||||I|||||||III O US005262305A United States Patent (19) 11 Patent Number: 5,262,305 Heller et al. 45) Date of Patent: Nov. 16, 1993

54 INTERFERANTELIMINATING 4,758,323 7/1988 Davis et al...... 435/26 BIOSENSORS 4,764,416 8/1988 Ueyama et al...... 428/212 4,776,944 10/1988 Janata et al...... 435/817 75) Inventors: Adam Heller; Ruben Maidan, both of 4,784,736 11/1988 Lonsdale et al. . ... 204/157.15 Austin, Tex. 4,795,707 1/1989 Niiyama et al...... 435/817 w 4,917,800 4/1990 Lonsdale et al. . ... 204/157.5 73) Assignee: E. Heller & Company, Austin, Tex. 4,919,767 4/1990 Vadgama et al...... 435/817 4,927,516 5/1990 Yamaguchi et al...... 435/817 21 Appl. No.: 753,812 4,938,860 7/1990 Wogoman ...... 435/817 22 Filed: Sep. 3, 1991 4,968,400 8/1988 Ueyama et al...... 428/212 a W. FOREIGN PATENT DOCUMENTS Related U.S. Application Data 127958A2 5/1984 European Pat. Off. . 63) Continuation-in-part of Ser. No. 664,054, Mar. 4, 1991. 12539 11/1984 European Pat. Off. . 184909 6/1986 European Pat. Off. . 51) Int. Cl...... C12Q 1/28; C12M 1/40; 24-1309 4/1987 European Pat. Off. . C07C 1/00; G01N 27/26 278647 8/1988 European Pat. Off. . 52 U.S. C...... 435/28; 435/288; 368209A1 6/1989 European Pat. Off. . 435/25; 435/291; 435/817; 435/26; 435/24; 390390A1 3/1990 European Pat. Off. . 435/14; 204/403; 204/153.1; 429/111; 2: OTHER PUBLICATIONS 58) Field of Search ...... 435/28, 25, 291, 817, Nagy, et al., "A New Type of Enzyme Electrode: The 435/14, 24, 190, 26, 288; 429/111, 40; 373/80; Ascorbic Acid Eliminator Electrode,' Life Sciences, 428/212; 204/157.15, 403 vol. 31, pp. 2611-2616, Pergamon Press, 1982. 4. Heller, A., "Electrical Wiring of Redox Enzymes,' (56. References Cited Reprinted from Accounts of Chemical Research, vol. U.S. PATENT DOCUMENTS 23, No. 5, 1990 (pp. 128-134). 4,098,574 7/1978 Dappen ...... 435/14 Japanese Patent No. 03028752 A2 to Omochi, et al., 4,168,205 9/1979 Danninger et al...... 435/25 "Method for Manufacture of an Electrode Containing 3: 5, 3. SA et F. Immobilized Enzyme and Interfering-Substance yawad Ty akamura et al...... 435/817 99. O 4,247,297 1/1981 Berti et al...... 435/24 Elias.st,” (Abstract) Chemical Ab 4,356,074 10/1982 Johnson ...... 435/190 s 4,375,399 3/1983 Havas et al ... 435/14 (List continued on next page.) 4,390,621 6/1983 Bauer ...... 435/14 4,404,066 9/1983 Johnson ...... 435/87 4,418,148 11/1983 Oberhardt ...... 435/14 4,427,770 1/1984 Chen et al. ... 435/14 Primary Examiner-Michael G. Wityshyn 4,461,691 7/1984 Frank ...... 429/11 Assistant Examiner-Louise N. Leary 4,476,003 10/1984 Frank et al...... 429/11 Attorney, Agent, or Firm-Pravel, Hewitt, Kimball & 4,524,114 6/1985 Samuels et al...... 429/40 Krieger 4,545,382 10/1985 Higgins et al...... 435/817 4,552,840 l/1985 Riffer ...... 435/28 (57) ABSTRACT 4,581,3364,619,754 10/19864/0886 NikiMalloy et al.et al...... 435/817 A biosensor including an interferant-aiminati eliminating cata 4,655,885 4/1987 Hill et all 429/43 lyst and method for analyzing an analyte in a biological 4,711,245 12/1987 Higgins et al..... 4378i, sample is disclosed. 4,717,673 1/1988 Wrighton et al...... 373/80 4,721,601 1/1988 Wrighton et al...... 429/23 20 Claims, 3 Drawing Sheets

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OTHER PUBLICATIONS Castner, et al., "Mass Transport and Reaction Kinetic Parameters Determined Electrochemically for Immobi German Patent No. 3934.299 C1, "Enzyme Electrodes lized Glucose Oxidase," Biochemistry, vol. 23, No. 10, Containing Oxidase and Peroxidase' to Schmid et al., pp. 2203-2210 (1984). (Abstract) Chemical Abstracts, 114(23):225208w. Abstract of U.S. Pat. No. 4,193,982, Mar. 18, 1980, Japanese Patent Application No. 02310457 A2, "En Avarmas, et al. zyme Biosensor For Micro Analysis of Body Fluid" Abstract of Brandt, et al., "Covalent Attachment of (Abstract), Chemical Abstracts, 114(21):2031.11a. Proteins to polysaccharide Carriers by Means of Benzo Foulds, et al., "Immobilization of Glucose Oxidase in quinone,' Biochim. Biophys. Acta, 386(1), 192-202 Ferrocene-Modified Pyrrole Polymers', Anal. Chem, (1975). vol. 60, No. 22, pp. 2473-2478 (1988). Abstract of Narasihan, et al., "p-Benzoquinone Activa Hale, et al., "A New Class of Amperometric Biosensor tion of Metal Oxide Electrodes for Attachment of En Incorporating a Polymeric Electron-Transfer Media zymes," Enzyme Microb. Technol, 7(6), 28-6 (1985). tor," J. Am. Chem. Soc., vol. III, No. 9, pp. 3482-3484 Abstract of Ikeda, et al., "Glucose Oxidase-Immobil (1989). ized Benzoquinone-carbon Past Electrode as a Glucose Foulds, et al., "Enzyme Entrapment in Electrically Sensor,” Agric. Biol. Chem, 49(2), 541-3 (1985). Conducting Polymers,” J. Chem. Soc., Faraday Trans. Abstract of U. Pat. No. 4,581,336, Apr. 8, 1986, Malloy, l, vol. 82, pp. 1259-1264 (1986). et al. Degani, et al., "Electrical Communication Between Abstract of European Patent No. 177,743 A2, Apr. 16, Redox Centers of Glucose Oxidase and Electrodes via 1986. Electrostatically and Covalently Bound Redox Poly Abstract of Bartlett, et al., "Modification of Glucose mers,” J. Am. Chem. Soc., vol. III, pp. 2357-2358 Oxidase by Tetrathiafulvalene,' J. Chem. Soc., Chem. (1989). Commun., (16), 1135-6 (1990). Bartlett, et al., "Strategies for the Development of Am Yao, "Chemically-Modified Enzyme Membrane Elec perometri Enzyme Electrodes,” Biosensors, vol. 3, pp. trode as an Amperometric Glucose Sensor,” Analytica 359-379 (1987/1988). Chemica Acta, vol. 148, pp. 27-33 (1983). Clark, et al., "Electrode Systems for Continuous Moni Abstract of Albery, et al., "Amperometric Enzyme toring in Cardiovascular Surgery,” Ann. N. Y. Acad. Electrodes, Part II. Conducting Salts as Electrode Ma Sci., vol. 102, pp. 29-(1962). terials for the Oxidation of Glucose Oxidase,' J. Elec Clark, et al., "Long-Term Stability of Electroen troanal. Chem. Interfacial Electrochem., 194(2), zymatic Glucose Sensors Implanted in Mice,” Trans. 223-35. Am. Soc. Artif. Inten. Organs, vol. 34, pp. 259-265 Dicks, et al., "Ferrocene Modified Polypyrrole with (1988). Immobilised Glucose Oxidase and its Application in Cass, et al., "Ferricinium Ion as an Electron Acceptor Amperometric Glucose Microbiosensors,' Ann. Biol. for Oxido-Reductases,' J. Electroanal. Chem., vol. 190, Clin., vol. 47, pp. 607-619 (1989). pp. 117-127 (1985). Yabuki, et al., "Electro-Conductive Enzyme Mem Alvery, et al., “Amperometric Enzyme Electrodes,” brane,” J. Chem. Soc., Chem. Commun., pp. 945-946 Phil. Trans. R. Soc. Lond, vol. B 316, pp. 107-119 (1989). (1987). Trojanowicz, et al., "Enzyme Entrapped Polypyrrole Scheller, et al., "Enzyme Electrodes and Their Applica Modified Electrode for Flow-Injection Determination tion," Phil. Trans. R. Soc. Lond, vol. B316, pp. 85-94 of Glucose,' Biosensors & Bioelectronics, vol. 5, pp. (1987). 149-156 (1990). Pollak, et al., "Enzyme Immobilization by Condensa Degani, Y. and Heller, A., “Direct Electrical Commu tion Copolymerization into Cross-Linked Polyacryl nication Between Chemically Modified Enzymes and amide Gels," J. Am. Chem. Soc., vol. 102, No. 20, pp. Metal Electrodes. 1. Electron Transfer from Glucose 6324-6336 (1980). (List continued on next page.) 5,262,305 Page 3

OTHER PUBLICATIONS Catalyst Based on Ruthenium (IV). pH 'Encapsulation' Oxidase to Metal Electrodes via Electron Relays, in a Polymer Film,” J. Am. Chem. Soc., vol. 103, pp. Bound Covalently to the Enzyme,” Journal of Physical 307-312 (1981). Chemistry, vol. 91, pp. 1285-1289 (1987). Denisevich et al., "Unidirectional Current Flow and Degani, Y. and Heller, A., "Direct Electrical Commu Charge State Trapping at Redox Polymer Interfaces on nication Between Chemically Modified Enzymes and Bilayer Electrodes: Principles, Experimental Demon Metal Electrodes. 2. Methods for Bonding Electron-- stration, and Theory," J. Am. Chem. Soc., vol. 103, pp. Transfers Relays to Glucose Oxidase and D-Amino-A- 4727-4737 (1981). cid Oxidase,” JACS, vol. 110, pp. 2615-2620 (1988). Abruna, et al., "Rectifying Interfaces Using Bartlett, et al., "Covalent Binding of Electron Relays to Two-Layer Films of Electrochemically Polymerized Glucose Oxidase,” J. Chem. Soc., Chem. Commun., pp. Vinylpyridine and Vinylbipyridine Complexes of Ru 1693-1704. thenium and Iron on Electrodes,' J. Am. Chem. Soc., Umana, U.S. Army Research Office Report No. ARO vol. 103, pp. 1-5 (1981). 23106.3-LS entitled "Protein-Modified Electrochemi Ellis, et al., "Selectivity and Directed Charge Transfer cally Active Biomaterial Surface,' dated Dec. 1988. Through an Electroactive Metallopolymer Film,” J. Samuels, et al., "An Electrode-Supported Oxidation Am. Chem. Soc., vol. 103, pp. 7480-7483 (1981). U.S. Patent Nov. 16, 1993 Sheet 1 of 3 5,262,305

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TIME (SECONDS)- 5,262,305 1. 2 which eliminates the necessity for oxidant (e.g. hydro INTERFERANTELIMINATING BIOSENSORS gen peroxide) to be present during bioassay. Preactiva tion of the catalyst is of particular utility in ex vivo This is a continuation-in-part application of pending applications, such as disposable biosensors and clinical U.S. application Ser. No. 07/664,054, filed Mar. 4, 1991. 5 analyzers. FIELD OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES This invention relates to biosensors, more particu FIG. 1 is a schematic view of an electrochemical larly to an improved biosensor and method for detect biosensor having a analyte sensing layer (layer I) on top ing an analyte which minimizes or eliminates false sig 10 of the electrode surface. nals caused by interfering substances in a biological FIG. 2 is a schematic view of an electrochemical sample. biosensor with a catalyst-containing layer (layer II) that eliminates interferants. BACKGROUND OF THE INVENTION FIG. 3 is a schematic view of an electrochemical Amperometric biosensors are capable of quantifying 15 biosensor with an catalyst containing layer that elimi trace amounts of biological analytes such as glucose, nates interferants (layer II) and an oxidant generating urea, cholesterol, etc. in biological fluids and foods. layer (layer III) covering the catalyst containing layer. Analyte may be electrooxidized directly at the ele FIG. 4 shows the results of a measurement performed crode, or an enzyme may be immobilized on an elec with an electrode of the type described in Example 1. trode such that the reaction product of the enzyme with 20 The signals (current as a function of time) obtained its substrate is detected by an electrical change, e.g. upon additions of ascorbate (0.1 mM) and glucose (2 change in current flow, at the electrode. The current mM) are shown in the absence of hydrogen peroxide generated at the electrode is a function of the quantity (a), and shown in the presence of hydrogen peroxide (b) of analyte in a sample. (0.1 mM). The accuracy of existing amperometric biosensors is 25 FIG. 5 shows the results of a measurement performed compromised by the presence of additional electrooxi with an electrode of the type described in Example 1. dizable compounds (hereinafter termed "interferants') The signals (current as a function of time) obtained present in a biological test sample. Examples of such upon additions of acetaminophen (0.1 mM) and glucose interferants include ascorbate, urate, bilirubin, cysteine (2 mM) are shown in the absence of hydrogen peroxide and acetaminophenol. 30 (a), and in the presence of hydrogen peroxide (0.1 mM) It would be of great utility to provide a biosensor (b). which would more accurately quantify an analyte with FIG. 6 is a schematic diagram of a multilayer elec out interference from other compounds present in a trode having a barrier layer between the catalyst con biological sample. taining layer and the analyte sensing layer. 35 FIG. 7 shows the results of measurements performed SUMMARY OF THE INVENTION with the hemin-containing electrode of Example 4. The The present invention solves the prior art problems of signals (current as a function of time) obtained upon interference by providing a biosensor having an inter additions of ascorbate (0.1 mM) are shown in the ab ferant-eliminating layer. The interferant-eliminating sence of hydrogen peroxide (a), and are shown in the layer includes a catalyst capable of oxidizing and 40 presence of hydrogen peroxide (b) (1.0 mM). thereby eliminating a plurality of interfering com FIG. 8 shows the results of experiments comparing pounds before they reach the sensor. Consequently, the the response of a preactivated biosensor of Example 5 inventive interferant-eliminating biosensors permit the with that of a non-preactivated biosensor. The signals desired analyte to be more accurately quantified in a (current as a function of time) obtained are shown for a biological sample. 45 non-preactivated biosensor (a) and a preactivated bio A biosensor of the present invention includes an elec sensor (b). trode capable of specifically sensing an analyte to be FIG. 9 shows the results of experiments comparing detected and an interferant-eliminating layer containing the preactivated biosensor of Example 6 with a non a catalyst. The analyte may be sensed directly through preactivated biosensor in measuring glucose in the pres electrooxidation on a metallic electrode or through SO ence of an interferant. The signals (current as a function sensing elements which are in electrical contact with of time) are shown for a non-preactivated biosensor (a) the electrode, whereas the catalyst is isolated from elec and a preactivated biosensor (b). A control signal ob trical contact with the sensing elements and/or elec tained in the absence of interferants is also shown (c). trode. The electrical isolation of the catalyst from the DETAILED DESCRIPTION OF THE electrode may be effected by means of an insulating 55 barrier layer between the catalyst and electrode or sens PREFERRED EMBOOIMENTS ing layer, or by selection of an operating potential for In its simplest embodiment, a biosensor of the present the bioassay at a level that prevents flow of current invention includes an electrode capable of specifically between the electrode and the catalyst. sensing an analyte to be detected, e.g., sensed directly In a preferred embodiment, a sensing layer contains a by electrooxidation of analyte on a metallic electrode, sensing enzyme. The sensing enzyme is preferably an and an interferant-eliminating layer. oxidoreductase such as glucose oxidase for the detec Referring to FIGS. 1-3 and 6, a biosensor of the tion of glucose, lactate oxidase for the detection of present invention may include an electrode 12 substan lactate, cholesterol oxidase for the detection of choles tially covered by a sensing layer 14. The sensing layer terol, and the like. Preferred catalysts are horseradish 65 contains a sensing enzyme and additional sensing ele peroxidase and iron (III) porphyrins such as hemin. ments required to achieve electrical contact with the In an alternative preferred embodiment, the catalyst electrode. The biosensor also includes an interferant is "preactivated", or raised to a higher oxidation state, eliminating layer 16 containing a catalyst which is capa 5,262,305 3 4. ble of oxidizing and thereby eliminating a plurality of agent, such as poly(ethylene glycol)diglycidyl ether, to interferants. The interferant-eliminating layer is isolated form a useful epoxy redox film. from electrical contact with the electrode, i.e., at a The catalyst mediates oxidation of an interferant in given applied potential at which components of the the presence of an oxidant to yield a non-interfering sensing layer are electrooxidized, or where analyte is compound which is no longer electrically active at the directly electrooxidized on the electrode, the catalyst is operating potential of the electrode and thus does not not electroreduced. interfere with the biosensor's function. The catalyst In the case of "electrical contact', current will flow may be a natural enzyme, e.g. horseradish peroxidase, in the external circuit as a result of an oxidation or or a synthetic catalyst such as an iron (III) porphyrin. reduction reaction in one or more layers of the sensor. O Suitable catalysts include horseradish peroxidase, cyto In the case of "electrical isolation', current in the exter chrome c peroxidase, chloroperoxidase, lactoperoxi nal circuit will not flow as a result of an oxidation or dase, thyroid peroxidase, Japanese radish peroxidase a, reduction reaction in the isolated layer. Japanese radish peroxidase c, myeloperoxidase, NADH The electrode may be made of any material known peroxidase, turnip peroxidase A, turnip peroxidase A2, for the manufacture of biosensing electrodes. Prefera 5 turnip peroxidase B, turnip peroxidase D, glutathione bly, the electrode is formed of solid material, e.g. glassy peroxidase, and transition metal porphyrins such as iron carbon. Additional suitable electrode materials include (III) porphyrins. A preferred iron (III) porphyrin is graphite, platinum, palladium, tin oxide, and conducting hemin, chloroprotoporphyrin IX iron (III). It is con organic salts. The electrode material itself may, in some templated that additional, known metalloporphyrins cases, be able to directly sense and detect the analyte, 20 may be useful as interferant-eliminating catalysts. for example, electrodes made of Group VIII metals or Horseradish peroxidase is a preferred catalyst be their oxides, such as palladium or ruthenium dioxide. cause interferants present in biological fluids, such as Alternatively, a special sensing layer must be applied to ascorbate, urate, acetaminophen, bilirubin, and cysteine, the electrode material. are rapidly oxidized by hydrogen peroxide in the pres 25 ence of horseradish peroxidase. The analyte sensor or sensing layer may be the elec Hemin is a preferred catalyst for several reasons, trode itself or may be a film applied to the electrode including its high stability, and high specific activity per which contains a specific enzyme which reacts with the unit mass. These characteristics enable longer storage analyte to be detected. It is intended that the term "sens times and lower production costs. However, due to the ing layer' include all the necessary sensing elements to 30 smaller size and greater diffusivity of this catalyst, care provide electrical contact of the sensing enzyme with must be taken to insure electrical isolation of the cata the electrode, permitting detection and transfer of a lyst from the electron relays of the sensing layer. This signal for analyzing an analyte. For example, this layer isolation may be achieved by immobilizing and/or will include the specific sensing enzyme and also elec cross-linking the catalyst in a polymer matrix in a man tron relays and/or diffusing redox mediators which 35 ner sufficient to prevent diffusion of the catalyst. An participate in transporting electrons from enzyme to example of a suitable polymer for immobilization of electrode. Any known method may be used to permit hemin is poly-(N-vinylimidazole) (PVI). The imidazole electrical contact between the sensing enzyme and the moieties in this polymer coordinate to the iron center of electrode. See, for example, U.S. Pat. No. 4,758,232 to the hemin, attaching it to the polymer matrix and in Davis et al., which is hereby fully incorporated by ref. creasing its activity. Additional imidazole containing erence. polymers or copolymers or polymers containing other Suitable sensing enzymes are oxidoreductases includ electron donating ligands may also be used to immobil ing dehydrogenases and oxidases. Examples of specific ize the catalyst. enzymes suitable for use are glucose oxidase for glucose The interferant-eliminating catalyst is isolated from analysis, lactate oxidase for lactate analysis, xanthine 45 electrical contact with the electrode. This isolation may oxidase for xanthine analysis, cholesterol oxidase for be achieved by retaining the catalyst in a discrete layer cholesterol analysis, pyruvate oxidase for pyruvate surrounding the sensing layer. For example, a soluble analysis, L-amino acid oxidase for L-amino acid analy catalyst in solution may be retained adjacent to the sis, D-amino acid oxidase for D-amino acid analysis, sensing layer within a dialysis membrane. Alternatively, alcohol oxidase for alcohol acid analysis, glycolate oxi 50 a catalyst may be immobilized within a polymer or dase for glycolate analysis, sarcosine oxidase for sarco copolymer matrix. An immobilized catalyst may be sine analysis, and other similar enzymes. In a preferred further restricted by cross-linking to form an insoluble embodiment, glucose oxidase is used as the sensing film, or by positioning a physical barrier between the enzyme in a biosensor for the measurement of glucose in sensing layer and the catalyst layer. a biological sample. 55 In a further embodiment, the desired electrical isola Mediators are electrochemically active fast redox tion may be achieved by selecting a catalyst which is couples that freely diffuse in solution. These include not electroreduced at a potential where the analyte is ions of conducting organic salts, quinones and their electrooxidized. At a given applied potential, the elec derivatives, organometallic complexes such as ferro trode itself, the sensing elements, and catalyst may pro cenes and their derivatives, and polypyridine complexes duce competing or non-competing oxidation and reduc of transition metals such as osmium or ruthenium and tion currents. Therefore, appropriate selection of the their derivatives. Electron relays are enzyme or polym catalyst, sensing elements and operating potential may er-bound fast redox couples. A preferred group of elec be such that at the operating potential of a bioassay the tron relaying redox polymers is based on derivatives of catalyst, oxidizable by the oxidant, is not in electrical a poly(vinylpyridine) complex of osmium bis (bipyri 65 contact with the electrode, whereas the sensing en dine) chloride. When the derivative is a polyamine, e.g., zyme, ususally reduced by the analyte, is in electrical the backbone contains pyridinium-N-ethylamines, the contact with the electrode. That is, the operating poten redox polymer may be crosslinked using a crosslinking tial is selected to maximize the direct or enzymatic 5,262,305 5 6 electrooxidation of the substrate while minimizing di be achieved by physical separation, e.g., by physical rect or catalyst-mediated electroreduction of the oxi barriers, immobilization of elements within a matrix, dant or of the preactivated catalyst. and/or matrix crosslinking. Electrical isolation of the The biosensor of the present invention minimizes or catalyst from diffusional redox mediators can be ef eliminates interference caused by reaction of other oxi fected through an ion exchange membrane. For exam dizable compounds present in a test sample with the ple, an anionic diffusional redox mediator will be re sensing elements. An interfering compound, upon react tarded by a polyanionic cation exchange membrane. ing with the catalyst, is quickly oxidized by the oxidant Likewise, a cationic diffusional redox mediator will be and prevented from reaching the sensing element in its retarded by a polycationic ion exchange membrane. electrochemically active state. The oxidant, e.g. hydro O Alternatively, and as discussed above, electrical isola gen peroxide, may be added to the assay solution or may tion may be achieved by selecting the components of be produced in situ by one or more oxidase enzymes, the biosensor and the operating potential to maximize provided the corresponding enzyme substrates are pres the response of the sensing enzyme while minimizing ent in the sample. These oxidase enzymes may be pro competing responses. In this embodiment, the catalyst vided in a separate oxidant-generating layer or the oxi 5 and sensing elements need not be provided in distinct dant-generating enzyme may be the same enzyme as layers. that used as the sensing enzyme. For example, in a glu In the method of the present invention, the inventive cose biosensor, glucose oxidase can both mediate the biosensor is used as a working electrode to detect and oxidation of glucose, permitting the analysis of glucose quantify the amount of an analyte in a biological sample. through electron relays coupled to the electrode and 20 These biosensors may be used in known electrochemi also mediate the oxidation of glucose by oxygen, gener cal sensing methods, including amperometric, coulo ating hydrogen peroxide which may be subsequently metric, potentiometric, conductometric or impedimet used with the catalyst for eliminating interferants. How ric methods. A preferred use is in amperometric meth ever, it is generally preferred that an oxidant-generating ods. enzyme be mixed with or located in close proximity to 25 For example, in an amperometric method, a sample is the interferant eliminating catalyst, e.g., in an adjacent placed in a three-electrode glass cell containing an elec layer, as shown in FIG. 3. trolyte and working, reference, and counter electrodes. In a preferred embodiment, the catalyst is "preac A saturated calonel electrode may be the reference tivated', thereby eliminating the requirement for oxi electrode. An electrochemical potential is applied to the dant present in the sample or an oxidant-generating 30 working electrode with respect to the reference elec system. The preactivated catalyst also reduces the ne trode and current flow is measured as a function of time. cessity of a multi-layer system, in an interferant The potential applied will depend on the redox poten eliminating biosensor. tial of the mediator or the relay of the sensing elements. Preactivation includes the preoxidation of the cata The measured current is a function of the concentration lyst by an oxidant, e.g. hydrogen peroxide, to form a 35 of the substances being oxidized. When the catalyst is stable, oxidized catalyst intermediate. The preactivated not active, both analyte and interferants are oxidized, catalyst thus has one or more centers with a higher and the measured current reflects the combined concen oxidation state. At the time of assay, and in the presence tration of analyte and interferants, resulting in inaccu of interferants, the catalyst will be regenerated and rate analyte measurements. When the catalyst is active, interferants oxidized. interferants are oxidized and do not reach the analyte A biosensor having a preactivated catalyst obviates sensing electrode or the elements of the analyte sensing the need for diffusing oxidant to be present during the layer in electrical contact with the electrode. The nea biochemical analysis or for the biosensor to include an sured current then more correctly reflects only the oxidant generating enzyme. A preactivated biosensor concentration of the analyte. allows the reaction with interferants to proceed rapidly 45 Under certain operating conditions the catalyst, cou until the catalyst in its reactive, oxidized form is ex pled with the electron relays present in the analyte hausted. When the oxidized catalyst is exhausted, it may sensing layer, may enhance the rate of electrochemical be regenerated, e.g., by brief exposure to an oxidant. reduction of the oxidant. This process may result in a The availability of a biosensor containing a preac reduction current superimposed on the oxidation cur tivated catalyst allows the prompt use of oxidizing 50 rent of the analyte and the net current will no longer be equivalents in the preactivated catalyst immediately proportional to the analyte concentration. This undesir when the analyte measurements begin. As discussed able situation may be prevented by making the potential further in Example 6, this permits a time window be of the working electrode sufficiently oxidizing such that tween the start of analyte measurement and the onset of reduction process will not proceed at a substantial rate, the interferant signal, allowing completion of the mea 55 whereas analyte electrooxidation will proceed rapidly. surement of the analyte prior to the onset of the interfer Alternatively, electrical contact between the catalyst ant signal. and the sensing elements may be physically prevented. As shown in FIGS. 1-3, biosensors of the present A barrier layer, fabricated of a polymer film such as invention may be multi-layered. An electrode 12 is sub a cellulose derivative, polycarbonate, or perfluorinated stantially covered by a sensing layer (layer I) 14 con membrane, may be used to effect such physical separa taining sensing elements in electrical contact with the tion. Alternatively, the analyte sensing layer matrix may electrode. An interferant-eliminating, catalyst-contain be crosslinked to make it less permeable to the catalyst. ing layer (layer II) 16 may be adjacent to the sensing A protein crosslinked by a protein cross-linking agent layer 14, or may be physically separated from the sens such as glutaraldehyde or cyanuric chloride may be ing elements by a barrier layer 20 as shown in FIG. 6. 65 used. An oxidant-generating layer 18 (layer III) may be adja In a further preferred embodiment, the catalyst is cent to the catalyst layer 16. Electrical isolation of the preactivated, i.e., brought to a higher-oxidation state. catalyst from the electron relays of the sensing layer can The catalyst can be preactivated, for example, by reac 5,262,305 7 8 tion with an oxidant to create a relatively long-lived I, 85 units/mg) solution (100 mg/ml in a 0.1M phos oxidized catalyst intermediate. Such preactivation obvi phate buffer pH 7.2 containing 5 mg/ml glutaralde ates the necessity for oxidant to be present in the sample hyde) on top of the barrier layer and was left for two during the bioassay. hours to set. The following specific examples illustrate how the Electrochemical measurements were performed with invention may be carried out but should not be consid a Princeton Applied Research Model 273 potentiostat/- ered as limiting the invention. galvanostat and recorded on a x-y-t chart recorder. Measurements were performed in a three-electrode cell EXAMPLE 1. containing 5 ml of an electrolyte solution (0.1M phos A piece of glassy carbon rod (10 mm long, 3 mm 10 phate buffer, pH 7.2 and 0.1M NaCl). The multilayer diameter, V25 Vitreous Carbon, Atomergic Chemetals biosensor was used as the working electrode, a satu Corp., Farmington, N.Y.) was sealed in a glass tube rated calomel electrode (SCE) was used as the refer with the insulating epoxy resin (Quick Stic-GC Elec ence electrode, and a platinum wire was used as the tronics, Rockford, Ill.). A copper wire was attached to counter electrode. The potential of the working elec the rear end of the rod with electrically conducting 15 silver epoxy (Epo-tek H2OE, Epoxy Technology, Inc., trode was held at 400 mV with respect to the SCE Billerica, Mass.). The top end of the glass tube was electrode and the current flowing was measured as a polished with sand paper (grit 600) until a smooth sur function of time. Five microliters of a stock solution of face was obtained, exposing a 3 mm disc of glassy car glucose or of the interferant were added to the electro bon (the electrode surface). The electrode surface was 20 lyte solution and rapidly mixed. Stock solutions of the pretreated by sequentially polishing with alumina pow interferant compounds (ascorbate, urate, bilirubin and der (5.0, 1.0 and 0.3 micrometer particle size). After acetaminophen) were freshly prepared because solu each polishing step the electrode surface was rinsed tions of these compounds are unstable in air. with deionized water, sonicated for 3 minutes and dried FIG. 4 shows the results of an experiment using a under a stream of nitrogen. 25 sensor as described above. In this experiment, glucose (final concentration 2.0 mM) and ascorbate (final con Synthesis of the Osmium Redox Polymer, ("POs-EA') centration 0.1 mM) were separately added and mixed Cis-bis (2,2'-bipyridine-N,N') dichloroosmium (II) rapidly. FIG. 4(a) shows the currents measured in the Lay, P. A.; Sargeson, A. M.; Taube, H., Inorg. Synth., absence of hydrogen peroxide. FIG. 4(b) shows the Vol. 24, 1986, pp. 29-306) (0.494 g, 0.864 mmol) and 30 currents measured in the presence of hydrogen peroxide poly(4-vinylpyridine) (PVP, Polysciences-Warrington, (0.1 mM). Pa., MW 50,000) (0.430 g, 4.09 milliequivalent) were FIG. 5 shows the results of a similar experiment with heated under nitrogen at reflux in 18 ml ethylene glycol different interferants. In this experiment, glucose (final for 2 h. After cooling to room temperature, 30 ml di concentration 2.0 mM) and acetaminophen (final con methyl formamide and 1.5g 2-bromoethylamine hydro 35 centration 0.1 mM) were separately added and mixed bromide (7.3 millimole) were added and the solution rapidly. FIG. 5(a) shows the currents in the absence of was stirred at 45° C. overnight. The crude polymer was hydrogen peroxide. FIG. 5(b) shows the currents mea precipitated by pouring the solution into rapidly stirred sured in the presence of hydrogen peroxide (0.1 mM). acetone. The hygroscopic precipitate was collected, As shown in FIGS. 4 and 5, when H2O2 is present the dissolved in H2O, filtered, and precipitated as the PF6 false signal from the interferant is substantially reduced. salt by addition of a solution of NH4PF6. The dry PF6 The glucose signal is not affected by the catalyst con salt (0.49 g) was then dissolved in 20 ml acetonitrile, taining layer that eliminates the interferants. Similar diluted with 50 ml H2O and stirred over 5.2 g anion results are obtained when uric acid or bilirubin are the exchange beads (Bio-RadAG1-X4, chloride form) for 2 interferants instead of ascorbate or acetaminophen. Sim hours. This solution was filtered, and then evaporated in 45 ilar results are also obtained when a mixture of interfer vacuum to about 10 ml. Concentrated HCl was then ants is present. It is anticipated that similar results will added to bring the solution to pH 2, and the solution be obtained from other interferants (such as cysteine) was dripped into rapidly stirred acetonitrile. The pre that are oxidized by hydrogen peroxide in the presence cipitate was filtered and dried in a vacuum desiccator. of the catalyst (e.g. peroxidase) containing layer. The Preparation of the analyte (glucose) sensing layer 50 results shown in FIGS. 4 and 5 are for experiments done (layer I) comprised preparing a solution containing 2 with separately added glucose and interferant. When a mg/ml osmium redox polymer (POs-EA), 0.12 mg/ml mixture containing glucose and one or more of the polyethylene glycol diglycidyl ether (Polysciences MW interferants is injected in the presence of H2O2 the cur 400), and 1 mg/ml glucose oxidase (Sigma type X, St. rent measured is equivalent to that measured for the Louis, Mo., 128 units/mg) in a 5 mM HEPES buffer 55 injection of glucose alone. This indicates that the inter solution, pH 7.8. A one microliter droplet of the solu ferant eliminating catalyst layer works as expected and tion was applied to the electrode surface. The electrode prevents multiple interferants from reaching the elec was left in a vacuum desiccator at room temperature for troactive layer. 48 hours to set. The glucose sensing layer was further The biosensor described in Example 1 is an embodi crosslinked by dipping the electrode in a 0.25% glutar ment appropriate for use where hydrogen peroxide may aldehyde solution for 5 seconds, followed by rinsing be added to the analyte test solution from an external with a phosphate buffer (pH 7.2, 0.1 molar (M) solution source. Examples of applications of this method include for 30 minutes. The purpose of this step is to avoid use for clinical analysis of samples (ex vivo), or analysis electrical contact between the electron relays in the of samples in industrial processes, such as in food and glucose sensing layer and the peroxidase in the adjacent 65 beverage preparation. This biosensor is particularly layer. appropriate in flow injection analysis where a pulse of The catalyst containing layer (layer II) was prepared an oxidant solution can preceed a pulse of the analyte by depositing 5 microliters of a peroxidase (Sigma, type solution. 5,262,305 9 10 The elimination of interferants described in these EXAMPLE 2 three examples is also beneficial in that such elimination An electrode was prepared as described in Example improves the stability of the sensor, the sensing layer of 1. On top of the catalyst containing layer a hydrogen which may be degraded by reaction with intermediates peroxide generating layer containing lactate oxidase 5 generated by the oxidation of interferants. Specifically, (LOD) was immobilized by crosslinking it with glutar the catalytic oxidation of urate ions improves the long aldehyde (FIG. 3). This layer was prepared by deposit term stability of the sensors. ing 5 microliters of LOD (Finnsugar, Schaumburg, Ill., 33 units/mg) solution (100/mg/ml in a 0.1M phosphate EXAMPLE 4 buffer pH 7.2 containing 5 mg/ml glutaraldehyde) on 10 Poly-(N-vinylimidazole) (PVI) prepared by the the catalyst layer. The sensor was then left for two method of Chapiro et al., Eur. Polym. J., 24: 1019-1028 hours to set. (1988) was dissolved in water (10 mg/ml). Hemin (bo The experimental use of this electrode was similar to vine, #85,856-0 Aldrich, Milwaukee, Wis.) was dis that described in Example 1 except that the oxidant was solved in a water/ethanol (25/75 by volume) solution generated from lactate and molecular oxygen. Thus, 15 containing NaOH (0.01M). The concentration of the hydrogen peroxide was not added to the analyte solu hemin in this solution was 5 mg/ml. tion. The results obtained with this electrode were simi A crosslinked hemin-containing gel was prepared by lar to the results described in Example 1. one of the two following methods and cured directly on The biosensor described in Example 2 is preferred the surface of a polished glassy carbon electrode similar when it is undesirable to add hydrogen peroxide to the 20 to the one described in Example 1: analyte test solution from an external source. Examples 1) A PVIgel was prepared using as a diepoxide cross of such cases include in vivo measurements, disposable linker polyethyleneglycol diglycidyl ether (PEGDGE), sensors for analysis of small amounts of analyte solution average molecular weight 400 (#08210 Polysciences (for example, blood drops), or industrial fermenters or Inc., Warrington, Pa.) (2 mg/ml solution in water). Ten reactors. 25 microliters of PVI solution were mixed with 2 ul of PEGDGE solution and coated on top of the electrode. EXAMPLE 3 After curing for 24 hours the crosslinked gel was An electrode was prepared as described in Example 1 dipped into the hemin solution for 5 minutes and then for the electrode surface pretreatment and analyte sens rinsed with water. In this step, hemin was complexed ing layer preparation. This layer was not further cross 30 into the PVI matrix, binding to the imidazole groups of linked with glutaraldehyde nor was it coated with a the polymer. barrier layer. The catalyst containing layer was coated 2) A gel was prepared by a one step procedure using directly on top of the analyte sensing layer by immobi the hemin as the crosslinker. Ten microliters of PVI lizing peroxidase on a hydrazide polymer matrix. Spe solution were mixed with 10 ul of hemin solution, ap cifically, the catalyst containing layer was immobilized 35 plied on top of the electrode, and allowed to dry for 2 as follows: Solution (A) comprised 5 mg/ml polyacryl hours. The resulting film was no longer water soluble amide hydrazide (Water soluble, Sigma #P9905) dis and contained the hemin necessary as catalyst for inter solved in water. Solution (B) comprised 2 mg peroxi ferant elimination. dase (Sigma, type VI, 260 units/mg) dissolved in 100 FIG. 7 shows the results obtained with a glassy car microliters 0.1M sodium bicarbonate solution. Sodium bon electrode that was directly coated with hemin by periodate (50 microliter of a 12 mg/ml solution) was the first method. The experimental measurement condi added to solution (B) and that solution was incubated in tions were similar to those described in Example 1. It the dark at room temperature (20-25 C.) for 2 hours. can be seen that injection of ascorbate resulted in an After the incubation, 7.5 microliter from Solution (A) oxidation current. When hydrogen peroxide was added were added to Solution (B) and 10 microliter of the 45 to the solution, the current decreased indicating elimi mixture were applied on top of the glucose sensing nation of the ascorbate due to its PVI-hemin catalyzed layer. The electrode was left to set for 2 hours and then oxidation by H2O2. used. In an alternative method, the crosslinked film could be formed without the polyacrylamide hydrazide EXAMPLE 5 polymer. SO The basis of the delayed elimination of interferants is It is understood that the above-described method demonstrated using an electrode produced as described using Solutions (A) and (B) are interchangeable with for Example 3. The experimental set-up was similar to similar procedures using glutaraldehyde (or similar that described for Example 1. compounds), and vice versa. The method using Solu Ascorbate (final concentration 0.1 mM) was injected tions (A) and (B) tends to be milder than other methods 55 into the cell and rapidly mixed to achieve an homogene because it tends to destroy less of compounds it ous solution. As shown in FIG. 8, when the electrode contactS. was not preactivated, injection of ascorbate resulted in This electrode was used in a manner similar to that a rapid rise in the oxidation current (false signal). When described for Example 1 except that a more oxidizing the same electrode was preactivated by its exposure to potential (500 mV v. SCE instead of 400 mV vs. SCE) 0.2 mM H2O2 for 10 seconds followed by rinsing with was applied to the working electrode. The results ob the buffer solution, injection of ascorbate resulted in no tained with this electrode were similar to the results response for over 100 seconds whereafter the rise of the described in Example 1. oxidation current remained slow. An advantage of this biosensor structure is that it is simpler, having one less layer than the biosensor of 65 EXAMPLE 6 Example 1 (i.e., does not entail application of an inter All experiments in this example were conducted in a mediate layer between the analyte sensing layer and the petri dish electrochemical cell (35 mm diameter)x10 catalyst containing layer). mm height). The working electrode was a glassy carbon 5,262,305 11 12 (vitreous carbon) rotating-disk electrode (3 mm diame from the scope and spirit of the invention as described ter) encapsulated in a teflon rod (12 mm diameter), above or claimed hereafter. mounted on a Pine Instruments (Grove City, Pa.) We claim: AFMSRX electrode rotator. A standard calomel elec 1. A biosensor comprising: trode was the reference electrode and a platinum wire an electrode on which an analyte is electrooxidized at was the counter electrode. The electrolyte solution (5 a given applied potential; and ml) contained phosphate buffer (pH 7.2, 0.1M) and an interferant-eliminating layer substantially covering NaCl (0.1.M). A model 273 potentionstat/galvanostat the electrode but electrically isolated therefrom at (EG & G Princeton Applied Research) using the model the given applied potential, comprising a catalyst 270 electrochemical analysis system software was used O which is capable of catalyzing substantial oxidation throughout the experiments. of a plurality of interferants but not substantial The electrodes were assembled in a fixed position oxidation of the analyte. while the petri dish was mounted on an adjustable base 2. A biosensor comprising: that allowed to change its position with respect to the an electrode on which an analyte is electrooxidized at electrodes. There were two possible positions: (1) when 15 a given applied potential; and the cell was raised the electrodes were immersed into an interferant-eliminating layer substantially covering the cell solution, (2) when the cell was lowered the the electrode but electrically isolated therefrom, electrodes were no longer in contact with the solution comprising a preactivated, oxidized catalyst which and were exposed to the air. is capable of catalyzing substantial oxidation of a Prior to the beginning of each measurement the cell plurality of interferants but not substantial oxida was lowered and the electrodes rinsed three times with tion of the analyte. buffer solution and dried by blowing air and rotating 3. The biosensor of claim 2, further comprising a the working electrode at 2500 RPM for 10 seconds to sensing layer in which analyte is electrooxidized, said expel any remaining buffer. Measurement was initiated sensing layer substantially covering and in electrical by rising the petri dish in such a way that the three 25 contact with the electrode. electrodes were submerged into the analyte solution 4. The biosensor of claim 3, wherein the sensing layer and immediately applying the working potential while includes a sensing enzyme which catalyzes oxidation of measuring the resulting current for several minutes. analyte. Between measurements the petri dish was lowered ex 5. The biosensor of claim 4, wherein the sensing en posing the electrodes that were again washed and dried zyme is an oxidoreductase. in the above described fashion. At this point the elec 6. The biosensor of claim 5, wherein said oxidoreduc trode was exposed to the oxidant for preactivation, tase is glucose oxidase, lactate oxidase, xanthine oxi washed and dried again. dase, cholesterol oxidase, pyruvate oxidase, L-amino FIG. 9 shows the results of experiments carried out acid oxidase, D-amino acid oxidase, alcohol oxidase, by the above-described manner with a working elec 35 urate oxidase, aldehyde oxidase, glycolate oxidase, or trode prepared in a similar way to that described in sarcosine oxidase. Example 3. In the first experiment, the electrode was 7. The biosensor of claim 6, wherein said oxidoreduc not preactivated and after rinsing and drying the elec tase is glucose oxidase. trode, the cell containing 0.5 mMascorbate and 5.0 mM 8. The biosensor of claim 2, wherein the preactivated glucose was raised. A potential of 400 mV vs SCE was catalyst is insulated from electrical contact with the applied to the working electrode and as a result a rapid electrode by a physical barrier. rise in oxidation current curve (a)) was observed. 9. The biosensor of claim 8, wherein the physical When the same experiment was repeated after the barrier is a membrane. working electrode had been preactivated by exposure 10. The biosensor of claim 9, wherein the membrane to H2O2 (0.2 mM), curve (b) was obtained. A control 45 is an ion exchange membrane. experiment was performed with a cell that did not con 11. The biosensor of claim 5, wherein relative redox tain ascorbate but only glucose (5.0 mM). The glucose potentials at the sensing layer and at the interferant only control experiment is shown in curve (c). In this eliminating layer are such that at the given applied case, the currents were the same for the preactivated potential, the oxidoreductase is in electrical contact and the non-preactivated electrode. 50 with the electrode and the interferant-eliminating layer These results are explained by the elimination of as is electrically isolated from the electrode. corbate in curve (b). For up to 50 seconds from the start 12. The biosensor of claim 2, wherein said catalyst is of the measurement, the current measured with the horseradish peroxidase, cytochrome c peroxidase, chlo preactivated electrode (b) in the presence of glucose roperoxidase, lactoperoxidase, thyroid peroxidase, Jap and interferant is similar to that measured when only 55 anese radish peroxidase a, Japanese radish peroxidase c, glucose is present (c). Only after this period, in which myeloperoxidase, NADH peroxidase, turnip peroxidase centers oxidized in the preactivation step are exhausted, A1, turnip peroxidase A2, turnip peroxidase B, turnip did the current in curve (b) start increasing to eventu peroxidase D, glutathione peroxidase, or a transition ally attain the level of curve (a). These experiments metal porphyrin. show that for the preactivated electrode the current 60 13. The biosensor of claim 12, wherein said catalyst is measured before the onset of the interferant ascorbate horseradish peroxidase, oxidation (50 seconds in this case) is solely a function of 14. The biosensor of claim 12, wherein said catalyst is to the glucose concentration and is not be affected by an iron (III) porphyrin. the presence of ascorbate. 15. The biosensor of claim 14, wherein said iron (III) Although the invention has been described with ref. 65 porphyrin is hemin. erence to its preferred embodiments, those of ordinary 16. A process for producing a preactivated interfer skill in the art may, upon reading this disclosure, appre ant-eliminating biosensor, said process comprising the ciate changes and modifications which do not depart steps of: 5,262,305 13 14 substantially covering an electrode on which an ana reacting the catalyst with an oxidant to increase the lyte is electrooxidized with an interferant-eliminat oxidation state of the catalyst and to form a stable, ing layer comprising a catalyst, said catalyst capa oxidized catalyst intermediate. ble of catalyzing substantial oxidation of a plurality 18. A process for analyzing analyte in the presence of of interferants but not substantial oxidation of the 5 a plurality of interferants in a test sample comprising the analyte; and steps of: reacting the catalyst with an oxidant to substantially contacting a sample with a preactivated, interferant eliminating biosensor having an electrode and an increase the oxidation state of the catalyst and to interferant eliminating layer; form a stable, oxidized catalyst intermediate. 10 substantially oxidizing interferants in the interferant 17. A process for producing a preactivated, interfer eliminating layer; and ant-eliminating biosensor, said process comprising the detecting the analyte at the electrode in the absence steps of: of substantial interference. substantially covering an enzyme electrode on which 19. The process of claim 18, wherein said contacting an analyte is electrooxidized with an interferant 15 is in the absence of oxidant in the sample. eliminating layer containing a catalyst, said catalyst 20. The process of claim 18, wherein said analyte is capable of catalyzing substantial oxidation of a glucose, lactate, xanthine, cholesterol, pyruvate, an L plurality of interferants but not substantial oxida or D-amino acid, alcohol, glycolate, or sarcosine. tion of the analyte; is 20

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