry the product, it was dissolved in a minimum amount of distilled water and then freeze-dried for forty-eight hours.
A crystalline material, compound (II), was obtained upon standing at 4C for forty-eight hours.
Example 3 Determination of redox Potentials The redox potentials of compound (I) and compound tII) were determined.
An electrochemical cell was set up using a gold working electrode, a saturated calomel reference electtode (SC~) and a platinum counter electrode, connected to a potentiostat. The clean electrochemical cell was filled with a solution of the ferrocene derivative (2 mM) and glucose ( ~0 mM) in lOOmM Tris at pH 10.15. T~e gkr/m~cS2471 page 21 final volume was 400 ~1. Two different buffers were used to make up the solution, but did not affect the eesults.
The voltage was swept from 0 to 600 mV at a rate of 5 mVsec . From the resultant voltammograms, the tedox potentials of compounds (I) and (II) were calculated.
For compound (I), the redox potential is around +390mV.
For compound (II), the redox potential is around +370 mV.
The redox potentials of compounds (I) and (II) were pH-independent in the range studied.
Example 4 PreParation o~ an alternative substrate, Compound (III) ~ à~ (III) 5g of the sodium salt of 2,6-dichloroindophenol was dissolved in water and hydrogen chloride gas was added until a deep blue precipitate was deposited. The solid was filtered, dissolved in dichloromethane and dried gkr/m~c52471 page 22 over magnesium sulphate, The solution was filtered and the solvent removed to yield 2,6-dichloroindophenol.
The 2,6-dichloroindophenol was dissolved in dry pyridine and one equivalent of phosphorus oxychloride was added dropwise with stirring. After L5 minutes, ice was added and the solution neutralized with sodium carbonate. The solvent was removed and the residue extracted with boiling ethanol. The ethanol was filtered and evaporated. The residue was washed with ethanol to yield the desired compound (III).
_xamPle S
Determination o~ Alkaline PhosPhatase Activity A two compartment electrochemical cell was u6ed. In addition to a 4mm diameter graphite disc working electrode, the cell contained a lcm platinum gauze counter electrode and a saturated calomel electrode ("SCE") as a re~erence.
D.C. cyclic voltammetry experiments were carried out using a BAS-100 Electrochemical Analysec.
Calf intestine alkaline phosphatase (supplied by Calzyme) was dialysed into O.lM Tris buffer, pH 10.15 The ~inal protein concentration was 0.86 mq/ml.
6~
gkr/m~cS2471 page 23 0.6ml of a 2mM substrate solution in O.lM Tris pH 10.15, containing lOmM MgC12 and SOmM NaCl, was placed in the sample compartment of the electrochemical cell and the D.C. cyclic voltammogram recorded. The peak current, if any, at 180mV was measured and thereafter, lOo~l of a suitably diluted stock solution of alkaline phosphatase was added and the cell contents incubated at room temperature for 15 minutes. After incubation the cyclic voltammogram was again recorded and the current at 180mV measured. This procedure was repeated for a range of alkaline phosphatase concentrations.
The peak current at L80mV due to the oxidation of ferrocene to the ferricinium ion in the phenolic product was used to estimate alkaline phosphatase activity.
The peak currents at 180mV as a function of alkaline phosphatase concentration are shown graphically in Figure 2.
An in-cell concentration of 1.23 x 10 mol alkaline phosphatase is equivalent to 8.6 x 10 moles of ~ yme in the electrochemical cell.
Accordingly, d very low amount of alkaline phosphatase can be determined using cyclic voltammetry under relatively miId conditions, with lS minutes incubation at room temperature (as opposed to the more common conditions IJse(i in immunoassays, with at least 30 ~z~
gkr/m~cS2471 page 24 minutes incubation at 37C).
~xamPle 6 AmPerometric END~B EnzYme Immunoassav Having demonstrated the inherent sensitivity of the electrochemical alkaline phosphatase assay, a detection system for enzyme immunoassay was tried. An ENDAB
enzyme immunoassay kit for unconjugated oestriol in serum was purchased from CMD (UK) Ltd.
The assay procedure supplied with the oestriol kit was followed until the second antibody precipitation step, and then modified to give an assay of this invention.
Thus, 50 ~l of each oestriol standard t0, l, 3, ~0, 20 and 40 ng ml ) were pipetted into the appropriate glass tubes. In order to allow for non-specific binding (~'NSB~), 50 ~l of the 0 ng ml standard was placed in the NSB tube. Thereafter, 25 ~l of the oestriol-enzyme conjugate was added to all the tubes, followed by 100 l~l of oestriol anti-serurn. Oestriol anti-serum was not added to the NSB tube, and instead 100 ~l of Background Reagent was added.
The reagents were mixed and incubated for 20 minutes at room temperature. l ml of the second antibody-separating reagent was then added to all gkr/m~cS2471 page 25 tubes. The tubes were centrifuged at 3000 tpm for 10 minutes and the supernatant discarded. The tubes were then blotted on absorbent paper to remove excess liquid.
At this point, instead of following the procedure specified with the kit for spectrophotometric determination of oestriol, the centrifugal pellet, which contained the antibody-bound oestriol-alkaline phosphatase, was resuspended in a solution containing the ferrocene linked substrate (0.6ml of a 2mM solution in O.lM Tris, pH 10.15 containing lO~M MgC12 and 50mM
NaCl). The solution was incubated at room temperature for 15 minutes. ~fter 15 minutes incubation, the solution was transferred to the sample compartment of the electrochemical cell.
The cyclic voltammogram of the solution was recorded over the range 0 to +650mV versus SCE, and the peak current at 180mV measured.
This procedure was carried out for each seru~ oestriol standard, that is, 0, 1, 3, 20 and 40 ng/ml.
The background current at 180mV for the substrate in the absence of any alkaline phosphatase, that is, the non-specific binding current, was 1.15~A. This value was subtracted from the results obtained using the seru~
gkr~m~c52471 page 26 standards.
The corrected peak current at 180mV for each serum standard was plotted on the standard--curve paper supplied with the kit to give Figure 3.
Plotting - ln i~io against oestriol concentration gave a straight line (y = 0.04 x + 0.43, r = 0.98).
This ~NDAB immunoassay with amperometric detection was achieved using shorter incubation times and lower temperatures than the standacd kit method.
xamPle 7 (a) Use of compounds (I) and (II) The same apparatus and voltage sweep was used as in Example 3.
In this experiment 1 ~1 of the enzyme glucose oxidase (GOD), 175 mg/ml, was added to the solutions as in Example 3, giving a einal volume o~ 0.35 ml.
The voltage sweep was carried out as before and the results, cuerent against voltage, were recorded.
It was found ~hat both compounds (I) and (II) will act l~S~
gkr/m~c52471 page 27 as mediators for the glucose oxidase enzymic reaction and that the resulting current produced at oxidising potentials had been amplified. This amplification is due to the catalytic reduction of the ferricinium ion by the enzyme and the subsequent reoxidation of ferrocene at the electrode.
Measurements against time of current at a constant potentia} of 450 mV were performed in the three-electrode system, with detection of hydrolysis of the phosphate group of the ferrocene derivative in the presence of a crude preparation of an acid phosphatase. 8 ~1 compound (II) (50 ~M in ethanol) and 50 ~1 glucose (lM) in 100 mM citrate buffer, pH
4.66, were used, giving a final volume of 400 ~1.
The samples were all stirred throughout the experiment.
Four sets of measurements were recorded:
(a) Compound (II) plus glucose, no acid phosphatase or glucose oxidase.
(b) As (a) but with the addition of 25 ~1 of 2 mM
glucose oxidase after ten minutes incubation of compound (II) and glucose at 37C. The incubation was used to show that any decrease in current occurring afte~
addition of acid phosphatase was not due to breakdown in ~,4~
gkr/m~cS2g71 page 28 solution of compound (II).
Thece was a clear difference in current between (a) and (b), due to the oxidase enzymic reaction acting through compound (II) as a media~or.
(c) As (b) but with 10 ~1 of crude acid phosphatase solution incubated with compound (II) and glucose for ten minutes at 37C prior to addition of glucose oxidase.
A decrease in current compared with (b) was observed, presumably due to reaction of acid phosphatase on compound (Il). which alters its mediator characteristics.
(d) As (c), but 30 ~1 of acid phosphatase solution was added.
A further decrease in current was observed, showing an 1~ inverse relationship between the acid phosphatase concentration and the catalytic current.
(b) Use of compound (III) In a similar manner to Example 7(a), the cyclic voltammogram o~ compound (II) with O.lM glucose was determined at pH ~.8 using a scan rate of 20mV/s, givin~
gkr/m~c52471 page 29 curves (a), (b) and (c) of Figure 4 respectively for the compound (IrI), the compound (III) after 5 minutes with alkaline phosphatase, and the compound (III) with alkaline phosphatase and glucose oxidase. Again the amplification was marked.
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of assay with the use of electrochemical detection of mediator level using a redox enzyme and redox sub-strate to detect conversion in an assay of a non-mediating com-pound to an effective mediator by an assay enzyme label.
2. A method of assay in which an enzyme label is employed, characterized in that the label is electrochemically detected with a redox enzyme and a redox substrate as a result of conversion by the labelling enzyme of a non-mediating substrate from an ineffective mediator to an effective mediator for mediat-ing electron transfer between the redox enzyme and an electrode.
3. A method of assay according to claim 2, which is an immunoassay.
4. A method of assay according to claim 3, which is an ELISA.
5. A method of assay according to claim 1, 2 or 3, in which in the electrochemical detection, an electrode is poised at a preselected potential which is intermediate between the redox potential of the substrate for the redox enzyme and the product, such that only the product having a redox potential lower than that at which the electrode is poised can undergo a redox reac-tion at the electrode.
6. A method of assay according to claim 1, wherein the redox substrate is a phenol derivative.
7. A method of assay according to claim 1, 2 or 3, wherein the labelling enzyme is a phosphatase.
8. A method of assay according to claim 6, wherein the redox substrate is the compound (I)
9. A system for detecting the activity of a first enzyme, comprising a) a freely diffusing reagent convertible by the first enzyme into a product exhibiting mediator activity thereby enabling detection of the product, and, b) detection means comprising a redox enzyme, a substrate for the redox enzyme, and an electrode, whereby, in the presence of an active form of the first enzyme, the reagent is converted into the product and a detectable change in mediated electron transfer to the electrode occurs.
10. A bioelectrochemical cell incorporating a substrate for an assay enzyme label whereby consumption of the substrate by said assay enzyme label produces a change in output from the bioelectrochemical cell enabling said enzyme to be detected, the cell comprising a second enzymic reaction system which provides an electrical output through conversion of the substrate to a mediator.
11. A method for detecting the activity of a non-redox enzyme, in the presence at least one electrode poised at a fixed potential, which method comprises:
a) treating a sample suspected of containing the non-redox enzyme with a reagent which has a redox potential higher than the poised potential of the electrode, the reagent being a substrate for the non-redox enzyme, and convertible by the activity of the non-redox enzyme into a product which has mediator activity and which has a redox potential lower than the poised potential of the electrode, and, b) treating the sample with a redox enzyme, and a substrate for the redox enzyme whereby in the presence of a mediator compound having a redox potential lower than the poised potential of the electrode, a measurable transfer of charge to the electrode occurs.
a) treating a sample suspected of containing the non-redox enzyme with a reagent which has a redox potential higher than the poised potential of the electrode, the reagent being a substrate for the non-redox enzyme, and convertible by the activity of the non-redox enzyme into a product which has mediator activity and which has a redox potential lower than the poised potential of the electrode, and, b) treating the sample with a redox enzyme, and a substrate for the redox enzyme whereby in the presence of a mediator compound having a redox potential lower than the poised potential of the electrode, a measurable transfer of charge to the electrode occurs.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB858527777A GB8527777D0 (en) | 1985-11-11 | 1985-11-11 | Enzymic assay procedures |
| GB8527777 | 1985-11-11 | ||
| GB8619113 | 1986-08-05 | ||
| GB868619113A GB8619113D0 (en) | 1986-08-05 | 1986-08-05 | Assay techniques |
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| Publication Number | Publication Date |
|---|---|
| CA1254616A true CA1254616A (en) | 1989-05-23 |
Family
ID=26289993
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000522577A Expired CA1254616A (en) | 1985-11-11 | 1986-11-10 | Electrochemical enzymic assay procedures |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US4830959A (en) |
| EP (1) | EP0223541A3 (en) |
| AU (1) | AU596127B2 (en) |
| CA (1) | CA1254616A (en) |
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| US10981172B2 (en) | 2013-03-14 | 2021-04-20 | Abbott Point Of Care Inc. | Thermal control system for controlling the temperature of a fluid |
| WO2016183493A1 (en) | 2015-05-14 | 2016-11-17 | Abbott Diabetes Care Inc. | Compact medical device inserters and related systems and methods |
| US10213139B2 (en) | 2015-05-14 | 2019-02-26 | Abbott Diabetes Care Inc. | Systems, devices, and methods for assembling an applicator and sensor control device |
| US10766795B2 (en) | 2015-10-27 | 2020-09-08 | Massachusetts Institute Of Technology | Electrochemical devices or systems comprising redox-functionalized electrodes and uses thereof |
| WO2018081653A1 (en) * | 2016-10-27 | 2018-05-03 | Massachusetts Institute Of Technology | Use of electrochemical devices or systems comprising redox-functionalized electrodes for bioseparation and/or biocatalysis |
| CN110461217B (en) | 2017-01-23 | 2022-09-16 | 雅培糖尿病护理公司 | Systems, devices, and methods for analyte sensor insertion |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4446231A (en) * | 1979-10-03 | 1984-05-01 | Self Colin H | Immunoassay using an amplified cyclic detection system |
| CA1218704A (en) * | 1983-05-05 | 1987-03-03 | Graham Davis | Assay systems using more than one enzyme |
| CA1220818A (en) * | 1983-05-05 | 1987-04-21 | Hugh A.O. Hill | Assay techniques utilising specific binding agents |
| GB8328520D0 (en) * | 1983-10-25 | 1983-11-23 | Serono Diagnostics Ltd | Methods of assay |
| DE3380098D1 (en) * | 1983-12-05 | 1989-07-27 | Battelle Memorial Institute | A method of immunoassay detection by reaction-rate potentiometry using fluoride ion-selective electrode |
| WO1985002627A1 (en) * | 1983-12-16 | 1985-06-20 | Genetics International, Inc. | Assay for nucleic acids |
| GB8432069D0 (en) * | 1984-12-19 | 1985-01-30 | Iq Bio Ltd | Apparatus for immunoassay |
| GB8504522D0 (en) * | 1985-02-21 | 1985-03-27 | Genetics Int Inc | Electrochemistry of mediators |
| WO1987002464A1 (en) * | 1985-10-14 | 1987-04-23 | Genetics International (Uk) Inc. | Enzyme-labelled assay |
-
1986
- 1986-11-10 CA CA000522577A patent/CA1254616A/en not_active Expired
- 1986-11-10 US US06/929,332 patent/US4830959A/en not_active Expired - Fee Related
- 1986-11-11 AU AU65099/86A patent/AU596127B2/en not_active Expired - Fee Related
- 1986-11-11 EP EP86308781A patent/EP0223541A3/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| US4830959A (en) | 1989-05-16 |
| AU596127B2 (en) | 1990-04-26 |
| EP0223541A3 (en) | 1989-08-23 |
| AU6509986A (en) | 1987-05-14 |
| EP0223541A2 (en) | 1987-05-27 |
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