CA2243970A1

CA2243970A1 - Electrodes and metallo isoindole ringed compounds

Info

Publication number: CA2243970A1
Application number: CA002243970A
Authority: CA
Inventors: Markas A.T. Gilmartin
Original assignee: CANADIAN BIOCONCEPTS Inc
Current assignee: Individual
Priority date: 1996-01-23
Filing date: 1997-01-23
Publication date: 1997-07-31
Also published as: EP0876603A1; WO1997027473A1; US5830341A; AU1298797A

Abstract

The present invention provides for electrode assemblies and methods in which electron transfer between a redox reaction product and an electrically conductive electrode material is facilitated by a metallo macrocyclic compound, preferably a metallo isoindole ringed compound and more preferably a ferro isoindole ringed compound. The redox reaction is usually catalysized by a redox enzyme, such as an oxidase.

Description

-ELECTRODES AND 3\IETALLO ISOIN13OLE RINGED COMPOUNDS

INTRODUC~TION
Te~hni~zll Field The present invention relates electrodes with metallo ringed compounds and methods of using the sarne. Such electrodes are particularly useful in cle~Llochemical assays of analytes using printed versions of the electrodes withoxidases.

BAC~GROUND
The rapid development of the m~-~ir~ and life sciences continues to create an increasing den~n~l for the rapid, accurate and sensitive detection of analytes.
Rapid and convenient detection of analytes is imperative in a myriad of situations, such as emclgellcy rooms ~e.g., for ~cet~minophen, aspirin or alcohol detection in 1~ overdose cases), law enfo.~,elllent situations (e.g. for illicit drug detection) and food factories (e.g., to detect microorg~nicm~, waste products or toxins in spoiled or inferior quality produce). Modern health care has also clem~ntled more home or point-of-care determinations of analytes for real-time diagnosis, patient management, and cost cont~inm~nt In the environmental fields, a typical ~sessm~nt of a sample, for inct~nre for a water quality sample, involves lengthyand involved separations, extractions and inr~lb~tions, and in some cases may take a day or more to complete. Such delays often lead to inaccurate results or the detrimental spread of pollution. Thus, new and efficient methods for performing a host of bioch~mir~l tests would be a welcome arrival in diagnostic-oriented fields.
Previously, hydrogen peroxide has been used extensively as an indicator molecule in analytical determinations. One significant development has been the co~ u-;~ion of anl~elolll~,L-ic hydrogen peroxide sensors. Although this appioach can provide an attractive route to measuring hydrogen peroxide, being simpler, ~aster and less expensive coll.pal~,d to chromatographic, and spectrophotometriccounterparts, there are signifir~nt drawbacl~s to contelllporary hydrogen peroxide sensors. Aulperollletric sensors configured for hydrogen peroxide detection are SlJ~ ~ TE SHEET (RULE 26) WO 97/27473 P~T/CA97/00044 2.
fraught with problems due to the high potentials needed to drive reactions that permit detection of the electro-chemical breakdown of hydrogen peroxide. This istrue of both oxidation and reduction modes for measuring hydrogen peroxide, unless sophicti- ~t~c~ surface design strategies are adopted to create sensory interfaces with more predictable properties. These approaches complicate sensor fabrication, lengthen response times and often cc,lllpl~ lllise both the reliability and storage characteristics of the devices. Thus, the search for a one-step method for the m~mlfactllre of chemi~ ~ly modified electrodes with adept hydrogen peroxide electro-ch~mi~tries has remained unsolved until the advent of the invention described herein.

SUMMARY OF THE INVl~NTION
Until the development of tne present invention, detection of analytes using electrodes, particularly electrodes ~le5ignl-rl for det~cting hydrogen peroxide in small samples, were beset with tet~hnir~l limh~inns such as inte.rtlellL~, electrode sensitivity, electrode selectivity, and high voltage requilelllel~L~. The present invention provides for electrode assemblies and methods in which electron transfer between a redox reaction product and an electrically conductive electrode material is facilitated by a metallo macrocyclic compound, preferably a metallo isoindoleringed compound and more preferably a ferro isoindole ringed compound. The redox reaction is usually catalysized by a redox enzyme, such as an oxidase.
The electrode assemblies of the invention can be configured with a macrocyclic compound in a variety of ways to facilitate 1) electrical contact between an electrode's electrode material and the metallo macrocyclic compound and 2) electrical contact between the metallo macrocyclic compound and a redox enzyme, such as an oxidase, if so desired. As described herein different electrodes and electrode assemblies can be achieved by 1) dispe.~illg an metallomacrocyclic compounds in electrode material, 2) applying an metallo macrocyclic compound to the surface of the electrode, 3) applying a layer over the electrodecomprising a metallo macrocyclic compound or 4) a combination thereof. Such assemblies permit low voltage measurements of analytes which reduces noise, increases selectivity and increases the signal to noise ratio.

SUBSTITUTE Sl{EET (RULE 26) 3.
Another em~odiment of the invention is an electrode assembly comprising 1) a first hydrogen peroxide electrode comprising a metallo isoindole ringed compound dispersed in an electrode material, and ~) an first enzyme layer comprising a first enzyme and in electrical contact with said first hydrogen peroxide electrode. Such an electrode provides for excellent hydrogen peroxide selectivity, especially for the detection of hydrogen peroxide at low voltages.
Preferably, the metallo isoindole ringed compound coordinates Fe, Ru, Rh, or Mn.
Another embodiment of the invention provides for an electrode, particularly a hydrogen peroxide electrode, that comprises a) a ferro isoindole ringed compound, b) an electrode, and c) an oxidase enzyme. The metallo isoindole ringed compound is provided in a sufficient amount to permit electron transfer between the electrode material and the oxidase product, usually hydrogen peroxide, and allows for detection of an electrical current related to catalysis by lS the oxidase.
The invention affords cletrcting analytes using a number of different assay formats. Generally, the method for electrically detecting the presence of an analyte comprises: 1) measuring an electrical current from a redox electrode co~ fisillg an electrode material and a ferro isoindole ringed compound in fluidco.""~ ic~tion with a redox enzyme, particularly a oxidase. The redox enzyme catalyses a redox reaction that corresponds to the presence of an analyte and produces a product that either 1) directly chemically oxidizes or reduces said ferro isoindole ringed compound or 2) indirectly oxidizes or reduces said ferro isoindole ringed compound. Because the present invention is particularly well suited for m~nnfartllre using printing techniques the methods and devices of the invention offer relia~le, simple and cost effective analyte ~ietermin~tions for a myriad of applications in the health care, en~dloll"lellLal, food, 7~grirll1tllre and law enforcement fields.

BRIEF DESCRIPI ION OF THE DRAW~NGS
FIG. 1 illustrates a dia~ .",1,~tir representation of a screen printed electrode assembly arrangement and geometry that can be used to measure SUts~ ~ .)TE SHEET (RULE 26) 4.
multiple analytes c;mlllt~n~ously. Connecting strip A can be inserted into a spade connector to a potentiostat during operation. An insulation strip B can be provided for by means of a 3mrn wide strip of tape or preferably an inc~ ting layer. The working area C can be used as a detection zone. D depicts an inert solid support, S such as PVC (polyvinyl chloride), for a screen printed electrode.
FIG. 2 illustrates an electrode material, such as graphite fibers or particles, dispersed with a ferro isoindole ringed compound and the sequence of electron transfer events in a proposed catalytic reaction for the reduction of hydrogen peroxide exploiting the elecko-reduction of a ferro isoindole ringed compound.
FIG. 3 illustrates DC (direct current) cyclic voltammograms showing the electro-activity of carbon electrodes, such as screen printed carbon electrodes (SPCEs), with a ferro isoindole ringed compound (such as C32Hl6N8Fe(II)) dispersed in the electrode material. The voltammograms reflect results in anoxygenic solutions. Voltammograms A and B are recorded in plain 0.1 mol drn~
3 (0. lM) glycine buffer (pH 9) with no hydrogen peroxide and in a sirnilar solution cont~ining 2.2 x 10-3 mol dm-3 hydrogen peroxide, respectively The response for an unmodified SPCE is shown in voltammogram C and is recorded in the same glycine buffer using electrodes similar to those electrodes for voltarnmograms Aand B, except that no ferro isoindole ringed compound is present. Electrode areas are 9 rnm2 and the scan rate is 20mV s-l. The re~n~ining instrumental parameters:
initial and switching potentials, current ranges, scan initiation and direction and galvo zero are shown in the figure.
FIG. 4 illustrates hydrodynamic voltammograms (~A (currents) and V
(volts)) of 2.2 x10-3 mol dm-3 hydrogen peroxide at unrnodif~ed, C32HI6N8Co(II),C32HI6N8Mn(III), and C32HI6N8Fe(II) SPCFs. The supporting electrolyte is the same as in FIG. 3. The instrl-m~nt~l parameters are the following: time base, O.5mm s-l, applied voltage; 50mV increments, and stirring rate; 4 (teflon coatedm~gr~ . stirrer).
FIG. 5 illustrates amperometric cathodic calibrations of hydrogen peroxide using C32HI6N8~e(1I) SPCEs. Il~Llull-ental parameters are the same as in FIG. 4 except the potential is fixed according to the graph legend SUBSTITUTE SHEEll (RULE 26) 5.
FIG. 6 illustrates amperometric interference test using SPCEs cont~ining C32H~6N8Fe(II). Arrows show responses to snrcessive additions of hydrogen peroxide, and the int~lre~nL~, L-ascorbic acid, uric acid and paracetamol all at 0.5 x 10-3 mol dm-3 final concentrations. ~lectrodes biased at -Q. lV (SCE) other conditions as in FIG. 5.

OESCR~PI'ION OF SPECIFIC EMBODIl\~ENTS
INT~ODUCTION
Until the development of the present invention, detection of analytes using electrodes, particularly electrodes designed for detecting hydrogen peroxide in small samples, were beset with technical limitations relating to interferents, electrode sensitivity, electrode selectivity, slow response times, m.~ tor le~rhing, complicated electrode m~mlf~rtllre, high voltage requirements and noisy signals that thwarted signal tr~n.c(l~lc~ion. The present invention provides for electrode assemblies and methods in which electron transfer between a redox reaction product and an electrically conductive electrode material is facilitated by a metallo macrocyclic compound, preferably a metallo isoindole ringed compound. The redox reaction is usually catalysized by an enzyme, such as an oxidase.
Metallo macrocyclic compounds, particularly ferro isoindole ringed compounds, on electrode surfaces can promote the catalytic reduction of hydrogenperoxide according to the following electro-chçmic~l reaction pathway:
e- + 2H+ ~ 2Fe2+ + HzO2 (chemical) ~ Fe3+ ~ 2H2O (1) e- ~ Fe3+ (electrorh~Tnic~ Fe2+ (2) In effect, the natural biological catalysts, peroxidases, can be replaced in reaction (1) by a ch~mir~l analog of the enzyme's prosthetic heme group. Thus, bio-~u~cepLibility to external factors is drastically minimi7rd with an attendant enh~nrrm~nt in the electrode's observed stability patterns and perfoTmance. As these devices can be created simply, reproducibly and on a large-scale basis they provide an elegant means of generating single-use hydrogen peroxide cathodes.
Catalytic carbon-based, electro-catalytic electrodes enable the selective determination of hydrogen peroxide. Other suitable electrode substrates (e.g., pl~tinl~rn, gold, and silver) can be used as well. The present hydrogen peroxide Sl,~ 111 ~JTE SHEET ~RULE 2Ei) w 097/27473 PCT/CA97/00044 6.
measurement system is based on the classical Fenton-~Iaber process whereby peroxide chemically oxidizes Fe2+ ions (or other metals). The resT-I~in~ trivalent species is electrochemically reduced by an a~.p~opLiate potential thus closing the catalytic cycle. One such lay-out is shown in FIG. 1 and FIG. 2, where the S electro-catalyst and n~ccc~ry electrode components are immobilized within a carbon-based matrix. This new catalytic reaction can be harnessed in two- and three-electrode formats. The former can be afforded by printing a pseudo-reference electrode (typically composed of Ag/AgCl in proportions ranging from 10-90%) in close proximity to the working catalytic electrode. This can be used to create a completely disposable assay assembly, which is an especially desirable feature for biom~rlic~l analysis where conformation to aseptic practice is crucial.
The biological components (i.e., enzymes) can be printed together or on top of this c.ht-mic~l sensing platform to render each electrode unique and specific to a particular task. Such assemblies can be used for single- or multi-parametermeasurement profiles. Other transition metal-based catalysts and modified catalysts can be used which are suitable for disparate applications, for exampleanalyzing organic-phase samples (e.g., oils, paints). Electrode assemblies can be operated in reductive or oxidative modes depending on the complexity of the matrix under e~min~tion. For in.ct~nre, simple media may be interrogated using peroxide anodes (e.g., oxidative tr~nC~ ction), on the other hand, more 'exotic'samples (such as blood and urine~ would be analyzed using peroxide-selective cathodes (e.g., cathodic tr~3n~ ction (reduction)).

ELECTRODE ASSEMBLnES
The electrode assemblies of the invention can be configured with a macrocyclic compound in a variety of ways to facilitate 1) electrical contact between an electrode's electrode material and the metallo macrocyclic compound and 2) electrical contact between the metallo macrocyclic compound and a redox enzyme, such as an oxidase, if so desired. As described herein different electrodes and electrode assemblies can be achieved by 1) dispersing an metallo macrocyclic compounds in electrode material, 2) applying an metallo macrocyclic compound to the surface of the electrode, 3) applying a layer over the electrode SUBSTITUTE SHEET (RULE 26) 7.
comprising a metallo macrocyclic compound or 4) a combination thereof. Such electrodes can be configured to measure a wide variety of analytes by providing a redox enzyme that senses the presence of an analyte in a sample, either directly(such as when the analyte is the redox enzyme's substrate) or indirectly (such as with an analyte binding moiety). Such assemblies also permit low voltage measurements of analytes which reduces noise, increases selectivity and increases the signal to noise ratio.

ELEC~RODES
One embodiment of the invention is an electrode assembly comprising 1) a first hydrogen peroxide electrode comprising a metallo isoindole ringed compounddispersed in an electrode material, and 2) an first enzyme layer comprising a first enzyme and in electrical contact with said first hydrogen peroxide electrode. Such an electrode provides for excellent hydrogen peroxide selectivity, especially for the tletection of hydrogen peroxide at low voltages. The catalytic oxidation of hydrogen peroxide can be clet.-ct~l at more positive voltages as well. The electrode preferably has a large surface area and a surface structure that the permits close association of a metallo isoindole ringed compound with a first enzyme in a first enzyme layer. Such close proximity facilitates electro-chPmic~l communication, in this case electron transfer between a metallo isoindole ringedcompound and a reaction product, such as hydrogen peroxide. Preferably, the metallo isoindole ringed compound coordinates Fe, Ru, Rh, or Mn.
A variety of electrode materials may be used in such electrodes, particularly materials that present a large surface area to a metallo isoindole ringed compound. Preferably, an electrode, especially an hydrogen peroxide selective electrode, will use electrode materials such as pl~timlm, gold, silver and carbon.
Such electrodes, particularly carbon or gold electrodes, can optionally comprise a second metallo macrocyclic compound, such as metallo isoindole ringed compound.
Preferably, electrodes, especially hydrogen peroxide electrodes (which could be used in disposable assay kits), are made of electrically conductive carbon.
In both the preferred and non-pre~erred embodiments the amount or concel,L.dtion SUt~ JTE SHEET (RULE 26) W097/27473 PCT/CAg7/00044 8.
of the metallo isoindole ringed compound provides for sufficient electron transfer to permit detection of an electrical current. Usually, the electrical current measured from either preferred or non-preferred embodiments of the invention in the presence of a metallo macrocyclic compound ~such as a metallo isoindole ringed compound) will be greater, less noisy and less prone to i~ r~l~ents, or more reproducible than in the absence of the metallo macrocyclic compound. For carbon based electrodes, preferably the metallo isoindole ringed compound is C32H,6N8M, where M is Fe or Mn. Such metallo isoindole ringed compounds are compatible with first enzymes that are redox enzymes. A particularly compatible combination in an electrode is a metallo isoindole ringed compound with the formula C32H,6N8Fe(TI) with a redox enzyme that is an oxidase, particularly an oxidase that catalyses hydrogen peroxide production.
In many applications it will desirable to measure more than one analyte.
For in~t~nee, a hydrogen peroxide electrode assembly can com~lise a plurality ofworking electrodes configured either as a two or three reference electrode system.
Such assemblies comprise at least one additional working electrode (in addition to the first electrode) comprising a second electrode which comprises a metallo isoindole ringed compound usually dispersed in an electrode material, and a second enzyme layer co~ ,ishlg a second en_yme in electrical contact with the second detection electrode. Each additional electrode is usually configured to separately measure tne production of a product (e.g. hydrogen peroxide) in response to an additional analyte and is electrically isolated from the other electrodes so as to ~ l.i,r cont~min~ti-ln of other electrical ~;ull~nL~ and to minimiz~? possible analyte assay reagent co..l;.".i.~tion.
One way the specificity of an electrode or an array of electrodes used in a multi-analyte detection system can be accomplished is through the use of analytebinding moieties. Analyte binding moieties are çh~mi~l structures that recogni_ea molecular surface or structure of an analyte, and som~otime~, when so desired, an analyte analog. Examples of analyte binding moieties include, but are limited topolynucleotides, single stranded oligonucleotides, antibodies, receptors, ligandbinding domains, sugar (such as complex carbohydrates), polymers, proteins, non-protein ligands, and recombinant proteins (such as receptors, antibodies and ligand SIIJ~ ITE SHEET (RULE 26) .
binding domains). For example, in a multi-analyte detection system, such as one that uses hydrogen peroxide electrodes, the first enzyme is attached to a first analyte binding protein that recognizes a first analyte (the analyte is also recognized by a second analyte binding moiety in the vicinity of the first electrode). The second enzyme is attached to a third analyte binding moiety thatrecognizes a second analyte in the vicinity of a second electrode. The third analyte binding moiety recognizes a different analyte the first analyte binding moiety. Thus, the third analyte binding moiety permits specific detection of theadditional analyte. Such multi-analyte analysis, as well as others, known in ~he art can be used with the invention described herein.
Analyte binding moieties can also recognize and bind to analyte analogs.
Analyte analogs mimic the structure and som~;m~-s the function or partial function of analytes. Analyte analogs include compounds or sllbst~n~es that act as an agonist or antagonist of receptors (or other analyte binding entities) for analytes.
Often an analyte analog will have a structure similar to the analyte, except forsome ch~mir~l modification that confers a different property (or properties) to the analyte analog compared to the analyte.
It should also be appreciated that multi-analyte systems can be used with different electrode embodiments described herein. Preferably, such electrodes will be covered by a enzyme layer and each electrode will have a different enzyme layer, wherein the enzyme may be the same or different from the other er~ymes used with the other electrodes and the analyte binding moiety may be the same ordifferent from the analyte binding moieties used with the other electrodes.
Preferably, each electrode will have a different analyte binding moiety in electrical contact with the electrode. Preferably, each enzyme layer will comprise a matrixwith an analyte binding moiety attached to the matrix. Such multi-analyte detection systems can measure a number of analytes depending on the number of electrodes, such as 4 to 10 or more.
Another embodiment of the invention provides for an electrode, particularly a hydrogen peroxide electrode, that colllplises a) a ferro isoindole ringed compound, b) an electrode, and c) an oxidase enzyme. The oxidase has sufficient enzymatic activity to catalyze hydrogen peroxide production in ~letec~hle amounts.

SUBSTITUTE SltEET (RULE 26~

W097/27473 PCT/C~97/00044 10.
As described herein any number oi oxidases known in the art or developed in the future may used. Preferably, such an electrode comprises electrically conductive carbon used with a ferro isoindole ringed compound, such as C32HI6N8Fe(II) or C32Hl6N8Fe(III). The metallo isoindole ringed compound is provided in a sufficient amount to permit electron transfer between the electrode material and the oxidase product, usually hydrogen peroxide, and allow for detection of an electrical current related to catalysis by the oxidase. Usually, the ferro isoindole ringed compound is dispersed in the electrically conductive carbon.
Preferably, the ferro isoindole ringed compound mass is at least 1% of the electrically conductive carbon mass. More preferably, the ferro isoindole ringedcompound mass is between 3 and 8% of the electrically conductive carbon mass.
Even though the metallo macrocyclic compounds described herein will tremendously improve the selectivity and sensitivity of electrodes, it can be desirable to include an additional selectively reducing h~ lr~L~ means. For in.ct~n~e, a hydrogen peroxide electrode can further comprise a selectively permeable layer either 1) between the oxidase and the hydrogen peroxide electrode or 2) covering both the oxidase and the hydrogen peroxide electrode. Thus, the selectively permeable layer can exclude small (100-800 mw), m~Ail-m (700-2500 mw) or (greater than 2250 mw3 large molecular weight interferents from said hydrogen peroxide electrode. If the selectively permeable membrane is between the oxidase and the electrode, it is preferably permeable to the oxidase's reaction product, while l~L~ldillg the passage of its substrate. If the selectively permeable membrane covers both the oxidase and the electrode, it is preferably permeable to the oxidase's substrate, by not large molecular weight components of the sample.When used in con3unction with an redox enzyme, such as an oxidase, the ferro isoindole ringed compound may not be dispersed in the electrically conductive carbon. Instead it can be present in a layer covering the electrode, such as on its surface. Alternatively, electrodes can have a ferro isoindole ringed compound both fli~persefl in the electrode material and coated on its surface.
Usually, the electrodes of the invention will be covered by a layer col~ isillg a matrix that permits electrical commnnir~tion with the sample or sample buffer. Electrical cl mmnni~tinn as used herein refers to the ability of SlJtfS ~ ITE SHEET (RULE 26~

11.
electrically conductive material or fluid to conduct an electrical current. It is uIlderstood that electrical commnnic-~ion refers to the abiliey of the matrix, or other material or liquid to conduct an electrical current when the matrix is wetwith a liquid, as well as referring to actual electrical co~ unication in the matrix S or other material. In the case of a matrix, electrical comm-lni~tion does not nPcecc~rily imply that the matrix is charged, only that the matrix will permit electrical commllni~tion. The matrix also usually permits fluid comm-lni~tion between an oxidase and a ferro isoindole ringed compound, so that electro-chemical commllnir~tion can occur between those two entities. It is understood that fluid col-",.ll.,ir~tion refers to the ability of a matrix or other structure to fluidly commlmiL~tP between two points when the matrix or other structure is wetwith a liquid, as well actual fluid commllnic~tion that occurs in the presence of a non-gaseous liquid.
A ~ relled type of electrode, especially for disposable test cards used in hand-held assay monitors, is a printed electrode. Printed electrodes usually comprise a metallo macrocyclic compound, such as a ferro isoindole ringed compound, dispersed in a printed electrode material. For such electrodes, the printed electrode material either 1) lacks cellulose acetate or 2) has less than .25%
(mass/mass) cellulose acetate conlpa.ed to the mass of electrically conductive carbon (or some other electrode material) when the electrode material colll~lises electrically conductive carbon (or some other electrode material). The electrodematerial usually co~ lises electrically conductive carbon printed on an inert solid support, as it is an in~L,ellsive way of producing electrodes and such electrodes are highly selective as described herein. In some cases, to ease the m~mlf~ lre of the electrode, it will be desirable to disperse an enzyme (such as a redox enzyme or an oxidase) in the electrode m~tPrial Preferably, such electrodes are screen printed. Screen printed electrodes usually have a ferro isoindole ringed compound mass that is at least 1% of the electrically conductive carbon mass. For enh~n~e-l detection properties the ferro isoindole ringed compound mass can be between 3 and 8% of the electrically conductive carbon mass.

SU~ )TE SHEET (RULE 26) 12.
~EECTRODE ~ATE~1ALS
A variety of electrode materials may be used in the electrodes of the invention, particularly materials that present a large surface area to a metalloisoindole ringed compound. Preferably an electrode, especially an hydrogen peroxide selective electrode, will use electrode materials such as pl~t~n~Tm, gold, silver and carbon. When carbon is used it is usually an electrically conductive carbon and the metallo isoindole ringed compound that provides for sufficient electron transfer to the carbon to permit detection of an electrical current. Carbon in various forms can be used such as fibers, pyrolytic carbon, and rhombohedral crystals. Particle sizes before being printed or applied are usually 0.1 and 50 ~L
(micron), preferably particles are between 0.1 and 1.0 ~.

ELECTRON TRANSFER CO~IPOUN~S
The electrodes and methods of the invention can be practiced with a varieLy of metallo macrocyclic compounds described herein and known in the art.
Particularly preferred are the metallo isoindole ringed compounds as they increase electrode selectivity and sensitivity without comprising stability or increasingnoise. Such metallo isoindole ringed compounds are particularly good for reducing the voltage required for measurements of redox reaction products, particularly hydrogen peroxide. Metallo macrocyclic compounds are usually selected for their ability to transfer electrons from a product of a redox reaction (catalyzed by aredox enzyme, such as oxidase) to the electrode material. This process can be judged by current voltages relationships in the presence and absence of the product and metallo macrocyclic compound as described herein. Based on this criteria, aswell the selectivity experiments described herein, a metallo macrocyclic compound can be selected to use with an electrode to measure a redox product of a redox enzyme catalysized redox reaction. Metallo isoindole ringed compounds include compounds with four isoindole rings (linked in a 16-membered ring) and denvatives thereof. Many of such compounds have the general formula C32Hl6N8M; where M is Fe, Ru, Rh or Mn and preferably where M is Fe or Mn (the forrnula and compounds of page 448 of Grant and Hackh's Chemical Dictionary, FditQrs R. Grant and C. Grant, McGraw-Hill Publishers 1987 is Sll,~ UTE SHEET (RULE 2Ç) CA 02243970 l998-07-2l 13.
herein incorporated by reference). Ferro isoindole ringed compounds include compounds of the formula C32HI6N8Fe(II) and C32Hl6N8Fe(III). The general str~lctural formula for C32HI6N8M is the following:

N
NH N
N M N
~N HN~
N~--~3 In addition porphyrins can be used in the present invention. Porphyrins of the following general formula can be used, as well as derivatives thereof:

~_NH N=~
'I H N
,~ N HN ~/
\~--N~

Pol~hy~ derivatives known tne art such as those of U.S. 4,957,615 issued September 18, 1990 and PCT application WO 93/15174 pu~lished August 5, 1993 (the compounds of which are herein incorporated by reference) can be used, especially those co~.l;.i"i~g Fe or Mn.
Derivatives of either metallo isoindo}e ringed compounds or porphyrins can be used or synthPsi7Pd (if not in existence) that selectively and catalytically ~ transfer electrons at a desired operating voltage. For example the rings of these compounds can be modified to include sllbstitllent groups that provide an electron donating species (or more than one species) (such as -CH3, -CH2CH3, an alkyl 3-8 Sl,,.~ ~ JTE SHEET (RULE 26) 14.
carbons in length or -NH2). Such modifications will lower the activation energy to reduce the metal coordinated by the coor~linzl~in~J rings. Thus, the redox potential can be manipulated to facilitate resolution of the oxygen and hydrogen peroxide reduction waves. In the case of hydrogen peroxide, the redox potential for reduction of hydrogen peroxide can be shifted away from the redox potential for oxygen to a more positive potential.
In electrodes with the metallo macrocyclic compound dispersed in the electrode material the amount (mass) of metallo macrocyclic compound used will generally be at least 1% of the mass of the electrode material, which is usuallycarbon. Usually the mass/mass ratio (metallo macrocyclic compound/electrode material) percent is between 1 to 15%, preferably between 2 to 12% and more preferably between 4 and 9%.

ELECTRODE ASSEMBLY LAYERS
The present invention provides for electrodes that can be optionally covered with an enzyme layer or a selectively permeable layer or a combination thereof.
The enzyme layer comprises a first enzyme in electrical contact with an electrode.
If the electrode has dispersed within it a metallo macrocyclic compound it will not be nPc~s~ry to include a macrocyclic compound in the enzyme layer, however it can be advantageous to so if diffusion times into the electrode material appear to be rate lim~ting. The selectively permeable layer (or layers) can be designed toretard interferents (i.e. compounds that interfere with the detection of electrical signals) or it can be designed to prevent sample components from increasing enzyme activity that is not associated with the presence of an analyte in the sample ~i e. it reduces enzyme activity background).
Normally, the first enzyme layer will comprise a redox enzyme (preferably an oxidase) that catalyses a reaction that results in a product that can be det~cte~l by the catalytic transfer of electrons from the product to the electrode material via a metallo macrocyclic compound. The first enzyme layer can also contain additional enzymes that form a series of coupled reactions (including redox reactions) and lllt;m~tely result in a product that can be electrically detectPdPre~erably, the last enzym~tir~l~y catalysized reaction will result in the production 51~5 ~ ITE SHEET (RULE 26) CA 02243970 l998-07-2l 15.
of hydrogen peroxide. If multi-analyte systems with multiple electrodes are used, the first enzyme layer of each electrode can contain a different enzyme to confer specificity for a particular analyte. For example, a first electrode is covered with glucose oxidase, a first enzyme, to directly measure glucose, a second electrode is covered with cholesterol oxidase, a second enzyme, to directly measure cholesterol and a third electrode is covered with lactate oxidase, a third enzyme, to directly measure lactate. Preferably, each electrode and its corresponding enzyme is electrically isolated from the other electrodes.
Preferably, an oxidase can be used in the first enzyme layer that produces hydrogen peroxide. Suitable oxidases include 6-hydroxy-D-nicotine oxidase, alcohol oxidase, aldehyde oxidase, allyl-alcohol oxidase, amine oxidase, bi}irubin oxidase, cholesterol oxidase, choline oxidase, cyclohexylamine oxidase, D-amino acid oxidase, D-aspartate oxidase, D-glllt~m~te acid oxidase, dihydroorotate oxidase, dimethyl-glycine oxidase, ethanolamine oxidase, g~l~rtose oxidase, glucose oxidase, glycolate oxidase, glycolate oxidase, glyoxylate oxidase, hexose oxidase, L-amino acid oxidase, ~-gluconolactone oxidase, L-glllt~m~te acid oxidase, L-ricin c~-oxidase, ~-sorbose oxidase, lactate oxidase, malate oxidase, N-methylamino acid oxidase, N6-methylrycine oxidase, nitroethane oxidase, nucleoside oxidase, oxalate oxidase, putrecine oxidase, pyranose oxidase, pyridoxine 4-oxidase, pyruvate oxidase, rathosterol oxidase, sarcosine oxidase, sulphite oxidase, tyramine oxidase, urate oxidase, urease and xanthene oxidase.
The oxidase, like any first enzyme, should provide sufficient activity amount of enzyme activity to permit detection of hydrogen peroxide (or other product~. Preferably, the enzyme purity is least 80% active enzyme. However, because the detection techniques and electrodes described herein are so sensitive and selective, enzyme preparations with less purity can be used, especially for the m~mlfarhlre of low cost disposab}e devices. Typically enzyme activity can range from 100 to 200,000 U/g. The amount of enzyme per electrode will typically vary depending on the activity, Lt;~ e~dture, ionic strength, substrate collcellLldtion' electrode size, cofactor conrpntration (if required) and pH.
Usually, less than a mg of enzyme will be required per electrode, preferably 10 to 500 ~g (micrograms) will be used. Electrodes can also be constructed with 1 to SlJes~ UTE SHEET (RULF 26) ~6.
10 mg per electrode or greater to boost sensitivity. The amount of enzyme used per electrode can also easily be tailored to each specific application and sample type by comparing the current of the electrode as a function of the amount of enzyme per electrode at a constant concentration or amount of substrate.
Alternatively, tne use of an enzyme layer may be not desired to facilitate m~mlf~ctnre of the electrode assembly, in which case the enzyme can be incorporated in the electrode material. If enzymes are dispersed in the electrode material, generally the electrode material will not be heated to temperatures that would denature either tbe lyophilized or dehydrated enzyme. Any of the suitable enzymes mentioned herein and known in the art for redox reactions can be used insuch electrodes. Any of the methods or electrode configurations can be suitably combined with such electrodes. Normally, methods that require ~tt~rhment of the first enzyme to an analyte binding moiety will not included in electrodes with dispersed enzymes.
Many embo~limPnt~ of the invention can utilize an enzyme layer with an analyte binding moiety. The enzyme can be attached to a first analyte binding mo~ety, which is often the case in sandwich assays. Usually, the enzyme is att~rhPcl to the analyte binding moiety with a covalent bond, however non-covalent bonds can be used (e.g. an avidin labeled enzyme and a biotin labelled antibody).
In some situations the first analyte binding moiety will not be bound to a matrix in the enzyme layer and is free to diffuse through the layer, such as in a sandwichassay. Often a second analyte binding moiety will be present in the enzyme layeror in a layer that is in fluid co.,.."~.~.;r~tion with the enzyme layer. In such cases the second analyte binding moiety is usually ~tt~-~ht~ to the matrix either covalently or non-covalently. Both the second and the first analyte binding protein recognize the analyte and can bind the analyte, usually at different positions on tne analyte. In some in.ct~nr.es, it will be preferable to use multi-sandwich assay in which the first enzyme is indirectly bound to the analyte through multiple binding moieties that bind the analyte in a multi-sandwich manner. Preferably, the firstanalyte binding moiety is an antibody and is covalently ~tt~rhPc~ to a first enzyme.
Redox enzymes, oxidases, and the oxidases specifically mentioned herein can be ~rhP-l to the first analyte binding moieties for use in assays, particularly SU~a ~ )TE SHEET (RULF 263 W097/27473 ~CT/CA97/00044 17.
sandwich assays. Dirr~,ent types of ~tt~chment, either non-covalent or covalent,to the matrix or the analyte binding moiety can also be combined.
If an interferent is a substrate for an enzyme, an enzyme that catalyzes the breakdown of the inte,rere.lt into an electro-inactive species can be included in the enzyme layer or in another layer in fluid commllnir~tion with the enzyme layer to reduce the }evel of the illielrelcllt. A scavenger layer comprising a second enzyme can be located between the electrode, particularly a hydrogen peroxide electrodeand the first enzyme layer. For example, the second enzyme can be L-ascorbic acid oxidase. The L-ascorbic acid oxidase has sufficient activity to reduce the amount of dissolved oxygen in the presence of its substrate and to reduce the amount of ascorbic acid present in the sample that would normally act as an hl~e, rt~ lL.
If signal amplif~lcation is desired, it can often be accomplished by adding a second enzyme to the enzyme layer that catalyzes the breakdown of a neutral product of the first enzyme into the same product that produces an electrical signal as the first enzyme. For example, the first enzyme is an oxidase which produces a hydrogen peroxide and a substrate for a second enzyme and the second enzyme produces hydrogen peroxide. Such an example is an oxidase that has lactate oxidase activity and produces pyruvate and a second enzyme that has pyruvate oxidase activity.
In another enzyme layer configuration, the enzyme layer colll~l.ses a matrix and the enzyme is attached to the matrix, either covalently or non-covalently. Preferably, the first the enzyme is covalently attached the matrix.
Redox enzymes, oxidases, and the oxidases specifically mentioned herein can be ~tt~r~ to the matrix for use in assays, particularly assays where the analyte is a substrate for the enzyme. In such as assays, preferably the enzyme is directly ~tt~f h~--l to the matrix, such as in a glucose assay where glucose oxidase is directly ~tt~- h~-l to the matrix.
The matrix can be made from a variety of matrix materials such as, but not limited to, cellulose, polycarbonate, perfluorinated material, polystrole, polyurethane, polysulphone and silica. Preferably, matrix materials are screen printed onto the electrodes. As described herein a selectively permeable layer SU~ JTE SHEET (RULE 26) CA 02243970 l998-07-2l 18.
between the matrix and the electrode can be desirably and is preferal~ly screen printed.
The enzyme layer can also be comprised of a first enzyme attached to an analyte or analyte analog. Preferably, an oxidase is ~tt~ch~(l to an analyte or an analyte analog. The enzyme attached to the analyte or analyte analog can preventthe analyte from binding to an analyte binding moiety. Such enzyme layers can bein assays where the analyte is prevented from binding to the analyte binding moiety which is ~tt~rh~ to a matrix in tne enzyme layer. Enzyme layers with a ~Irst enzyme ~tt~h~(l to an analyte or analyte analog are particularly useful for competition or displacement assays of the analytes. In such assays, the assay isusually designed to measure a decrease the current in the presence of the analyte compared to the absence of the analyte. The matrix can provide for electrical communication with the electrode, usually a hydrogen peroxide electrode, in the presence of a liquid cont~ininP~ a sufficient amount electrolytes to provide forelectrical commllnic~tion. The matrix in such devices, and other devices described herein, can be part of a fluid guidance pathway that is in fluid col"".~ tion with a waste reservoir. Usually, tne enzyme used in the enzyme layer is provided as alyophilized enzyme and is dry and covers ~e electrode.
As described herein, electrodes of the invention can include a layer (or layers) to exclude various assay or sample components. For inct~n~e, hydrogen peroxide perrneable membranes may be used to isolate the working electrode from potential i~ relGll~. A garnut of membrane materials and compositions can be used to elimin~t,- chpmir~l 'cross-talk' (electro-t~h~mi~l and biophysica}
hlLt;l~:lellL5) by redox-active co-existing compounds encountered in complex samples or assays components (Gilmartin, M. A. T., and Hart, J. P., Analyst, 1994, 119, (11), 2431; Lonsdale, H. K., Cross, B. P., Graber, F. M; ~ril~5tto~r7, C. E. In "Permselective Membranes," Rogers, C. E., Ed.; Marcel Dekker: New York, 1971; pp 167-187; Wang, J.; and Lu, Z.; Anal. Chem.; 1990, 62, 826-g29;
V~lg~m~, P., and Crump, P. W., Analyst, 1992, 117, 1657; Keedy, F. H., and V~A~m~, P., Biosensors & Bioelectronics, 1991, 6, 491; Sitt~mr~l~m, C~., and Wilson, G. S., Anal. Chem., 1983, 55, 1608; Johnson, J. M., Halsall, H. B., and Hein~m~n, W. R., Ana~. Chem., 1982, 54, 1377; and Ernr, S. A., and Yacynych, SU~ I l l ~JTE SHEET (RULE 26) 19.
A. M., }~lectroanalysis, 1995, 7, (10), 913; which are herein incorporated by reference). These may be categorized into three distinct groups narnely, electro-polymerized, solvent-cast and composite membranes. These membranes are polymeric in nature and may be deposited or laid down over electrodes, creating mesh-type, cross-linked or continuous phase interfaces. Such membranes prevent unwanted species from interacting with the electrode while ret~ining the electrode's heterogeneous electron transfer characteristics.
Membranes can be deposited using electro-polymerization. This technique permits 'all-~hPmir~l' in situ membrane synthesis (Emr, S. A., and Yacynych, A.
M., Electroanalysis, 1995, 7, (10), 913; ~eider, G. ~., Sasso, S. V., Huang, K-M., Yacynych, A. M. and Wieck, H. J. Anal. Chem., 1990, 62, 1106; and Sun, Z., and Tachikawa, H., Anal. Chem., 1992, 64, 1112, which are herein incorporated by reference). Therefore, such techniques are applicable to coatingcomplex surfaces that are also in close proxirnity with one another in order to facilitate electro-chemi~l and electron ~ldr~rel. This provides a particularly desirable method for m~mlf~c~llring mini~h-rized and multi-analyte electrodes.
The membranes are generally 'grown' from the oxidation of monomers (e.g., minobenzene and pyrrole) and may be insoluble, con-l~lctin~ or inc~ tin~ in nature. Oxidation is accomplished by the electrode current. Such modifications can be used in the preparation of electrodes that work in conjunction with enzymes (e.g. redox or oxidase), as the catalyst may be simply entrapped in the deposited membrane. In particular, immobilization of enzymes within con-lrlctingpolymers formed during the oxidation of the monomer are especially advantageous, facilit~tin3~ the control of enzyme deposition and its spatial distribution, while m~int~inin~ low instr~lm~nt~tion costs.
Solvent casting of layers can be used with the electrodes of the invention and involves the formation of a layer by evaporation of a polymer solution placed on an electrode surface (T~t~llrn~, T., Okawa, ~., and Watanabe, T., Anal.
Chem., 1989, 61, 2352, which is herein incorporated by re~erence). Casting is usually used with simple, two--lim~n~ional structures. Layers (cast or otherwise) can include matrices with polarity (hydrophobic/hydrophilic), size-exclusion structures (e.g. porous polymer and fibers), and permeable selective layers. For SU~ JTE SHEET (RULE 26) 20.
example, cellulose acetate (CA~ can be used to select on the basis of si~e, and po}yurethane can be used to select on the basis of polarity, in order to re3ect inte~le~ . Polyelectrolyte coatings can be used to selectively exclude certain compounds on the basis of charge. For example, perfluorosulphonate ionomers, such as Nafion, are strong acid cation exchanger polymers and therefore, have a tendency to repel anionic species, while allowing the passage of cations (in particular, divalent hydrophobic cations) to the electrode surface. Other examples include polyester-sulfonic acid~, zeolites and F.~ctm~n AQ-SSD polymers (Wang, J., Rayson, G. D., ~u, Z., and Wu, H., Anal. Chem., 1990,~2, 1924; ~olinson, D. R., Nowak, R. J., Welsh, T. A., and Murray, C. G., Talanta, 1991, 38, (1), 27; and Koopal, C. ~., Feiters, M. C., Nolte, R. J. M., Bioe}ectrochem. &
Bioenerg., 1992, 29, 159, which are herein incorporated by reference). Because the deposition of casted layers introduces further electrode complexity, such casted layers are not preferred for printed electrodes.
Composite selective iayers can be created by combining two or more different types of polymers to produce an anti-inte.îe.c~llce barrier. For example agarose/latex porous membranes offer exclusion to only macromolecular species and thus would be useful as an external interface (Koopal, C. G., Feiters, M. C., Nolte, R. J. M., Bioelectrochem. & Bioenerg., 1992, 29, lS9 and ~3enmakroha, Y., {~hristie, I., Desai, M., and Va~sg~m~, P., in Analyst, l99S, which are herein incorporated by reference). Sulphonated polyether-ether sulphone composites alsoprovide charge separation characteristics. The marriage of uncharged polysulphone backbone and charged pendent sulphonated groups produces a hydrogen peroxide-specific layer.
ELECr~ODE MANUFAC~
The electrodes and electrode assemblies of the invention can be easily m~m-f~rtured using a variety of techniques. Because the electrodes can be easilymassed produced and the same type of electrode (e.g. hydrogen peroxide electrode) can be used for the assay of many different analytes by simply ch~nging the enzyme or analyte binding moiety or both that are in association with the electrode, the present invention provides for a universal platform on which assays SIJ~S 111 ~JTE SHEET (RULE 26) CA 02243970 l998-07-2l 21.
can be performed. Mass producible electrodes and electrode assemblies can be used as universal platforms for monitoring biochemical reactions such as, but not limited to, enzymatic, affinity and receptor-oriented arrangements. These disposable strips or test cards may be fabricated using highly cost- and labor-effective techniques based on, but not limited to, a common printing theme encaps~ ting screen-, air-brush of inkjet printing techniques. The electrodes are particularly suitable as base tr~nctlllrers for the assembly of single-use electrochemical assays geared for kit format applications akin to over-the-counter tests available from pharmacists. Such kits can include any of the electrodes, enzymes, analyte binding moieties and reagents described herein and known in theart to be suitable for such applications. A typical electrode arrangement is depicted in ~IG. 1, however, the dimensions and configuration of the structures can be modified depending on, for in~t~nf e, the screen type, ink composition and printing aL,~aldL~ls. Thus, strips or test cards could take the form of simple, single monolithic surface through to multi-strip and interrligit~te~l array cor~lgurations by exploiting the inherent versatility of screen-printing, for example.
The invention also encomr~cses electrode designs based on three-~lTmf n~i~nal polishable or renewable macro-electrodes, such as solid and paste-type materials. Such modifications can be performed after the electrodes are applied to a solid support. The e1ectrodes can also be printed as peroxide-specific, catalytic micro-electrodes. These are devices bearing at least one dimension in the micrometer range.
Generally, carbon electrodes are made with a metallo macrocyclic compound, such as ferro isoindole ringed compound, using an amount (mass) that is at least l % of the electrically conductive carbon mass. Usually, in such electrodes the metallo macrocyclic compound mass is between l and 15% of the electrically conductive carbon mass, preferably between 2 and 12% and more preferably between 3 and 8%. Preferably such electrodes are screened printed.
Other techniques for applying electrode material can be used so long as the particle size of the carbon is made small enough to provide an increased surfacearea that presents a surface to allow for electron transfer between the metallo macrocyclic compound and the electrode material. Usually the carbon particles SUBSTITUTE SHEET ~RULE 26) 22.
are mill rolled or sonicated or both to break the carbon into particles less the500~. Preferably, the carbon particles are less than 50~, more preferably less than lQ~L, and most preferably less than 1,u. Often the electrically conductive carbon is a plurality of bead- or roll- milled particles, or ultra-sonicated particles and such particles can be applied to the solid support using printing techniques.
Particles of electrode material, whether or not they are used for printing carbon based electrode, preferably can pass a screen with 1.0 ~ holes. More preferably the particles of electrode material can pass through a .25 ~ screen. The electrode assemblies, especially the said printed electrode will usually have a thickn~ss 1~ between 1,000 and 10 ~ with respect to the plane of the solid support, andpreferably between 200 and 20~L. Preferably, the electrodes are made with an inter ligit~tp(1 pattern. The electrodes usually comprise a surface area that fits on test card. The electrodes will usually have a worlcing area with a width between 1 rnrn to 20 ~nm wide.
Screen printing techniques can also be used to provide the enzyme layer, other layers ~liccllCce~l herein, coatings of enzyme without a matrix, matrices,insulation layers, fluid g~ n~e pathways and any other reagent n~cecc~ry for theassay. Such techniqll~ s can be readily applied to the present invention (U.S.
Patent Application Serial No. 08/512,358, filed August 8, 1995, which is herein incorporated by reference).
Printing techniques can provide for any desired pattern of fluid guiding pathways Generally, a deflned printed pattern of a fluid guiding pathway refers to a pattern of printed material that will perrnit directional fluid transport.
Furthermore different "inks" or "pastes" can be applied to produce fluid guiding~dlllwdys that can include fluid guiding pathway portions having different flow properties. C~,n~r~lly, the term paste relates to materials at a concentration of 10 to 60% ~w~v) ypically in water or an organic solvent. Preferably pastes are usedin the 20 to 40% (w/v) range. Printing compositions usually range from 1 to 25 poise at 25~C~ preferably from 4 to 18 poise at 25~C and more preferably at 6 to12 poise at 25~C. The term "flow properties" may refer to properties affecting liquid flow along a fluid guiding pathway or to properties affecting the conveyance of solutes by carrier liquids as in chromatography. Materials can be used to 513~ ~ JTE SHEFT ~RULE 2~) W097127473 PCT/CA971~0044 23.
change solute/solvent RF values. ~F value refers to the ratio of the ~lict~n(~e moved by a particular solute to that moved by a solvent front. For example, printed fluid guiding pathways can be m~mlf~tured with a printed layer or layerscomprised of two or more different materials ("multi-material") providing different rates of fluid transport. Multi-material fluid guiding pathways can be used when it is desirable to modify retention times of reagents in lluid guiding pathways (such as reduction of retention times, as ~ cll~ce~l herein for conjugates, or to increase retention times to allow reactions to occur, e.g., antibody-analyte interactions), prevent non-specific binding, improve the assay sensitivity or improve the reproducibility of analyte assays. Such multi-material fluid guiding pathways can be constructed as a heterogenous printed layer, where a plurality of materials are mixed together and then printed. Multi-material fluid guiding pathways can be also made by printing multiple layers of materials with different flow properties on top of one another. Multi-material fluid guiding pathways are examples of some types of ~low accelerators further described herein.
Printed fluid guiding pathways can be provided with regions cont~ininsg reagent substances, by including reagent substances in the "inks" used to produce them or by a sllbseq l~n~ printing step. A pl~relled printing technique to use is screen printing. Such regions are usually referred to as reagent zones. Non-irnmobili~ed reagents are preferably printed on materials that release the reagents ~uickly to the fluid flow to allow for rapid hydration of reagents and less reagent retention. Inert materials are especially suitable for this purpose.
Screen printing techniques are also preferably used for printing fluid guiding ~a~lw~ys, conductivity strips, electrodes and their associated conduction t~acks, and for printing reagents at specific fluid guiding pathway locations. Air-brushing may also be used to print fluid guiding pathways. Ink jet printing can be used for printing reagents but is not generally suitable for depositing particulate material greater than 20-5,u.
~uch fluid g~ n~e pathways, when so desired, can an improve the control 3~ of the flow of liquid and the subsequent timing of reagent delivery in an assay device. Geometrically defined and printed ~luid guiding pathways can be used to guide the fluid flow and can be made of disparate chromatographic materials (such SlJr~5 1 1 1 UTE SHEET (RULE 2~i) 24.
as cellulose, silica gel, silica including silica modified to increase the hydrophobicity of the silica, starch, agarose, ~gin~tF~, carrageenin or polyacrylic polymer gel and mixtures of such materials~ to facilitate ~ pliate retention of individual reagents and to assist and enhance the sequential delivery of the assay components. Different physical, as well as ~h.Q~i~l, properties can be used to affect transit times, e.g., densely packed small particles produce longer transit times than loosely packed larger particles of the same material. Regularly shaped particles can be deposited to form a closely packed regular structure which facilitates the passage of proteinaceous material.
Generally, printed fluid guiding pathways, especially screen printed fluid pathways, comprise thin layers of matrix sufficiently thick so as to provide enough fluid for a ~l~tPct~ble signal and to ensure uniforrnity. Typically, a single printed layer can vary from 5 to 500~L in thirkn~s.s and preferably, 5 to 100,~b or 40 to 100~ in thirkn~oc.c and more preferably 40 to 100~L in thi~ n~oss Fluid guiding pathways can be made of multiple layers or single layers wherein the total layerthickn--sc is usually 50 to ~i00,u and preferably, 75 to 300~.
For printed electrodes, a variety of electrode assemblies can be used, including two- and three-electrode-based assemblies. Two-electrode assemblies are preferred because of the ease of operation and printing of the electrodes and conduction tracks. Working electrodes can be catalyzed carbon based, such as rhodir~ized carbon electrodes like MCA 4 (MCA, Cambridge, United Kingdom).
Other electrodes based on a combination of carbon and transition metals, prcferably pl~timlm, can be used to facilitate low potential oxidation of enzymeproduct. Such electrodes help reduce background noise associated with measuring assay products, such as hydrogen peroxide, at higher voltages (600-700mV versus AglAgCI reference electrode~ required by other types of electrodes, e.g., pure carbon or pure platinum group based electrodes. Generally, it is advantageous toincorporate in a two-electrode system an auxiliary/reference of sufficient size so as not to limit the current required for the potentiostat. For reference electrodes, Ag/Ag~l is typically used in the range of 10 to 90% Ag, although for disposable test cards, 10% is preferable. Electrodes are preferably printed so as to maximize t~ction, for in.ct~n-~e ~y locating the electrode broadside to the fluid transport, as Sl~ JTE SHEET (RULE 26) 25.
well as creating an inter~ligit~tin~ pattern between the reference and working electrodes.

ANALYTE ~ETECI~ON M ETHO~S
The invention affords detechng analytes using a number of different assay formats. Virtually any assay that can use electro-chemical detection as a means for detecting an analyte can be adapted for use with the invention's electrodes and electrode assemblies. Generally, the method for electrically f~et~cting the presence of an analyte comprises: 1) measuring an electrical current from a redox electrode comprising an electrode material and a ferro isoindole ringed compound in fluid commllnie~tion with a redox enzyme, particularly a oxidase. The redox enzyme catalyses a redox reaction that corresponds to the presence of an analyte and produces a product tnat either 1) directly f h~mic~lly oxidizes or reduces said ferro isoindole ringed compound or 2) indirectly oxidizes or reduces said ferro isoindole ringed compound. The present invention can also be used witnout an enzyme for the direct measurement of analytes that decompose under suitable voltages ~e.g.
direct measurement of hydrogen peroxide).
Methods of detection will often include applying a voltage field between the redox electrode and a reference electrode assembly in either a reduction or oxidation mode. The mode can be selected so as to avoid reduction or oxidation current peaks associated with unwanted species (non-analytes). For example, the applying step can comprise applying a voltage field between tne redox electrode and the reference electrode assembly to permit electro-reduction of the ferro isomdole ringed compound from C32HI6N8Fe(I~I) to C32HI6N8Fe(II). Usually, the 2~ method includes coll.palillg the electrical current in the absence of the analyte and in the presence of the analyte. The method can also include qll~ntit~tin~ the amount of the analyte in relation to the amount of the electrical current. Standard curves can be established as a reference point and kits can include standards, as well as negative and positive controls to assure the accuracy of the measurements.
The L~ref~ll.,d voltage field is between -50 and 450 mV, especially when hydrogen peroxide is being measured either directly as an analyte or as a reaction product that is related to the amount of analyze present in the sample. Preferably, the SUCS;~ JTE SHEET (RULE 26) 26.
voltage field corresponds to a current peak or steady state current associated with reduction or oxidation of the product of a redox reaction, such as reduction o~
hydrogen peroxide.
The methods can also include the step of conS~ting the analyte with a matrix that provides for huid cornmunication with a redox enzyme, such as an oxidase. For example, the cont~f~ting step can comprises 1) cont~rrtng the analyte with an analyte attached to an oxidase or an analyte analog attached to the oxidase.
The analyte and either 1) the analyte ~tt~h~d to the oxidase or 2~the analyte analog ~rf~h~Cl to the oxidase are recognized by an analyte binding moiety. Suchmethods can ~e applicable to competitive or displacement assays. ~uch methods can be suitable combined with other methods and devices taught herein and known in the art.
Alternatively, the cont~ting step can comprise conr~rting the analyte with an analyte binding moiety ~ ch~r~ to a matrix. The method can further include 1~ cont~rting the analyte with a second analyte binding moiety ~tt~eh~rl to a redox enzyme, such as oxidase. Such methods are applicable to measuring the analyte ina sandwich assay. The analyte can also be measured in a multi-sandwich assay if so desired by including additional reagents such as secondaly antibodies, oEigonucleotide probes or their equivalent in the assay. Usually the first and second analyte binding moiety will an antibody, although only one of the bindingmoieties might be an antibody and the other could be a receptor.
The analyte can be any one of number o~ different ch.omic~l entities including a nucleic acid, simple sugar, carbohydrate, protein, recombinant protein, drug, polymer, antigen, antibody, cell, lipid, polynucleotide, oligonucleotide, biologically active molecule endogenously produced in an organism and biologically active molecule exogenously produced outside an org~ni~m Although the analytes might often be proteins, analytes will also often be non-proteins.
Analyte proteins can be virtually any protein from an organism (e.g. a bacteria, fungus or virus) or syn~h~si7~fi in vitro using manual or automated methods. A protein analyte can be from many org~ni~m~, including, but not lirnited to, A, B and C hepatitis virus, R~7cil~ anthracis, Bovine rhinotracheitis, br~chernatis, Brucella abortus, Campylobacter jejuni, chlamyd~ia, Clostridium SIJ~S ~ JTE SHEET (RULE 26) W O g7127473 PCT/CA97/00044 27.
boh~limlm, coronavirus, cytomegalo virus, Ebola virus, Eschenchia coli, group A
strep, herpes virus, HIV, human papilloma virus, Lassa fever virus, Leptospira interrogans, ~isteria monocytogenes, Maedi visna virus, Mycobactenum ~uberc~osis, Mycoplasma bovis, Neisseria gonorrhoea, Salmonella, Staphylococus aurens, Stephanuras dentatus, swine fever virus, Taxoplasma gondii, Treponema pa~lidum, Vibrae epp., Yibno cholerae, Yellow fever virus, Yersinia enterocolitica, and Yersinia pestis. The methods can also be applied to measuring an antibody or other serological marker associated with an olg~ lll, such as those org~ni~mc described herein. Usually the analyte is antibody that recognizes an protein or sugar moiety of a microorganism.
Analytes may not be proteins such as ,B-inhibin, 17~-methyltestosterone, 17,B-estradiol, 2,4-D, 2,4,5-T, 3-Acetyl deoxynivalenol, a~et~min- phen, alachlor, aldrin, amikacin, allliLli~Lyline7 amph.ot~minf~, atrazine, bacillus thuringensis toxin, barbiturates, BAY SIR 8514, benzodiazepines, bilirubin, caffeine, cannabinoids, ca~balllazepine, calllalllazeL~ine, chloramphenicol, chlorosulfuron, cocaine metabolics, cyanzine, cyclosporin, cyclosporine, DDT, deoxynlvalonel, deoxyverrucarol, desipramine, diacetoxyscil~ellol, dibenzofilrans, dichlorfop-methyl, dieldrin, diethylstilbestrol, difubenzuron, digitoxn, dioxins, disopyramide, endosulfon, estrogen, ethosuximide, flec~ini-l~, gentamycin, group A
~0 tricho~h~ce~Ps, hCG, hexoestrol, imipramine, iprodione, kanamycin, kepone,lllt~ni7ing hormone, lidocaine, malete hydrazide, metalaxyl, m~th~Aone, methamphetamine, methaqualone, methotrexate, N-acetylproc~in~mide, neitilmicin, nortriptyline, opiates, oxfendazole, paraoxon, paraquat, parathion,polychlorinated benzene, pentachlorophenol, phencyclidiine, phenobarbital, phenytoin, primidone, proç~in~mirle, proge~Lelul1e, qll;ni~lin~, roridin A, rubratoxin B, S-bioallethin, sterigmatocystin, streptomycin, T-2 tetranltetracetate, terhutryn, testosterone, theophyilline, thyroxine, tobl~llycin, trenbolone, triadimefon, triazine, valproic acid, valproic acid, vancomycin, warfarin, zearalenone and zeronat.
3~ In many embodiments of the invention the analyte will be the substrate for the redox erzyme, as in the case of many of the oxidases described herein. ~or example, the analyte can be glucose, cholesterol, lactate, oxalate, pyruvate, Sl,~ 1 l l ~JTE SHEET (RULE 26) 28.
bilirubin, galactose, sulphite, xanthene, and glnt~m~t~ Analytes can also be oxidase substrates for oxidases described herein. The electrode assemblies and methods described herein can provide for a biochemical 'snapshot' of the state of key physiological metabolites including, but not exclusively, glucose, cholesterol, lactate, and urate. These analytes could be measured singly, using a single apposite enzyme, or on a multi-analyte basis using a disposable strip cont~inin~ a combination of peroxide-producing enzymes. Also such methods can be used for agro-food applications, such as the crisp industry, which need to monitor glucose levels in potatoes as a consequence of their effect on the aesthetics of the product.
The present invention can be used to measure analytes in a variety of samples, such as those samples commonly found in the various fields described herein. For example, in the health care field analytes can be measured, normallywithout pretreatment, from such fluids as blood, urine, extracellular fluid, Iymph, diluted feces, sweat, mucus and saliva The enzyme layer mentioned herein can contain components from such sarnples during usage, particularly analytes. The enzyme layer can also contain predetermined amounts of analytes that are expected in a sample to use as control in a kit or as a calibration sample.
The present invention also provides for the detection of pathogenic micro-or~ni~ms. This provides for rapid and to provide unequivocal idenrific~tion of the etiological agents of infectious ~ e~ces including, but not limited to, AIDS(~IV, especially types I and II), h~p~titic (strains A, B and C), tuberculosis, chlamydia and gonorrhea. The surface antigens of such pathogens can also be ~letecte-l by assaying antibodies to such pathogens using a surface antigen (or a portion thereof that bind to the antibody) ~tt~.'h.-.1 to a matrix, applying the sample with the antibody to be rlet~ct~ (analyte) and then applying an anti-antibody ~tt~cll~fl an enzyme, such as an oxidase, to detect the binding of the antibody that recognizes the pathogen.
The present invention can also be sued to monitor therapeutic drugs as a safety support m.srl~ to a~;cu~llpally drug delivery programs. A selection of these pharTn~re~ltic~ls includes theophylline, salicyclic acid, digoxin, gentarnicin, n~tilmicin, tobramycin, vanomycin, and ~cet~min~>phen.

SlJ~ JTE SHEET (RULE 26) 29.
Detection of prescribed abused and il}icit substances such as barbiturates, benzodiazepines, cannabinoids, cocaine metabolites, opiates, methadone, amphet~min~, methamphct~minP and anabolic agents (such as 17B-estradiol, oestrogen, testosterone and pro~ el~ne) also be accomplished using the present invention.
Ch~mic~l and biological pollutants such as pesticides (organophosphorous, organochloro) heavy metals (lead, c~millm, mercury) and compounds such as dioxins, PCBs, atrazine, Bacillus thuringensis toxin, DDT can also be det~c.
using the present invention.
Diagnostic tests for livestock-, food- and water-borne diseases an be made using the present invention. Examples include Brucella abortus, Escherichia coli(strain EC10) and Vibrio cf~olerae.
The present invention also allows for the detection of toxins such as Clostr~dium ~o~ulinum neurotoxin ~A - E), Staphylococcus aureus enterotoxin (A -E) and aflatoxins (B1, B2).
The electrodes can also be used without enzymes to detect the non-enzymatic detection of breakdown products in paints and oils.
The invention also includes in vivo electroch~mir~l applications such as, but not restricted to, monitoring brain chemistry, the neuronal propagation and dysfilnction, and the detection of ~l~m~ging free radicals in tissues (e.g. for use in diagnosing arthritis and plant cell damage) witn micro-electrodes.

SUBSTITUTE SHEET (RULE 26) 30.
EXAMPLES = ~ -~HEMICALS AND REAGENTS
AIl chemicals are of reagent grade and obtained from ~3DH, (now Merck, Poole, Dorset, UK) unless stated otherwise. The ink (low resi.ct~nr.e carbon based particles), template (stainless steel mesh (100 counts)), apposite solvent system, e.g. (cyclohexanone solution with an alcohol) and facilities for screen-printing are kindly provided l~y Gwent Electronic Materials (GEM, Pontypool, IJK).
C32~II6N8Fe(II) is purchased from Kodak (Rochester, NY, USA). Ascorbic acid and hydrogen peroxide are obtained from Aldrich (Poole, Dorset, UK). Cysteine, reduced glutathione and uric acid are obtained from Sigma (St. Louis, MO, USA).
Solutions of ascorbic acid, paracetamol, glutathione and cysteine are prepared in 0.05 mol dm~3 phosphate prior to use. Uric acid is dissolved in 50 cm3 of 0.05 mol dm~3 sodium hydroxide by 20 minl7t~s sonication with a Decon FS100 sonicator ~Ultrasonics, Sussex, UK). The supporting electrolyte used throughout is phosphate buffer, which is prepared from stock solutions of 0.5 mol dm~3 of sodium dihydrogen-ortho-phosphate and ortho-phosphoric acid. These are mixed to give a buffer of the required pH and diluted with water, de-ionized with an R0200-Stillplus HP system (Purite, Oxfordshire, Thame, UK), to yield the desiredconcentration The stability of hydrogen peroxide is followed by titrating against acidified potassium perm~ngan~t~- which is also obtained from Aldrich as a 0.1N
volumetric standard in water.

VOLTA~U~ETRY A*~D A~PERO~ETRY
Cyclic volt~mmt~try is performed using a Metrohm E612 VA-scanner ~l~erisau, Switzerland) in conjunction with an E611 VA-detector; these are conn~ctP-l to a Linseis LY18100 x-y plotter (Recorder Laboratory Services, Surrey, UK) to record the voltammograms. A one-compartment cell is employed incorporating an SPCE as a working electrode and platinum and saturated calomel electrodes (~;CE) as counter and reference electrodes, respectively. Solutions are ebulliated with nitrogen for anaerobic experiments. Hydrodynamic conditions are achieYed using a stirrer and m~gn~-tic stirrer bar.

S(J~ 1 l l ~JTE SHEET (RULE 26) 31.
Oxygen and nitrogen are delivered via a system of pipes and the partial pressures of the former gas monitored using a portable oxygen meter (Oxymon, UK). This instrument ~ t~ct~rl the oxygen concentration amperometrically at -0.7V vs AglACl.

EXA~PLE 1 - EL~C~RODE FABRUCATION
Working electrodes, which in these examples are used to measure hydrogen peroxide, can be prepared as follows. A 5% (m/m) C32H,6N8Fe(II) loading (5g of C32HI6N8Fe(II) per 100g of car~on ink) is mixed with a base carbon ink (code:
D14, Gwent Electronic Materials, Pontypol, UK) for 10 minnt~c by hand using a mortar and pestle and then passed through a roll mill three times with a decreasing gap to make a homogenous ink. Me~-h~ni~l crushing and milling to a particle sizeof 10,u - 100~ (micron) are also acceptable. An inert lmm tnick, PVC support (ADP, Bristol, UK) is cle~n~ with etnanol, allowed to dry and cut to size 1~ (a~plo~ ately 50 x 50 m~n) with a guillotine. The homogeneous ink is then deposited onto the printing template (lcm before electrode heads) and forced through a stainless steel mesh (100 counts per inch) in one pass to produce the desired electrode pattern on the underlying PVC backing material. A semi-automatic printer (Dek, Dorset, UK) can be used in conjunction with a stainless steel screen ~10() counts per inch).
Unrnodified electrodes are m~mlf~ct~lred in a similar manner except that the ferro isonidole ringed compound is omitted at the ink mixing stage. Usually, no firing processes are used to fix the electrodes to the support. Test strips are wrapped in tissue (to minimi7~ surface cont~min~tion) and left to dry in a cabinet possessin~ suitable air-llow properties. Once dry, all electrodes are individually cut to size from the solid support. Usually, a conductive strip is trimrned to 15 mm and the working area ~9 mm2) of the electrode isolated by a tnin strip of inclll~tin~ tape (~S Components). FIG. 1 shows an example of a electrode assembly that can be used to measure up to five different analytes. The electrodes are then inserted into spade connectors which in turn are conn~cte-l to the potentiostat via coaxial cables.

SUBSTITUTE SHEET (RULE 26) W 097~27473 PCT/CA97/00044 32.
EXAMPLE 2: DETECTION OF HYDROGEN PEROXIDE REDUCTION USING

The current reduction peak for hydrogen peroxide can be measured using cyclic voltammograms. Cyclic voltarnmograms are initially recorded in plain solutions of 0. lmol dm-3 glycine buffer ~pH 9.0) with umnodi~led and C32Hl6N8Fe(II) SPCEs (screen printed carbon electrodes). The electrodes are thenused to measure reduction of hydrogen peroxide in the same solution cont~inin~
various concentrations of hydrogen peroxide. The voltam~netric conditions are asfollows: initial potential, -0.8 V; scan rate, 20 mV s-l and switching potential, +
lC) 1.0 V. All experiments are performed in triplicate (unless stated otherwise), using a fresh printed strip for each run; results represent the mean values for each parameter studied. All results are quoted with reference to SCE, where positive current represents the reduction of hydrogen peroxide.
FIG. 3 shows cyclic voltammograms can be recorded using umnodified and C32Hl6N8Fe(II) SPCEs in 0.1 mol dm--3 glycine buffer (pH 9) and in the presence of 2.2 x 10-3 mol dm~3 hydrogen peroxide (without stirring). The redox processes, denoted Ia and Ic, correspond to the respective oxidation and reduction of Fe2+ ~FeII) and Fe3+ (FeIII) ions, respectively, for modified electrodes. Theredox potential of the macrocyclic bound Fe2+1Fe3+ of the C32H~6N8Fe retained inthe complex printing ink matrix is -25OmV (SCE) in glycine buffer. The overYoltage associated with the reduction of hydrogen peroxide is dr~m~tir~lly shifted more anodic (ca 0.5V) with C32Hl6N8Fe(II) SPCEs (lA) when compared to the direct electrochemical reduction of hydrogen peroxide at unmodified SPCEs (lC). This inrli~ ~t~s that a catalytic process is present, as no a~p,~iable Faradaic activity is seen with non-met~llice~l C32HI6N8 only strips (not shown). The increase in current that occurs at the foot of the FeZ+/Fe3+ peak observed in plain solutions ~lB~. Tn~ t-os the active species and is consistent with the presence of divalent metal ion l~ re~ lg electrons to hydrogen peroxide in hydrogen peroxide cont~inin~ solutions (lA). The benefits of such catalysis are ap~a~ t when the electrodes are evaluated using amperometric tr~ncd-lction. The greatly reduced operating potential produces an unexpected irnprovement in the selectivity of the electrodes and electrode assemblies, especially at voltages less than -SUBSTITUTE SHEET (RULE 26) w 097127473 PCT/CA97/00044 33.
300mV. Such working electrodes demonstrate a superior selectivity towards hydrogen peroxide, especially the reduction of hydrogen peroxide at negative potentials (such as -350mV to -50mV).
.

EXA~PLE 3: HYDRODYNA~IC VOLTAMU~ETRY
Hydrodynamic volt~mm.otry can be used to optimize the voltage required to activate the reduction of hydrogen peroxide (with stirring). Hydrodynamic voltammograms are obtained for 2.89 x 10-3 M hydrogen peroxide solutions.
SPCEs are immersed in 0.1 mol dm~3, pH 9 0 glycine buffer solutions (20 cm3).
The applied potentials of the working electrodes are then increased manically in 50 m~ steps The resulting steady-state cathodic current responses are plotted versus applied voltage in FIG. 4 ~ach experiment is performed in triplicate with a fresh electrode and the results represent the mean current values FIG. 4 shows that raw data can be obtained from hydrodynamic vol~mn~.otric experiments using a variety of modified and unmodified electrodes.Clearly, the most ef~lcient vehicle for selective peroxide deterrnination can obtained using C32Hl6N8Fe(II) SPCEs, followed by C32H,6N8 Mn(III) and Co~II).
The current density with Fe(II) is at least two times the current density with Mn(III) and at least four with Co(II) Little activity is obtained with strips lacking an organometallic catalyst, such as a metallo isoindole ringed compound.

EXA~PLE 4: A~MpERo~nETRuc CALIBRATION AND INTERFERENCE STUD
Ampelu,lleLly at a fixed potential in a stirred solution can be used to evaluate the electrode characteristics of hydrogen peroxide-specific electrode assemblies. A baseline is established in 20 cm3 of 0.1 mol dm~3 glycine buffer (pH 9.2). The current will increase in the cathodic response, following (10-100 ml} additions of an 2.9 x 10-3 M hydrogen peroxide stock solution (approximately145 ~M final concentration). The current can be linearly recorded over a wide range of micromolar to millimolar concentrations.
FIG. ~ shows a typical alllpe-ul~letric calibration can be obtained for hydrogen peroxide (at hydrogen peroxide reduction voltages), using the C3~HI6N8Fe(II) SPCEs described herein. Such results show that C32Hl6N8Fe(II) SU~;~ JTE SHEET (RULE 26) CA 02243970 l998-07-2l 34.
provides a useful analytical signal for measuring the reduction of hydrogen peroxide, which is a product of many oxidase catalyzed reactions. TABLE 1 shows calibration data for the catalytic reduction of hydrogen peroxide using C32Hl6N8~e(II) modified screen printed electrodes using different voltages. The experimental conditions are the same as I~IG. 5. ~SD refers to the relative standard deviation.

E:app Response Factor Correlation RSD I~lte~ce~t (m~ (~A/~mol dm3) (%) (nA) Q 0.004 0.983 7.9 0.89 -50 0.008 0.995 6.~ 1.52 -103 0.014 0.998 8.1 1.54 0.021 0.999 23.1 0.00 -200 1.601 0.983 27.2 -54.2 A}nperometry can also be used to test the selectivity of the electrode or electrode assemblies to a number of naturally-occurring or synthetic compounds that may ~amper hydrogen peroxide analyses. A baseline is established as described herein using a glycine buffer and several selected interferents can beintroduced in the solution in contact with the electrode to yield micromolar or millim~lar final concentrations.
~IG. 6 shows that a lcL~IesellL~Li~e amperogram can be perfor~ned with an C3~l6N8Fe(II) SPCE biased at -0. lV. Such results show the analytical performance of such electrodes is surprisingly excellent, producing a rapid response time (10s at 95 % of the maximum steady-state current), and accurate responses to the analyte over a wide dynamic range (hydrogen peroxide additions were 1 x 10-6 30 x 1~3 mol dm~3), r = 0.999, n = 3 (slope of hydrogen peroxide concentration and current)?. In particular, additions of three notorious i"-~lr~L~ s, ascorbic acid, uric acid and ~el;.,.,;,-ophen (paracetamol) at concentrations well above physiological concenLI~tions can show no appreciable 3~) current responses. Such experiments can further substantiate the highly selective SUBSTITUTE SHEET (RULE 26) 35.
sensing capabilities of this system. For confirrnatory purposes C~f~l~ce, an enzyme that specifically degrades hydrogen peroxide, can be introduced into the solution.
The resulting dramatic tlimimltion in the signal provides conelusive evidence that ~ hydrogen peroxide is the molecule being detected.
S The eleetrodes described herein are surprisingly stable under normal operating and storage eonditions, as there is no appreciable loss of catalytic eapability during operation or storage. E~min~tion of the efficiency of the electrodes over a four month period, i.e., the mean slopes of three separate ealibrations, will show that they retain full reaetivity, which is a particularly desirable feature for mass produced electrodes that desirably have a long shelf life.
~he effieacy of ink mixing and eleetrode printing for the relative standard deviation of inter- and intra-bateh eleetrode fabrieation, can be determined voltarnmetrieally, and is 4.9% and 6.5% (n = 12), respectively (slopes of hydrogen peroxide ealibrations). Thus, sueh eleetrodes will reduee m~m-~etllringvariability of electrodes, particularly screen printed electrodes.

SUBSTITUTESHEET(RULE26) 36.
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All publications and patent applications mentioned in this herein are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually in~1ir~tf~d to be incorporated by reference.
The invention now being fully described, it will be apL,alcnt to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

SIJ~ JTE SHEET (RULE 26)

Claims

41 .
WHAT IS CLAIMED IS:

1. An hydrogen peroxide electrode assembly comprising:
a first hydrogen peroxide electrode comprising a metallo isoindole ringed compound dispersed in an electrode material, and an first enzyme layer comprising a first enzyme and in electrical contact with said first hydrogen peroxide electrode, wherein said metallo isoindole ringed compound coordinates Fe, Ru, Rh, or Mn.

2. The hydrogen peroxide electrode assembly of claim 1, wherein said electrode material is a material selected from the group consisting of platinum,gold, silver and carbon, wherein said carbon or gold electrodes can optionally comprise a second metallo isoindole ringed compound.

3. The hydrogen peroxide electrode assembly of claim 2, wherein said electrode material further comprises electrically conductive carbon and said metallo isoindole ringed compound provides for sufficient electron transfer to permit detection of an electrical current.

4. The hydrogen peroxide electrode assembly of claim 3, wherein said metallo isoindole ringed compound is selected from the formula C32H16N8M; where M is Fe or Mn and said first enzyme is a redox enzyme.

5. The hydrogen peroxide electrode assembly of claim 4, wherein said metallo isoindole ringed compound is of the formula C32H16N8Fe(II) and said redox enzyme is an oxidase.

6. The hydrogen peroxide electrode assembly of claim 5, wherein said oxidase catalyses hydrogen peroxide production.

7. The hydrogen peroxide electrode assembly of claim 6, wherein said oxidase is an enzyme selected from the group consisting of 6-hydroxy-D-nicotine oxidase,alcohol oxidase, aldehyde oxidase, allyl-alcohol oxidase, amine oxidase, bilirubin 42.
oxidase, cholesterol oxidase, choline oxidase, cyclohexylamine oxidase; D-amino acid oxidase, D-aspartate oxidase, D-glutamate acid oxidase, dihydroorotate oxidase, dimethyl-glycine oxidase, ethanolamine oxidase, galactose oxidase, glucose oxidase, glycolate oxidase, glycollate oxidase, glyoxylate oxidase, hexose oxidase, L-amino acid oxidase, L-gluconolactone oxidase, L-glutamate acid oxidase, L-ricin .alpha.-oxidase, L-sorbose oxidase, lactate oxidase, malate oxidase, N-methylamino acid oxidase, N6-methylrycine oxidase, nitroethane oxidase, nucleoside oxidase, oxalate oxidase, putrecine oxidase, pyranose oxidase, pyridoxine 4-oxidase, pyruvate oxidase, rathosterol oxidase, sarcosine oxidase, sulphite oxidase, tyramine oxidase, urate oxidase, urease and xanthene oxidase, wherein said first enzyme provides sufficient activity to permit detection of hydrogen peroxide.

8. The hydrogen peroxide electrode assembly of claim 7, wherein said oxidase has glucose oxidase activity.

9. The hydrogen peroxide electrode assembly of claim 8, wherein said oxidase is attached to a first analyte binding moiety

10. The hydrogen peroxide electrode assembly of claim 9, wherein said enzyme layer comprises a matrix and a second analyte binding moiety attached to said matrix.

11. The hydrogen peroxide electrode assembly of claim 10, wherein said first analyte binding moiety is an antibody and is covalently attached to said first enzyme.

12. The hydrogen peroxide electrode assembly of claim 6, wherein said first enzyme is attached to a first analyte binding moiety.

43.

13. The hydrogen peroxide electrode assembly of claim 12, wherein said enzyme layer comprises a matrix and a second analyte binding moiety attached to said matrix, wherein said second analyte binding moiety recognizes said analyte.

14. The hydrogen peroxide electrode assembly of claim 13, wherein said first analyte binding moiety is an antibody and is covalently attached to said first enzyme.

15. The hydrogen peroxide electrode assembly of claim 14, wherein said oxidase produces a substrate for a second enzyme, wherein said second enzyme produces hydrogen peroxide.

16. The hydrogen peroxide electrode assembly of claim 15, wherein said oxidase has lactate oxidase activity and produces pyruvate and said second enzyme has pyruvate oxidase activity.

17. The hydrogen peroxide electrode assembly of claim 6, wherein said oxidase is attached to said matrix.

18. The hydrogen peroxide electrode assembly of claim 8, wherein said oxidase is glucose oxidase and said glucose oxidase is directly attached to said matrix.

19. The hydrogen peroxide electrode assembly of claim 18, wherein said matrix is selected from the group consisting of cellulose, polycarbonate, perfluorinated material, polystrole, polyurethane, polysulphone and silica.

20. The hydrogen peroxide electrode assembly of claim 19, wherein said matrix is screen printed.

21. The hydrogen peroxide electrode assembly of claim 18, further comprising a selectively permeable layer between said matrix and said electrode.

22. The hydrogen peroxide electrode assembly of claim 20, further comprising a reference electrode for said hydrogen peroxide electrode.

23. The hydrogen peroxide electrode assembly of claim 22, wherein said enzyme layer comprises substances found in blood or urine.

24. The hydrogen peroxide electrode assembly of claim 23, wherein said first hydrogen peroxide electrode comprises a surface area that fits on test card.

25. The hydrogen peroxide electrode assembly of claim 24, wherein said first hydrogen peroxide electrode and said enzyme layer are screen printed.

26. The hydrogen peroxide electrode assembly of claim 24, wherein said first hydrogen peroxide electrode comprises a working area with a width between 1 mm to 20 mm wide.

27 The hydrogen peroxide electrode assembly of claim 26, wherein said first hydrogen peroxide electrode is in a voltage field, wherein said voltage field has a voltage between 450 mV and -50 mV using an Ag/AgC1 reference electrode, wherein said voltage field permits measurement of hydrogen peroxide reduction.

28. The hydrogen peroxide electrode assembly of claim 27, further comprising a scavenger layer comprising a second enzyme and said scavenger layer is locatedbetween said first hydrogen peroxide electrode and first enzyme layer.

29. The hydrogen peroxide electrode assembly of claim 28, wherein said second enzyme is L-ascorbic acid oxidase, wherein said L-ascorbic oxidase has sufficient activity to reduce the amount of dissolved oxygen in the presence of a substrate for said L-ascorbic acid oxidase.

30. The hydrogen peroxide electrode assembly of claim 5, further comprising at least one additional hydrogen peroxide electrode assembly comprising:

45.
a second hydrogen peroxide electrode comprising a metallo isoindole ringed compound dispersed in an electrode material, and a second enzyme layer comprising a second enzyme and in electrical contact with said second detection electrode;
wherein each said additional hydrogen peroxide electrode is configured to separately measure hydrogen peroxide production in response to an additional analyte.

31. The hydrogen peroxide electrode assembly of claim 30, wherein said second enzyme is attached to a third analyte binding moiety that recognizes a second analyte and said third analyte binding moiety is different from said first analyte binding moiety, wherein said third analyte binding moiety permits detection of said additional analyte.

32. The hydrogen peroxide electrode assembly of claim 31, wherein said second enzyme layer comprises a matrix and a fourth analyte binding moiety attached to said matrix, wherein said fourth analyte binding moiety binds to said additional analyte and said fourth binding moiety is attached to said matrix to permit electrical contact with said second hydrogen peroxide electrode and to minimize electrical contact with said first hydrogen peroxide electrode.

33. The hydrogen peroxide electrode assembly of claim 32, further comprising four to ten hydrogen peroxide electrodes, wherein each said hydrogen peroxide electrode assembly separately measures hydrogen peroxide.

34. An hydrogen peroxide electrode assembly comprising:
a) a ferro isoindole ringed compound, b) an electrode, and c) an oxidase, wherein said oxidase has sufficient activity to catalyse hydrogen peroxide production in detectable amounts.

46.

35. The hydrogen peroxide electrode of claim 34, wherein said electrode comprises electrically conductive carbon and said ferro isoindole ringed compound is C32H16N8Fe(II) and said C32H16N8Fe(II) is in amount to provide for sufficientelectron transfer to permit detection of an electrical current related to catalysis by said oxidase.

36. The hydrogen peroxide electrode of claim 35, wherein said ferro isoindole ringed compound is dispersed in said electrically conductive carbon.

37. The hydrogen peroxide electrode of claim 36, wherein said ferro isoindole ringed compound mass is at least 1% of said electrically conductive carbon mass.

38. The hydrogen peroxide electrode of claim 37, wherein said ferro isoindole ringed compound mass is between 3 and 8% of said electrically conductive carbon mass.

39. The hydrogen peroxide electrode of claim 36, further comprising a selectively permeable layer either 1) between said oxidase and said hydrogen peroxide electrode or 2) covering both said oxidase and said hydrogen peroxide electrode, wherein said selectively permeable layer excludes medium molecular weight interferents from said hydrogen peroxide electrode.

40. The hydrogen peroxide electrode assembly of claim 39, wherein said electrode is screen printed.

41. The hydrogen peroxide electrode assembly of claim 40, wherein said hydrogen peroxide electrode has a thickness between 1,000 and 50 µ.

42. The hydrogen peroxide electrode assembly of claim 41, wherein said oxidase is attached to an analyte or an analyte analog and said oxidase can prevent said analyte from binding to an analyte binding moiety.

47.

43. The hydrogen peroxide electrode assembly of claim 42, wherein said analyte binding moiety is attached to a matrix and said matrix electrically communicates with said hydrogen peroxide electrode in the presence of a liquid containing a sufficient amount electrolytes to provide for electrical communication.

44. The hydrogen peroxide electrode assembly of claim 43, wherein said matrix is part of a fluid guidance pathway that is in fluid communication with awaste reservoir.

45. The hydrogen peroxide electrode assembly of claim 41, wherein said ferro isoindole ringed compound is also present in a layer covering said electrode.

46. The hydrogen peroxide electrode of claim 35, wherein said oxidase is dry and covers said electrode.

47. The hydrogen peroxide electrode assembly of claim 46, wherein said ferro isoindole ringed compound is not dispersed in said electrically conductive carbon and is present in a layer covering said electrode.

48. The hydrogen peroxide electrode assembly of claim 47, further comprising a matrix, wherein said matrix permits fluid communication between said oxidase and said ferro isoindole ringed compound.

49. The hydrogen peroxide electrode assembly of claim 48, wherein said ferro isoindole ringed compound is present on a surface of said hydrogen peroxide electrode or said ferro isoindole ringed compound is present in said matrix and said matrix permits electrical communication between said ferro isoindole ringedcompound and said electrode.

50. The hydrogen peroxide electrode assembly of claim 49, wherein said oxidase is covalently attached to said matrix.

48.

51. The hydrogen peroxide electrode assembly of claim 49, wherein said oxidase is noncovalently attached to said matrix.

52. The hydrogen peroxide electrode assembly of claim 51, wherein said oxidase is attached to said matrix through a first analyte binding moiety.

53. The hydrogen peroxide electrode assembly of claim 51, wherein said oxidase is attached to said matrix through a second analyte binding moiety, wherein said first analyte binding moiety is covalently attached to said matrix

54. The hydrogen peroxide electrode assembly of claim 52, wherein said oxidase is attached to a second analyte binding moiety and said second analyte binding moiety is attached to said matrix through an analyte that is attached to said matrix through said first analyte binding moiety.

55. The hydrogen peroxide electrode assembly of claim 54, wherein said ferro isoindole ringed compound is a ferrous isoindole ringed compound.

56. A method for electrically detecting the presence of an analyte comprising:
measuring an electrical current from a redox electrode comprising an electrode material and a ferro isoindole ringed compound in fluid communication with an oxidase, wherein said oxidase catalyses a redox reaction that corresponds to the presence of an analyte and produces a product that either 1) directly chemicallyoxidizes or reduces said ferro isoindole ringed compound or 2) indirectly oxidizes or reduces said ferro isoindole ringed compound.

57. The method of claim 56, further comprising:
applying a voltage field between said redox electrode and a reference electrode assembly.

49.

58. The method of claim 57, wherein said applying step further comprises applying said voltage field between said redox electrode and said reference electrode assembly to permit electroreduction of said ferro isoindole ringed compound from C32H16N8Fe(III) to C32H16N8Fe(II).

59. The method of claim 58, further comprising:
comparing said electrical current in the absence of said analyte and in the presence of said analyte.

60. The method of claim 59, further comprising:
quantitating the amount of said analyte in relation to the amount of said electrical current.

61. The method of claim 59, wherein said voltage field is between -50 and -450 mV.

62. The method of claim 61, wherein said product is hydrogen peroxide.

63. The method of claim 62, wherein said analyte is a substrate for said oxidase.

64. The method of claim 63, wherein said analyte is glucose.

65. The method of claim 56, further comprises:
contacting said analyte with a matrix that provides for fluid communication with said oxidase.

66. The method of claim 65, said contacting further comprises:
contacting said analyte with an analyte attached to said oxidase or an analyte analog attached to said oxidase, wherein said analyte and either 1) saidanalyte attached to said oxidase or 2) said analyte analog attached to said oxidase are recognized by an analyte binding moiety.

50.

67. The method of claim 66, wherein said analyte is measured in a competitive or displacement assay.

68. The method of claim 67, further comprises:
applying a voltage field between said redox electrode and said reference electrode assembly to permit electroreduction of said ferro isoindole ringed compound from Fe(III) to Fe(II).

69. The method of claim 65, further comprises:
contacting said analyte with an analyte binding moiety attached to said matrix.

70. The method of claim 69, further comprises:
contacting said analyte with a second analyte binding moiety attached to said oxidase.

71. The method of claim 70, wherein said analyte is measured in a sandwich assay.

72. The method of claim 70, wherein said analyte is measured in a multi-sandwich assay.

73. The method of claim 71, wherein said analyte binding moiety is an antibody.

74. The method of claim 73, wherein said analyte is a protein.

75. The method of claim 74, wherein said protein is a protein from an organism selected from the group consisting of B and C virus, Bacillus anthracis, Bovine rhinotracheitis, brachematis, Brucella abortus, Campylobacter jejuni, chlamydia, Clostridium botulinum, coronavirus, cytomegalo virus, Ebola virus, Escherichia coli, group A strep, hepatitis A, herpes virus, HIV, human papilloma 51.
virus, Lassa fever virus, Leptospira interrogans, Listeria monocytogenes, Maedi visna virus, Mycobacterium tuberculosis, Mycoplasma bovis, Neisseria gonorrhoea, Salmonella, Staphylococus aurens, Stephanuras dentatus, swine fever virus, Taxoplasma gondii, Treponema pallidum, Vibrae epp., Vibrio cholerae, Yellow fever virus, Yersinia enterocolitica, and Yersuia pestis.

76. The method of claim 74, further comprising applying a voltage field that corresponds to a current peak associated with reduction of said product.

77. The method of claim 71, wherein said analyte is an antibody that recognizes an protein or sugar moiety of a micoorganism.

78. The method of claim 71, wherein said analyte is not a protein.

79. The method of claim 71, wherein said analyte is an analyte selected from the group consisting of .beta.-inhibin, 17.alpha.-methyltestosterone, 17.beta.-estradiol, 2,4-D, 2,4,5-T, 3-Acetyl deoxynivalenol, acetaminophen, alachlor, aldrin, amikacin, amitriptyline, amphetamine, atrazine, bacillus thuringensis toxin, barbiturates,BAY SIR 8514, benzodiazepines, bilirubin, caffeine, cannabinoids, carbamazepine,carhamazepine, chloramphenicol, chlorosulfuron, cocaine metabolics, cyanzine, cyclosporin, cyclosporine, DDT, deoxynlvalonel, deoxyverrucarol, desipramine, diacetoxyscirpenol, dibenzofurans, dichlorfop-methyl, dieldrin, diethylstilbestrol, difubenzuron, digitoxn, dioxins, disopyramide, endosulfon, estrogen, ethosuximide, flecainide, gentamycin, group A
trichothecenes, hCG, hexoestrol, imipramine, iprodione, kanamycin, kepone, LH, lidocaine, lidocaine, malete hydrazide, metalaxyl, methadone, methamphetamine, methaqualone, methotrexate, N-acetylprocainamide, neitilmicin, nortriptyline, oestrogen, opiates, oxfendazole, paraoxon, paraquat, parathion, polychlorinated benzene, pentachlorophenol, phencyclidiine, phenobarbital, phenytoin, primidone,procainamine, progesterone, quinidine, roridin A, rubratoxin B, S-bioallethin, sterigmatocystin, streptomycin, T-2 tetranltetracetate, terhutryn, testoterone, 52.
theophyilline, thyroxine, tobramycin, trenbolone, triadimefon, triazine, valproic acid, valproic acid, vancomycin, warfarin, zearalenone and zeronat.

80. A printed electrode comprising:
a ferro isoindole ringed compound dispersed in a printed electrode material, wherein said printed electrode material either 1) lacks cellulose acetate or 2) has less than 25% (mass/mass) cellulose acetate compared to the mass of electricallyconductive carbon when said electrode material comprises electrically conductivecarbon.

81. The printed electrode of claim 80, wherein said electrode material comprises electrically conductive carbon printed on a solid support.

82. The printed electrode of claim 81, wherein said printed electrode further comprises an enzyme dispersed in said electrode material.

83. The printed electrode of claim 82, wherein said printed electrode is a screen printed electrode.

84. The printed electrode of claim 83, wherein said ferro isoindole ringed compound mass is at least 1% of said electrically conductive carbon mass.

85. The printed electrode of claim 84, wherein said electrically conductive carbon mass is between 3 and 8% of said ferro isoindole ringed compound mass.

86. The printed electrode of claim 85, wherein said printed electrode is a screen printed electrode and said electrically conductive carbon is a plurality of ultrasonicated particles.

87. The printed electrode assembly of claim 86, wherein said printed electrode has an interdigitated pattern and a thickness between 1,000 and 50 µ with respect to the plane of said solid surface.

53.

88. The printed electrode of claim 81, wherein said electrically conductive carbon is a plurality of particles and said plurality of particles can pass through a 1.0 µ screen.

89. The printed electrode of claim 88, wherein said printed electrode is a screen printed electrode.

90. The printed electrode of claim 89, wherein said plurality of particles can pass through a .25 µ screen.

91. The printed electrode of claim 89, wherein said printed electrode furthercomprises a printed oxidase on a surface of said printed electrode.