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WO1989005454A1 - Materiau d'electrode - Google Patents

Materiau d'electrode Download PDF

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Publication number
WO1989005454A1
WO1989005454A1 PCT/GB1988/001087 GB8801087W WO8905454A1 WO 1989005454 A1 WO1989005454 A1 WO 1989005454A1 GB 8801087 W GB8801087 W GB 8801087W WO 8905454 A1 WO8905454 A1 WO 8905454A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
reaction
graphite material
amount
bonded graphite
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1988/001087
Other languages
English (en)
Inventor
Marco Fabio Cardosi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxoid Ely Ltd
Original Assignee
Novo Biolabs Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Biolabs Ltd filed Critical Novo Biolabs Ltd
Publication of WO1989005454A1 publication Critical patent/WO1989005454A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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

  • ELECTRODE MATERIAL This invention relates to biochemical assays, and in particular to immunoassays , and assays involving hybridisation of nucleotide sequences, for example so-called "DNA probe” assays.
  • Such assays are widely used in a variety of fields, for example to diagnose various clinical conditions, by the detection of various substances, such as hormones, antigens, and cancer markers.
  • a wide range of formats have been proposed for biochemical assays, of which the best known are probably the so-called “sandwich” assays, and “competition” assays. Whatever ' the format, such
  • assays typically function by localising an amount of a detectable label, which is dependent upon the amount of a target substance present in a sample.
  • the localisation is usually carried out by causing a labelled substance to take part in a specific binding reaction on a surface.
  • the labelled substance can then be determined by a variety of methods.
  • biochemical assays in particular immunoassays, involved detection by means of radioisotopes, or fluorescence. Later assays have utilised enzymes as labels, and a number of detection systems have been developed for quantifying accurately the amounts of enzymes which become bound during assays .
  • Electrode materials have been proposed in the past for such electrochemical detection systems.
  • the electrode most commonly proposed is platinum, because of its general chemical inertness.
  • platinum electrodes are susceptible to the absorption of proteins which are generally to be found in solution under the reaction conditions in which biochemical assays take place. Such protein absorption can effectively block the electrode, such that it is impossible to obtain consistent results, without complicated cleaning procedures.
  • graphite materials have also been proposed as electrode materials in immunoassays.
  • Such graphite materials can provide electrodes which, initially at least, show relatively good kinetic performance, such that substantially reduced overpotential is required in order to obtain a reaction rate which is diffusion limited, as compared with glassy carbon.
  • Such materials have been found to deteriorate rapidly, in the environment of a biochemical assay, such that their performance is not reproducible.
  • U.K. Patent Specification No. 0247850 describes a type of electrode for amperometric measurements, using electrodes.
  • the electrodes consist of an enzyme, immobilised or absorbed onto the surface of electrically conductive support members, consisting of a porous layer of resin bonded carbon or graphite particles, having a finely divided platinum group metal, particularly platinum or palladium, intimately mixed with the particles , to form a porous substrate layer, on which the enzyme is adsorbed or immobilised.
  • the electrode surface is a heterogeneous layer of resin-bonded carbon or graphite particles, with the platinum group metal dispersed substantially uniformly throughout the layer.
  • the preferred synthetic resin employed is a hydrophobic resin, particularly polytetrafluoroethylene. The presence of the platinum graph metal is essential to the functioning of the electrodes described in this reference.
  • a non-porous resin-bonded graphite material substantially free of platinum group metals can provide a performance which is very much superior to known electrode materials in a biochemical assay.
  • a method of carrying out a biochemical assay comprises carrying out a specific binding reaction to localise an amount of a substance capable of causing or affecting the generation of a component to a redox couple able to take part in a redox reaction at an electrode, and measuring electrochemically the amount of the said component which takes part in the said reaction at the electrode, characterised in that electrode surface is formed of a non-porous resin-bonded graphite material substantially free of platinum group metals.
  • a test kit for carrying out a biochemical assay comprising a reaction surface, means for localising on the surface an amount of a labelled substance dependent upon the amount of a target substance in a sample, the labelled substance being capable of causing or affecting the generation of a component of a redox couple able to take part in a redox reaction at an electrode, and at least one electrode at which the said component may be determined to provide a measure of the target substance in the sample, characterised in that the electrode surface is formed of a non-porous resin-bonded graphite material.
  • the resin-bonded graphite material may be formed of graphite which has been bonded with a thermoplastic or thermosetting resin, in such a way as to present a substantially non-porous surface, and in particular a surface which does not absorb proteins and the like materials to a significant extent.
  • the preferred resin-bonded graphite material is formed by a hot-pressing process.
  • a particularly preferred material are those materials sold by Morganite Electrical Carbon Limited, Swansea, UK, under the references RH 708 and HY67.
  • the resin-bonded graphite material is a non-hydrophobic material (i.e. is a hydrophilic material).
  • the electrode material should have good electrical conductivity, rapid polarisation properties, good reproducibility, low surface porosity, low levels of electrochemically active impurities and chemical inertness in the test solution.
  • the main impurities of the Morganite RH 708 material are -quartz, -haematite and calcium aluminate which are generally inert under the conditions of the assay.
  • the electrode material used in accordance with the present invention is preferably used in conjunction with a silver/silver halide, preferably a silver/silver chloride cathode.
  • the electrode material used in accordance with the present invention is particularly suitable for use in the electrochemical interconversion of ferricyanide ion and ferrocyanide ion.
  • the working potential of the electrode for such an interconversation is preferably about +450 mV when used With a Ag/AgCl2 counter electrode.
  • the electrode material of the present invention is particularly suitable for the electrochemical determination of immunoassays and the like, of the kind described in International Patent Application No. WO/J03837..
  • EXAMPLE A cover was constructed for a 8 well polystyrene microplate (Nunc-Denmark) .
  • the cover also served as a mounting support for a number of pairs of electrodes.
  • a pair of electrodes was provided for each well of the microplate, one electrode of each pair being formed of resin-bonded graphite material (Morganite HY67), the other electrode of the pair being a silver/silver chloride electrode.
  • the silver/silver chloride electrodes were formed by depositing electrochemically a 20 micrometer layer of silver on a 0.5 mm copper film, laminated to a plastics support. The silver was then anodised in lOOrrtM KCL for 10 minutes.
  • the electrodes of each pair were connected to a suitable edge connector on the cover, to enable electrode pairs to be supplied with a suitable potential, and for the resulting current between the electrodes to be measured.
  • TSH thyroid stimulating hormone
  • Carbon anodes were made by machining cylindrical electrodes of length 2.5 cm and radius 0.25 cm from blocks of Morganite RH 708 phenolic resin-bonded graphite and then fixing dome shaped brass caps on top with an electrically conducting silver loaded epoxy resin. The electrodes were then sonicated in purified water to give a clean graphite surface free of resin and debris. This combination of machining followed by ultrasonic cleaning gave a surface which was essentially graphite crystals embedded in resin.
  • a cathode was pressed from a single strip of stainless steel in the form of an eight-toothed comb.
  • the comb was cleaned ultrasonically for 30 minutes in chromic acid to remove any surface deposits and to improve its corrosion resistance.
  • the carbon anodes ' and stainless steel comb were then assembled in a plastic body to form an electrode array which formed a cover for an eight well microtitre strip (Nunc-Denmark) .
  • Each carbon anode had a central plastic sleeve which helped eliminate any variation caused by differences in the heights of the meniscus.
  • the array was calibrated in a standard solution containing 10 M CaCl2.
  • the electrode array was then used to determine ferrocyanide produced by the reduction of ferricyanide.
  • the measurement protocol was in two parts. The first part, a "pretreatment phase", involved applying five voltage steps (0 - 800 mV) of five second durations to each electrode with a two second delay between each pulse. This routine improved reproducibility, possibly by generating a stable and consistent diffusion layer at the surface of each electrode.
  • the voltage was returned to 0 mV and the soltuion left for 10 minutes without any mixing, to accumulate ferrocyanide.
  • the potential was then stepped to 800 V and the resultant current was measured for 15 seconds.
  • the charge passed during the electrolysis was calculated after ignoring the first 3 seconds (20%) of the transient, thereby allowing the non Faradaic component of the response and any instability to iR effects to be resolved from the purely Faradaic current.
  • NAD + was assayed using the electrode array in the following manner.
  • An amplifier solution of the general kind discussed in EP-A-0132968 , EP-A-0183383 , EP-A-023513 and PCT/GB87/00899 was prepared as follows. Alcohol dehydrogenase (2.0 mg) and diaphorase (1.5 mg) were dissolved in water to give a final protein concentration of 0.35 mg/ l. The ratio of the two enzymes was adjusted to give a maximum rate of ferricyanide reduction at 25 ⁇ C when 0.20 ml of enzyme solution were added to 1.8 ml of 25 mM diethanolamine pH 8.8 containing 10 mM CaCl2, 4 per cent (v/v) ethanol and 100 nM NAD + .
  • One unit of amplifier was arbitrarily defined as the concentration of enzyme which in the presence of 100 nM NAD + caused the reduction of 70 uM ferricyanide in 10 minutes at 25 ⁇ C under the above conditions.
  • 1 ml of l ⁇ amplifier and 1 ml of 50 mM diethanolamine pH 8.8 containing 20 mM CaCl2_ 4 per cent (v/v) EtOH and 20 mM ferricyanide were mixed together in a suitable vessel. Aliquots (240 ul) of the diluted amplifier were then dispensed into the wells of the test strip together with samples of
  • NAD + prepared in purified water. After mixing, the electrode array was placed into the wells and the measurement routine initiated.
  • TSH Assay Procedures Each well of the microtitre plate was coated with a mouse monoclonal antibody to the beta subunit of TSH. A second mouse monoclonal antibody to a different epitope of intact TSH was conjugated to alkaline phosphatase.
  • the Faradaic current resulting from the diffusion controlled oxidation of ferrocyanide at the electrode surface was integrated for 12 seconds and the charge passed during this time used to construct a calibration curve by a four parameter logistic fit.
  • the precision (intra assay) of the electrochemical assay over the range 0 - 25 ml.0/1 was assessed by measuring each TSH standard stample eight times (i.e. one sample per 8 well strip) as described above. The results are summarised below.
  • the electrochemical immunoassay compared favourably with other TSH assays in terms of its sensitivity.
  • the limited dynamic range of the electrochemical assay described (up to 25 ml.0/1) is in part due to the amount of ferricyanide ion that can be added to the amplifier solution without inhibiting the enzymes.
  • This limitation may be overcome by the selector of other, more suitable enzyme or by varying the experimental conditions, i.e. amplifier strength, immunoincubation time, amplification time and/or dephosphorylation time.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Méthode d'exécution d'essais biochimiques, comportant d'une part l'exécution d'une réaction de liaison spécifique afin de localiser une quantité de substance capable de provoquer ou d'affecter, directement ou indirectement, la formation d'un composant d'un couple d'oxydoréduction, en mesure de prendre part à une réaction d'oxydoréduction au niveau d'une électrode, et d'autre part la mesure électrochimique de la quantité dudit composant prenant part à ladite réaction au niveau de l'électrode. La surface de l'électrode est formée d'un matériau en graphite, hydrophile et non poreux, lié avec de la résine synthétique et ne comportant pratiquement pas de métaux du groupe du platine. La réaction qui se produit au niveau de l'électrode est l'interconversion de l'ion ferricyanure et de l'ion ferrocyanure.
PCT/GB1988/001087 1987-12-11 1988-12-09 Materiau d'electrode Ceased WO1989005454A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB878729002A GB8729002D0 (en) 1987-12-11 1987-12-11 Electrode material
GB8729002 1987-12-11

Publications (1)

Publication Number Publication Date
WO1989005454A1 true WO1989005454A1 (fr) 1989-06-15

Family

ID=10628378

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1988/001087 Ceased WO1989005454A1 (fr) 1987-12-11 1988-12-09 Materiau d'electrode

Country Status (4)

Country Link
EP (1) EP0391963A1 (fr)
AU (1) AU2820989A (fr)
GB (1) GB8729002D0 (fr)
WO (1) WO1989005454A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516644A (en) * 1991-07-29 1996-05-14 Mochida Pharmaceutical Co., Ltd. Electrochemical immunochromatographic assay
WO1998002399A1 (fr) * 1996-07-12 1998-01-22 Wolf Bertling Procede et dispositif de purification et d'enrichissement de molecules
DE19530376C2 (de) * 1995-08-18 1999-09-02 Fresenius Ag Biosensor

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003837A1 (fr) * 1984-12-19 1986-07-03 Iq (Bio) Limited Procede et appareil d'analyses biochimiques
EP0230472A1 (fr) * 1985-06-21 1987-08-05 Matsushita Electric Industrial Co., Ltd. Capteur biologique et son procede de fabrication
WO1987007295A1 (fr) * 1986-05-27 1987-12-03 Cambridge Life Sciences Plc Electrodes a enzymes immobilises
EP0266432A1 (fr) * 1986-04-22 1988-05-11 Toray Industries, Inc. Micro-electrode pour analyses electrochimiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003837A1 (fr) * 1984-12-19 1986-07-03 Iq (Bio) Limited Procede et appareil d'analyses biochimiques
EP0230472A1 (fr) * 1985-06-21 1987-08-05 Matsushita Electric Industrial Co., Ltd. Capteur biologique et son procede de fabrication
EP0266432A1 (fr) * 1986-04-22 1988-05-11 Toray Industries, Inc. Micro-electrode pour analyses electrochimiques
WO1987007295A1 (fr) * 1986-05-27 1987-12-03 Cambridge Life Sciences Plc Electrodes a enzymes immobilises

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516644A (en) * 1991-07-29 1996-05-14 Mochida Pharmaceutical Co., Ltd. Electrochemical immunochromatographic assay
US6218134B1 (en) 1991-07-29 2001-04-17 Mochida Pharmaceutical Co., Ltd. Process for specific binding assay for measuring the amount of analyte in a liquid test sample
DE19530376C2 (de) * 1995-08-18 1999-09-02 Fresenius Ag Biosensor
WO1998002399A1 (fr) * 1996-07-12 1998-01-22 Wolf Bertling Procede et dispositif de purification et d'enrichissement de molecules
US6479644B1 (en) 1996-07-12 2002-11-12 Wolf Bertling Method for purifying and enriching molecules

Also Published As

Publication number Publication date
GB8729002D0 (en) 1988-01-27
EP0391963A1 (fr) 1990-10-17
AU2820989A (en) 1989-07-05

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