JPS6017360A - Inspecting method using special binder - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

The present invention relates to assay methods, particularly for complex molecules or part-mol structures that are commonly biologically occurring and often in complex mixtures with other similar molecules.
(7) Concerning detection, measurement and monitoring. The method of the invention uses specific binding agents, ie complex molecular species that react specifically with one or more active sites of only a given type of molecule, usually based on electrostatic or hydrophobic effects. One feature of the present invention is the use of immunoassay methods that can be performed in vivo or ex vivo, i.e., using naturally occurring or non-naturally occurring plasma, serum, whole blood, urine, interstitial fluid, brain, etc., such as cerebrospinal fluid or synovial fluid. These are immunoassays and methods that involve reacting large antibody or antigen molecules that have been artificially produced in body fluids (or produced as monoclonal antibodies) with small ligand molecules that normally react specifically. A ligand is a species that is not itself antigenic, but can be made antigenic by cross-linking to a suitable carrier. Another aspect of the invention is to Second, the reaction of enzymes with inhibitors or cofactors; receptors and hormones. , reactions with vitamins, toxins or growth factors; reactions with complex carbohydrates and lectins; reactions with nucleic acids and proteins; reactions with macromolecules with aromatic groups and dyes; and macrotetratiles that interact with metals. and other types of reactions in which specific non-immune binding occurs, such as the reaction between a ligand and an antiligand. The present invention also relates to devices and materials useful in the above-described methods, either neat or as a conveniently prepared kit of parts for use. Known immunoassay methods These methods are based on antibody folds, a competitive reaction in which the combined labeled ligand is replaced by a labeled substance in the assay sample; Ligands that are not labeled with T) are most closely related in that they are proportional to T). For example, when testing drugs, such methods include: a) Radioimmunoassay (RIA): In this method, the drug or other substance to be tested is exposed to 8T (, 1
4 (3, 1811, 75So, 853O, and 126E (7)) and quantified using a scintillation counter. RIA is a so-called heterogeneous immunoassay, and The reason for this is that a separation step is necessary to remove the labeled drug that has been displaced from the drug bound to the antibody before immobilization.
It is a powerful method with a sensitivity on the order of ~10-10M. Medications to which this method may be applied include painkillers such as aspirin, antibiotics such as adriamycin and gentamicin, painkillers such as phenytoin,
These include anti-tumor drugs such as methotrexate. , b) Spin immunoassay method: In this method the label is a stable free radical whose unpaired electron can be detected by its characteristic spin resonance. In this case, the free spin −
The ESR spectrum of the labeled ligand is different from the ESR spectrum of the bound spin-labeled ligand (the narrower and higher beats).Therefore, there is no need to separate the bound ligand from the free ligand, and This type of immunoassay method can be described as a homogeneous immunoassay method.The sensitivity of this method is at most in the range of 10-4 to 10-molar.An important limitation of this method is the high cost of measurement. One example of this method is to label morphine with a nitroxytospin. This compound gives eight narrow ESR lines in solution, but , one wide peak (
C) Homogeneous enzyme immunoassay method (also known as enzyme-linked immunodetection method - EL ISA): The method involves binding the enzyme and the drug to be assayed in such a way that the enzyme activity remains unchanged, which can be difficult to achieve due to the relatively large size of the enzyme. However, drug-enzyme conjugate) (c
When a drug is conjugated to a conjugated antibody, a structural (morphological) change occurs, which in turn reduces the enzyme activity, or the active position of the enzyme is blocked by steric hindrance. . Therefore, if free drug is present in the sample, this drug will bind to the antibody, thus releasing the fermentation-drug conjugate. The activity of the enzyme is therefore proportional to the amount of free drug present in the sample. Since the immunoassay method is a method for assaying enzyme activity, separation of free drug from bound drug is unnecessary. Additionally, the label used in this assay provides some degree of amplification, since one molecule of unlabeled drug can release an enzyme molecule, and this enzyme molecule These assays unfortunately often require colorimetric measurements of enzyme activity. The assay method is heroin, methatone, cocaine,
For narcotics like THClLSD, S'['P and POP, for antiasthmatics like theophylline, for antiarrhythmics like lidocaine, CardiOaCtiVe ar
ug), thyroid hormones such as thyroxine, and drugs used for therapeutic control such as ethosuximide. d) Fluorescence-induced transfer immunoassay method: This method uses two types of labels and is based on the fluorescence-induced transfer phenomenon;
This method allows rapid detection using low drug concentrations. In one variation of this method, the drug is labeled with a fluorophore, such as fluorescein-1;
The antibody is labeled with a receptor (quencher) such as D-damine. (drug) (s-identifying antibody) when the average distance between the quencher and fluorophore in the complex is close enough to allow dipole-dipole coupling and energy transfer. When a complex is formed, quenching of the fluorescence occurs. Addition of unlabeled drug to this assay mixture reduces the extent of quenching. Unfortunately, this method also requires expensive equipment and highly trained personnel. Known DNA (RNA) probe technologies have similarities in that DNA (RNA) polymers are not easily detected by their inherent biochemical activity. It is therefore necessary to mark DNA (RNA) polymers with certain signal-generating chemical or biochemical species; such methods include: a) Avidin-biotin reaction: This technique This is based on the affinity of avidin, a middle glycoprotein, for biotin. Biotin (vitamin W) is a single biological subunit of DNA polymers and can be covalently bound to the nucleotide G. Since denatured subunits are still capable of performing classical binding reactions between complementary strands of duplex DNA, such subunits can be synthesized and incorporated into NA probes. To detect the presence of such probes with short double-stranded regions formed after exposure to a complementary DNA sample, first remove the unbound probe from the DNA.
Must be separated from the A sample/bound probe complex. This is usually accomplished by carrying out a binding reaction under conditions in which the DNA sample is immobilized on a substrate, followed by washing, although centrifugation may also perform a similar effect. Bound probe is detected by adding a fluorescent marker-labeled antibody or enzyme-conjugated avidin. One problem with the above-mentioned method is that the oligonucleotide probes (20 nucleotides) have only a small number of biotin-bound positions (oiotinylated sj, te), which limits the number of avidin legs that can be bound. Attempts have been made to add long "tails" of up to a few dozen bases to 70-nucleotide DNA, with some success.In this case, it is the tail that needs to be identified. This method has a DNA resolution of lo 18 g (r6so
up to 1 OL of one gene
One copy gusset can be detected. Initially the marker for avidin was horseradish peroxidase, a fairly short-lived enzyme, but this method has now been expanded to include alkaline phosphatase. Unfortunately, this method is generally difficult to establish as a new diagnostic system because the biotin-bound probe DNA is difficult to produce or the S tail interferes with sensitivity. b] Radioisotope Vote Method: This method is the first method to detect specific DNA sequences. , DNA probe 8H, 1
composed of nucleotides with 40 or 8 Qp;
The binding of the probe to the target sequence is caused by the D
It is detected by measuring the NA duplex emission by autoradiography. In this method, the target DNA is digested with a restriction endonuclease, the fragments are separated electrophoretically on an agarose gel, and then "blotted" on a nitrocellulose filter.
Typically, a nitrocellulose filter binds the target DNA and holds it in place during exposure to the radioactive probe. Unfortunately, it can take several weeks to obtain an autoradiograph of a particular nitrocellulose area, and the reagents used in this method are destructive and potentially dangerous. C) Other methods: Other methods involve indirectly detecting specific DNA sequences of interest by using restriction enzyme fragment length polymorphism J (HELP). This method can be used to detect fetal disease by examining small amounts of fetal DNA, approximately 100 μg, removed from the fetal trophoblast. Chemiluminescence detection has been used with varying degrees of success. One purpose of the DNA probe technology and enzyme detection/assay technology that has been developed so far is to detect the "genetic diseases" of the gods and errors built into the substances that cause them. there were. These diseases include common goiter (iodothi, rosine dehalogenase deficiency), maple molasses (α-ketodecarboxylase deficiency), xatinuria (xanthine oxidase deficiency), and methemoglobinemia (methemoglobin reductase deficiency). A complete list of the 85,000 conditions caused by defective genes can be found in MangukkuThink (M
Mendelian inheritance in men (Me
Inheritance in Man
) shown in J. However, as can be seen from the above description, highly trained personnel (currently used or susceptible to the action of light) or (
A definite disadvantage is that it requires some or all expensive equipment (for autoradiography, ESR measurements or low-level light detection)15. Known assay methods for binding reactions other than antigen/antibody and DNA/complementary DNA are generally similar to those described above and suffer from similar problems. The present invention aims to overcome the above-mentioned drawbacks by using a simple electrochemical measurement technique and is related to the invention described in European Patent Application No. 82805597. It is an object of the present invention to provide a method for detecting, measuring or monitoring specific binding agents that does not have the above-mentioned drawbacks. Another object of the present invention is to provide apparatus and reagents for use in carrying out the method of the present invention. Prior applications relating to all of the above relate to the use of mediator compounds to transfer a charge to the electrode and to directly measure the progress of an enzyme's catalytic reaction on its specific substrate by means of a suitable electrode. . The present invention provides the following benefits: (a) the effective level of the mediator is changed; (b) the effective level of the enzyme is changed; (C) both are changed;
T! It is based on the finding that a measurable difference is achieved when the effective surface of the poles is varied, and furthermore, a measurable effect on the ligand/antiligand is achieved by a specific binding reaction between the ligand and antiligand. It is based on the finding that said changes can be brought about in such a way that they can be related to the occurrence or extent of binding. Accordingly, in one aspect, the present invention provides that (a) at least 100% of the reactive species, including a ligand and an antiligand,
a specific binding reaction stage @ associated with an enzyme/mediator system electrochemically coupled to a substrate at a level at which the enzyme is catalytically active, such that said binding reaction electrochemical availability of one component
the electrochemical availability), and then detecting or measuring the catalytic effect on the electrochemical availability to obtain a signal, and deducing the presence or dispersion of the reactive species from this signal. The method of the present invention involves the use of a ligand and an antiligand, such as an antigen/antibody or a DNA target sequence/DNA probe sequence; a substrate; an enzyme specific for the substrate; and a mediator compound. The reaction can be carried out in the entire solution, i.e. it can be a homogeneous reaction, or it can be carried out at one solid surface,
For example, it can be a heterogeneous reaction. The solid surface used in such a method can be the wall of the containing container or the electrode surface, and the electrode itself can be a simple carbon electrode, or it can be a gold or other metal electrode. or can be more or less sophisticated, coated with various components, in particular mediator, antibody or antiligand and substrate, and possibly one or more of the substrates.
There are many alternatives to implementing the invention described above. These methods and their utility and advantages are detailed below by way of example. The invention, in all its aspects, can take advantage of certain features of the method and materials, in particular the properties of the enzyme/substrate on the one hand and of the mediator on the other hand. The enzyme/substrate pair exists solely as an indicator of the presence or extent of a specific binding reaction. Theoretically, the enzyme/substrate pair is therefore a pair that is independent of the reaction atmosphere, i.e. its electrochemical or other effects cannot be confused with the front effects of components naturally present in the total test mixture. Can be paired. Of course, if the method chosen relies in whole or in part on reducing the enzyme level, i.e. the electrochemical availability of the enzyme, then the enzyme chosen will not be affected by specific binding reactions that affect the above-mentioned availability. It is necessary to make it such that it has a positive impact. Enzyme/substrate pairs whose electrochemical properties have been studied in conjunction with mediator compounds are: pyruvate oxidase, pyruvate L-amino acid oxidase, L-amino acid aldehyde oxidase, aldehyde xanthyl oxidase, xanthine glucose oxidase, glucose glycose acid. Oxidase Glycolate Sarcosine Oxidase Sarcosine Lactate Oxidase Lactate Glutathione Reductase NA D (P) H Riboamide Dehydrogenase NA DHPQQ# Cumulative Glucose Dehydrogenase Glucose Methanol Dehydrogenase Methanol and Other Alkanols Methylamine Dehydrogenase Methylamine Heme Containing Enzyme Lactate Dehydrogenase Lactate (Yeast Cytochrome B 2) Horseradish Peroxidase Hydrogen Peroxide Yeast Cytochrome C Hydrogen Peroxide Among these are enzyme/substrate pairs that have been characterized in great detail and which give a good and preferably linear response over the expected measurement range. It is clearly advantageous to use Glucose enzymes have been carefully studied for testing in vivo sensing of glucose (eg in diabetics). N ADP H-independent glucose dehydrogenases, such as Acinetobactyl, Calcoa 7-F-ri7. (aci, netobac
It has been found that enzymes such as those obtained from P. ter caicoaceticus are useful for this purpose and can be used in the present invention. Glucose oxidase undergoes a well-known reaction and forms a useful marker for the occurrence or extent of specific binding reactions in the present invention. Furthermore, this enzyme is stable during storage, can be used industrially, and is inexpensive. Although glucose oxidase may be preferred to some extent, other enzymes from the list above may also be used to advantage in certain variations of the invention. In particular, the kinetics of glucose oxidase may be useful. Mediators indicated for use in the above-mentioned patent applications include polypyrogens, polyunyls, promanyls, etc., but also compounds commonly known as "metallocenes", especially those containing at least two organic rings. and one metal atom, and the organic ring is a charge conjugated system, and the metal atom is a compound that is in covalent atomic contact with each of the rings. Ferrocenes (bis-cyclopentagenyl iron and its derivatives) belong to the last group mentioned above and are superior to other mediators used in conjunction with oxygen/substrate reactions aimed at charge transfer. Ferrocene (bis75cyclopentadienyl iron: F8C
The unique structure and properties of p, ) and its derivatives have generated much theoretical and experimental research. Ferrocene was first synthesized in 1951 and was the first example of the metallocene compounds that are well known today. Although ferrocenes were known to have limited value in spectrophotometric assays due to their low solubility in aqueous solutions and low extinction coefficients, ferrocenes are useful in bioelectrochemical systems. The ferrocenes found to be compatible include: (a) a wide range of redox potentials that can be varied by substitution of the all-enabled cyclopentadienyl ring; (b) electrochemically reversible, sexually active has →-electron redox properties; and (Ql 1)H-independent redox potential and slow autooxidation properties of the reduced state. As derivatives of such general classes of 7-erocenes, i.e. monomers and polymers substituted in one or both rings, the following individual 7-erocenes have been found to be advantageous: =1.1L dimethylferrocene +00 I,D-ferroceneacetic acid 124 8 8
70 H)-Roxyethylferrocene 161 S-Ferrocene 165 1. D 885 Ferrocene monocarboxylic acid 275 S 420 Ferrocene 1.1'-dicarbonel 885 S-chloroferrocene 845 1. D -Methyltrimethylaminofersen 400 8 - (In the above table, S is water-soluble, 1.D is insoluble in water, and 8% detergent (T
EO is a value expressed in mv with respect to standard calomel monopole (hereinafter abbreviated as SOE), and E is a measured value expressed in mv. ) pH of various 7 erocenes shown in the table above =
The E values for SOE in phosphorus@soil buffer in 7 are in the potential range of 100-400 mV. This E0
The trend in values is consistent with what would be expected based on substitution effects. Generally, electron donating groups stabilize the positive charge and therefore promote oxidation more than electron withdrawing groups. As detailed below, in certain instances there may be a chemical bond between either the ligand or the enzyme and the mediator. This is particularly useful for monitoring therapeutic agents where it is not anticipated that ferrocene or other mediators can be chemically coupled to the ligand. Example 1: Phenobarbital phenytoinproamide theophylline is combined with a mediator to form an assay system that can determine whether the drug concentration is within a suitable therapeutic range. There may be cases where it is possible to do so. Agents other than those mentioned above can also be used. Ferrocene-modified electrodes can be manufactured in several different ways. The simplest method is to deposit hydrophobic tF[K, such as Eva, ferrocene, vinylferrocene and 1,1'-dimethylferrocene, onto the platinum or graphite surface by evaporation from an organic solvent solution. Alternatively, the substituted derivative can be covalently attached to the hydroxyl group on carbon or platinum, as shown in the following formula: In the case of such specific variations, one or two
Incorporating more than one thiol group allows ferrocene to bind directly to all electrodes.

[It means rubbing. The electrode surface can also be coated with a functionalized polymer to which substituted ferrocene is covalently bonded, such as polytyramine, as shown in the following formula: Alternatively, vinylferrocene can be polymerized (vinylferrocene ) : (OH
2-OHO6H,Fecp)n can be obtained and deposited on the wheel 11 by either solvent evaporation or electrodeposition. Ferrocene-carboxylic acids have a combination of practical properties that make them suitable for widespread use in the present invention, and they can be chemically linked to ligands, antiligands, and/or enzymes via their carboxyl groups, as described below. ``I found that it gave me an opportunity to meet.'' Several methods within the scope of the present invention have been found to be particularly useful as practical assay methods. These methods include: ■, immunoassay methods for detecting or measuring the presence of a desired ligand, such as an antigen or antibody, in a mixture; ■; An analyte (analyte) that can be chemically bonded to an enzyme and that can undergo a specific binding reaction to a ligand.
A homogeneous immunoassay method for detecting or measuring the amount of l:yte). In, a homogeneous nucleic acid probe technology in which specific binding to complementary sequences affects the electrochemical binding of one or more species in a mixture. ■ A heterogeneous immunoassay method that is related to, but superior to, the known ELISA method. (1) An immunoassay method in which the surface of an appropriate electrode is coated or modified by a specific binding reaction so as to affect the transfer of charge to one electrode. Each of the above methods will be described below. ■ Homogeneous immunoassay method for ligands In one aspect, the method comprises: a) a ligand-antiligand pair capable of specifically binding together; and b) a doliplet of a mediator, an enzyme, and a corresponding substrate. and when the substrate is converted to the product by the enzyme, a charge is generated and this charge is transferred to the electrode via the mesoator, with at least a portion of one of the pair , at least a portion of one of said triblends is chemically cross-linked so that at least a portion of said charge transfer occurs when said ligand and antiligand pair are bound together. This is a test system designed to prevent this. In a first example of this form, the invention provides an unknown quantity of a ligand species to be measured, and an antiligand species for said ligand species, which is itself chemically bound to said mediator compound; Enzyme only in bound state/
A known excess of the antiligand available for charge transfer from the substrate reaction is subjected to a specific binding reaction, resulting in an electrochemical reaction of the excess unbound mediator/anti-tsuga 21 species. A method as described above for determining the amount of said ligand species by activity. In a second example of this form, the invention provides an unknown amount of a ligand species to be measured, and an antiligand species for said ligand species, which is itself chemically bound to said enzyme, and whose binding A specific binding reaction with a known excess of said antiligand species available for enzyme/substrate reactions only in the state of A method as described above for determining the amount of said ligand species by the electrochemical activity of the mesoator. The term “ligand species” is used here because (a
) the original antigen or antibody, i.e. serum naturally occurring or artificially produced in the human or animal subject, for assay or in fully purified or partially purified form; whole blood, urine, interstitial fluid,
antigens or antibodies present in the brain as cerebrospinal fluid or synovial fluid or as monoclonal antibodies, and (bl antigens/antibodies, e.g. by known amounts of its own antiligand known to be in excess). like generating,
This is to encapsulate a partially reacted form of an antigen/antibody or a complementary 1 derivative substance. Thus, for example, the present invention in this form may include: (a) (1) a mixture containing a sample containing the ligand to be assayed, a known amount of antilica (11) produced in a known excess, and a mediator; By mixing the compound with a known excess of the chemically bound ligand,
leaving all of the free ligand binding sites occupied and some ligand binding mediator electrochemically available; (1) mixing the enzyme and its substrate in a liquid mixture; (C) contacting said enzyme/substrate mixture with the mixture from step (a), and then (dl) contacting the resulting mixture with a sensor electrode, such that the charge transferred to said sensor electrode is available. This homogeneous enzyme immunoassay method is characterized in that the unbound amount varies by the Gantt binding mediator B (and therefore the deviation of the initial ligand I is allowed. The characteristic steps (11 and (11) are sequential steps). Of course, the order of other steps can be slightly changed.In particular, enzymes, substrates and electrodes can be combined;
The mixture produced by addition of step (a), i.e., the mixture of step (a), can be formed in a pre-existing liquid system of the enzyme substrate mixture, etc. Other stepwise methods are also used to add unknown amounts of mediator-bound substances. It is conceivable if it can be made into a final measuring form that can be coupled to the
If the ligand/antiligand has several binding positions, the distribution of binding and/or the equilibrium binding of different species can be predicted to some extent, then the mathematical derivation is a simple subtraction. Not necessarily expected. These characteristics can be measured and described. In this aspect of the invention, a useful enzyme-substrate system is glucose/glucose oxidase. , Homogeneous Enzyme Immunoassay Method for LIJ Gantt Subjects In a first example of this form, the present invention provides the method described above, in which (,1) (al unknown amount of ligand species X and (
(11) A certain amount of species X-E (where X is chemically bonded to enzyme E without destroying the enzyme activity) is mixed in a solution, and (11) this mixture is mixed in the presence of the sensor electrode. is brought into contact with the substrate S for the enzyme and the mediator compound M to transfer the measured charge to the sensor electrode depending on an enzymatically catalyzed reaction, (liD antiligand A
is brought into contact with the solution to cause a competitive, competitive coupling reaction represented by the following reaction formulas (1) and (n): to convert the enzymatically active species X-E into an enzymatically inactive species. A-
X-E, thereby changing the degree of reaction and the measured charge at the electrode, and then GV) It is. In a variation of the method described above, the mediator M can be chemically bound to the ligand analyte X in step (11), and the mediator M can be contacted with the enzyme E and the substrate S, ) can occur, i.e., a reaction that replaces E with M throughout. For example, a "dry strip" test is developed to measure levels of analyte X. In some cases, and to a lesser extent, the complex A'-X=E (or A-X=M)
Starting from , it is possible to operate only by a slow displacement reaction expressed in the following reaction formula: % formula % . In one form, the present invention comprises: (a) covalently bonding W analyte X and enzyme E to produce an oxygenically active species X-E; reacting with a reactive ligand A to produce an enzymatically inactive species A-X-E in which the specific binding site is completely occupied; (0) the enzymatically inactive species A-X-E; is brought into contact with the species X to be assayed to cause a competitive specific binding reaction with the species X,
Achieve equilibrium expressed by the following formula;
This mixture is then contacted with a substrate for X-E, and the enzymatically active species X-E present is transferred by a mediator compound from the resulting enzymatically catalyzed reaction to the sensor electrode. This is an immunoassay method for species binding the enzyme and analyte in close proximity to the active site of the enzyme (X-E) such that Ligand A effectively blocks or causes a morphological change in the active site; It turns out that is preferable. Alternatively, the ligand analyte X can be attached to a prosthetic group of the enzyme. Again, the exact order of steps used and! and the presence of additional steps can be modified by those skilled in the art. It is also possible to operate using the A-X-E complex arranged on the electrode itself, and thus by contacting the analyte It may be possible to cause enzymatic activity to occur in the presence of . In this general form of the invention, the amount of enzymatically active species is determined. This is useful for any additional features mentioned above and detailed below in which the enzyme and mediator are chemically linked. In this form, the invention provides for chemically linking at least one of a mediator and an enzyme to a nucleic acid probe sequence such that the specific binding of said nucleic acid Pro-1 sequence to a target sequence in the single-stranded nucleic acid material to be investigated is achieved by the enzyme. In a method as described above, affecting the electrochemical availability of said chemically bound species to be detected by a sensor electrode in the presence of a substrate, thus making the presence of said target sequence detectable. be. The nucleic acid sequence can be RNA, such as Metsenger RNA, but is usually DNA. In other words, this form of the present invention: (a) supplies a single-stranded nucleic acid substance to be investigated with respect to a predetermined target sequence; (b) supplies a probe substance having a nucleic acid sequence complementary to the target sequence; (c) The following eight types of treatments; Middle: A treatment in which the probe is bound to the enzyme and the enzyme-bound probe is added to a solution containing both a substrate and a mediator for the enzyme. , (+l) chemically bonding said probe and mediator and adding said mediator-bound probe to a solution containing both a substrate and an enzyme for said substrate; and 11ii) combining said probe 1 with a mediator/enzyme. (d) performing one treatment selected from the group consisting of: chemically bonding the probe to the combination and adding the thus denatured probe to a solution containing a substrate for the enzyme; contacting a solution containing a chemically bound Pro-1 sequence with a sensor electrode to transfer charge by the mediator from the N% substrate reaction catalyzed by the enzyme to the electrode, and then (e) said solution A nucleic acid characterized in that the specific binding reaction between the probe and the target that affects the availability of an enzyme, a mediator, or a combination thereof is indicated by a change in charge transfer by contacting the nucleic acid with the heavy chain substance. This is a method for detecting target sequences in substances. Probe materials can be naturally occurring DNA fragments or synthetic materials. Changes in the process order can be easily made. Although the sensor electrode itself can also contain a mediator or enzyme, it is usually preferred that both the probe and target sequences are present in solution. The mediator can be indirectly bound to the probe sequence by the linker group G, and a substance reactive with the linker group can be present on the electrode. In this case, the entire complex is present on the electrode, and if the target sequence is present and bound to the probe sequence, a change in current occurs. IV, a heterogeneous enzyme immunoassay method derived from the ELISA method In this form, the present invention involves immobilizing the ligand to be assayed on a suitable surface and then adding an excess of a suitable antiligand which is chemically bound to the enzyme. specific binding reaction with the amount, then removing this excess amount, adding a substrate for said immobilized enzyme, and then contacting said ? This method is characterized in that the charge transferred to the jl pole is proportional to the amount of enzyme present. In a preferred embodiment, the invention provides: (a) immobilizing the ligand to be assayed on a suitable surface, for example by binding said ligand to an antiligand species previously bound to said surface; (b) said ligand is brought into contact with an excess amount of enzymatic active species consisting of an antiligand chemically bonded to the enzyme to cause a specific binding reaction with the immobilized ligand, thereby immobilizing an amount corresponding to the amount of the ligand to be assayed. (d) contacting said immobilized enzyme with a substrate for said enzyme and a charge transferable mediator to generate said immobilized enzyme and a charge transferable mediator; 1 in contact with the mediator! A method for detecting heterogeneous enzyme binding, characterized in that the pole emits a charge corresponding to the amount of the immobilized enzyme as a signal, and from this, the initial amount of the ligand to be assayed is determined. The above steps can be rearranged and the mediator can be present in solution or on the electrode. (2) Enzyme immunoassay method on the electrode surface In this form, the present invention provides a method for conducting a specific binding reaction on the surface of the sensor electrode to at least partially block the surface or changing the characteristics of the surface. The above method is characterized in that the presence or extent of a specific binding reaction is determined by the presence or amount of a decrease in detected charge. - In a preferred embodiment, the invention provides: (~ selecting a liquid medium containing the ligand to be assayed; Additionally, C) the liquid medium contains (1) an antiligand capable of a specific binding reaction with the ligand to be assayed, and (II)
contacting a sensor electrode having on its surface a combination of said enzyme and <+++> mediator compound, thus transferring a charge from said enzyme to said electrode when said enzyme is catalytically active to generate a signal; (d) comparing said signal with the signal received in the absence of said ligand to determine the amount of said ligand present;
This immunoassay method is characterized in that it is determined as a function of the blocking state or morphological change on the electrode surface caused by the specific binding reaction. The ligand may be an antigen that is immunologically reactive with the antiligand and that occurs in biochemical or medical tests such as those exemplified above. Its general properties are that, like large protein-like molecules,
It has the effect of sealing off a portion of the electrode surface when the ligand reacts with the antiligand on the electrode surface. This causes a decrease in the current (11), and by observing this and measuring its magnitude, the presence of the ligand can be determined or its level can be determined. Relatively small molecules, 1-
that is, combining nitroglycerin with a relatively large molecule such as bovine albumin, injecting this combination into an animal subject to produce a nitroglycerin-sensitive antiligand, and then This antiligand can also be bound to an electrode. In such a case, the small bound nitroglycerin molecule would probably be
Sequestering the electrode surface causes a morphological change in the antiligand (especially when the concentration of antibody and/or nitroglycerin is relatively high) and thus changes the signal. The substrate material could theoretically be any of the substrates mentioned above. The reason for this is that the basic enzyme/substrate reaction has wide flexibility when using such electrodes. However, it is advantageous to use a simple, readily available and proven substrate, and for this purpose barrel glucose is preferably used. The enzyme present on the electrode may be any enzyme, but the 7-Rabin protein and quinone protein shown above are particularly preferred. However, since the preferred substrate is glucose, the preferred enzymes are glucose oxidase or glucose hydrogenase, such as those obtained from Acinetobactyl Calcoacetyphus, the latter being preferred since its turnover number is 100 times greater than the former. The foregoing discussion has described the invention using several enzyme/mediator binding compounds. In such a form of carrying out the invention, more than one ``Ndeutsch-type'' unit, such as ferrocene, can be present in the enzyme up to a concentration at which molecular deformation and loss of enzyme activity are observed. For example, glucose oxidase and about 8 to 12 7-erocene units are still enzymatically active and can be attached to a carbon electrode using known methods, such as carbodiimide linkages. - The denatured enzyme is not only enzymatically active but also electrochemically active. Although the above description relates to assay methods, it will be clear to those skilled in the art that kits comprising modified electrodes and suitably arranged components containing such electrodes also form part of the invention. In particular, it is possible to use strip test electrodes that contain all the necessary components of the present invention and are formed so that they simply need to be immersed in the test liquid. Hereinafter, the present invention will be explained based on examples and drawings. Example 1 Assay using glucose oxidase and 7-erocene In Figure 1, a patient's swab containing the antigen to be assayed (1) is mixed with a solution of antibody (2) of known concentration in buffer. This antigen is, for example, a drug. Antigen (1) in the serum binds to the solvated antibody at a specific antigen binding site (8), producing an antigen/antibody complex (4). Not all possible antigen binding sites on the antibody are occupied, leaving a large number of free binding sites W(5). The number of free positions is a function of the number of antigens (1) in the serum. The mediator-bound antigen (6) is added in excess to the mixture obtained from the first step. In this example, the mediator (7) is a heptadro7ine derivative chemically bound to the antigen (8). However, other metallocenes can be substituted. The ferrocene-conjugated antigen occupies the 7-lead position (5) of the antibody (2) through the antigen/antibody binding reaction.
The heptadrocene-binding antigen (6) is present in excess of the number of free positions σ), and therefore some of the heptadrocene-binding antigen (9) remains in the solution. Glucose and glucose oxidase (referred to as G'OD) are added in excess to the resulting mixture, and the current generated by the redox action of COD on glucose is transferred to an electrode (E
). Only the 7-rocene-conjugated antigen (9 complexes) that is not bound to the antibody (2) is present at the electrode (K
) can be coupled with GOD to 11L vapor chemically.
The φ winding does not allow coupling of the COD to the electrode due to steric hindrance from the significantly larger antibody (2). By measuring the electrochemical effect of the mediator present after addition of the enzyme (COD) and its substrate,
Calculate how much known amount of mediator (6) is bound to antibody (2), and how much free antigen (1) is bound to antibody (2).
must have been present in the original sample. Electrode (E) may be gold, carbon, platinum or any suitable material. Can be manufactured from materials. The addition of COD and glucose is optional to provide for the specific chosen mediator to couple the enzyme/substrate reaction to the electrode. Substitutions can be made by adding other enzyme/substrate bears. Neither the enzyme nor the substrate (both present in excess), ie neither COD nor glucose in this example, are limiting factors. The limiting factor is the amount of mediators that are free to spread if free locations are occupied. Example 2 A diagram showing this test is shown in Figure 2. All assays were carried out at room temperature (l
The assay was carried out in 50 mM Lys/HOI buffer at pH 7,5 with an excess of substrate (10 omM') at -25°C. No correction was made for unspecific binding. All solutions were degassed and ferrocene monocarboxylic acid was used as a mediator. The electrodes used were pyrolytic graphite ground between runs using a slurry of 0.3 alumina in H20. The components are not fixed, and the eight 1! A ls cell was used. The total sample volume was 1-. glucose oxidase labeled T4 thyroxine and T,
Antibodies to thyroxine were procured by Corning Glass, Inc. (Metfield, Mass,
U, S, A). Tris/HOl 1) I(7,5) Add 7ethene monocarboxylic acid to the sample solution drawn from the buffer. 1.66
It showed a current of μA. Adding an excess of 100 mM glucose reduced this current to 1.468 μA. Upon addition of 50 μl of GOD mT4, the current increased to 2.10 μA due to the catalytic activity of glucose oxidase due to the available glucose. The electrons removed from glucose oxidase were transferred to the electrode via the mesoie. When antibody was added to COD-labeled T4, this current was 1.59
decreased to μA. These results indicate that the addition of antibody inhibits the activity of GOD-labeled T4, making it difficult for assay emplacement and/or quantification. This produces detectable and measurable changes in the 1Rit stream used to provide the measurements. This is shown in Figure 2, which shows the competitive reaction between the enzyme-bound assay and the free assay for the binding position of the antibody. The obtained electrons are transferred to the electrode by the mediator compound, and the mediator is regenerated on the electrode surface. Example 8 (1) In Figure 3a, glucose oxidase (G
OD ) exhibits a catalytic action to convert glucose to gluconic acid and releases electrons that reduce the erocene mediator F from the oxidized state to the reduced state. When the reduced ferrodinium is oxidized with an electrode and the 4-pass current is measured, it is proportional to the amount of glucose present. DNA fragment D, derived from a naturally occurring DNA sequence or synthesized, is attached to GOD by any suitable method. (It) Steady state current is obtained in the presence of excess glucose. The DNA sample to be assayed for a specific target sequence complementary to that of DNA7ragmen)D is converted to single strands by any suitable method and then added to the reaction mixture. DNA fragment) D is mixed with the target sequence that complements it, inhibiting the enzyme reaction between glucose and the enzyme. Therefore, the production amount of glucose S to glucose acid is reduced, and the coupling reaction of ferrocene is reduced. The change in the rate of decrease of 7erocene is reflected in the decrease in current at the electrode. The change in current is proportional to the amount of DNA fragment D binding to the target DNA, and thus proportional to the amount of target DNA present. , Example 3b Mediator bound to DNA prologue
1(I) In Example 8b, the redox active 1M 7erocene F
directly to the cleaved DNA used as a probe. The generation of mediator-bound DNA probes is based on the droplet short response of mediator F, or the assayed D
It does not interfere with the binding interaction of the Mediator DNA probe with the complementary target sequence contained in the NA. (Add the old Mediator DNA probe to the assay mixture and measure the resulting amperometric response. If single-stranded genetic material is present that complements the probe, the probe in the sample DNA is binds to the complementary sequence in the mediator DN, which significantly reduces or completely inhibits the amperometric response.
The A-blob target DNA complex is not amperometrically active. The decrease in the initial amperometric response is
Generated mediator DNA probe/Tarkera) DN
is proportional to the amount of the A complex, and therefore the amount of mediator D
In this example, the enzyme activity does not change, but the extent transferred to the electrode changes. (I) In this example (see Figure 8C), the 7erocene DNA probe contains one or more linker groups L (for example, biotin can be used). 1. Linker group L is placed on the electrode surface. . Recognizing electrochemically active substances R (e.g. 7(II)
When the Mediator Linker-DNA probe is treated with a mixture of single-stranded genetic material, the Mediator Linker-DNA probe binds to any complementary sequence present. The initial current decreases upon binding of the electrochemically active substance R to the mediator linker-DNA probe/target DNA complex Y-i. The decrease in current is again a known DNA! ...proportional to the amount of single-stranded sequence added, along with the target sequence complementary to the probe sequence. In this example, DNA! Although it has been described in terms of deoxyribonucleic acid (deoxyribonucleic acid), it can also be applied in the form of RNA such as Metsusenja RNA. Example 4 As shown in Figure 4, when antibodies are placed in a polyester or polystyrene container (11), some of the antibodies (12) bind to the walls of the container. The container is treated as described below to promote antibody binding and/or to prevent non-specific binding of subsequent antibody (12). This treatment to avoid or reduce non-specific binding may include coating the container (11) with bovine serum albumen. The antigen sample binds to the antibody itself, which is added at 12a and bound to the wall of the container. Antibody\bound oxygen, in this example also antibody (12)
Addition of glucose oxidase (15) bound to the antigen/antibody binding reaction proceeds. To ensure binding, different antibodies can bind different proportions of antigen 12a. The vessel is washed to remove excess antigen-enzyme complex ←÷φ, and a solution containing soluble ferrocene (16) is added along with an aliquot of glucose substrate. Reference electrode (17), e.g. calomel, silver-silver chloride, gold,
A platinum or any other suitable noble metal electrode is placed in the solution along with an electron transfer electrode (18) with or without a ferrocene shell. The current generated at the electrode (18) is read via line (19). The current is proportional to the activity of glucose oxidase (15) and thus has a stoichiometric relationship to antibody glucose binding and therefore to the sample antigen 12a to which it binds. If desired, the mediator can be coupled to the antibody and the enzyme added along with the substrate. Other changes are also possible. Example 5 - This example is shown diagrammatically in Table 5. (I) In this example, a ligand is assayed. An antiligand 2o that can specifically bind to a ligand is immobilized on the electrode surface E. In the absence of ligand 21, mediator 2 in solution
2 is free and transfers charge from the enzyme ENZ to the electrode surface when the enzyme is catalytically active on the substrate S. (■) However, if the ligand 21 is in solution, the result indicates the presence of the ligand, since the ligand will likely occlude part of the electrode surface when bound to the antiligand, thus lowering the current; Measure the level of ligand in that range. This test system has a large beam Gantt, for example, I
Of value if it is an immunoglobulin such as gE.且↓ Preparation of Modifying Enzyme Ferrocene mole carboxylic acid was coupled to glucose oxidase using isobutyl chloroformate. The terminal amine of the protein was included for reaction with the 7-erocene carbonic anhydride. The reaction was carried out as follows. Ferrocene monocarboxylic acid (
1 mM in 8 ml of dry tetrahydrofuran (285 ■)
The solution was stirred and cooled (-8°C) and to this solution was added isobutyl chloroformate (0.13ml) and triethylamine (0°14ml) with constant stirring. Care must be taken to avoid containing water of reaction at this stage to prevent hydrolysis of the anhydride. The drying tube was attached to the apparatus or sealed and was previously purged with argon. The mixture was stirred for 80 min at -8"C, then warmed to room temperature and further stirred. The resulting ferrocene carbonic anhydride was added drop by drop to the glucose oxidase cooling (2 °C)
solution (o,t M NaHOO8 solution in 50 ml of 150
1v). Since ferrocene is not added, 1) H is 0.
8 using I M NaHOO8. The reaction mixture was stirred for 24 hours at 4°C and then centrifuged. This removed a large amount of unreacted ferrocene and some precipitated enzyme. Then protein I) H8,5
The mixture was dialyzed into a borate buffer solution (0.2M boric acid, 0.05M borax, 0.2M boric acid, 0.05M borax). Characteristics of the Modifying Enzyme Since about 10% of glucose oxidase can be used for modification with amino acid residues, it is expected that many 7epsens will be coupled to each enzyme molecule. The following method was used to measure the number five. The iron content of the modifying enzyme was determined by atomic absorption. Spectroscopic techniques were used to determine protein content. This spectroscopic analysis shows that the organic dye Coomassie Blue, at 465 nm, ... ~595 nm, when bound to proteins at acidic pH.
It is based on the shift in absorbance in nm. The 20% aqueous dye solution was filtered. A standard solution of modified glucose oxidase (1,4~/mJ) was prepared and this concentration of glucose oxidase was mixed with Coomassie blue solution. The optical density at 5 fl 5 nm was then measured for each concentration of enzyme, and a standard curve was obtained for the unmodified enzyme (Figure 4.2). The protein concentration of the modifying enzyme can thus be assessed by finding the optical density of the sample and comparing it to a standard curve. In the prepared samples, tampa! [-The concentration of phosphorus is 11
．． It was 04 μM. The iron concentration was 87.51 μM. Therefore, it can be inferred that an average of eight 7-erocenes are coupled to each molecule of glucose oxidase. Electrochemistry of Modified Enzymes From the preparation of the enzymes described above, an 11 μM enzyme solution in borate buffer is obtained. To investigate the electrochemistry of modifying enzymes,
A cyclic voltamodrum was obtained using a conventional 8-electrode cell. The experiment was conducted as follows. 800 PL cells
of modified enzyme (]]μM). The reference arm was previously filled with pH 8 borate buffer. Representative diffusion-controlled kinetics show that the ferrocene molecule (linked to the enzyme via the amide) behaves as a reversible one-electron mediator, exhibiting type II kinetics. In the absence of glucose, the enzyme converts to its oxidized form, enzyme-FA
D exists, and no catalytic reaction is observed. However, β
-D-When one glucose is added, the enzyme becomes -F A
It exists as D H2 and the catalytic reaction... takes place between this reduced form and the 7-erysinium ion at an oxidizing potential. The second-order rate constant K for the reaction between ferrocene and the octamate form of glutenose oxidase is 8.6 x 105 m-1.
It was calculated as 8-1. If we compare this value with that obtained for the solution kinetics of the same system, (1,15X 1
0'm-1B-''), modification is observed to increase the reaction rate.Also, the enzymes in the above table were electrochemically coupled via the 7 erocene monocarboxylic chain.Each of these enzymes was Sigma Chemical Company (St. Louis) was used without purification. In general, the techniques exemplified can be applied to a number of ligands, and there exists a "specific binding partner" that binds specifically to the ligand. do. An example of this interaction is shown below. IJ Cant Specific Binding Antiligand Antibody Hapten Antibody Raised to Hapten Conjugate Antibody DNA Probe or Antibody Fragment to Polypeptide Chain Protein or Protein Fragment 7 Bacterial Fragment Antibody This ligand is (a) immunological Protein or polypeptide chain (b) is a habuten with a molecular weight of 100 to 2,000. In the latter case, the hapten can be conjugated to a protein (eg, bovine serum albumin) to raise antibodies to the hapten (0) for immunization with carbohydrates or other organic molecules. Ligand Examples Hormone Examples Tumatropin Placental Lactogen Thyroid Stimulating Hormone Tissue Hormone Insulin Follicle Stimulating Hormone Glucagon Gonadotropin Leutinizing Virus Retrovirus Herpes Virus Hepatitis Virus Enzymes Serum Enzymes (Amaliase, Cholinesterase, etc.) Cytochrome Prostatic Acid Phosphatase Monoamine Oxidase Bacteria and their DNA fragments containing fragments, cell surface labels and cell surface polysaccharides immunoglobulin IgG, IgM, IgA,
IgE, IgD and each of these 7 fragments Fa
b and F. Examples of blood coagulation factor habten ligands Vitamins A, B, O, D, E, folic acid drugs (a
) Aminoglycoside examples amikacin, gentamicin, netilmicin, tobramycin, sisomicin, kanamycin (b) Nucleotide examples FAD, NAD. ADP (0) Steroid examples Cortisol, tetosterone (e) Other phenobarbital lidocaine, amphetamines, catechloamines, caffeine, digoxin, quinidine, shisopyramide, theophylline, cannapinoids, opiates, palpyrate, benzodiazepines, methatone (f) Antiepileptic drugs, phenytoin, brimidone, fuemebarbital, carbamazepine (g) antineoplastic agents methotrexate, midomycin C bleomycin, ifosfamide, cyclofoisfamide, cisplatinum agents vinblastine vincristine As mentioned above, both heterogeneous and homogeneous , many test charts can be developed. These include: (1) Binding to ferrocene or Gantt. All components diffuse freely. (11) Ligand bound to ferrocene. 7 erocene acts by covalently bonding to the m-polar surface. An example of a group used to act on ferrocene is "'C-(OH)"
There are NHt Rike-(0H2) nOOOH Sono and others 2 n 2. (Ill) Same as (11) above, but has glucose oxidase adsorbed, cross-linked, or immobilized on the m-pole surface. 0v) Ferrocene O that binds to and acts on COD to produce a COD-ferro7ine-ligand complex. (V) Ferrocene that binds to and acts on COD. G alone
'Ligand that binds and acts on OD. 7 erocene can in turn be attached to the electrode surface if desired. (Vl) Ferrocene-free, GoD-bound ligand. -Yu Ferrocene bound to a complex of FAD or an enzyme and bound to a ligand. FAD-Fe4sen+7Gand complex←+-)=X is generated. FAD-7Edasen-ligand complex
If it does not contain , it only binds to the apoenzyme. A competitive test demonstrating this is shown. (COD (7 enzyme))-X-Ab-AggAbA9+
(GOD Apo-X) The X in each S combination is FAD-7-Ligand complex-GOD. A variety of electrochemical techniques can be used to determine electrochemical changes, such as differential pulse portammetry, cyclic portammetry, or square wave portammetry. To minimize response time, kinetic amperometric measurements are preferred rather than end point measurements. Furthermore, according to the invention it is necessary to represent a thiol or a similar sulfur derivative of 7-erocene in the mediator, whereby the mediator is bonded directly to a noble metal electrode or similar. The thiol group can be attached directly or indirectly to one ring of the ferrocene structure, for example by a lower alkyl group containing 1 to 6 carbon atoms. J, Ohem,
SOo, 69 g (1,958) Simple thio-/I/(ferrocene)-8 by /x and boson
H can be used. Also, alkylthio-/L/
7 Erocenylthiobutane, i.e. (ferrocene) −
0,H8-SH is valid. Still other double-linked 4-thiol-like compounds include, for example, 1.28, trithia-
(8)-y is a tetracenophane in which the two rings are connected by a chain of sulfur atoms (it is a mixture of substances with different numbers of chain sulfur atoms). Prepare a gold electrode, for example, in a bath of this type of compound...
The mediator ferrocene structure is immersed in a liquid to bond the mediator ferrocene structure to the conductive metal. An example of the production of this type of material is shown below. Example 7 The method is shown below, but no product was evident from sublimation of the crude mixture. The sublimated material with the most obvious (ie most odoriferous) potential of 1... was subjected to silica chromatography (30 cm x 2 cm column) flushed with hexane to yield 8 products. 1 Contains no sulfur by analysis. 2, O: 48.72, H: 2.88, S+88.05,
CIoH8FeS8 is O: 42.9B, S: 84
．． 42 is required. Yield 0.45g. One complex, 8.011 g of oyster molecule, was not tested other than mass spectrometry. This analysis did not show a $7 eronanophane with ] sulfur atoms. Upper ball ferrocentiobencune (7 erocenylthiobutane) 1
, 7xaSe/Ilbutyric acid (J, Am, Gh8m, 800
．． 1957゜79.8420) Fo-Co -OH, -OH, -0H2-000H Prepared by the method of the above publication 2.7 Erocenylbutyric acid Fo-OH, -OH, -0H2-0H2-C00H Prepared by Talemensen reduction (dish 3. Ferrocenylbutanol Fo-0H2-OH, -0H2-0H2-OH, -OH
The acid (2) was dissolved in ether (distilled from sodium/potassium) and treated with lithium aluminum hydride in a nitrogen atmosphere. Upon completion of the reaction, excess lithium aluminum hydride was destroyed using ethyl acetate and then water. Separate the organic phase;
The aqueous phase was washed with ether (2X20+!Lt). The organic phases were combined and dried (Mg5O, ). -After the
The solvent was removed on a rotary evaporator. A two-component red oil was obtained. Silica chromatography C
C80C'12 Effluent with 1 ether and hexadiene, alcohol and ester, Fo-OH2-OH2-OH2-OH, -0
000H8 was obtained. 4,7 Erocenylthiobutane (3) ('400 mg') was dissolved in pyridine, hydroxylated, dried over IJ and cooled in a water bath. Tosyl chloride (1 L) was added and the solution was stirred until clear and then left at 4°C for 24 hours. The mixture containing solid pyridine hydrochloride is placed in ice/water to precipitate the tosylate. This was passed through water to obtain a yellow solid. This dry part is 1. R. The spectrum gave the characteristics of tosylate. The remainder (0.65%, still containing moisture) was diluted with ether/I/:methanol (1:1.
) and while stirring, add sodium hydrogen sulfide XH,
0 (1.6 liters) was added and the mixture was maintained at 5°C. After 30 minutes, the water bath was removed and the mixture was allowed to warm to room temperature. After 8 hours, thin layer chromatography (silica, 1: I Et
, On hexane) indicated that the reaction was complete. The mixture was dried on a rotary eno<dζ rotor, then dissolved in a minimum amount of Et20/hexane (1:11),
Silica chromatography (60-120 mesh, 2
It was flushed through 6 columns (5 x 2 cm columns) with It2O2/hexanes (1.:11). Approximately 150 ml was collected as the thiol ran off very quickly. Fo-OH,-0)(2-OH2-OH,-(
Yield of 3H2-3H200 g. Principles of flood electrochemical enzyme immunoassay 1. -111111116-10 -The principle of this enzyme-linked immunoassay is illustrated schematically below. 2 FeD + 2 Ab − 2 AbFeD2 F
eD + 2D+ 2Ab - AbFeD + Ab
D+D+FeDFFeD+D+Al1-A
D-drug Ab-antibody FeD-ferrocene-drug in the bFaD+D formula Conjugate ferrocene to the therapeutic agent in question. Without this antibody, the ferrocene-drug conjugate is free to mediate between glucose oxidase and the electrode. Since excess glucose is used, the enzyme is not substrate limited. The presence of this antibody binds the ferrocene-X agent conjugate to the antibody so that the ferrocene is no longer free to mediate between the enzyme and the electrode. However, when the intact drug is present in the sample, ferrocene
1. can compete with the drug Funshugate and prevent the binding of the drug-7 erosene conjugate, leaving the intermediary between glucose and m4dA free. The ideal 7erocene-labeled therapeutic in question would not need to alter the binding properties observed from natural drugs for its antibodies. That is, the antibody needs to bind labeled as well as unlabeled drug. Although the presence of Fersen-labeled drugs can be detected using direct electrochemical techniques, the use of enzyme-linked systems provides a "built-in" means of amplification that can be easily measured at very low concentrations. (a) Preparation of ferroceneacetonitrile tJ! (Compound I) Dimethylamine methyl 7 erocene methiodide (9
) and potassium cyanide (109) were dissolved in 100 ml of water and refluxed for 2 hours. The mixture was then cooled and extracted with ether (3 X 150 TR1) and the combined extracts were washed with water (6 X 100 rnl).
. After drying the ether phase with sodium sulfate, the solution was filtered and the solvent was removed in vacuo, yielding a yellow crystalline powder (
J, Org, Ohem, 28 (1958) 658]'. The product has an I, R, spectrum of 2240 Cm-' (
-ON expansion/contraction), 1.002cm-'7x Losene (0-H
)be >)' and 1.108 Cm'-'' has the characteristics of an asymmetric term pulse. Its melting point is 77-78°C. (b) Preparation of 7-eroceneacetic acid (compound n) Nitrile (
6,209) was dissolved in ethanol (100g) and added to a solution of potassium hydroxide (18g) in water (150M) and the solution was refluxed for 18 hours and then cooled. The bulk of the solvent was removed on a rotary evaporator to a final volume of 10
It was set to 0m. This 1.. solution is dissolved in ether (a x 1.
50 at) and the aqueous phase, then filtered and acidified (with orthophosphoric acid 85 to pH, o)
. Filter the yellow precipitate, dry it with phosphoric acid, and use a desiccator.
), gave a yellow powder, 4.54g10 Analysis = tm value 0:59.05% H; 4.96% actual value C: 58,76% H: 4.99% Melting point 158
~155℃ (lit, 152~156°) (C) 1t2
tBtθ, 7-bentahydro 1,8-dimethyl-2,
Preparation of 6-dioxopurine-8-methylferrocene (compound ■) Ferrocene acetic acid (5.1 g) and 5,6-diamino-1,
8-Dimethyluracil hydrate (3,5777) was dissolved in dimethylaniline (3Q m
1) under nitrogen. After 18 hours, the solution was cooled, aqueous sodium hydroxide (8%, 36 ml) was added, and the solution was steam distilled. At the end, the vessel contents were filtered and then oxidized to p) 14.5 using acetic acid. The precipitate formed was filtered under vacuum and dried over phosphorus pentoxide in a vacuum desiccator. Yield 1,679, brown powder. Touch point: 80℃. Mass spectrometry molecular exhibition-378 Trace analysis Calculated value 0 for C□8H□8N, O, Fe
: 57.14chi, H: 4°76%, N! 14
, 81chi actual measurement value 0: 58.00%,) I: 5.
15%, N: 18.80fr, R, 700~8
QQem-", 160 (1-1750 cm-17-ofyline. 1000 cm"", 1100 cm-11 monkeys were substituted with a 7-functional locene derivative. From the mass spectrometry data, this substance was 10""8 am Hg to volatilize the sample. It is non-volatile as it required a current of 0.6 amperes at pressure. (d) Preparation of ferrocene ethylamine (compound ay>0
．． 859 LiAlH4 as 4 Q rnl (7):
The mixture was gently refluxed in a c-f solution for 1 hour. Ferrocenylacetonitrile (z
, 1) were added at a rate to achieve reflux while maintaining N2 pressure. After continuing to reflux for 2 hours, the mixture was cooled and
+n1 (7) water added, then 1 g of 10% Na, O
H% Finally 5 ml of water was added. The ether layer was decanted from the solids and the solids were washed with 8 x 10 ml of ether. The combined organic phases were treated with HOI gas and N
The yellow salt of 7-erocene ethylamine hydrochloride was separated by decantation under 2. solid (still soaked in ether)
was added to 2N NaOH and the mixture was extracted with ether. The ether phase was then dried over Mg5O, and the solvent was evaporated in vacuo to yield a dark brown oil. (e) 1,2,8,6,7-bentahydro1,3-dimethyA/-2,6-thioxoprine-8-butyric acid (compound V) 4,5-diamino-1,8-dimethylpyrimidine-2,5 -dione (19,5.9 mmol) and glutaric anhydride (11,849, 11.8 mmol) in 10IIIt of N,N-dimethineaniline using a Dean static trap for 2.5 hours under N2. It refluxed. 5I++/ of solvent was added and reflux continued for an additional 0.5 hour. The reaction mixture was cooled and filtered. The solid material was washed with benzene and recrystallized from water to yield 600 nm of white crystals. Melting point 288-240'C. Molecular weight from mass spectrometry, 266°C11H14”404 moleculesff1266mff1 analysis
Actual value 0: 50.04%, H: 5.14'%,
N: 21.12% calculated value 0:49.62%, H
: 5.26 yen, N: 21.05 Article (f) 1, 2, 8
,6,7-bentahydro1,8-dimethyl-2,6-
Thioxopurine-8-(N(2-ethylferrocenylcobutanamide) (Compound #!l') Compound V (181m!
; 1,0.5 mmol) and compound N (1,15 m
9, 0.05 mm%/l/) was dissolved in 5 ml of N,N-dimethylformamide. Dicyclohexylcarbodiimide (120 μ, 0.6 mmol) was added to the stirred solution at room temperature. I stirred it continuously for 4 hours. The reaction was complete as determined by thin layer chromatography. The plate was washed with cyclohexane-ethyl acetate-methanol 4:2:1.
Experiments were conducted using the following solvent systems. The reaction mixture was poured into 50 m/ml of ether. The precipitated material was filtered and washed thoroughly with ether, yielding 220 mg of a brown powder. Melting point: 216-220°C. Molecular weight from mass spectrometry: 477. 0.8H, 71J, 08 Fe molecular weight: 477. Observed trace analysis value: 0: 56.80. H: 5.42%, N:
14°28% Calculated value C: 57.86%, H: 5.66
%, N: 14゜67chi1, R, spectrum is 700-8
00cm, theophylline at 1600-175Qcm, and characteristic peaks of the signal due to the ferrocene component substituted with one ring at OnCm''''1. (Antigenic ferrocenteophylline conjugate Ill and ■ of the conjugate
The immunotechniques-two-antibody enzyme immunoassay kit was used to determine the antigenic properties. This is a heterogeneous enzyme immunoassay, based on the principle of competitive binding, in which a theophylline ferrocene conjugate or standard in the sample competes with the theophylline enzyme Funshugate for specific antibodies. The separation step was performed using dual antibody immunoprecipitation. Enzyme activity determined in globules shows 0-4op for theophylline.
Obtained in the therapeutic range of g/go problems. Typically, the standard curve is shown as a plot of either the absorbance of the sample expressed as plank or the absorbance at 4001 m versus the logarithm of the concentration. However, a more useful method is to perform a logit transformation of the data. Plot %A/Ao against the natural logarithm of concentration to obtain a straight line. To test the crosslinking reactivity of compounds,
These logito transformations require obtaining parallel straight lines. The data are shown in Tables 7A and 7B. FIG. 6 shows standard curves for theophylline and Compound 2. Compound ■ was more antigenic to theophylline antibodies than theophylline itself. %A/Ao-50 (i.e. logito%A/Ao-0)
A comparison of the results to shows that compound ■ is 7.6 times more antigenic than theophylline. Similarly, in experiments with compound ■, approximately 4
It was found to be 00 times more antigenic (data not shown). Table 7A (μ9m 0 - - 2.5 88 1.99 5.0 81 i, 45 10.0 67 0.71 20.0 49 -0.04 40.00 85.5 -0.60 Logi ) b-In (b/100-b) b-'AA
/A. r--0,998 gradient--0,96 Table 7B (Theophylline) 0.25 84 1.66 0.50 78 1.22 1.00 69,6 (1,88 2,5050,10,004 6 ,0038゜2 -0.70 7.50 25.6 -1.07 10.00 18.0 -1.52 15.00 1.5.9 -1.66 20.00 12,4 -1.96 r - 0,997 gradient - 0,95 glucose oxidase (FC1, 1, 8, 4> was from Beichringer Mannheim. D-glucose (AnalaR) was from BDH. All bath solutions was prepared from Alistair grade reagents in high purity water (millibo/I/).The supporting electrolyte was 0°1.M K2HPO, adjusted to the required 1)H using Hai6. Equipment, O, cyclic portammetry experiment, 9.5
The experiment was carried out using two 1σ11 cells with a working capacity of m/. In addition to the 4 gold disc working electrodes, the cell also has]
cm" platinum gauze counter electrode and a saturated calomel electrode as m 14C4 were included. Total positions refer to the saturated calomel electrode (S, 0.E, ). For cyclic portammetry, Brian X-Y 2 for auto-electrode potentiostat
A 600OA+3 chart recorder was used. Procedure Compounds m and ■ were insoluble in aqueous solution, so the required amount was dissolved in a small amount of dimethylformamide (DMF) and this solution was added to the buffer solution t7 to give a final DMF concentration of 10% (V
:V) was given. In this method, it was possible to keep the compound and M in aqueous solution for electrochemical experiments. , Results Under the experimental conditions used in this study, the compound ■
and ■ showed a voltamodrum consistent with a reversible one-electron redox agent at a gold electrode. Compound me E H-105mv Compound M, Ey, -15omv Compound Highly anti-ophyllin antibody. Because it was found that theophylline is a
This compound is only 7.6 times more antigenic than theophylline and therefore requires as much antibody as is required by compound ■ to block the electrochemical reaction. I don't. However, compound (1) and compound (2) acted as mediators for the glucose oxidase reaction. In a study where the peak current changes as the concentration of compound ■ increases, a glucose oxidase coupling reaction was performed. The results are summarized in Table 70. Table 70 Concentration of compound ■ (μM) IP binding IP non-binding (μA
) (μA) 20 0.8 9.1 40 0.62 25.1 (100,8908 80, 1,13148 1001,86177 5 mys) was used for the binding and non-binding experiments. As such, a linear relationship between peak current and concentration exists for bound reactions. However, such a relationship is not evident for unbound reactions. This is probably due to the very small peak current generated by this reaction. This may be due to an error in the evaluation of (see drawing). ・(j) Electrochemically bound glucose observation procedure for compound ■ Phosphate buffer p containing 10% (v:v) DMF
The compound group prepared in H7,1 was diluted by adding 100 Itλ (200μ of stock solution! 50μ to the sample) of phosphate buffer or 50μl of undiluted rabbit antiserum (Immunological Techniques). From this solution 150 μl was placed into a 0.5 ml electrochemical cell containing 250 μl of buffer and 50 μl of 1M glucose solution. Non-bonded reaction glue, O, cyclic voltam duram,
Recordings were made using a scan rate of 5 mVs-'. after that,
50μ of 3m91m/glucose oxidase solution was added, the solution was degassed with argon, and the portumgram of the enzymatic catalytic regeneration of 7erocene from the ferridinium ion was recorded. The final compound m concentration was 24 μM. Additionally, for compounds with equal concentrations of theophylline,...
Samples were prepared containing theophylline at a concentration 10 times that of Compound (2) and Compound (2). In both cases, bound and unbound portamograms were recorded. Finally, theophylline (240/jM) and the compound ■
After incubation with 50 pl of undiluted rabbit antiserum (24 μM) for 15 minutes at room temperature, the samples were removed and voltamodalum was scored in the presence or absence of glucose oxidase. Results The unbound cyclic voltammodalum of a 24 μM solution of the compound showed the expected behavior of an electron redox agent (Ey, −195 mV, Ep −60 mV, 1p −25 μA). Addition of glucose oxidase to the solution produced the portamo7g change outlined above. No peak was observed, and Owashi's catalytic current (0.3μA)
flowed at the oxidation potential. When compound ① was pre-challenged with rabbit antiserum, voltamodalum showed no reversible behavior, showing only an anodic peak at 240 my and no peak at reduced potentials. This peak was shown because the antiserum itself contains a known component. When glucose oxidase is added,
The catalytic current was not observed, and in fact the portamogram was identical to that of the unbound reaction. Since Compound (1) was completely bound to the antibody, it could not participate in the electron transfer reaction at the gold electrode and could not act as a mediator for glucose oxidase. Addition of free theophylline to a solution of Compound 1 at the above concentration did not have any effect on the electrochemical behavior of the ferrocene-drug conjugate in the presence and absence of glucose oxidase. Theophylline (240
μM) and compound ■ (24 μM) using rabbit antiserum, the recorded portamogram was
It was revealed that compound ■ freely participates in electron transfer reactions at the electrode and can act as a mediator for glucose oxidase. Indeed, the catalytic current generated (0.8 μA) was identical to that obtained in the absence of theophylline and antiserum. This indicates that theophylline completely binds to the antibody and thus generates a free compound in solution that can participate in the enzyme-linked reaction, thus providing a basis for assaying theophylline in serum. In one form of this assay, the theophylline/ferrocene funshugate, theophylline antibody, glucose oxidase, etc. can alternatively be used to separate (by spatial location or physical state) at least the conjugate from the antibody and the glucose oxidase from the glucose. ,
A drying process in which glucose is placed on the active electrode) contains the IJ tube. The above components are an example of the present invention. Upon addition of a biological fluid containing an unknown theophylline level, a competitive binding reaction occurs as described above, the current decreases, and the assay proceeds for measurement as described above. A preferred structure is one with 7 points relative to the antibody binding site.
A competitive binding reaction occurs between the erocene/theophylline conjugate and the theophylline to be assayed on the other hand. For this purpose, the following steps were performed. (A) An unknown amount of theophylline is used. (B) Mixing the conjugate therewith. , (0) adding the mixture of cA) and (B) to excess glucose (glucose oxidase system in the presence of a sensor electrode, e.g. a gold electrode in a 2-8 electrode system)
. ) to measure the catalytic current. (g)) A known amount of antibody is added to cause a competitive binding reaction between theophylline and the conjugate. (F) [Measuring the drop in flow, or rate of decline.

[Brief explanation of the drawing]

Figures 1+2+8a-30, 4 and 5 are diagrams showing the assay method according to the present invention, Figure 4.2 is a graph showing the standard curve for unmodified enzyme, and Figure 6 is a graph showing the standard curve for the unmodified enzyme. 1 is a graph comparing the antigenicity of drug/medicine conjugates. 1... Antigen 2... Antibody 3... Antigen binding site 4... Antigen/antibody complex 5.
...7-like binding position 6...mediator-binding antigen 7...mediator 8...antigen 9...ferrocene-binding antigen 11...container 12...antibody 12a...antigen 15...・Glucose oxidase 16... Ferrocene 17... Reference electrode 18...
Electron transfer electrode 19... Current line 20 Antiligand E... Electrode 22... Mediator GOD... Glucose oxidase F... Ferrocene mediator D... DNA fragment L... Linker group R. ...Electrochemically active substance S...substrate ENZ...enzyme. Engraving of drawings (no changes in content) FIG, 1 FIG, 2 +jwoI=t F,, 3c. FIG, 4゜(GB) ■8333651 ■January 19, 1984 ■United Kingdom (GB) ■8401399 @ February 29, 1984 Φ United Kingdom (GB) [Yes] 8405262 ■ February 29, 1984 ■ United Kingdom (G B) @8405263 414- Procedural amendment (method) August 3, 1980 Director General of the Patent Office Shiga Half 1, Indication of the case 1982 Patent Application No. 90831 2, Title of the invention, Specific binding agent Testing method used 4, Agent procedure amendment Written on August 3, 1980, Commissioner of the Patent Office Shiga Half 1, Indication of case Patent application No. 90831 of 1983 2, Name of the invention Test using a specific binding agent Method 4, Agent 5, Subject of amendment “Detailed description of the invention” column 1 of the specification
, "Table 5" in the first line of page 67 of the specification is corrected to "Figure 5." 2. r 2 FeD + 2 A on page 81, line 14
t) −2AbF8DJ as “Fep + Ab * A
Corrected to bFeD J. 8, r 2FeD+2D+2Ab on page 81, line 15
-AbFeD+ 71bD -1-D + FeD J
Delete. 4, page 81, line 16 (7) rFeD+D+Ab-
AbFeD+DJ r FeD+D+Ab d AbF
eD+D Correct to J. 5. On page 90, lines 11 to 13 of the same page, "Ferrocene...determined."
+11 and ■ were assayed to determine antigenic characteristics. ” is corrected.

Claims (1)

[Claims] L (a) At least one specific binding reaction step between a reactive species, including a ligand and an antiligand, electrochemically coupled to a substrate at a level at which the enzyme is catalytically active. the binding reaction is carried out to affect the electrochemical availability of at least one component of the enzyme/mediator thread, and then (bJ M distribution electrochemical availability An assay method characterized by obtaining a signal by detecting or measuring the effect on the reactive species, and determining the presence or amount of the reactive species from this signal.2 Claims: The specific binding reaction is carried out in a solution. The method according to claim 1. & The method according to claim 1, wherein the specific binding reaction is carried out on a solid surface. 1. It has been found that there is an excess of the antiligand species that is itself chemically bound to the mediator compound and is available for charge transfer from the enzyme/substrate reaction only in that bound state.
2. The method according to claim 1, wherein the amount of the ligand species is measured by the electrochemical activity of the resulting excess amount of unbound mediator/antiligand species by causing a specific binding reaction with a known amount of the ligand species. a(a)(1) Mix a sample containing the ligand to be assayed with a known amount of antiligand known to be in excess; The resulting mixture is mixed with a known excess of the ligand chemically bound to the mediator compound to determine the free ligand binding capacity. (b) mixing an enzyme and a substrate therefor in a liquid mixture; (C) mixing said enzyme/substrate mixture with contacting said mixture from step 1a) and then (d) contacting the resulting mixture with a sensor electrode, such that the charge transferred to said sensor electrode varies with the amount of unbound ligand binding mediator available; A homogeneous enzyme immunoassay method characterized in that a deviation in the initial Gantt amount is therefore allowed. The method according to claim 5, wherein the mediator is 7-erocene. 7. The ferrocene is carboxylferrocene. A method according to claim 6. & A method according to any one of claims 4 to 6, wherein the substrate/enzyme system is selected from glucose/glucose oxidase and glucose/glucose dehydrogenase. (1) (a) Ligand building X to be tested for unknown quantity and (b)
A quantity of species X-E (where X is chemically bonded to enzyme E without destroying enzyme activity) is mixed in a solution (11) and this mixture is placed in the presence of a sensor electrode. In contact with the substrate S Ig for the enzyme and the mediator compound M, the sensor 1! measures a charge depending on the enzymatically catalyzed reaction. the enzymatically active species The enzymatically inactive species A-
Claim 1, characterized in that the concentration of the analyte X is measured from the decrease or rate of decrease of the charge (φ) by converting it into No. (1) (a) An unknown amount of the ligand species X to be assayed and (b) a certain amount of the species (11) This mixture is applied to the substrate S in the presence of the sensor 1! pole.
and a catalytically active enzyme E thereto to transfer a charge to the sensor electrode for measurement in dependence on an enzymatically catalyzed reaction, The species X
Converting the -M moiety to an inert rod A-X-M, thereby changing the extent of the reaction and the measured charge at the electrode, and then determining the concentration of the ligand analyte X from the decrease or rate of decrease in (1v)'aL charge. A method according to claim 1, characterized in that: IL (a) covalently bonding analyte X and enzyme E to produce an enzymatically active species X-E; (b) forming an appropriate reactivity with the enzymatically active species X-E;・
reacting with ligand A to produce an enzymatically inactive glaze A-X-E in which the specific binding sites are completely occupied; (C) assaying this enzymatically inactive species A-X-E; Bringing it into contact with the target species X to cause a competitive specific binding reaction with the species X,
Achieve equilibrium expressed by the following formula: % to be able to measure the amount of N(X to be tested) from the total amount of the species X-E;
(I:i) this mixture is then contacted with a substrate for X-E, and the enzymatically active species X present is transferred by a mediator compound from the resulting enzymatically catalyzed reaction to the sensor electrode; -Measure the entire view of B,
An immunoassay method for species X, characterized in that the amount of species X is determined from the measurement results. 1λ. The method of claim 9, wherein the mediator is ferrocene. 13. The method of claim 12, wherein the 1 & 7 erocene is carboxyl 7 erocene. 14. The method according to any one of claims 9 to 11, wherein the substrate/enzyme system is selected from glucose/glucose oxidase and glucose/glucose dehydrogenase. 15. At least one of the mediator and the enzyme is chemically bound to the nucleic acid probe sequence, so that specific binding of the nucleic acid probe sequence to the target sequence in the single-stranded nucleic acid material to be investigated is achieved in the presence of the enzyme substrate. influencing the electrochemical availability of the chemically bound species as detected by the sensor electrode;
2. A method according to claim 1, whereby the presence of the target sequence can be detected. 1a(al) Supplying a single-stranded nucleic acid substance to be investigated to a predetermined target sequence; (b) Selecting a probe substance having a nucleic acid sequence complementary to the target sequence; (0) The following eight types of treatments (11) Chemically combining the probe p-1 with an enzyme and adding the enzyme-bound probe to a clear liquid containing both a substrate and a mediator for the enzyme. and adding this mediator-bound probe to a solution containing both a substrate and an enzyme for said substrate, and chemically coupling the probe to the mediator/enzyme combination and thus 2. Perform one treatment selected from the group consisting of: adding the denatured probe 1 to a solution containing a substrate for the enzyme; contacting a solution containing the sensor electrode with the substrate reaction catalyzed by the enzyme;
(e) contacting said solution with said -heavy chain substance to produce an enzyme;
Detection of a target sequence in a nucleic acid substance, characterized in that a specific binding reaction between the dip and the target, which affects the availability of a mediator or a combination thereof, is indicated by a change in the amount of charge transfer. Method. 17. Binding the mediator indirectly to the probe array through a linker group, causing a substance reactive with the linker group to be present on the electrode and binding to the probe array to produce a 'mm lightning current change. The method according to claim 15 of the patent claim. 18. The method according to any one of claims 15 to 17, wherein the mediator is 7-erocene. 19. The method according to any one of claims 15 to 17, wherein the ferrocene is carboxyl-7 erocene. 20. Any one of claims 15 to 17, wherein the substrate/enzyme thread is selected from glucose/glucose oxidase and glucose/glucose dehydrogenase. The method described here. 2L The ligand to be assayed is immobilized on a suitable surface, then subjected to a specific binding reaction with an excess amount of an appropriate antiligand chemically bonded to the enzyme, after which this excess amount is removed and the immobilization is carried out. 2. The method of claim 1, wherein a substrate for the enzyme is added and then brought into contact with the mediator and sensor II electrodes such that the charge transferred to said electrodes is proportional to the amount of enzyme present. (a) Immobilizing the ligand to be assayed on a suitable surface; (b) contacting said ligand with an excess amount of an enzyme-active species consisting of an antiligand chemically bound to the enzyme to combine with the immobilized ligand; (0) removing an excess amount of unimmobilized chemically bound enzyme/antiligand; Then ((1) the immobilized enzyme is brought into contact with a substrate for the enzyme and a charge mobility mediator, and an electrode in contact with the mediator detects a charge corresponding to the amount of the immobilized U violet as a signal, 23. A method for detecting heterogeneous enzyme binding, characterized in that the initial amount of the ligand to be assayed is determined. ε & The method according to claim 22, wherein the mediator is 7-erocene. 24. The method according to claim 22, wherein the 7-erocene is 24. The method of claim 23, wherein the substrate/enzyme system is selected from glucose/glucose oxidase and glucose/glucose dehydrogenase. G. The method according to this invention. 2a. Detecting the presence or extent of the specific binding reaction by causing the specific binding reaction to occur on the surface of a sensor electrode and at least partially blocking or altering the properties of the surface. 27. The method of claim 1, wherein the method is determined by the presence or amount of a decrease in charge. 27. (a) selecting a liquid medium containing the ligand to be assayed; (b) adding an enzyme to said liquid medium. (0) adding a known amount of substrate 1 capable of carrying out the reaction catalyzed by the enzyme;
11) contacting a sensor electrode having a combination of said enzyme and (m ) a mediator compound on its surface, thus transferring a charge from said enzyme to said electrode when said enzyme is catalytically active; emitting a signal, and then (d) comparing said signal with the signal received in the absence of said ligand to determine the amount of said ligand present;
An immunoassay method, characterized in that the difference in Q to the front viewing wing λ that exists as a function of the blocking state or morphological change on the electrode surface caused by the specific binding reaction is determined. 21L. The method of claim 26, wherein the mediator is 7erocene. 28. The method of claim 27, wherein the ferrocene is carboxylferrocene. 29. A method according to any one of claims 26 to 28, wherein Bo, the substrate/enzyme system is selected from glucose/glucose oxidase and glucose/glucose dehydrogenase. 81. The method according to any one of claims 1 to 8, wherein the enzyme and the mediator are chemically bonded together. B& In determining the presence of a selected ligand species present in a mixture of compounds, 1) reacting said ligand species with an antiligand to form a ligand/antiligand complex; 11) allowing the enzyme to catalyze the reaction; 111) exposing the mixture of the enzyme and the electron transfer mediator to an electrode having a conductive surface to transfer the electrons; Electrochemical control of the rate of the reaction catalyzed by the enzyme by allowing a transfer mediator to transfer electrons between the electrode surface and the enzyme at a rate corresponding to the rate of the reaction catalyzed by the enzyme. A method for measuring the presence of a selected ligand species, characterized by detecting the presence of a selected ligand species. 8 & Claims FI in which the chemically linked mediator analyte conjugate is FOOH,-theophylline! I. Method according to paragraph 1O. 84 On the electrode surface, (a) a combination of a ligand species and a mediator), (b) an antiligand for this, (0) an enzyme substrate, and (d) an enzyme that is catalytically active for the substrate are supported. , an electrode having a structure in which, by spatial arrangement or physical state, at least the conjugate is separated from the antiligand and the enzyme is separated from its substrate. 8B: Bringing the electrode according to claim 84 into contact with a medium containing an unknown amount of the ligand species to be assayed, and measuring the signal or change in the signal received from the electrode to determine the unknown amount. Test method.