WO1989005454A1

WO1989005454A1 - Electrode material

Info

Publication number: WO1989005454A1
Application number: PCT/GB1988/001087
Authority: WO
Inventors: Marco Fabio Cardosi
Original assignee: Novo Biolabs Limited
Priority date: 1987-12-11
Filing date: 1988-12-09
Publication date: 1989-06-15
Also published as: EP0391963A1; AU2820989A; GB8729002D0

Abstract

A method of carrying out a biochemical assay, comprising carrying out a specific binding reaction to localise an amount of a substance either directly or indirectly 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 measuring electro-chemically the amount of the said component which takes part in the said reaction at the electrode. The electrode surface is formed of a hydrophilic non-porous resin-bonded graphite material substantially free of platinum-group metals. The reaction which takes place at the electrode is the interconversion of ferricyanide ion and ferrocyanide ion.

Description

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. Early 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 .

More recently still, attention has been paid to the possibility of detecting biochemical assays electrochemically. A number of examples of such techniques are to be found, for example in EP 121385, WO 86/03837, EP 125139 and WO 86/03837.

A variety of 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. In practical application however, it is found that 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.

Proposals have also been made for the use of carbon as the material for the electrode at which the redox reaction is carried out. In particular, glassy carbon (amorphous carbon) has been proposed. It is found that, although glassy carbon is relatively inert, the kinetics at such a surface of the electrochemical redox reactions involved in typical biochemical assays are poor, such that typically an overpotential of approximately 250 mV is required, in order to obtain diffusion-limited reaction at the electrode. This need for a substantial overpotential increases greatly the likelihood of other reactions occurring at the electrode, in addition to the reaction which it is desired to measure. This effect makes glassy carbon a far from ideal material. I-n addition, extensive pretreatment of the glassy carbon is required, in order to obtain a usable electrode material.

Various forms of graphite 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. However, 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.

Both because the material employed in GB-A-0247850 employs a platinum group metal deposit on its surface, and because of the inherent absorbency of the material, it requires extensive pretreatment, and suffers from the same disadvantages as the materials noted above, that it tends to be easily blocked by materials present in the assay, and can also require an undesirable overpotential.

We have now discovered that a non-porous resin-bonded graphite material substantially free of platinum group metals (specifically, Pt and Pd) can provide a performance which is very much superior to known electrode materials in a biochemical assay.

Accordingly, in a first aspect of the invention, there is provided a method of carrying out a biochemical assay, which method 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. In a further aspect of the invention, there is provided 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.

It is particularly preferred that the resin-bonded graphite material is a non-hydrophobic material (i.e. is a hydrophilic material).

It is also desirable that 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..

The invention is described in the following example. ^J

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.

An immunoassay for thyroid stimulating hormone (TSH) was conducted in the wells of the microplate, so as to bind to the walls of the wells an amount of alkaline phosphatase dependent upon the amount of TSH present in the initial sample, by the method disclosed in International Patent Application No. WO 86/03837.

A potential of 450 mV was applied between the two electrodes of each pair, and the resulting current was measured, using an analogue-to-ditigal convertor, and microcomputer, as disclosed in International Patent Application No. WO 86/03837. Samples containing different amounts of TSH were employed as shown in Table 1. The charge passing between the electrodes in a 12 second period was measured, and the results are shown in Table 1. Well No.

1 2 3 4 5 6 7 8

Repeated tests showed that the electrode materials were able to provide reproducible determinations of the amounts of TSH present in the original samples, and furthermore that complex treatments were not required between tests, all that was required being washing of the electrodes with distilled water.

Example II

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. At the start of each assay, the array was calibrated in a standard solution containing 10 M CaCl2. 8 mM potassium ferricyanide, 0.3 mM potassium ferrocyanide and 25 mM KC1. During this procedure, the voltage was pulsed from 0 to 800 mV and the current transient integrated over a 12 second period. This process was repeated 3 times, with a one minute delay between the pulses, in a quiet solution and the mean charge passed by each electrode calculated and used to generate calibration factors which were used in subsequent measurements.

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. After this step, 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.

Substantially improved results were obtained when the resin bonded graphite material was employed in the presence of a divalent cation, such as Mg"^■"^■", Ba⁺⁺ or, preferably, Ca⁺⁺. This is believed to be because the surface of the carbon carries a high density of negative charge which is effectively quenched by the presence of the divalent cation.

NAD⁺ Assay Procedures 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^βC 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^βC under the above conditions. 1 ml of lϋ 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.

Plotting of charge against NAD⁺ yielded a linear response to NAD+ over the range 0 - 180 nM, with a regression of y = 1.02X + 24.9, r = 0.999 as long as the concentration of the cycling intermediates remain well below the K_m values of the two enzymes (9.1 uM for diaphorase and 93 uM for alcohol dehydrogenase) . The limit of detection for NAD+, defined as the value corresponding to 2 SD above the mean of the zero calibrator, was 500 pM (CV% = 1.0, SD = 0.24, mean = 24.9, n •^*■^*■ 3). Although it is possible to increase the sensitivity of the assay by using a more concentrated amplifier solution (2U or 3U) , it was found that the lu amplifier was suitable for this application. Furthermore, it is also possible to increase the sensitivity of the assay by extending the incubation time beyond 12 minutes i.e. for as long as the amplifier components remain active, although this may not always be a practical solution.

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.

For routine applications the following protocol was followed. To each well of the microtitration strip, 75 ul of enzyme-antibody conjugate and 25 ul of TSH standard (or serum sample) were added. The solutions were mixed for 30 seconds and then incubated for 30 minutes at room temperature. The wells were emptied an washed three times with 250 ul of diluted wash buffer. After the final wash, the wells were tapped firmly onto adsorbent tissue to remove all wash buffer. 120 ul of substrate solution containing 0.1 mM NADP⁺, 50 mM diethanalamine, 20 mM CaCl2 and 4% v/v ethanol at pH 8.8 was then added to each well and the plate incubated for exactly 20 minutes at room temperature. After this time, 120 ul of lϋ amplifier in purified water containing 10 mM potassium ferricyanide were added to each well. After mixing, the electrodes were inserted into the wells and the measurement routine initiated.

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.

TSH

(milli-int.units/L): 0.0 0.3 0.8 1.5 5.0 12.5 15.0 25.0

% Response CV . 2.1 2.7 4.8 4.3 4.6 3.7 4.6 4.2

% Dose CV : 0.0 8.5 6.9 4.7 2.5 6.3 9.2 15.8 The working range of the assay was determined graphically as 0.2 - 18.5 ml.0/1 (defined as the range where the percentage dose coefficient of variation (CV) is less than 10) .

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.

Claims

1. A method of carrying out a biochemical assay, which method comprises carrying out a specific binding reaction to localise an amount of a substance either directly or indirectly 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 measuring electro-chemically the amount of the said component which takes part in the said reaction at the electrode, characterised in that the electrode surface is formed of a non-porous resin-bonded graphite material substantially free of platinum-group metals.

2. A method as claimed in Claim 1, wherein the resin-bonded graphite material is a hydrophilic material.

3. A method as claimed in Claim 1 wherein the reaction which takes place at the electrode is the interconversion of ferricyanide ion and ferrocyanide ion.

4. A method as claimed in Claim 3, wherein the standard working potential of the electrode for the ferricyanide/ferrocyanide interconversion, using a silver/silver chloride cathode, is about 450 mV.

5. A method as claimed in Claim 1, wherein the resin-bonded graphite material is used in conjunction with a silver/silver halide counter-electrode, or a stainless steel counter electrode.

6. A test kit for carrying out a biochemical assay, comprising:- a reaction surface, means for localising on the reaction 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, substantially free of platinum group metals.

7. The use of a non-porous resin-bonded graphite material substantially free of platinum group metals as an electrode material in a biochemical assay.

8. The use of a non-porous resin-bonded graphite material substantially free of platinum group metals as an electrode material in an immunoassay or a nucleic acid hybridisation assay.