CA2196380A1 - Electrodes and their use in analysis - Google Patents
Electrodes and their use in analysisInfo
- Publication number
- CA2196380A1 CA2196380A1 CA002196380A CA2196380A CA2196380A1 CA 2196380 A1 CA2196380 A1 CA 2196380A1 CA 002196380 A CA002196380 A CA 002196380A CA 2196380 A CA2196380 A CA 2196380A CA 2196380 A1 CA2196380 A1 CA 2196380A1
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- CA
- Canada
- Prior art keywords
- polymer
- electrode
- enzyme
- solution
- film
- 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.)
- Abandoned
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Classifications
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
Abstract
A method for indicating the concentration of a substance in solution comprises passing an alternating voltage between a first electrode structure having coated thereon a polymer and a second separate counter electrode in the solution. The polymer is in one of an oxidised and a reduced state, between which states its conductivity varies. Changes in the conductivity of the polymer coating are measured, the measurement being representative of the state of the polymer and thereby of the concentration of the substance in the solution. Electrodes for use in this method are also described.
Description
W096/04398 ~ 3 ~ ~ r~ ,J'0l8l8 ELECTRODES AND THEIR USE IN ANALYSIS
This invention relates to electrochemistry and in particular to novel electrode structures and their use in analysis for the detection of enzymes or their substrates.
The invention is of particular utility in sensors for biomedical applications.
There is a ~on~;nlling need for improved ;rmlln~n~a techniques particularly for the detection of low levels of analytes in small samples.
In recent years, a number of electrochemical techni~ues have been developed in the hope of avoiding the generally conventional use of radioisotope labels for the detection of low levels of analytes. Direct amperometric measurements of NADH and of phenol has been reported by Wright et ~1 Anal.
Chem., 58(1986) 2995 and by Wehmeyer et al Anal. Chem., 58(1986) 135 employing enzyme-labelled antigens. The use of redox-labelled drug conjugates, specifically ferrocenyl-lidocaine for similar direct amperometric mea~uL~ -nt.C has also been reported by Di Gleria et al Anal. Chem. 58(1986) 1203. These direct amperometric measurement techniques tend to suffer from a sensitivity limitation and in general the techni~ues are limited to low micromolar concentration ranges.
Cham~ers and Walton in J. Electxoanal. Chem., 250(1988) 41~-425 report use of a poly(vinylferrocene)-modified glassy carbon electrode used as a charge-accumulating device to a accumulate charge from a solution of glucose/glucose oxidase via the int~rr~~i~y of a redox-labelled antigen, which is cycled repeatedly between the enzyme and the electrode.
Chambers and Walton were able reproducibly to detect 1,1'-dimethylferrocene-3-ethan-1-ol-2-amine at nanomolar W096/04398 ~ ~ PCT/GB9~/0l8l8 ~
concentrations. Although Chambers and Walton identified 8-ferrocenyl-theophylline as their preferred redox-active mediator for further work, they found that it possessed a significant disadvantage in that it appeared to be bound unspecifically to anti-theophylline antibodies and so was not ideal for use in a competitive immllnn~say.
A different approach has been adopted by 3artlett and ;3irkin, (Anal. Chem. 1993, 65, 1113-1119), namely the production of a DC bioelectrochemical transistor in the form of an enzyme switch. In this prior enzyme switch system, to provide a switch responsive to glucose, glucose oxidase was immobilized in a thin insulating film of poly~l,2-~;sm;nnhenzene) deposited on top of a poly~aniline) film deposited across a gap between two carbon electrodes. The change in ~nn~llct;vity of poly~aniline) between its oxidised and reduced states is measured by applying a small DC
voltage from one carbon electrode to the other across the film and is used to measure the presence of redox mediator shuttling charge between the enzyme and polymer film. One drawback of this system is the need to form a polymer film which bridges the two electrodes, taking into account the minimum distance which can be tolerated between the electrodes.
The present invention has arisen from the work of the present inventors in seeking to improve upon the prior techniques.
As will be seen from the detailed description below, the methods and apparatus of the present invention draw upon the technology developed in connection with the prior enzyme switches, but apply this technology in a quite new way, namely in an alternating current system using a single polymer coated electrode.
In acco~dance with a first aspect of the present ~ W096l04398 ~ 3~s r~ i616 invention, there is provided a method for indicating the r~nr~ntr~tion of a substance in solution comprising passing an alternating voltage between a first electrode structure having coated thereon a polymer, the polymer being in one of an oxidised and a reduced state between which states its conductivity varies, and a second separate counter electrode in said solution; and measuring the change in c~n~nct;vity of the polymer coating, such measurement being representative of the state of the polymer and thereby of the ~n~ntration of said substance in the ~olution.
For use in the above method the invention also provides an electrode structure forming a single electrode in a circuit comprising an electrode having coated thereon a polymer film the polymer being in one of an oxidised and a reduced state between which states its conductivity varies.
In a preferred : 'lo~ir~nt of the invention, the method is used to provide an indication of the concentration of an enzyme substrate in solution. The enzyme is preferably immobilised in a film overlying the polymer coating and is involved in redox reactions with its substrate present in the solution. As the enzyme/substrate system undergoes redox reaction, charge is transferred to or from the polymer film bringing about a change in the oxidation state of the polymer. This change in oxidation state leads to a variation in the conductivity of the polymer. The rate at which this change occurs is dependent on the concentration of the substrate in solution so that by measuring the impedance of the system over time, the concentration of the substrate may be determined. Alternatively, a substrate may be ; r~hi 1; ~ed on the polymer coating and the electrode is used to indicate the concentration of an enzyme in solution.
While in preferred embodiments, the electrode finds use in an enzyme system, we have also found that the method allows for direct measurement of substrates which undergo W096/04398 2 t ~ r ~ ol8l8 ~
redox reactions. For example, NADH and L-ascorbic acid have been oxidised in the presence of a polymer film which undergoes reduction and thus changes from an insulating to a conducting state This changes the conductivity of the film as outlined above to measure directly the concPntr~t;nn of a particular substance in solution. Alternatively, the system may be used to identify changes in the redox condition of the polymer film which are not a direct result of an enzyme/substrate system i.e. the background activity action, to provide a more accurate indication of the enzyme or substrate concentration.
Redox mediators may also be provided in the solution or immobil;q~ nn the support to transfer charge to or from the polymer film. As the substance or the substrate in the presence of=the enzyme undergoes reductlon or oxidation, the polymer sup~ort will also undergo oxidation or re~l-ctinn either directly or through the use of mediators.
The method may also be carried out in the absence of the sub3tance whose rnn~ntration i9 to be determined either with the enzyme electrode or base polymer film electrode to monitor ba~hy,~lld charges in the polymer conductivity which are not related to the cnnn~ntration of the substance of interest.
The invention is described in detail with particular reference to the glucose/glucose oxidase system, employing poly~aniline) as the conductive polymer. The invention is not so limited but may be applied broadly to other enzyme/substrate systems and may also be used with other polymers with changing conductivity depending on their redox state.
The use of a single electrode associated with a polymer film allows for smaller electrodes to be produced having thinner coatings of polymer film. This has the advantage in ~ W096io4398 21 9 ~ 3 ~ ~ ~ PCT/Gs95/01818 that it i8 not necessary to form the film as a gap between two electrodes which may be difficult due to limitations in the minimum size gap that can be formed or tolerated without short-circuiting. Furthermore, the system i5 more sensitive when smaller quantities of polymer are used. Since the change in oxidation state of the polymer has a direct effect on conductivity, if less charge transfer is required to switch the polymer film from an insulating to a conducting state, the system becomes more sensitive to smaller quantities of substrate.
The electrode structure of the invention can be in the form of a micro electrode which enables commercial application of the technique as a bio-sensor where very small sample volume are involved, such as lO ~l. Analyte may be present in micromolar concentrations or less. The sensitivity of the system may allow concentrations in the femtomolar range to be detected, particularly where polymer films of thickness in the range O.l~m to l~m are applied to the electrode.
The invention has been described with reference to poly(aniline) which is insulating in its oxidised state and conducting in a reduced state. It may be readily returned to its oxidised state by holding the potential of the electrode at +0.4V vs. saturated calomel electrode (SCE) in a clean pH5 buffer solution.
The invention is hereinafter described in more detail by way of example only with reference to the ~com~nying drawings in which:-Fig. l is a drawing illustrating the experimental setup for use of an electrode structure in accordance with the present invention;
Fig. 2 is a circuit diagram of the system of Fig. l;
W096/04398 2 1 9 ~ 3 ~ b PCT/GB9~01818 ~
Fig. 3 is an exploded view of a carbon microband electrode used in the present application;
Fig. ~ ls a greatly enlarged schematic representation of the cross section of an electrode of the present application;
Fig. 5 18 a schematic diagram of a reaction scheme of the present application;
Fig. 6 is a schematic diagram of an alternative reaction scheme;
Fig. 7 shows measurements of impedance against time for one example of electrode structure in accordance with the present invention when exposed to different concentrations of glucose in solution;
Fig. 8 shows how the glucose ~nnr~n~ration is related to the switching rate (defined as the transient maximum gradient divided by the impedance change over the time period recorded) as derived f rom Fig. 7 for the electrode structure concerned;
Fig. g shows measurement of impedance against time for an example of electrode structure in accordance with the present invention when exposed to difference concentrations of ~-ascorbic acid in solution;
Fig. 1~ shows how the ascorbic acid concentration is related to the switching rate as derived from Fig. 9 for the electrode structure concerned; and Fig. 11 is a schematic representation of the cross section of an alternative electrode of the present application.
~ W096io4398 21 ~3~0 PCT/GBg5/0l8l8 The method of the present application involves forming a circuit comprising an electrode having coated thereon a polymer film and a counter electrode, an AC voltage being passed through the circuit to establish the impedance of the system between the electrodes. The c~n~ t~n~ of the polymer film alters depending on the oxidation state of the polymer, which is in turn altered in response to oxidization or re~ t;~n rP~rti~n~ occurring to substrates in the solution in which the electrodes are placed.
Representative circuit diagrams are shown in Figs. 1 and 2. The polymer coated electrode (1 of Fig. 1) is represented by the equivalent circuit of a resistance Rp which ranges from 50 ohms=to 100 kiloohms and a capacitance Cp in parallel. A blocking capacitor Cb may be mounted in series with the electrode to prevent any unwanted DC signal affecting the oxidation state of the polymer. The resistance Ru is nn~mr~nnated and represents the resistance of the solution (2) and of the counter electrode (3) which may suitably be formed of platinum gauze. An alternating voltage V~ is connected across the counter electrode and the blocking capacitor. The change in conductivity of the polymer film is readily detectable and the rate at which the film is switched from an insulating state to a conducting state (or vice versa) is dependent upon the c~n~ntration of the substrate in the solution which is undergoing redox reaction.
In its simplest form, a polymer film is formed over a carbon electrode. A suitable polymer is poly(aniline) which is insulating in its oxidised state and condu~ting in a reduced state. The change in the oxidation s~ate of the poly(aniline) film is brought about by redox reactions occurring in the solution and/or at the polymer/solution interface. Mediators may be added to shuttle charge from the redox reactions occurring in the solution and the polymer film. An example of the reaction in its simplest W096~4398 ~ 9 6 ~ r~ 616 ~
form is shown at Fig. 6 where the n~i~tinn of L-ascorbic acid leads to direct oxidation products while at the same time the poly(aniline) film is reduced from an insulating to cnn~ncting state. Similar results may be seen with NADH.
In an alternative aspect of the invention, the electrode is used in an enzyme system to measure the nnnr~ntr~inn of a substrate. In a preferred embodiment, the enzyme is immobilized in a thin insulating film on the polymer film. Such a system is the glucose/glucoseoxidase system exemplified by Fig. 5 where oxidation of glucose leads to reduction of the glucose oxidase enzyme. A
mediator may be included such as tetrathiafulvalene or ferrocyanate [Fe(CN) 6]
While it is not essential for the enzyme to be bound in a separate layer over the polymer film, it is preferable.
Binding the enzyme to the polymer electrode allows smaller quantities of enzyme to be used and aids eage of h~n~ling the device, and thus increases the reproducibility of results obtained with the device.
In a preferred aspect of the invention, the polymer electrode is formed as a number of carbon microband gap electrodes on which a polymer film is applied. These microband electrodes may be produced by screen printing onto a PVC card and are shown schematically in Fig. 3.
Typically, each card may have a number carbon microband gap electrodes formed thereon which are subsequently separated.
Firstly, a carbon electrode pattern (4) is printed onto a PVC card (5). Subsequently two layers of dielectric (6) are printed followed by a second carbon electrode layer (7) which lies directly over the first carbon electrode layer (4). Two more layers of dielectric (8) are applied to this second electrode layer (7). After each printing step, the PVC card is placed in an oven at 55~C until the freshly applied layeir has dried. Typically, this takes about 30 to ~ W~96/04398 Ll 9 6 3 8b 60 minutes. The carbon layers are determined to be dry when the resistance of the carbon track (measured with a Digital Volt Meter) reached a minimum constant value of typically of about one kiloohm. Eig. 3 shows an exploded view of a carbon microband single electrode having two carbon layers.
A single carbon layer could also be used.
To expose the microband- electrodes, the finished structures are sheared across the two carbon layers to expose the edges of the two carbon print layers. This is achieved by freezing the electrodes in liquid nitrogen and then fracturing them to avoid deformation and problems with shorting which can occur if the electrodes are cut mechanically to expose the carbon microband. The carbon microband electrodes are approximately 4.5 millimetres in length and lO to 15 ~m wide separated by a 20 ~m gap.
A layer of poly(aniline) (PANI) is then grown on to the carbon microband electrode by holding the potential of the carbon electrode for 20 seconds at +0.9 v vs. S OE in a solution containing 200 ~ of aniline and 5 cm3 of NaHSO~
acidified to a pH of approximately 0 with 0.5 cm3 of ~n~nt~ated H,SOi, suitably at 95-98~ AR grade. Each of the above reagents may be obtained from Aldrich Chemical Co.
;llingh~m, Dorset. The typical total charge passed during this deposition was 3mC, and corresponds to a film thickness in the region of 20~m.
Fig. 4 shows a schematic view of the electrode, having two carbon microband electrodes (4,7) having a poly(aniline) film (9) applied thereto. An enzyme (lO) such as glucose oxidase i8 immobilised in an insulating film (ll) applied to the surface of the poly(aniline) film (9). In the method of the invention, this electrode structure i8 used as a ~ingle electrode.
While the electrode described above has a 20~m polymer 3 ~.Q
W096/04398 ~' ' film and a 20~m gap, single electrodes for use in the present invention may be fabricated with significantly thinner coatings of polymer in the region of 0.1 to 1 ~m enabling a wider range of polymer films to be applied and enhanced sensitivity.
Though it has been found that the use of poly(aniline) provides good results, this conductive polymer is pH
~p~n~nt, The invention is not limited to the use of this specific polymer but can employ any other polymer which has an oxidized and a reduced state and is electrically conductive in one of these states Among the wide variety of electrically conductive polymers usable in practice of the present invention for appropriate systems are: -Poly(aniline) and derivatives thereof;
Poly(thiophene) and derivatives thereof;
Poly(pyrrole) and derivatives thereof; and Poly(pyridine) and derivatives thereof.
Re~erence may be made to the speci~ication o~
International Patent Application PCT/~S88/02319 published under No: W0 89/01694 o~ Allied Signal, Inc which de9cribe9 the manufacture and u~3e of ~hPr~77y stable ~orm3 of electrically~ conductive polyaniline. The polyaniline is rendered conductive by the use of a dopant solute which upon addition to the polyaniline ionize3 the polymer with concomitant formation of an ionic dopant solute species. In general, suitable compounds for addition to the polymer have the formula:
[R - SO3-] n.M-n.
where:
R is an organic radical;
M~n is a species having a positive charge equal to n, n being 1 to 4 In the preferred arrangement M is univalent and preferabry hydrogen.
~ W096104398 ~ 1 9 638~
Similar such dopants may be incorporated in the polytthiophene), poly(pyrrole), poly(indole) and poly(pyridine) systems. A wide variety of suitable monomers may be used for forming the polymers. The selection of individual monomers will have an effect on the film-forming properties as will the length of the polymer backbone, as will be well understood by polymer chemists and would be a matter of appropriate adjustment to provide a polymer of desired physical properties. By way of example, typical monomers are set out below for each of the abovenoted polymer systems:
Poly(aniline) NR2R4 (R3)n (m + n) is no greater than 5 Poly(pyrrole) l~R2 H N H
R
Poly(thiophene) 1 ~ ~2 W096/04398 2 ~ 9 63~O E~~ A8l8 ~
Poly(indole) )m (R1 )n R3 (m + n) is no greater than 4 Poly(pyridine) (R~(H)m (m + n) is no greater than 5 R, Rl, R2, R3 and Rj are suitable organic radicals the selection of which will be well understood by polymer chemists. Most preferably, each of R, Rl, Rz, Rl and R~ will be lower alkyl (i.e., l to 4).
There is a oubstantial ease in detection by connecting the single electrode in a simple alternating current circuit. From the above, it is clear that by reference to a single electrode, a microband electrode structure may be used. By virtue of the presence of the counter electrode and the construction of the device, it is in effect a single electrode. ~o special reference electrode is required. Any appropriate counter electrode may ~e employed to complete the circuit since its resistance is in effect subsumed within the unc~pPnq~ted resistance Ru and what is being detected is the change in conductivity of the conductive polymer electrode.
;6~80 W096/04398 P~ 01818 An appropriate enzyme for the detection of glucose, an analyte of particular interest, is glucose oxidase. This enzyme is irreversibly damaged at a pH below 5 so that the enzyme is incompatible with the conditions in which the poly(aniline) film is grown. To produce an electrode structure in accordance with the present invention, the enzyme glucose oxidase is entrapped in an insulating poly(1,2~ m;nrhenzene) film which is electrochemically deposited on top of the poly(aniline) film. The poly(1,2-~;~minr,henzene) film i9 chosen because it can readily be electrochemically polymerized at a potential where poly(aniline) is conducting. It produces a highly active enzyme film. The practical steps performed in order to provide the enzyme coating consist of transferring the poly(aniline)-coated electrode to a solution of citric acid and Na2HPO; solution at pH 5 containing 0.5 mol dm~3 Na2SO~ , mmol dm~3 1,2-~;~m;nrhenzene, and 167 ~mol glucose oxidase. The electrode is left in solution for six minutes to allow adsorption of the enzyme to the poly(aniline) surface. The poly(1,2~ m;nohrn7ene) film was then deposited by holding the potential of the electrode at +0.4 V vs. SCE for 15 minutes.
Where [Fe(CN) 6] 3 is used as a mediator, the electrode structure is placed in a stirred solution rrnt~in;nr~ 3cm3 [Fe(CN)6] AT 50 mmolar in pH 5.0 buffer. The final electrode structure is stored at room temperature in pH 5.0 solution Ferrocyanate ([Fe(CN) 6] 3~~ acts as a redox mediator shuttling charge between the enzyme and conducting polymer, as shown schematically in Fig. 5. In its oxidized state at +0.5 V vs. SCE and at pH 5.0, poly(aniline) is insulating.
On the addition of glucose, the film is reduced through the following reactions:
~ g 3 8 0 W096/04398 ~ ''s P~~ 0.
~-D-glucose + GOx(ox) --> GOx~red) + gluconolactone GOx(red) + 2[Fe(CN) 6] 3~+ --~ GOx(ox) + 2[Fe(CN) 6~ 2-[Fe(CN) 6] 2- + PANI(ox) --> [Fe(CN) 6] 3~+ + PANI(red) where GOx(ox) and GOx(red) represent the oxidized and reduced forms of the enzyme, [Fe(CN)6]3~ and [Fe(CN)6]'~ are the oxidized and reduced forms of the mediator, and PANI(ox) and PANI(red) represent the oxidized, insulating state on the one hand and a reduced and conducting state on the other hand of the poly(aniline) film.
It may be desirable to place the electrode in solutions not cnn~;n;ng glucose first to establish whether there are any background redox reactions occurring, affecting the conductivity of the polymer which are not directly related to the enzyme/substrate reactions.
Tetrathiafulvalene ~TTF) may also be used as a mediator and is applied by placing the polymer coated electrode structure in a stirred solution ~nnt~ining 3cm3 pH 5.0 buffer, 50~1 of DM 50, 12~1 of Triton X100 and solid TTF for at least two hours. The final electrode structure is stored at room temperature in TTF nont~inin~ solution. TTF- and TTF
represent the oxidised and reduced forms of the mediator respectively.
The graphs illustrated in Figs. 7 and 8 show the results obtained in measuring impedance of the poly(aniline) glucose oxidase electrode structure as against time with different ~on~ntrations of glucose in the solution. The switching rate of the device, defined as the transient maximum i~r~n~ gradient divided by the ; ~ nce change over the 180s of measurement, is plotted against glucose concentration in Fig. 8 from which it will be clear that there is a ready relationship between the switching rate and W096/04398 2 ~ 9 6 3 8 o PCT/GB9S/01818 the glucose concentration. As a result, measurement of the switching rate for any unknown glucose concentration can readily identify the concentration.
As an alternative to the enzymatic systems it is possible to employ bare poly(aniline~ (PANI) to measure L-ascorbic acid. Again the same AC principle was employed, however, the fabrication of the device was much simpler requiring only the potentiostatic growth of poly(aniline) on an electrode. Figure 9 shows the responses to L-ascorbic acid down to physiological ranges and Figure l~ shows the corresponding switching rate.
In this system, as with the others, the response to L-ascorbic acid is concentration ~p~n~nt The system now is much simpler as demonstrated in Figure 6.
This very simple system may be used to remove interference caused by L-ascorbic acid. The signal produced by ascorbic acid on a blank electrode could be used to adjust the sensor to the actual substrate concentration. In particular, L-ascorbic acid is a minor intereferent to the glucose system.
In a very similar way to L-ascorbic acid, NADH can be detected by the AC switch. Again only a bare poly(aniline) film is required and the oxidation of NADH on poly(aniline) shows the response of a single bare device to NADH. The system involves the reduction of poly(aniline) film from its insulating to conducting state.
Integration of the enzymatic response is performed during the process as each substrate molecule reacts and alters the redox potential and thus the impedance of the conducting polymer. The oxidation state change remains even if the structure is removed from the analyte solution. In effect, the polymer film records in a memory fashion any W O 96/04398 2 1 9 6 ~ ~ 16 PC~r/GB95/01818 enzymatic reactions that have taken place. Accordingly, slow enzymatic reactions which would otherwise prove extremely difficult to accurately measure amperometrically can readily be detected and the concentration of the analyte estimated using the inherent integrating property of the conducting polymer in the single electrode device o~ the present invention.
The results presented above have been obtained using an electrode having a 20~m polymer film and have shown the effectiveness of mea~ ntq in an AC circuit. ~sing the polymer coated electrode as a single electrode, much smaller electrodes could be used re~uiring only a very thin film of polymer in ~he range of 0.1 to l~m. In view of the m~rh~ni cm by which the oxidation changes in the polymer lead to conductivity charges which can be correlated to substance r~rrPntration, such thinner films greatly enhance the sensitivity of the system to lower concentrations, down to femtomolar ranges. Furthermore, the presence o~ a thinner coating which does not need to bridge a gap between electrodes increases the flexibility of the system and extends the range of polymers which can be used.
Miniat~rization of the device is pos8ible down to microelectrode dimensions since a large electrode area is not reriuired even ~or detection of low level analyte cr,nr~ntrations.
Repeated electrochemical oxidation of the polymer after each measurement can be used to reset the polymer to its original oxidation state. This means that a single electrode structure may be used to make a series of measurements on the same solution. This increases accuracy of use since the estimate of concentration can be given as the average =of numerous separate responses using the same device.
096l04398 9 ~iJ.
The use of a specific redox mediator such as ferrocene or TTE is not essential in the system. The enzyme itself may be modified by incorporating a chemically modified :~
covalent redox group in the enzyme, thereby removing the need for the homogenous mediator. ~=
The invention is applicable to other enzyme systems such as horseradish peroxidase and NADH dependent enzymes.
While the invention has been described by reference to a single polymer coated electrode, devices could be constructed where a plurality of electrodes are formed on a single base card, each being used independently of the others to complete a circuit. Different polymer coatings or enzyme systems may be applied to the different electrodes so that se~uential AC interrogation of each electrode using the same counter electrode will provide information for a number of different substances or substrates in the solution.
Thus, a single device may be provide for a number of substances using the same basic circuitry for each test.
Fig. 11 shows an example of such a device wherein two carbon electrode~ (12, 13) have coated therein a polymer film (14, 15) which may be the same or different. A
separate enzyme system (16, 17) may be applied to each polymer film such as horse radish peroxidase in any insulating film and glucose oxidase in an insulating film.
Each electrode (12, 13) is connected in the AC circuit separately to obtain a reading for the electrode/polymer/enzyme system employed. Additional electrodes could be provided with the same or different polymer films and with or without an enzyme system.
This invention relates to electrochemistry and in particular to novel electrode structures and their use in analysis for the detection of enzymes or their substrates.
The invention is of particular utility in sensors for biomedical applications.
There is a ~on~;nlling need for improved ;rmlln~n~a techniques particularly for the detection of low levels of analytes in small samples.
In recent years, a number of electrochemical techni~ues have been developed in the hope of avoiding the generally conventional use of radioisotope labels for the detection of low levels of analytes. Direct amperometric measurements of NADH and of phenol has been reported by Wright et ~1 Anal.
Chem., 58(1986) 2995 and by Wehmeyer et al Anal. Chem., 58(1986) 135 employing enzyme-labelled antigens. The use of redox-labelled drug conjugates, specifically ferrocenyl-lidocaine for similar direct amperometric mea~uL~ -nt.C has also been reported by Di Gleria et al Anal. Chem. 58(1986) 1203. These direct amperometric measurement techniques tend to suffer from a sensitivity limitation and in general the techni~ues are limited to low micromolar concentration ranges.
Cham~ers and Walton in J. Electxoanal. Chem., 250(1988) 41~-425 report use of a poly(vinylferrocene)-modified glassy carbon electrode used as a charge-accumulating device to a accumulate charge from a solution of glucose/glucose oxidase via the int~rr~~i~y of a redox-labelled antigen, which is cycled repeatedly between the enzyme and the electrode.
Chambers and Walton were able reproducibly to detect 1,1'-dimethylferrocene-3-ethan-1-ol-2-amine at nanomolar W096/04398 ~ ~ PCT/GB9~/0l8l8 ~
concentrations. Although Chambers and Walton identified 8-ferrocenyl-theophylline as their preferred redox-active mediator for further work, they found that it possessed a significant disadvantage in that it appeared to be bound unspecifically to anti-theophylline antibodies and so was not ideal for use in a competitive immllnn~say.
A different approach has been adopted by 3artlett and ;3irkin, (Anal. Chem. 1993, 65, 1113-1119), namely the production of a DC bioelectrochemical transistor in the form of an enzyme switch. In this prior enzyme switch system, to provide a switch responsive to glucose, glucose oxidase was immobilized in a thin insulating film of poly~l,2-~;sm;nnhenzene) deposited on top of a poly~aniline) film deposited across a gap between two carbon electrodes. The change in ~nn~llct;vity of poly~aniline) between its oxidised and reduced states is measured by applying a small DC
voltage from one carbon electrode to the other across the film and is used to measure the presence of redox mediator shuttling charge between the enzyme and polymer film. One drawback of this system is the need to form a polymer film which bridges the two electrodes, taking into account the minimum distance which can be tolerated between the electrodes.
The present invention has arisen from the work of the present inventors in seeking to improve upon the prior techniques.
As will be seen from the detailed description below, the methods and apparatus of the present invention draw upon the technology developed in connection with the prior enzyme switches, but apply this technology in a quite new way, namely in an alternating current system using a single polymer coated electrode.
In acco~dance with a first aspect of the present ~ W096l04398 ~ 3~s r~ i616 invention, there is provided a method for indicating the r~nr~ntr~tion of a substance in solution comprising passing an alternating voltage between a first electrode structure having coated thereon a polymer, the polymer being in one of an oxidised and a reduced state between which states its conductivity varies, and a second separate counter electrode in said solution; and measuring the change in c~n~nct;vity of the polymer coating, such measurement being representative of the state of the polymer and thereby of the ~n~ntration of said substance in the ~olution.
For use in the above method the invention also provides an electrode structure forming a single electrode in a circuit comprising an electrode having coated thereon a polymer film the polymer being in one of an oxidised and a reduced state between which states its conductivity varies.
In a preferred : 'lo~ir~nt of the invention, the method is used to provide an indication of the concentration of an enzyme substrate in solution. The enzyme is preferably immobilised in a film overlying the polymer coating and is involved in redox reactions with its substrate present in the solution. As the enzyme/substrate system undergoes redox reaction, charge is transferred to or from the polymer film bringing about a change in the oxidation state of the polymer. This change in oxidation state leads to a variation in the conductivity of the polymer. The rate at which this change occurs is dependent on the concentration of the substrate in solution so that by measuring the impedance of the system over time, the concentration of the substrate may be determined. Alternatively, a substrate may be ; r~hi 1; ~ed on the polymer coating and the electrode is used to indicate the concentration of an enzyme in solution.
While in preferred embodiments, the electrode finds use in an enzyme system, we have also found that the method allows for direct measurement of substrates which undergo W096/04398 2 t ~ r ~ ol8l8 ~
redox reactions. For example, NADH and L-ascorbic acid have been oxidised in the presence of a polymer film which undergoes reduction and thus changes from an insulating to a conducting state This changes the conductivity of the film as outlined above to measure directly the concPntr~t;nn of a particular substance in solution. Alternatively, the system may be used to identify changes in the redox condition of the polymer film which are not a direct result of an enzyme/substrate system i.e. the background activity action, to provide a more accurate indication of the enzyme or substrate concentration.
Redox mediators may also be provided in the solution or immobil;q~ nn the support to transfer charge to or from the polymer film. As the substance or the substrate in the presence of=the enzyme undergoes reductlon or oxidation, the polymer sup~ort will also undergo oxidation or re~l-ctinn either directly or through the use of mediators.
The method may also be carried out in the absence of the sub3tance whose rnn~ntration i9 to be determined either with the enzyme electrode or base polymer film electrode to monitor ba~hy,~lld charges in the polymer conductivity which are not related to the cnnn~ntration of the substance of interest.
The invention is described in detail with particular reference to the glucose/glucose oxidase system, employing poly~aniline) as the conductive polymer. The invention is not so limited but may be applied broadly to other enzyme/substrate systems and may also be used with other polymers with changing conductivity depending on their redox state.
The use of a single electrode associated with a polymer film allows for smaller electrodes to be produced having thinner coatings of polymer film. This has the advantage in ~ W096io4398 21 9 ~ 3 ~ ~ ~ PCT/Gs95/01818 that it i8 not necessary to form the film as a gap between two electrodes which may be difficult due to limitations in the minimum size gap that can be formed or tolerated without short-circuiting. Furthermore, the system i5 more sensitive when smaller quantities of polymer are used. Since the change in oxidation state of the polymer has a direct effect on conductivity, if less charge transfer is required to switch the polymer film from an insulating to a conducting state, the system becomes more sensitive to smaller quantities of substrate.
The electrode structure of the invention can be in the form of a micro electrode which enables commercial application of the technique as a bio-sensor where very small sample volume are involved, such as lO ~l. Analyte may be present in micromolar concentrations or less. The sensitivity of the system may allow concentrations in the femtomolar range to be detected, particularly where polymer films of thickness in the range O.l~m to l~m are applied to the electrode.
The invention has been described with reference to poly(aniline) which is insulating in its oxidised state and conducting in a reduced state. It may be readily returned to its oxidised state by holding the potential of the electrode at +0.4V vs. saturated calomel electrode (SCE) in a clean pH5 buffer solution.
The invention is hereinafter described in more detail by way of example only with reference to the ~com~nying drawings in which:-Fig. l is a drawing illustrating the experimental setup for use of an electrode structure in accordance with the present invention;
Fig. 2 is a circuit diagram of the system of Fig. l;
W096/04398 2 1 9 ~ 3 ~ b PCT/GB9~01818 ~
Fig. 3 is an exploded view of a carbon microband electrode used in the present application;
Fig. ~ ls a greatly enlarged schematic representation of the cross section of an electrode of the present application;
Fig. 5 18 a schematic diagram of a reaction scheme of the present application;
Fig. 6 is a schematic diagram of an alternative reaction scheme;
Fig. 7 shows measurements of impedance against time for one example of electrode structure in accordance with the present invention when exposed to different concentrations of glucose in solution;
Fig. 8 shows how the glucose ~nnr~n~ration is related to the switching rate (defined as the transient maximum gradient divided by the impedance change over the time period recorded) as derived f rom Fig. 7 for the electrode structure concerned;
Fig. g shows measurement of impedance against time for an example of electrode structure in accordance with the present invention when exposed to difference concentrations of ~-ascorbic acid in solution;
Fig. 1~ shows how the ascorbic acid concentration is related to the switching rate as derived from Fig. 9 for the electrode structure concerned; and Fig. 11 is a schematic representation of the cross section of an alternative electrode of the present application.
~ W096io4398 21 ~3~0 PCT/GBg5/0l8l8 The method of the present application involves forming a circuit comprising an electrode having coated thereon a polymer film and a counter electrode, an AC voltage being passed through the circuit to establish the impedance of the system between the electrodes. The c~n~ t~n~ of the polymer film alters depending on the oxidation state of the polymer, which is in turn altered in response to oxidization or re~ t;~n rP~rti~n~ occurring to substrates in the solution in which the electrodes are placed.
Representative circuit diagrams are shown in Figs. 1 and 2. The polymer coated electrode (1 of Fig. 1) is represented by the equivalent circuit of a resistance Rp which ranges from 50 ohms=to 100 kiloohms and a capacitance Cp in parallel. A blocking capacitor Cb may be mounted in series with the electrode to prevent any unwanted DC signal affecting the oxidation state of the polymer. The resistance Ru is nn~mr~nnated and represents the resistance of the solution (2) and of the counter electrode (3) which may suitably be formed of platinum gauze. An alternating voltage V~ is connected across the counter electrode and the blocking capacitor. The change in conductivity of the polymer film is readily detectable and the rate at which the film is switched from an insulating state to a conducting state (or vice versa) is dependent upon the c~n~ntration of the substrate in the solution which is undergoing redox reaction.
In its simplest form, a polymer film is formed over a carbon electrode. A suitable polymer is poly(aniline) which is insulating in its oxidised state and condu~ting in a reduced state. The change in the oxidation s~ate of the poly(aniline) film is brought about by redox reactions occurring in the solution and/or at the polymer/solution interface. Mediators may be added to shuttle charge from the redox reactions occurring in the solution and the polymer film. An example of the reaction in its simplest W096~4398 ~ 9 6 ~ r~ 616 ~
form is shown at Fig. 6 where the n~i~tinn of L-ascorbic acid leads to direct oxidation products while at the same time the poly(aniline) film is reduced from an insulating to cnn~ncting state. Similar results may be seen with NADH.
In an alternative aspect of the invention, the electrode is used in an enzyme system to measure the nnnr~ntr~inn of a substrate. In a preferred embodiment, the enzyme is immobilized in a thin insulating film on the polymer film. Such a system is the glucose/glucoseoxidase system exemplified by Fig. 5 where oxidation of glucose leads to reduction of the glucose oxidase enzyme. A
mediator may be included such as tetrathiafulvalene or ferrocyanate [Fe(CN) 6]
While it is not essential for the enzyme to be bound in a separate layer over the polymer film, it is preferable.
Binding the enzyme to the polymer electrode allows smaller quantities of enzyme to be used and aids eage of h~n~ling the device, and thus increases the reproducibility of results obtained with the device.
In a preferred aspect of the invention, the polymer electrode is formed as a number of carbon microband gap electrodes on which a polymer film is applied. These microband electrodes may be produced by screen printing onto a PVC card and are shown schematically in Fig. 3.
Typically, each card may have a number carbon microband gap electrodes formed thereon which are subsequently separated.
Firstly, a carbon electrode pattern (4) is printed onto a PVC card (5). Subsequently two layers of dielectric (6) are printed followed by a second carbon electrode layer (7) which lies directly over the first carbon electrode layer (4). Two more layers of dielectric (8) are applied to this second electrode layer (7). After each printing step, the PVC card is placed in an oven at 55~C until the freshly applied layeir has dried. Typically, this takes about 30 to ~ W~96/04398 Ll 9 6 3 8b 60 minutes. The carbon layers are determined to be dry when the resistance of the carbon track (measured with a Digital Volt Meter) reached a minimum constant value of typically of about one kiloohm. Eig. 3 shows an exploded view of a carbon microband single electrode having two carbon layers.
A single carbon layer could also be used.
To expose the microband- electrodes, the finished structures are sheared across the two carbon layers to expose the edges of the two carbon print layers. This is achieved by freezing the electrodes in liquid nitrogen and then fracturing them to avoid deformation and problems with shorting which can occur if the electrodes are cut mechanically to expose the carbon microband. The carbon microband electrodes are approximately 4.5 millimetres in length and lO to 15 ~m wide separated by a 20 ~m gap.
A layer of poly(aniline) (PANI) is then grown on to the carbon microband electrode by holding the potential of the carbon electrode for 20 seconds at +0.9 v vs. S OE in a solution containing 200 ~ of aniline and 5 cm3 of NaHSO~
acidified to a pH of approximately 0 with 0.5 cm3 of ~n~nt~ated H,SOi, suitably at 95-98~ AR grade. Each of the above reagents may be obtained from Aldrich Chemical Co.
;llingh~m, Dorset. The typical total charge passed during this deposition was 3mC, and corresponds to a film thickness in the region of 20~m.
Fig. 4 shows a schematic view of the electrode, having two carbon microband electrodes (4,7) having a poly(aniline) film (9) applied thereto. An enzyme (lO) such as glucose oxidase i8 immobilised in an insulating film (ll) applied to the surface of the poly(aniline) film (9). In the method of the invention, this electrode structure i8 used as a ~ingle electrode.
While the electrode described above has a 20~m polymer 3 ~.Q
W096/04398 ~' ' film and a 20~m gap, single electrodes for use in the present invention may be fabricated with significantly thinner coatings of polymer in the region of 0.1 to 1 ~m enabling a wider range of polymer films to be applied and enhanced sensitivity.
Though it has been found that the use of poly(aniline) provides good results, this conductive polymer is pH
~p~n~nt, The invention is not limited to the use of this specific polymer but can employ any other polymer which has an oxidized and a reduced state and is electrically conductive in one of these states Among the wide variety of electrically conductive polymers usable in practice of the present invention for appropriate systems are: -Poly(aniline) and derivatives thereof;
Poly(thiophene) and derivatives thereof;
Poly(pyrrole) and derivatives thereof; and Poly(pyridine) and derivatives thereof.
Re~erence may be made to the speci~ication o~
International Patent Application PCT/~S88/02319 published under No: W0 89/01694 o~ Allied Signal, Inc which de9cribe9 the manufacture and u~3e of ~hPr~77y stable ~orm3 of electrically~ conductive polyaniline. The polyaniline is rendered conductive by the use of a dopant solute which upon addition to the polyaniline ionize3 the polymer with concomitant formation of an ionic dopant solute species. In general, suitable compounds for addition to the polymer have the formula:
[R - SO3-] n.M-n.
where:
R is an organic radical;
M~n is a species having a positive charge equal to n, n being 1 to 4 In the preferred arrangement M is univalent and preferabry hydrogen.
~ W096104398 ~ 1 9 638~
Similar such dopants may be incorporated in the polytthiophene), poly(pyrrole), poly(indole) and poly(pyridine) systems. A wide variety of suitable monomers may be used for forming the polymers. The selection of individual monomers will have an effect on the film-forming properties as will the length of the polymer backbone, as will be well understood by polymer chemists and would be a matter of appropriate adjustment to provide a polymer of desired physical properties. By way of example, typical monomers are set out below for each of the abovenoted polymer systems:
Poly(aniline) NR2R4 (R3)n (m + n) is no greater than 5 Poly(pyrrole) l~R2 H N H
R
Poly(thiophene) 1 ~ ~2 W096/04398 2 ~ 9 63~O E~~ A8l8 ~
Poly(indole) )m (R1 )n R3 (m + n) is no greater than 4 Poly(pyridine) (R~(H)m (m + n) is no greater than 5 R, Rl, R2, R3 and Rj are suitable organic radicals the selection of which will be well understood by polymer chemists. Most preferably, each of R, Rl, Rz, Rl and R~ will be lower alkyl (i.e., l to 4).
There is a oubstantial ease in detection by connecting the single electrode in a simple alternating current circuit. From the above, it is clear that by reference to a single electrode, a microband electrode structure may be used. By virtue of the presence of the counter electrode and the construction of the device, it is in effect a single electrode. ~o special reference electrode is required. Any appropriate counter electrode may ~e employed to complete the circuit since its resistance is in effect subsumed within the unc~pPnq~ted resistance Ru and what is being detected is the change in conductivity of the conductive polymer electrode.
;6~80 W096/04398 P~ 01818 An appropriate enzyme for the detection of glucose, an analyte of particular interest, is glucose oxidase. This enzyme is irreversibly damaged at a pH below 5 so that the enzyme is incompatible with the conditions in which the poly(aniline) film is grown. To produce an electrode structure in accordance with the present invention, the enzyme glucose oxidase is entrapped in an insulating poly(1,2~ m;nrhenzene) film which is electrochemically deposited on top of the poly(aniline) film. The poly(1,2-~;~minr,henzene) film i9 chosen because it can readily be electrochemically polymerized at a potential where poly(aniline) is conducting. It produces a highly active enzyme film. The practical steps performed in order to provide the enzyme coating consist of transferring the poly(aniline)-coated electrode to a solution of citric acid and Na2HPO; solution at pH 5 containing 0.5 mol dm~3 Na2SO~ , mmol dm~3 1,2-~;~m;nrhenzene, and 167 ~mol glucose oxidase. The electrode is left in solution for six minutes to allow adsorption of the enzyme to the poly(aniline) surface. The poly(1,2~ m;nohrn7ene) film was then deposited by holding the potential of the electrode at +0.4 V vs. SCE for 15 minutes.
Where [Fe(CN) 6] 3 is used as a mediator, the electrode structure is placed in a stirred solution rrnt~in;nr~ 3cm3 [Fe(CN)6] AT 50 mmolar in pH 5.0 buffer. The final electrode structure is stored at room temperature in pH 5.0 solution Ferrocyanate ([Fe(CN) 6] 3~~ acts as a redox mediator shuttling charge between the enzyme and conducting polymer, as shown schematically in Fig. 5. In its oxidized state at +0.5 V vs. SCE and at pH 5.0, poly(aniline) is insulating.
On the addition of glucose, the film is reduced through the following reactions:
~ g 3 8 0 W096/04398 ~ ''s P~~ 0.
~-D-glucose + GOx(ox) --> GOx~red) + gluconolactone GOx(red) + 2[Fe(CN) 6] 3~+ --~ GOx(ox) + 2[Fe(CN) 6~ 2-[Fe(CN) 6] 2- + PANI(ox) --> [Fe(CN) 6] 3~+ + PANI(red) where GOx(ox) and GOx(red) represent the oxidized and reduced forms of the enzyme, [Fe(CN)6]3~ and [Fe(CN)6]'~ are the oxidized and reduced forms of the mediator, and PANI(ox) and PANI(red) represent the oxidized, insulating state on the one hand and a reduced and conducting state on the other hand of the poly(aniline) film.
It may be desirable to place the electrode in solutions not cnn~;n;ng glucose first to establish whether there are any background redox reactions occurring, affecting the conductivity of the polymer which are not directly related to the enzyme/substrate reactions.
Tetrathiafulvalene ~TTF) may also be used as a mediator and is applied by placing the polymer coated electrode structure in a stirred solution ~nnt~ining 3cm3 pH 5.0 buffer, 50~1 of DM 50, 12~1 of Triton X100 and solid TTF for at least two hours. The final electrode structure is stored at room temperature in TTF nont~inin~ solution. TTF- and TTF
represent the oxidised and reduced forms of the mediator respectively.
The graphs illustrated in Figs. 7 and 8 show the results obtained in measuring impedance of the poly(aniline) glucose oxidase electrode structure as against time with different ~on~ntrations of glucose in the solution. The switching rate of the device, defined as the transient maximum i~r~n~ gradient divided by the ; ~ nce change over the 180s of measurement, is plotted against glucose concentration in Fig. 8 from which it will be clear that there is a ready relationship between the switching rate and W096/04398 2 ~ 9 6 3 8 o PCT/GB9S/01818 the glucose concentration. As a result, measurement of the switching rate for any unknown glucose concentration can readily identify the concentration.
As an alternative to the enzymatic systems it is possible to employ bare poly(aniline~ (PANI) to measure L-ascorbic acid. Again the same AC principle was employed, however, the fabrication of the device was much simpler requiring only the potentiostatic growth of poly(aniline) on an electrode. Figure 9 shows the responses to L-ascorbic acid down to physiological ranges and Figure l~ shows the corresponding switching rate.
In this system, as with the others, the response to L-ascorbic acid is concentration ~p~n~nt The system now is much simpler as demonstrated in Figure 6.
This very simple system may be used to remove interference caused by L-ascorbic acid. The signal produced by ascorbic acid on a blank electrode could be used to adjust the sensor to the actual substrate concentration. In particular, L-ascorbic acid is a minor intereferent to the glucose system.
In a very similar way to L-ascorbic acid, NADH can be detected by the AC switch. Again only a bare poly(aniline) film is required and the oxidation of NADH on poly(aniline) shows the response of a single bare device to NADH. The system involves the reduction of poly(aniline) film from its insulating to conducting state.
Integration of the enzymatic response is performed during the process as each substrate molecule reacts and alters the redox potential and thus the impedance of the conducting polymer. The oxidation state change remains even if the structure is removed from the analyte solution. In effect, the polymer film records in a memory fashion any W O 96/04398 2 1 9 6 ~ ~ 16 PC~r/GB95/01818 enzymatic reactions that have taken place. Accordingly, slow enzymatic reactions which would otherwise prove extremely difficult to accurately measure amperometrically can readily be detected and the concentration of the analyte estimated using the inherent integrating property of the conducting polymer in the single electrode device o~ the present invention.
The results presented above have been obtained using an electrode having a 20~m polymer film and have shown the effectiveness of mea~ ntq in an AC circuit. ~sing the polymer coated electrode as a single electrode, much smaller electrodes could be used re~uiring only a very thin film of polymer in ~he range of 0.1 to l~m. In view of the m~rh~ni cm by which the oxidation changes in the polymer lead to conductivity charges which can be correlated to substance r~rrPntration, such thinner films greatly enhance the sensitivity of the system to lower concentrations, down to femtomolar ranges. Furthermore, the presence o~ a thinner coating which does not need to bridge a gap between electrodes increases the flexibility of the system and extends the range of polymers which can be used.
Miniat~rization of the device is pos8ible down to microelectrode dimensions since a large electrode area is not reriuired even ~or detection of low level analyte cr,nr~ntrations.
Repeated electrochemical oxidation of the polymer after each measurement can be used to reset the polymer to its original oxidation state. This means that a single electrode structure may be used to make a series of measurements on the same solution. This increases accuracy of use since the estimate of concentration can be given as the average =of numerous separate responses using the same device.
096l04398 9 ~iJ.
The use of a specific redox mediator such as ferrocene or TTE is not essential in the system. The enzyme itself may be modified by incorporating a chemically modified :~
covalent redox group in the enzyme, thereby removing the need for the homogenous mediator. ~=
The invention is applicable to other enzyme systems such as horseradish peroxidase and NADH dependent enzymes.
While the invention has been described by reference to a single polymer coated electrode, devices could be constructed where a plurality of electrodes are formed on a single base card, each being used independently of the others to complete a circuit. Different polymer coatings or enzyme systems may be applied to the different electrodes so that se~uential AC interrogation of each electrode using the same counter electrode will provide information for a number of different substances or substrates in the solution.
Thus, a single device may be provide for a number of substances using the same basic circuitry for each test.
Fig. 11 shows an example of such a device wherein two carbon electrode~ (12, 13) have coated therein a polymer film (14, 15) which may be the same or different. A
separate enzyme system (16, 17) may be applied to each polymer film such as horse radish peroxidase in any insulating film and glucose oxidase in an insulating film.
Each electrode (12, 13) is connected in the AC circuit separately to obtain a reading for the electrode/polymer/enzyme system employed. Additional electrodes could be provided with the same or different polymer films and with or without an enzyme system.
Claims
1 A method for indicating the concentration of a substance in solution comprising passing an alternating voltage between a first electrode structure having coated thereon a polymer, the polymer being in one of an oxidised and a reduced state between which states its conductivity varies, and a second separate counter electrode in said solution; and measuring the change in conductivity of the polymer coating, such measurement being representative of the state of the polymer and thereby of the concentration of said substance in the solution.
2 A method according to Claim 1 wherein a first measurement is made in the absence of the substance in solution to provide a background reading of any interfering substances in said solution.
3 A method according to Claim 1 wherein an enzyme is:
immobilised on the polymer coating, and the method is for indicating the concentration of a substrate for said enzyme in the solution.
4 A method according to Claim 3 wherein said first electrode comprises a mediator associated with the polymer or enzyme coating, for transfer of charge between the substrate and the polymer film.
A method according to Claim 3 or Claim 4 wherein a first measurement is made in the absence of said enzyme to provide a background reading of any interfering substances in said solution.
6 A method according to Claim 1 or 2 wherein a substrate for an enzyme is immobilised on the polymer coating and the method is for indicating the concentration of the enzyme in the solution.
7 A method according to any preceding claim wherein the polymer comprises poly(aniline).
8 A method according to any preceding claim wherein the polymer film has a thickness in the range of 0.1 to 1 µm.
9 A method according to any preceding claim wherein the first electrode structure comprises a plurality of electrodes each using a discrete polymer coating thereon, and an alternating voltage is passed through each electrode separately to measure the state of the conductivity of each polymer coating and thereby the concentration of a plurality of substances in solution.
An electrode structure forming a single electrode in a circuit for use in the method of any preceding claim comprising an electrode having coated thereon a polymer film the polymer being in one of an oxidised and a reduced state between which states its conductivity varies.
11 An electrode structure according to Claim 10 comprising an elongate electrode having an exposed face over which the polymer is coated.
12 An electrode structure according to Claim 10 or Claim 11 wherein the polymer film is covered by a composition in which an enzyme is immobilised.
13 An electrode according to any of Claims 10 to 12 wherein said electrode comprises a carbon microband electrode having one or more carbon electrodes inter-layered with insulating dielectric layers printed on a substrate, the polymer being coated on the exposed ends of carbon of said microband electrode.
14 An electrode according to any of Claims 10 to 13 wherein said polymer film has a thickness in the range 0.1 to 1 µm.
An electrode structure according to any of Claims 10 to 14 and comprising a plurality of electrodes each having coated thereon a discrete polymer film.
16 An electrode structure according to Claim 15 wherein an enzyme is overcoated onto one of the discrete polymer films.
2 A method according to Claim 1 wherein a first measurement is made in the absence of the substance in solution to provide a background reading of any interfering substances in said solution.
3 A method according to Claim 1 wherein an enzyme is:
immobilised on the polymer coating, and the method is for indicating the concentration of a substrate for said enzyme in the solution.
4 A method according to Claim 3 wherein said first electrode comprises a mediator associated with the polymer or enzyme coating, for transfer of charge between the substrate and the polymer film.
A method according to Claim 3 or Claim 4 wherein a first measurement is made in the absence of said enzyme to provide a background reading of any interfering substances in said solution.
6 A method according to Claim 1 or 2 wherein a substrate for an enzyme is immobilised on the polymer coating and the method is for indicating the concentration of the enzyme in the solution.
7 A method according to any preceding claim wherein the polymer comprises poly(aniline).
8 A method according to any preceding claim wherein the polymer film has a thickness in the range of 0.1 to 1 µm.
9 A method according to any preceding claim wherein the first electrode structure comprises a plurality of electrodes each using a discrete polymer coating thereon, and an alternating voltage is passed through each electrode separately to measure the state of the conductivity of each polymer coating and thereby the concentration of a plurality of substances in solution.
An electrode structure forming a single electrode in a circuit for use in the method of any preceding claim comprising an electrode having coated thereon a polymer film the polymer being in one of an oxidised and a reduced state between which states its conductivity varies.
11 An electrode structure according to Claim 10 comprising an elongate electrode having an exposed face over which the polymer is coated.
12 An electrode structure according to Claim 10 or Claim 11 wherein the polymer film is covered by a composition in which an enzyme is immobilised.
13 An electrode according to any of Claims 10 to 12 wherein said electrode comprises a carbon microband electrode having one or more carbon electrodes inter-layered with insulating dielectric layers printed on a substrate, the polymer being coated on the exposed ends of carbon of said microband electrode.
14 An electrode according to any of Claims 10 to 13 wherein said polymer film has a thickness in the range 0.1 to 1 µm.
An electrode structure according to any of Claims 10 to 14 and comprising a plurality of electrodes each having coated thereon a discrete polymer film.
16 An electrode structure according to Claim 15 wherein an enzyme is overcoated onto one of the discrete polymer films.
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| GB9415499A GB9415499D0 (en) | 1994-08-01 | 1994-08-01 | Electrodes and their use in analysis |
| GB9415499.4 | 1994-08-01 |
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|---|---|
| CA2196380A1 true CA2196380A1 (en) | 1996-02-15 |
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| CA002196380A Abandoned CA2196380A1 (en) | 1994-08-01 | 1995-08-01 | Electrodes and their use in analysis |
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| EP (1) | EP0775214B1 (en) |
| JP (1) | JPH10503846A (en) |
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| DE (1) | DE69531897D1 (en) |
| GB (1) | GB9415499D0 (en) |
| WO (1) | WO1996004398A1 (en) |
Families Citing this family (107)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9415499D0 (en) * | 1994-08-01 | 1994-09-21 | Bartlett Philip N | Electrodes and their use in analysis |
| US6413410B1 (en) | 1996-06-19 | 2002-07-02 | Lifescan, Inc. | Electrochemical cell |
| AUPN363995A0 (en) | 1995-06-19 | 1995-07-13 | Memtec Limited | Electrochemical cell |
| AUPN661995A0 (en) | 1995-11-16 | 1995-12-07 | Memtec America Corporation | Electrochemical cell 2 |
| US6863801B2 (en) * | 1995-11-16 | 2005-03-08 | Lifescan, Inc. | Electrochemical cell |
| GB9607898D0 (en) * | 1996-04-17 | 1996-06-19 | British Nuclear Fuels Plc | Improvements in and relating to sensors |
| JP3460183B2 (en) * | 1996-12-24 | 2003-10-27 | 松下電器産業株式会社 | Biosensor |
| US6036924A (en) | 1997-12-04 | 2000-03-14 | Hewlett-Packard Company | Cassette of lancet cartridges for sampling blood |
| US8071384B2 (en) | 1997-12-22 | 2011-12-06 | Roche Diagnostics Operations, Inc. | Control and calibration solutions and methods for their use |
| US6391005B1 (en) | 1998-03-30 | 2002-05-21 | Agilent Technologies, Inc. | Apparatus and method for penetration with shaft having a sensor for sensing penetration depth |
| JP2002537031A (en) | 1999-02-18 | 2002-11-05 | バイオバルブ テクノロジーズ インコーポレイテッド | Electroactive pores |
| US7166475B2 (en) * | 1999-02-26 | 2007-01-23 | Cyclacel Ltd. | Compositions and methods for monitoring the modification state of a pair of polypeptides |
| GB2350677A (en) * | 1999-06-04 | 2000-12-06 | Cambridge Life Sciences | Enzyme detection |
| US20050103624A1 (en) | 1999-10-04 | 2005-05-19 | Bhullar Raghbir S. | Biosensor and method of making |
| WO2001038873A2 (en) * | 1999-11-24 | 2001-05-31 | Biotronic Technologies, Inc. | Devices and methods for detecting analytes using electrosensor having capture reagent |
| DE10022750A1 (en) * | 2000-05-10 | 2001-11-22 | Wolfgang Schuhmann | Preparing surface with immobilized recognition elements, useful e.g. in biosensors, by electrodeposition of resin from emulsion containing active substances |
| US8641644B2 (en) | 2000-11-21 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
| US7025774B2 (en) | 2001-06-12 | 2006-04-11 | Pelikan Technologies, Inc. | Tissue penetration device |
| DE60234598D1 (en) | 2001-06-12 | 2010-01-14 | Pelikan Technologies Inc | SELF-OPTIMIZING LANZET DEVICE WITH ADAPTANT FOR TEMPORAL FLUCTUATIONS OF SKIN PROPERTIES |
| EP1404234B1 (en) | 2001-06-12 | 2011-02-09 | Pelikan Technologies Inc. | Apparatus for improving success rate of blood yield from a fingerstick |
| US9226699B2 (en) | 2002-04-19 | 2016-01-05 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling module with a continuous compression tissue interface surface |
| US7749174B2 (en) | 2001-06-12 | 2010-07-06 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device intergrated onto a blood-sampling cartridge |
| US7981056B2 (en) | 2002-04-19 | 2011-07-19 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
| US9427532B2 (en) | 2001-06-12 | 2016-08-30 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| ATE485766T1 (en) | 2001-06-12 | 2010-11-15 | Pelikan Technologies Inc | ELECTRICAL ACTUATING ELEMENT FOR A LANCET |
| US7682318B2 (en) | 2001-06-12 | 2010-03-23 | Pelikan Technologies, Inc. | Blood sampling apparatus and method |
| US8337419B2 (en) | 2002-04-19 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
| CN1920548B (en) | 2001-10-10 | 2013-05-29 | 生命扫描有限公司 | Method for manufacturing electrochemical cell |
| US7285198B2 (en) * | 2004-02-23 | 2007-10-23 | Mysticmd, Inc. | Strip electrode with conductive nano tube printing |
| US7232451B2 (en) | 2002-04-19 | 2007-06-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8267870B2 (en) | 2002-04-19 | 2012-09-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling with hybrid actuation |
| US8579831B2 (en) | 2002-04-19 | 2013-11-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US8360992B2 (en) | 2002-04-19 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7648468B2 (en) | 2002-04-19 | 2010-01-19 | Pelikon Technologies, Inc. | Method and apparatus for penetrating tissue |
| US9314194B2 (en) | 2002-04-19 | 2016-04-19 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| US7547287B2 (en) | 2002-04-19 | 2009-06-16 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7909778B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7976476B2 (en) | 2002-04-19 | 2011-07-12 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
| US7674232B2 (en) | 2002-04-19 | 2010-03-09 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US9795334B2 (en) | 2002-04-19 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7331931B2 (en) | 2002-04-19 | 2008-02-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7297122B2 (en) | 2002-04-19 | 2007-11-20 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7226461B2 (en) | 2002-04-19 | 2007-06-05 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device with sterility barrier release |
| US7491178B2 (en) | 2002-04-19 | 2009-02-17 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7901362B2 (en) | 2002-04-19 | 2011-03-08 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7892183B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
| US8702624B2 (en) | 2006-09-29 | 2014-04-22 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
| US7892185B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
| US7229458B2 (en) | 2002-04-19 | 2007-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8784335B2 (en) | 2002-04-19 | 2014-07-22 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling device with a capacitive sensor |
| US8221334B2 (en) | 2002-04-19 | 2012-07-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7175642B2 (en) | 2002-04-19 | 2007-02-13 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
| US9248267B2 (en) | 2002-04-19 | 2016-02-02 | Sanofi-Aventis Deustchland Gmbh | Tissue penetration device |
| US7371247B2 (en) | 2002-04-19 | 2008-05-13 | Pelikan Technologies, Inc | Method and apparatus for penetrating tissue |
| US7717863B2 (en) | 2002-04-19 | 2010-05-18 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7291117B2 (en) | 2002-04-19 | 2007-11-06 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8574895B2 (en) | 2002-12-30 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
| EP1628567B1 (en) | 2003-05-30 | 2010-08-04 | Pelikan Technologies Inc. | Method and apparatus for fluid injection |
| DK1633235T3 (en) | 2003-06-06 | 2014-08-18 | Sanofi Aventis Deutschland | Apparatus for sampling body fluid and detecting analyte |
| WO2006001797A1 (en) | 2004-06-14 | 2006-01-05 | Pelikan Technologies, Inc. | Low pain penetrating |
| US7645421B2 (en) | 2003-06-20 | 2010-01-12 | Roche Diagnostics Operations, Inc. | System and method for coding information on a biosensor test strip |
| US7452457B2 (en) | 2003-06-20 | 2008-11-18 | Roche Diagnostics Operations, Inc. | System and method for analyte measurement using dose sufficiency electrodes |
| US8679853B2 (en) | 2003-06-20 | 2014-03-25 | Roche Diagnostics Operations, Inc. | Biosensor with laser-sealed capillary space and method of making |
| US8071030B2 (en) | 2003-06-20 | 2011-12-06 | Roche Diagnostics Operations, Inc. | Test strip with flared sample receiving chamber |
| US7488601B2 (en) | 2003-06-20 | 2009-02-10 | Roche Diagnostic Operations, Inc. | System and method for determining an abused sensor during analyte measurement |
| US7718439B2 (en) | 2003-06-20 | 2010-05-18 | Roche Diagnostics Operations, Inc. | System and method for coding information on a biosensor test strip |
| US20070264721A1 (en) * | 2003-10-17 | 2007-11-15 | Buck Harvey B | System and method for analyte measurement using a nonlinear sample response |
| US7645373B2 (en) | 2003-06-20 | 2010-01-12 | Roche Diagnostic Operations, Inc. | System and method for coding information on a biosensor test strip |
| PT1639352T (en) | 2003-06-20 | 2018-07-09 | Hoffmann La Roche | Method and reagent for producing narrow, homogenous reagent strips |
| US8148164B2 (en) | 2003-06-20 | 2012-04-03 | Roche Diagnostics Operations, Inc. | System and method for determining the concentration of an analyte in a sample fluid |
| US8058077B2 (en) | 2003-06-20 | 2011-11-15 | Roche Diagnostics Operations, Inc. | Method for coding information on a biosensor test strip |
| US8206565B2 (en) | 2003-06-20 | 2012-06-26 | Roche Diagnostics Operation, Inc. | System and method for coding information on a biosensor test strip |
| US20040262170A1 (en) * | 2003-06-27 | 2004-12-30 | Steris Inc. | Sensor for sensing a chemical component concentration using an electroactive material |
| US8282576B2 (en) | 2003-09-29 | 2012-10-09 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for an improved sample capture device |
| EP1680014A4 (en) | 2003-10-14 | 2009-01-21 | Pelikan Technologies Inc | Method and apparatus for a variable user interface |
| US7822454B1 (en) | 2005-01-03 | 2010-10-26 | Pelikan Technologies, Inc. | Fluid sampling device with improved analyte detecting member configuration |
| EP1706026B1 (en) | 2003-12-31 | 2017-03-01 | Sanofi-Aventis Deutschland GmbH | Method and apparatus for improving fluidic flow and sample capture |
| EP1713926B1 (en) | 2004-02-06 | 2012-08-01 | Bayer HealthCare, LLC | Oxidizable species as an internal reference for biosensors and method of use |
| US8828203B2 (en) | 2004-05-20 | 2014-09-09 | Sanofi-Aventis Deutschland Gmbh | Printable hydrogels for biosensors |
| EP1765194A4 (en) | 2004-06-03 | 2010-09-29 | Pelikan Technologies Inc | Method and apparatus for a fluid sampling device |
| US9775553B2 (en) | 2004-06-03 | 2017-10-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
| US7569126B2 (en) | 2004-06-18 | 2009-08-04 | Roche Diagnostics Operations, Inc. | System and method for quality assurance of a biosensor test strip |
| MXPA06014896A (en) * | 2004-07-01 | 2007-08-20 | Vivomedical Inc | Non-invasive glucose measurement. |
| US20070027383A1 (en) * | 2004-07-01 | 2007-02-01 | Peyser Thomas A | Patches, systems, and methods for non-invasive glucose measurement |
| US8652831B2 (en) | 2004-12-30 | 2014-02-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte measurement test time |
| KR101321296B1 (en) | 2005-07-20 | 2013-10-28 | 바이엘 헬스케어 엘엘씨 | Gated amperometry temperature determination |
| JP5671205B2 (en) | 2005-09-30 | 2015-02-18 | バイエル・ヘルスケア・エルエルシー | Gated voltammetry |
| US8529751B2 (en) | 2006-03-31 | 2013-09-10 | Lifescan, Inc. | Systems and methods for discriminating control solution from a physiological sample |
| CN101055262A (en) * | 2006-04-11 | 2007-10-17 | 禅谱科技股份有限公司 | Discard type electrochemical sensing test piece and its making process |
| US20070235330A1 (en) * | 2006-04-11 | 2007-10-11 | Zensor Corp. | Electrochemical sensor strip and manufacturing method thereof |
| US8778168B2 (en) | 2007-09-28 | 2014-07-15 | Lifescan, Inc. | Systems and methods of discriminating control solution from a physiological sample |
| WO2009076302A1 (en) | 2007-12-10 | 2009-06-18 | Bayer Healthcare Llc | Control markers for auto-detection of control solution and methods of use |
| US8603768B2 (en) | 2008-01-17 | 2013-12-10 | Lifescan, Inc. | System and method for measuring an analyte in a sample |
| WO2009126900A1 (en) | 2008-04-11 | 2009-10-15 | Pelikan Technologies, Inc. | Method and apparatus for analyte detecting device |
| US8551320B2 (en) | 2008-06-09 | 2013-10-08 | Lifescan, Inc. | System and method for measuring an analyte in a sample |
| US9375169B2 (en) | 2009-01-30 | 2016-06-28 | Sanofi-Aventis Deutschland Gmbh | Cam drive for managing disposable penetrating member actions with a single motor and motor and control system |
| US20110168575A1 (en) * | 2010-01-08 | 2011-07-14 | Roche Diaagnostics Operations, Inc. | Sample characterization based on ac measurement methods |
| US8965476B2 (en) | 2010-04-16 | 2015-02-24 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| JP5831862B2 (en) * | 2010-07-15 | 2015-12-09 | 国立研究開発法人産業技術総合研究所 | Electrochemical devices using porous electrodes |
| US8706425B2 (en) | 2011-07-20 | 2014-04-22 | Veolia Water Solutions & Technologies Support | Method for validation of polymer aqueous solutions concentration and activation in water treatment applications and polymer make-up unit therefor |
| US9459233B2 (en) | 2012-06-25 | 2016-10-04 | Steris Corporation | Amperometric gas sensor |
| US10598624B2 (en) | 2014-10-23 | 2020-03-24 | Abbott Diabetes Care Inc. | Electrodes having at least one sensing structure and methods for making and using the same |
| US9956817B2 (en) * | 2015-02-05 | 2018-05-01 | Goodrich Corporation | Wheel assembly tie bolt retention system |
| US10876144B2 (en) | 2017-07-14 | 2020-12-29 | American Sterilizer Company | Process for determining viability of test microorganisms of biological indicator and sterilization detection device for determining same |
| US10900062B2 (en) | 2017-07-14 | 2021-01-26 | American Sterilizer Company | Process for determining viability of test microorganisms of biological indicator and sterilization detection device for determining same |
| US10889848B2 (en) | 2017-07-14 | 2021-01-12 | American Sterilizer Company | Process for determining viability of test microorganisms of biological indicator and sterilization detection device for determining same |
Family Cites Families (6)
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|---|---|---|---|---|
| WO1990010655A1 (en) * | 1989-03-13 | 1990-09-20 | Allage Associates, Inc. | Surface functionalized and derivatized conducting polymers and methods for producing same |
| US5250439A (en) * | 1990-07-19 | 1993-10-05 | Miles Inc. | Use of conductive sensors in diagnostic assays |
| US5202261A (en) * | 1990-07-19 | 1993-04-13 | Miles Inc. | Conductive sensors and their use in diagnostic assays |
| NL9002764A (en) * | 1990-12-14 | 1992-07-01 | Tno | ELECTRODE, FITTED WITH A POLYMER COATING WITH A REDOX ENZYM BOND TO IT. |
| JPH08505476A (en) * | 1993-03-05 | 1996-06-11 | ユニヴァーシティー オブ ウーロンゴング | Pulsed electrochemical detection method using electroactive polymer electrode |
| GB9415499D0 (en) * | 1994-08-01 | 1994-09-21 | Bartlett Philip N | Electrodes and their use in analysis |
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- 1995-08-01 EP EP95927815A patent/EP0775214B1/en not_active Expired - Lifetime
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| US5849174A (en) | 1998-12-15 |
| EP0775214B1 (en) | 2003-10-08 |
| ATE251673T1 (en) | 2003-10-15 |
| AU3182395A (en) | 1996-03-04 |
| EP0775214A1 (en) | 1997-05-28 |
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