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WO1986004926A1 - Analyse de substrats degradables par detection electrochimique d'especes redox - Google Patents

Analyse de substrats degradables par detection electrochimique d'especes redox Download PDF

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Publication number
WO1986004926A1
WO1986004926A1 PCT/GB1986/000095 GB8600095W WO8604926A1 WO 1986004926 A1 WO1986004926 A1 WO 1986004926A1 GB 8600095 W GB8600095 W GB 8600095W WO 8604926 A1 WO8604926 A1 WO 8604926A1
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Prior art keywords
redox
enzyme
species
assay
mediator
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PCT/GB1986/000095
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English (en)
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Calum Jack Mcneil
Monika Joanna Green
Hugh Allen Oliver Hill
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Genetics International Inc.
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Publication of WO1986004926A1 publication Critical patent/WO1986004926A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • 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

Definitions

  • the present invention concerns the electrochemical detection of redox species in a medium and the use of such detection to determine the occurence of a condition under which such species are liberated, produced, sequestered or discharged into a medium.
  • the present invention is concerned with the electrochemistry of mediators in combination with degradable substrates, such as polymers, and with an electrochemical assay which is in one aspect particularly concerned with an assay for the presence of, or amount of, polysaccharide complexes in a sample, but which extends a general assay for detecting the presence of, monitoring the level of or determining the concentration of compounds containing polymeric structures as set forth below and including but not limited to; celluloses, pectins, galactosides, hyaluronic acids, lipids, peptides, nucleic acids and specific binding partners in immunoassay.
  • an assay system for enzymes having a polymerase or depolymerase activity and for other chemical or physical conditions resulting in the making or breaking of chemical bonds, or changes in physical state or condition.
  • ⁇ -Amylase (1, 4- ⁇ -D-glucan glucanohydrolase, E.C.3.2.1.1.
  • an enzyme which catalyses the hydrolysis of 1,4- ⁇ -glycosidic linkages of polysaccharides (e.g. starches and glycogen) and of oligosaccharides to yield maltose and other simple cleavage products.
  • ⁇ -amylase in serum or urine is due to at least two distinct isoenzymes; a first ⁇ -amylase from the pancreas and a second from the saliva. This endoenzyme is extensively measured in clinical laboratories for the diagnosis of acute pancreatitis and obstruction of the pancreatic duct. The determination of ⁇ -amylase activity is also important for the food industry, especially for starch converting plants, breweries and other fermentation facilities.
  • amyloclastic i.e. starch-iodine
  • saccharogenic i.e. saccharogenic
  • chromogenic i.e. chromogenic
  • Hydrogen peroxide produced by the oxidation of glucose by glucose oxidase is determined by a polarographic electrode of the type used in the Yellow Springs Glucose Analyser.
  • Electroanal. Chera. 194 123-130 (1985)] is based upon the electrochemical detection of hydrolysis products from modified blue starch, the subs-trate normally used for chromogenic determination of ⁇ -amylase activity.
  • Simionescu discusses the attachment of ferrocene carboxylic acid to cellulose and to Polyvinylacetate by ester linkages, and the attachment of ferrocene carboxaldehyde to polyvinyl pyrrolidinone via an aldol condensation.
  • I t is suggested by Simionescu that the adduct acts as an effective slow-release agent for the treatment of anaemia.
  • Enzyme/substrate pairs whose electrochemical behaviour in association with mediator compounds have been studied by the Applicants include those listed in Table 1.
  • Oxidase Pyruvates L-Amino Acid Oxidase L-Amino Acids Aldehyde Oxidase Aldehydes Xanthine Oxidase Xanthines Glucose Oxidase Glucose Sarcosine Oxidase Sarcosine
  • Ferrocenes have: (a) a wide range of redox potentials accessible through substitution of the cyclopentadienyl rings which can be functionalised;
  • Eo is in mV vs a standard calomel electrode, ⁇ is measured in cm -1 M -1 1 -1 .
  • mediator compounds include those listed in Table 3. It should be noted that not all mediator compounds will mediate effectively between a particular redox enzyme and an electrode surface. The selection of the optimum mediator is to some extent determined by the particular enzyme in use, under a given set of chemical and/or physical conditions. TABLE 3 Mediator Compounds
  • Viologens and their polymers
  • the present invention concerns the use of an electro-chemical detection of redox species in a medium to determine the occurence of a condition under which such species are liberated, produced, released or sequestered, wherein such production or release is enabled by the digestion, build-up or disruption of a macroraolecular species
  • digestion includes the breakage of chemical bonds under a range of conditions, including enzymic digestion of the macromolecular species, build-up includes the polymerisation of a number of monomers, sub-units or oligomers into a macromolecule, and, disruption includes the breakage of bonds by physical processes such as the disruption of lysosomes (including liposomes) or micelles.
  • an assay system comprising; a) a macroraolecular species comprising at least in part a component, activatable on digestion or disruption of the macroraolecular species to produce, liberate, sequester or release directly, or facilitate indirect production, liberation, sequestration or release of redox species within a medium and, b) electrode means responsive to the presence of the redox species, for electrochemical detection or measurement of the redox species in the medium, whereby the said detection or measurement is related to the digestion or disruption of the macroraolecular species, as enabled, on occurence of a predetermined condition, thereby being capable of a response to the presence type or amount of the macromolecular species, and/or of the said presence or amount of digestion or disruption and/or of circumstances affecting the indirect production of the redox species.
  • This aspect of the invention provides for the direct or indirect production or release of a redox species following the digestion or disruption of the macroraolecular species, and may be employed to determine when conditions occur under which such a macromolecular species is digested or disrupted. For example, it is possible to determine the occurence of a condition which causes a macromolecular species composed of a plurality of linked subunits, to separate into individual sub-units, by labelling some or all of the individual components with a redox species. The redox behaviour of the separate labelled sub-units differs from that of the complexed sub-units and separation may be detected thereby.
  • this aspect of the present invention is seen to comprise a system capable of detecting a redox species in combination with a redox species bound to or held inactive as a component of a macromolecule. On the occurrence of a pre-determined condition vnicn releases the redox compound from its inactive condition or location, a detectable signal is produced.
  • the redox species may be chemically linked to a solid substrate, or linked to a colloidally or otherwise dispersed, or disolved molecular species inhibiting the redox activity of the species. Such linkage prevents free movement of the redox species and so alters the electrochemical response of the species.
  • the redox species may be chemically linked to a suitably large molecule, to prevent the passage of the redox species across a molecular exclusion membrane or other "molecular filter".
  • Suitable solid substrates include the wall of an assay vessel, or a suitable insert, such as a region of an electrode other than the working electrode surface or a mass of beads.
  • this aspect of the invention comprises an assay system including; a redox enzyme, a mediator compound capable of facilitating the transfer of charge from the redox enzyme to the electrode, and, a substrate for the redox enzyme; wherein; a) the component comprises one of said redox enzyme or said mediator, and, b) the macromolecular species comprises a large-molecule immobiliser material linked to the component, whereby upon digestion or disruption of the macromolecular species, the redox enzyme, substrate and mediator interact to provide the redox species; at the electrode.
  • the macromolecular species includes a redox active component which will facilitate the transfer of charge from the enzyme to an electrode, when the component is released from the macromolecule. Consequently, the present invention provides method of assay including the steps of; a) preparing a mixture containing known amounts of; i) a redox enzyme, ii) a mediator compound capable of transferring charge from the redox enzyme to an electrode on occurence of a redox reaction, and, iii) a substrate for the redox reaction, wherein at least one of components i) or ii) is linked to a macromolecule, whereby charge transfer to the electrode surface is restricted, b) treating the mixture with an agent suspected of being capable of effectively liberating the component linked to the macromolecule, and, c) measuring the charge transfer to the electrode surface, whereby the presence of the agent may be determined.
  • one of the components i) or ii) is absent from the medium. being linked to the macromolecule.
  • the enzyme/ mediator/ substrate system is completed, allowing charge transfer to the electrode to produce a detectable signal.
  • the macroraolecular species is a polymer, in which some or all of the monomeric sub-units are labelled with a component of the enzyme/ mediator/ substrate system, and the agent capable of liberating the component can be a depolymerase enzyme suspected of being in the sample.
  • an assay system comprising; a) a reagent system capable of facillitating the production or release of redox species within a medium in the presence of a component produced or released from a macromolecular species by digestion or disruption of the macroraolecular species, and, b) at least one electrode responsive to the presence of the redox species, for electrochemical detection of the redox species in the medium.
  • the analyte of the assay system may be the macromolecular species itself, and the assay system is such that in the presence of the macroraolecular species, the reagent system produces redox species which can be detected at the electode.
  • the present invention extends to an assay system including either; a redox enzyme or a mediator corapound capable of facilitating the transfer of charge from the redox enzyme to the electrode, and a substrate for the redox enzyme; wnerein; a) the component comprises one of said redox enzyme or mediator, b) the system further comprises a further enzyme to produce or release the component in the presence of the macromolecular species, wherein on treatment of the assay system with a sample containing the macromolecular species charge is consequently transferred via the mediator compound to the said electrode.
  • the component of the redox enzyme/ substrate/ mediator system is released when the macroraolecular species is acted upon by the further enzyme.
  • the macromolecular species may be a modified polymer, such as a polysaccharide
  • the further enzyme may be a polysaccharide depolymerase, such as amyloglucosidase or ⁇ -amylase.
  • the present assay systems may be employed in methods of assay for specific binding reactions, such as in methods of immunoassay.
  • the invention provides a method of assay including the steps of; a) preparing a mixture containing known amounts of; i) a redox enzyme, ii) a mediator compound capable of transferring charge between the redox enzyme and an electrode surface on occurence of a redox reaction, iii) a substrate for the redox reaction, and, iv) a ligand labelled with an enzyme having a depolymerase activity on a polymer, wherein, at least one of components i) or ii) is linked to the polymer whereby charge transfer is restricted until the component is liberated into the mixture, b) treating the mixture with a sample suspected of containing an antiligand capable of a specific binding reaction with the ligand, and, c) measuring the pertubation of charge transfer, whereby the presence of the reactive species may be determined.
  • a method suitable for the detection of a specific binding reaction in which the ligand is labelled with an enzyme having a depolymerase activity.
  • This activity is detected by the liberation from a macroraolecular substrate by the enzyme of a component of the redox enzyme/ mediator/ substrate system.
  • charge transfer occurs at the electrode.
  • the extent to which this charge transfer occurs is determined by the activity of the depolymerase, especially in the instance where the product of the depolymerase is a mediator to the redox enzyme.
  • the depolymerase activity is affected with a consequent effect on the transfer of charge to the electrode.
  • the specific binding reaction may be detected indirectly.
  • the macromolecule can be a polymer molecule, by which term we intend to include homopolymers, copolymers or large-molecular chains with related but different units e.g. protein, other polypeptide-linked substances and nucleic acids.
  • the immobiliser can comprise a liposome, attackable by a ohospnolipase.
  • the present invention extends to an assay system for an enzyme having a polymerase activity comprising; a) a quantity of monomer or oligomer capable of being condensed into a polymer, b) an enzyme having a redox activity on the said monomer or oligomer, c) a mediator corapound capable of transferring charge from the enzyme (b) to an electrode when the enzyme (b) is ⁇ atalytically active, wherein on treatment of the assay system with a sample containing a polymerase capable of polymerising the said oligomers or monomer, the rate of redox activity of the enzyme (b), and consequently the amount of charge transferred via the mediator compound to the said electrode, is reduced by the polymerisation of the monomer or oligomer.
  • the term "depolymerase” is intended to mean any enzyme which is capable of degrading a polymer into monomers or oligomers or a mixture of products. This includes, out is not limited to hydrolase enzymes, peptidases, hyaluronidases, galactosidases, and nucleases. "Polymerase” has the reverse meaning, and includes both those polymerase enzymes which use a template (such as the enzymes involved in RNA and DNA synthesis) and those enzymes which while manufacturing polymers do not require a template.
  • the mediator compound is chemically attached to the polymer, so that its "mediator” or charge transfer ability is reduced or eliminated until the polymer is attacked, it has been found preferable that the mediator is a metallocene. and more preferably ferrocene or a derivative thereof. It should however be noted that other, non-metal, mediators as listed above in Table 3 can also oe used, provided that the mediator activity is recovered when the mediator is released from the immobilising large molecule.
  • Ferrocene and derivatives thereof are particularly suitable as mediators in the present invention, as they have been shown to retain their activity even when considerable modification and/ or substitution of the ferrocene molecule has been performed.
  • the depolymerase activity may be that of a hydrolase and particular utility has been found with those embodiments in which the depolymerase is specific to a polysaccharide.
  • the second enzyme is preferably selected such that it has a a redox activity on the oligo- and mono-saccharide fragments produced by the action of the depolymerase.
  • the redox enzyme is glucose oxidase or glucose dehydrogenase.
  • amyloglucosidase (E.C.3.2.1.3), hydrolyses ⁇ -1,4 and ⁇ -1,6-glucan linkages in polysaccharides to produce monomers quantitatively.
  • the polymer substrate is a polysaccharide, amyiose, which has been modified by covalent attachment of ferrocene via an ester linkage. After digestion of the polymer by the hydrolytic enzyme, the products can mediate between glucose oxidase and an electrode, in the presence of a glucose substrate, and generate a measurable electrical signal.
  • ⁇ -amylase acts on a second ferrocene-labelled insoluble substrate to produce soluble oligomers which can be detected amperometrically.
  • the cleavage products are capable of acting as electron acceptors for glucose oxidase in the enzyme catalysed oxidation of glucose, thus giving rise to an amplification of the observed curren.t.
  • polymer-bound ferrocene is electrocheraically silent, that is. it cannot act as an electron acceptor for glucose oxidase and does not exhibit reversible or quasi-reversible electrochemistry.
  • Figure 1 shows a schematic drawing of an assay system in which the analyte is a polymer, and in which the substrate of the enzyme from which charge transfer occurs is a monomer or an oligomer of the polymer,
  • Figure 2 shows an assay system in which the analyte is a depolymerase, and in which a mediator compound is immobilised as a constituent of a polymer until monomers and oligomers having mediator activity are released by the action of the depolymerase,
  • Figure 3 shows an assay system in which the analyte is a reagent capable of releasing immobilised enzyme into the free portion of the assay system, and in which both substrate and a mediator for that enzyme are present in the free portion
  • Figure 4 shows in schematic form a system in which the release of the mediator compound into the assay medium is accompanied by the cleavage of a specific type of chemical bond
  • Figure 5 shows an extension of the technique illustrated in figure 5, in which a molecular exclusion membrane is employed
  • Figure 6 shows in schematic form the operation of a assay system in which the electrochemical response of the mediator compound is determined directly.
  • Figure 7 shows a) DC cyclic voltammogram of 3mg of a ferrocene derivative of a polysaccharide in 1ml K 2 HPO 4 :NaCl buffer, pH 7.0 @ 37°C, b) DC cyclic voltammogram of the solution (a) after incubation at 37°C for 10min. with
  • Figure 8 shows a) DC cyclic voltammogram of 3mg of a ferrocene derivative of a polysaccharide in 1ml
  • Figure 9 Shows a calibration plot of ⁇ -amylase concentration, against measured current.
  • Figure 10 Shows a calibration plot of IgG- ⁇ -amylase concentration, against measured current.
  • FIG 1 there is shown a schematic diagram of an assay system in which a polymeric species (A n ) is the analyte. and the substrate for an enzyme/ mediator system comprises a monomer or oligomer of the polymer (A n ).
  • the polymeric species (A n ) is degraded by a site or component specific depolymerase enzyme (Q) to produce shortened polymers (A m ) and monomers (S) possibly also together with diraers, trimers, etc. as discussed above.
  • Q site or component specific depolymerase enzyme
  • the structure of the polymer (A n ) is known and the action of the enzyme (Q) is accurately predicted, it is possible to design a system in which either or both of the oligomers (A m ) and the monomer (S) are a substrate for the redox enzyme (E), which is usually an oxidase, and to which redox enzyme a mediator such as ferrocene is available.
  • E redox enzyme
  • any free monomer (S) is pre-existing in the assay mixture, it is possible to perform a corrective assay for the polymer using the differential between the current in the presence and the absence of the depolymerase ( Q). It is envisaged that such measurements could be made simultaneously in a system which has more than one sensing electrode and a plurality of compartments separated by suitable molecular-exclusion membranes.
  • the illustrated system can also be used to assay for the depolymerase (Q), by providing an excess of polymer (A n ) in the assay mixture and omitting the depolymerase (Q).
  • the polymer is not a substrate of the enzyme (E) no current will be detected at the electrode until digestion of the polymer (A n ) occurs, thus the presence of the depolymerase (Q) can be detected.
  • an assay system contains substrate (S), enzyme (E), and mediator, but the depolymerase (Q) is replaced by a polymerase, which has the same substrate (S) as the enzyme (E) and which produces a polymer which is not a substrate for the enzyme (E), then a competitive reaction will occur between the consumption of substrata (S) at tne enzyme (E) and the polymerisation of (S) into an inactive species.
  • the current generated by such a systera would be changed if any of the rate or concentration parameters, such as activity of polymerase or enzyme (E), or such as availiabillity of substrate (S) were altered.
  • Figure 2 Assay for Depolymerase.
  • Figure 2 shows an assay systera in which the analyte is a depolymerase (R), and in which a mediator compound is immobilised as a constituent of a polymer (B n ) until monomers and oligomers having mediator activity are released by the action of the depolymerase (R).
  • mediator is liberated and is free to tranfer charge from the enzyme E to the electrode (1).
  • the substrate (S) and the product (P) of the enzyme (E) are not determined by the nature of the depolymerase. and consequently a wide range of enzymes, substrates and mediators can be employed with this particular embodiment of the invention.
  • a variant of this technique is directed at the assay of a further analyte and causes a change in the behavior of the assay system shown in the figure by its effect upon at least one of the rate or concentration parameters, such as activity of depolymerase (R).
  • the depolymerase (R) is covalently attached to one or other of a pair of specific binding partners such as antigen/antibody.
  • the depolymerase (R) with antibody attached retains its activity and the systera function in an identical fashion to that described above.
  • This technique could be used to assay antibody concentration or, in sandwich assays or hetergeneous competitive assays, to assay or monitor antigen concentration. This configuration is particularly suitable for the estimation of high molecular-weight antigens.
  • the liberation of the immobillised component accompanies the disruption of a large transfer restraining molecule or aggregation of molecules.
  • the immobiliser can comprise a liposome containing and confining therewithin a mediator compound and attackable by a phospholipase to release the mediator into the assay system and to thus allow electron transfer to occur.
  • the analyte is a reagent capable of releasing immobilised enzyme (E) into the free portion of the assay system, and in which both substrate (S) and a mediator for enzyme (E) are present in that free portion.
  • the enzyme (E) may be immobilised in several ways.
  • the enzyme (E) can be contained within a liposome (L) and released by the action of a by a phospholipase (T) on the liposome.
  • Figures 4a and 4b show a systera which comprises an assay for a peptide bond hydrolysing condition.
  • the enzyme (E) may perform its catalytic activity upon the substrate (S), in this instance present in solution.
  • a mediator (F) is attached via a linker group L 1 , to a structure L 2 including a peptide bond P.
  • the mediator/L 1 conjugate may freely diffuse and transfer charge from the enzyme to the electrode, thereby giving a detectable signal, and indicating that the peptide bond P has been broken.
  • Figure 5 shows an alternative system to that shown in Figure 4 which makes use of a molecular exclusion membrane (3) to separate the mediator (M) which is bound to a large molecule (X), and the enzyme (E), both of which are too large to pass through the membrane (3).
  • breakage of the linkage between the mediator (M) and the large molecule allows the mediator to diffuse through the membrane and mediate the transfer of charge from the enzyme E to the electrode.
  • FIG 6 a system is shown which lacks the redox enzyme generally referred to as E in the other figures.
  • the only enzyme which is required in the assay systera is that which depolymerises a polymer (C n ) which has mediator attached thereto.
  • mediator is liberated by a depolymerase (R). It is possible to obtain a response to the presence of the electrochemically active monomer at the electrode (1), from which the activity of the depolymerase can be determined. It should again be noted that presence of depolymerase (R) in the assay system for even a short time will cause a permanent change in the electrode response.
  • EXAMPLE 1 Preparation of 6-O-ferrocene-amylose
  • 6-O-ferrocene-amylose was prepared as described in Methods in Carbohydrate Chemistry Vol IV p 300 for the preparation of 6-O-tosyl-amylose.
  • the centrifuged product was washed with twice with each of methanol, water and acetone and finally again with methanol to leave a clean solid and colourless supernatant.
  • EXAMPLE 2 Electrochemistry of ferrocene-araylose in a three electrode cell with a graphite working electrode
  • the cell contained a 1 cm 2 platinum gauze counter electrode and a saturated calomel reference electrode. All potentials were referred to the saturated calomel electrode (SCE).
  • SCE saturated calomel electrode
  • the buffer used for .electrochemical experiments was 0.1M phosphate buffer (pH 7.0) containing 50mM glucose.
  • the ferrocene-amylose conjugate (3.0 mg) was suspended in phosphate buffer (10 mis) and sonicated for 2 mins to produce a finely dispersed suspension. After sonication, the suspension was placed in the sample compartment of the electrochemical cell and mixed thoroughly using a magnetic stirrer.
  • the d.c. cyclic voltammogram of the suspension was recorded over the potential range 0 to +650 mV vs SCE.
  • the cyclic voltammogram of the ferrocene-amylose complex suspended in buffer showed no direct electrochemistry at the working electrode.
  • Amyloglucosidase (1 mg) was added to the electrochemical cell and the sample was thoroughly mixed. The cyclic voltammogram of the solution was recorded at 5 and 15 mins after the addition of amyoglucosidase. On addition of amyloglucosidase, electrochemistry due to the release of 6-O-ferrocsnoyl glucose from the complex under the action of the enyzme, was observed.
  • EXAMPLE 4 Electrochemistry of ferrocene-amylose in the presence of amyloglucosidase, glucose oxidase and glucose.
  • glucose 50 ⁇ l of a 1M solution in acetate buffer, pH 4.8
  • glucose oxidase 50 ⁇ l of a 3 rag ml -1 solution in acetate buffer, pH 4.8
  • the following examples (5-10) relate to the action of ⁇ -amylase in hydrolysing amylose polymer and derivates thereof.
  • ⁇ -araylase was conjugated to bovine IgG using glutaraldehyde.
  • ⁇ -amylase (24rag) and IgG (10rag) were dissolved in 2ml phosphate buffer pH 6.8 (0.1M) to this 60 ⁇ l of a 10% aqueous solution of glutaraldehyde was added with gentle stirring. The solution was left to stand for 30min. The reaction was terminated by the addition of 100 ⁇ l of a 1% ethanolamine solution.
  • the conjugate was purified using FPLC ["Fast Protein Liquid Chromatography"; trade-name, of Pharmacia] using a gelfiltration column.
  • the IgG content of the conjugate was assessed using goat-antibovine IgG labelled with alkaline phosphatase.
  • EXAMPLE 6 Three-electrode system, electrochemistry of 6-O-ferrocene-amylose in the presence of hog-pancreas ⁇ -amylase.
  • EXAMPLE 7 Three-electrode system, electrochemistry of 6-O-ferrocene-amylose in the presence of hog pancreas ⁇ -araylase, GOD and glucose
  • FIG. 8c shows a cyclic voltammogram as for example 6, but with the addition of 40 ⁇ l of a 1M solution of glucose and 10 ⁇ l of a 2.3mM solution of glucose oxidase.
  • the catalytic current from the breakdown products was estimated as 3 ⁇ A at 500mV. It should be noted that in the absence of of ⁇ -amylase, the species with a redox peak at 550mV did not show catalytic behaviour when glucose oxidase was added to the solution.
  • EXAMPLE 8 Three-electrode system, electrochemistry of 6-O-ferrocenoyl-amylose in the presence of salivary ⁇ -araylase, GOD and glucose
  • FIG. 8a shows a DC cyclic voltammogram of 3mg of a ferrocene derivative of a polysaccharide in lml K 2 HPO 4 :NaCl buffer @ 37°C with 50 ⁇ l of human saliva.
  • Figure 8b shows a cyclic voltammogram as shown in figure 8a, but with the addition of 40 ⁇ l of a 1M solution of glucose and 10 ⁇ l of a 2.3mM solution of glucose oxidase.
  • Normal human serum has an ⁇ -araylase activity of about 1500 U/l. Therefore the effect of 1 U/ral ⁇ -amylase (final concentration) on 3mg amylose-ferrocene polymer was investigated under the conditions described above. The catalytic current measured under these conditions was approximately 90nA,
  • Ferrocene-amylose polymer (3 mgs/ml) was sonicated in phosphate buffer pH 7.0 (0.1M) containing 50mM glucose. One ml of this suspension was added to an electrochemical cell, and varying amounts of ⁇ -amylase were added. After a 10 minute incubation, excess glucose oxidase was added and the catalytic current measured at +550mV vs SCE a dose/response curve was obtained. The response curve is shown in figure 9.
  • Example 10 Dose/response curve to antibodies labelled with ⁇ -amylase.

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Abstract

Utilisation d'une technique de détection électrochimique d'espèces rédox (MR) dans un milieu pour déterminer l'existence d'une condition dans laquelle ces espèces sont produites ou libérées, cette production ou libération étant provoquée par la digestion ou la dissociation d'une espèce macromoléculaire (An). L'invention décrit un procédé d'analyse d'un polymère, d'une enzyme de dépolymérase et d'un agent liant sécifique marqué, où un polymère (An) est marqué avec un composant d'un système médiateur/enzyme/substrat qui reste inactif jusqu'au moment où il est libéré en solution. Des électrodes dans le milieu déterminent l'existence d'une condition dans laquelle des espèces rédox sont produites par le système médiateur/enzyme/substrat ou libérés en solution. Lorsqu'on utilise l'activité de médiateur, par ex., du ferrocène en glucose oxydase, on prévoit une étape d'amplification.
PCT/GB1986/000095 1985-02-21 1986-02-21 Analyse de substrats degradables par detection electrochimique d'especes redox WO1986004926A1 (fr)

Applications Claiming Priority (2)

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GB8504522 1985-02-21
GB858504522A GB8504522D0 (en) 1985-02-21 1985-02-21 Electrochemistry of mediators

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WO1986004926A1 true WO1986004926A1 (fr) 1986-08-28

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002464A1 (fr) * 1985-10-14 1987-04-23 Genetics International (Uk) Inc. Analyse avec marquage par enzyme
WO1989001047A1 (fr) * 1987-08-03 1989-02-09 Imperial College Of Science, Technology & Medicine Dosage enzymatique
EP0223541A3 (fr) * 1985-11-11 1989-08-23 MediSense, Inc. Procédés électrochimiques d'essai enzymatique
WO1994028414A1 (fr) * 1993-05-29 1994-12-08 Cambridge Life Sciences Plc Capteurs bases sur la transformation de polymeres
US5427912A (en) * 1993-08-27 1995-06-27 Boehringer Mannheim Corporation Electrochemical enzymatic complementation immunoassay
DE19622458A1 (de) * 1996-05-24 1997-11-27 Senslab Ges Zur Entwicklung Un Enzymatisch-elektrochemischer Einschritt-Affinitätssensor zur quantitativen Bestimmung von Analyten in wäßrigen Medien und Affinitätsassay
US5919642A (en) * 1994-12-19 1999-07-06 Boehringer Mannheim Corporation Competitive binding assays having improved linearity
USRE36268E (en) * 1988-03-15 1999-08-17 Boehringer Mannheim Corporation Method and apparatus for amperometric diagnostic analysis
US6262264B1 (en) 1998-06-01 2001-07-17 Roche Diagnostics Corporation Redox reversible imidazole osmium complex conjugates
EP0442969B1 (fr) * 1988-11-14 2002-02-27 I-Stat Corporation Biocapteurs entierement microfabriques et procede de production et utilisation de ces capteurs
US6576102B1 (en) 2001-03-23 2003-06-10 Virotek, L.L.C. Electrochemical sensor and method thereof
WO2003021010A3 (fr) * 2001-08-31 2003-12-04 Univ California Procede pour la detection specifique de marqueurs a activite d'oxydoreduction et utilisation de ceux-ci pour l'electrophorese capillaire en gel et le sequencage d'adn
US6849216B2 (en) 2001-03-23 2005-02-01 Virotek, L.L.C. Method of making sensor
US7390391B2 (en) 2001-09-14 2008-06-24 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
WO2011158736A1 (fr) * 2010-06-15 2011-12-22 株式会社マイクロブラッドサイエンス Procédé de détection/quantification de molécules très rapide et d'une grande sensibilité par mesure de charges faisant appel à une enzyme capable de générer de l'électricité et pièce et dispositif de détection associés

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE20017268U1 (de) * 2000-10-07 2001-03-29 DECHEMA Gesellschaft für Chemische Technik und Biotechnologie eV, 60486 Frankfurt Elektrochemische Messzelle zur Bestimmung der Zellzahl und Aktivität von biologischen Systemen
GB0205455D0 (en) * 2002-03-07 2002-04-24 Molecular Sensing Plc Nucleic acid probes, their synthesis and use
GB2419880B (en) * 2004-10-22 2008-04-30 E2V Tech Monitoring enzyme-substrate reactions

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4233403A (en) * 1978-02-27 1980-11-11 E. I. Du Pont De Nemours And Company Amylase assay
EP0034122B1 (fr) * 1980-02-12 1983-09-21 Pentapharm A.G. Dérivés de tripeptides et leur utilisation dans la détermination d'enzymes
EP0089626A2 (fr) * 1982-03-19 1983-09-28 G.D. Searle & Co. Procédé pour la préparation de polypeptides à l'aide d'un polymère d'acides aminés chargés et une exopeptidase
EP0125136A2 (fr) * 1983-05-05 1984-11-14 MediSense, Inc. Systèmes d'essai utilisant plus d'une enzyme
EP0142301A2 (fr) * 1983-10-25 1985-05-22 Serono Diagnostics Limited Procédés d'essai
EP0149339A2 (fr) * 1983-12-16 1985-07-24 MediSense, Inc. Essai pour acides nucléiques
EP0150602A2 (fr) * 1983-12-20 1985-08-07 Genetics International, Inc. Composés de bore et carbone dans des électrodes de transfert d'électrons

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1226036A (fr) * 1983-05-05 1987-08-25 Irving J. Higgins Materiel d'analyse et ses electrodes-sondes
CA1219040A (fr) * 1983-05-05 1987-03-10 Elliot V. Plotkin Mesure de reactions catalysees par des enzymes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4233403A (en) * 1978-02-27 1980-11-11 E. I. Du Pont De Nemours And Company Amylase assay
EP0034122B1 (fr) * 1980-02-12 1983-09-21 Pentapharm A.G. Dérivés de tripeptides et leur utilisation dans la détermination d'enzymes
EP0089626A2 (fr) * 1982-03-19 1983-09-28 G.D. Searle & Co. Procédé pour la préparation de polypeptides à l'aide d'un polymère d'acides aminés chargés et une exopeptidase
EP0125136A2 (fr) * 1983-05-05 1984-11-14 MediSense, Inc. Systèmes d'essai utilisant plus d'une enzyme
EP0142301A2 (fr) * 1983-10-25 1985-05-22 Serono Diagnostics Limited Procédés d'essai
EP0149339A2 (fr) * 1983-12-16 1985-07-24 MediSense, Inc. Essai pour acides nucléiques
EP0150602A2 (fr) * 1983-12-20 1985-08-07 Genetics International, Inc. Composés de bore et carbone dans des électrodes de transfert d'électrons

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002464A1 (fr) * 1985-10-14 1987-04-23 Genetics International (Uk) Inc. Analyse avec marquage par enzyme
EP0223541A3 (fr) * 1985-11-11 1989-08-23 MediSense, Inc. Procédés électrochimiques d'essai enzymatique
WO1989001047A1 (fr) * 1987-08-03 1989-02-09 Imperial College Of Science, Technology & Medicine Dosage enzymatique
USRE36268E (en) * 1988-03-15 1999-08-17 Boehringer Mannheim Corporation Method and apparatus for amperometric diagnostic analysis
EP0442969B1 (fr) * 1988-11-14 2002-02-27 I-Stat Corporation Biocapteurs entierement microfabriques et procede de production et utilisation de ces capteurs
WO1994028414A1 (fr) * 1993-05-29 1994-12-08 Cambridge Life Sciences Plc Capteurs bases sur la transformation de polymeres
US5427912A (en) * 1993-08-27 1995-06-27 Boehringer Mannheim Corporation Electrochemical enzymatic complementation immunoassay
US6110696A (en) * 1993-08-27 2000-08-29 Roche Diagnostics Corporation Electrochemical enzyme assay
US5919642A (en) * 1994-12-19 1999-07-06 Boehringer Mannheim Corporation Competitive binding assays having improved linearity
DE19622458A1 (de) * 1996-05-24 1997-11-27 Senslab Ges Zur Entwicklung Un Enzymatisch-elektrochemischer Einschritt-Affinitätssensor zur quantitativen Bestimmung von Analyten in wäßrigen Medien und Affinitätsassay
DE19622458C2 (de) * 1996-05-24 1998-03-26 Senslab Ges Zur Entwicklung Un Enzymatisch-elektrochemischer Einschritt-Affinitätssensor zur quantitativen Bestimmung von Analyten in wäßrigen Medien und Affinitätsassay
US6221238B1 (en) 1996-05-24 2001-04-24 Ufz-Umweltforschungszentrum Leipzig-Halle Gmbh Enzymatic-electrochemical one-shot affinity sensor for the quantitative determination of analytes for aqueous media and affinity assay
US6294062B1 (en) 1998-06-01 2001-09-25 Roche Diagnostics Corporation Method and device for electrochemical immunoassay of multiple analytes
US6262264B1 (en) 1998-06-01 2001-07-17 Roche Diagnostics Corporation Redox reversible imidazole osmium complex conjugates
US6352824B1 (en) 1998-06-01 2002-03-05 Roche Diagnostics Corporation Redox reversible bipyridyl-osmium complex conjugates
US7045310B2 (en) 1998-06-01 2006-05-16 Roche Diagnostics Operations, Inc. Redox reversible bipyridyl-osmium complex conjugates
USRE40198E1 (en) 1998-06-01 2008-04-01 Roche Diagnostics Operations, Inc. Method and device for electrochemical immunoassay of multiple analytes
US6576102B1 (en) 2001-03-23 2003-06-10 Virotek, L.L.C. Electrochemical sensor and method thereof
US6849216B2 (en) 2001-03-23 2005-02-01 Virotek, L.L.C. Method of making sensor
WO2003021010A3 (fr) * 2001-08-31 2003-12-04 Univ California Procede pour la detection specifique de marqueurs a activite d'oxydoreduction et utilisation de ceux-ci pour l'electrophorese capillaire en gel et le sequencage d'adn
US7390391B2 (en) 2001-09-14 2008-06-24 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
USRE44522E1 (en) 2001-09-14 2013-10-08 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
USRE45764E1 (en) 2001-09-14 2015-10-20 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
WO2011158736A1 (fr) * 2010-06-15 2011-12-22 株式会社マイクロブラッドサイエンス Procédé de détection/quantification de molécules très rapide et d'une grande sensibilité par mesure de charges faisant appel à une enzyme capable de générer de l'électricité et pièce et dispositif de détection associés

Also Published As

Publication number Publication date
GB2173313A (en) 1986-10-08
AU5516686A (en) 1986-09-10
EP0229765A1 (fr) 1987-07-29
GB8604368D0 (en) 1986-03-26
GB8504522D0 (en) 1985-03-27
JPS62501932A (ja) 1987-07-30
CA1239191A (fr) 1988-07-12

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