CA2401365A1 - Reagents and methods for detecting analytes, and devices comprising reagents for detecting analytes - Google Patents
Reagents and methods for detecting analytes, and devices comprising reagents for detecting analytes Download PDFInfo
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- CA2401365A1 CA2401365A1 CA002401365A CA2401365A CA2401365A1 CA 2401365 A1 CA2401365 A1 CA 2401365A1 CA 002401365 A CA002401365 A CA 002401365A CA 2401365 A CA2401365 A CA 2401365A CA 2401365 A1 CA2401365 A1 CA 2401365A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
Abstract
Reagents for detecting an analyte are described. A reagent comprises (a) an enzyme selected from the group consisting of a flavoprotein, a quino-protein, and a combination thereof; and (b) a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof. In addition, reagents having good stability to radiation sterilization are described. Electrochemical sensors and sampling devices comprising such reagents, methods of producing a sterilized device including such rea-gents, and methods for detecting an analyte which utilize such reagents are described as well.
Description
REAGENTS AND METHODS FOR DETECTING ANALYTES, AND
DEVICES COMPRISING REAGENTS FOR DETECTING ANALYTES
BACKGROUND
The present invention relates to reagents, methods and devices for s measurement of analytes and, more particularly, to reagents, methods and devices for the measurement of glucose in the blood.
The monitoring of certain analyte concentrations in the body enables early detection of health risks, and identifies the need for the introduction of therapeutic measures. One of the most commonly monitored analytes is glu to cose, the blood concentration of which is important in determining the appro-priate dosages of insulin for diabetics. Various methods have been devel oped for monitoring glucose levels in the blood, including the use of electro chemical biosensors. Electrochemical biosensors are based on enzyme-catalyzed chemical reactions involving the analyte of interest. In the case of 15 glucose monitoring, the relevant chemical reaction is the oxidation of glucose to gluconolactone. This oxidation is catalyzed by a variety of enzymes, some of which may contain a bound coenzyme such as nicotinamide adenine dinu-cleotide (phosphate) (NAD(P)), while others may contain a bound cofactor such as flavin adenine dinucleotide (FAD) or pyrroloquinolinequinone (PQQ).
DEVICES COMPRISING REAGENTS FOR DETECTING ANALYTES
BACKGROUND
The present invention relates to reagents, methods and devices for s measurement of analytes and, more particularly, to reagents, methods and devices for the measurement of glucose in the blood.
The monitoring of certain analyte concentrations in the body enables early detection of health risks, and identifies the need for the introduction of therapeutic measures. One of the most commonly monitored analytes is glu to cose, the blood concentration of which is important in determining the appro-priate dosages of insulin for diabetics. Various methods have been devel oped for monitoring glucose levels in the blood, including the use of electro chemical biosensors. Electrochemical biosensors are based on enzyme-catalyzed chemical reactions involving the analyte of interest. In the case of 15 glucose monitoring, the relevant chemical reaction is the oxidation of glucose to gluconolactone. This oxidation is catalyzed by a variety of enzymes, some of which may contain a bound coenzyme such as nicotinamide adenine dinu-cleotide (phosphate) (NAD(P)), while others may contain a bound cofactor such as flavin adenine dinucleotide (FAD) or pyrroloquinolinequinone (PQQ).
2 o In biosensor applications, the redox equivalents generated in the course of the oxidation of glucose are transported to the surface of an elec-trode whereby an electrical signal is generated. The magnitude of the electri-cal signal is then correlated with concentration of glucose. The transfer of re-dox equivalents from the site of chemical reaction in the enzyme to the sur-2 5 face of the electrode is accomplished with the use of electron transfer me-diators.
A significant problem with the use of electron transfer mediators in bio-sensors is the instability of these compounds upon exposure to common envi-ronmental conditions such as temperature and moisture. As a result, the number of mediators suitable for use in glucose biosensors is quite limited.
U.S. Pat. No. 5,520,786 ('786) to Bloczynski et al. describes families of phenothiazine and phenoxazine compounds suitable for use as electron transfer mediators with the enzymes dihydronicotinamide adenine dinucleo-tide (NADH), NADPH, and analogs thereof. Cofactor based enzymes such as FAD-glucose oxidase and PQQ-glucose dehydrogenase have several ad-vantages over NAD-based enzymes, including lower cost, higher enzyme ac-tivity, increased stability, and bound as opposed to readily dissociable cofac-1 o tor.
Electron transfer mediators previously used with FAD-glucose oxidase and PQQ-glucose dehydrogenase include quinones, phenzine methosulfate, dichlorophenolindophenol and ferricyanide. Unfortunately, these compounds have proven to be highly susceptible to the environmental agents described above, and result in biosensor reagents of low stability. Thus, mediators are needed which exhibit good stability upon exposure to commonly-encountered environmental agents, and which can be used in flavoprotein- and quinopro-tein-based systems.
In addition to the need for biosensor reagents that are stable to the en-2 o vironmental agents described above, it would be desirable to provide biosen-sor reagents that are stable to the radiation conditions commonly employed in lancet sterilization. Reagents stable to such radiation sterilization could be incorporated into highly user-convenient units in which lancet and biosensor are combined.
2 s The present invention is directed to electron transfer mediators for use in flavoprotein- and quinoprotein-based biosensor reagents, which exhibit im-proved stability to both environmental interferents and to radiation steriliza-tion.
A significant problem with the use of electron transfer mediators in bio-sensors is the instability of these compounds upon exposure to common envi-ronmental conditions such as temperature and moisture. As a result, the number of mediators suitable for use in glucose biosensors is quite limited.
U.S. Pat. No. 5,520,786 ('786) to Bloczynski et al. describes families of phenothiazine and phenoxazine compounds suitable for use as electron transfer mediators with the enzymes dihydronicotinamide adenine dinucleo-tide (NADH), NADPH, and analogs thereof. Cofactor based enzymes such as FAD-glucose oxidase and PQQ-glucose dehydrogenase have several ad-vantages over NAD-based enzymes, including lower cost, higher enzyme ac-tivity, increased stability, and bound as opposed to readily dissociable cofac-1 o tor.
Electron transfer mediators previously used with FAD-glucose oxidase and PQQ-glucose dehydrogenase include quinones, phenzine methosulfate, dichlorophenolindophenol and ferricyanide. Unfortunately, these compounds have proven to be highly susceptible to the environmental agents described above, and result in biosensor reagents of low stability. Thus, mediators are needed which exhibit good stability upon exposure to commonly-encountered environmental agents, and which can be used in flavoprotein- and quinopro-tein-based systems.
In addition to the need for biosensor reagents that are stable to the en-2 o vironmental agents described above, it would be desirable to provide biosen-sor reagents that are stable to the radiation conditions commonly employed in lancet sterilization. Reagents stable to such radiation sterilization could be incorporated into highly user-convenient units in which lancet and biosensor are combined.
2 s The present invention is directed to electron transfer mediators for use in flavoprotein- and quinoprotein-based biosensor reagents, which exhibit im-proved stability to both environmental interferents and to radiation steriliza-tion.
SUMMARY
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this sum-mary. By way of introduction, the presently preferred embodiments described s herein are directed towards remedying the aforementioned stability problems of electron transfer mediators and enzyme biosensors.
Briefly stated, a composition aspect of the present invention is directed to a reagent for detecting an analyte, comprising (a) an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination to thereof; and (b) a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and a combination thereof.
A first apparatus aspect of the present invention is directed to an elec-trochemical sensor comprising: (a) a working electrode having a surface; and (b) a second electrode coupled to the working electrode. The surface of the 15 working electrode is coated with a solution or mixture of a reagent comprising an enzyme selected from the group consisting of a flavoprotein, a quinopro-tein, and a combination thereof; and a mediator selected from the group con-sisting of a phenothiazine, a phenoxazine, and a combination thereof.
A second apparatus aspect of the present invention is directed to a de-2 o vice for measuring an analyte, comprising (a) a lancet; and (b) a sampling chamber connected to the lancet. The sampling chamber comprises a rea-gent comprising an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof;
and (b) a mediator selected from the group consisting of a phenothiazine, a 2 s phenoxazine, and a combination thereof.
A first method aspect of the present invention is directed to a method of producing a sterilized device for measuring an analyte, comprising (a) pro-viding a device in accordance with the present invention, and (b) irradiating the device with E-beam or gamma ray radiation.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this sum-mary. By way of introduction, the presently preferred embodiments described s herein are directed towards remedying the aforementioned stability problems of electron transfer mediators and enzyme biosensors.
Briefly stated, a composition aspect of the present invention is directed to a reagent for detecting an analyte, comprising (a) an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination to thereof; and (b) a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and a combination thereof.
A first apparatus aspect of the present invention is directed to an elec-trochemical sensor comprising: (a) a working electrode having a surface; and (b) a second electrode coupled to the working electrode. The surface of the 15 working electrode is coated with a solution or mixture of a reagent comprising an enzyme selected from the group consisting of a flavoprotein, a quinopro-tein, and a combination thereof; and a mediator selected from the group con-sisting of a phenothiazine, a phenoxazine, and a combination thereof.
A second apparatus aspect of the present invention is directed to a de-2 o vice for measuring an analyte, comprising (a) a lancet; and (b) a sampling chamber connected to the lancet. The sampling chamber comprises a rea-gent comprising an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof;
and (b) a mediator selected from the group consisting of a phenothiazine, a 2 s phenoxazine, and a combination thereof.
A first method aspect of the present invention is directed to a method of producing a sterilized device for measuring an analyte, comprising (a) pro-viding a device in accordance with the present invention, and (b) irradiating the device with E-beam or gamma ray radiation.
A second method aspect of the present invention is directed to a method for detecting an analyte which undergoes a chemical reaction, the method comprising (a) providing an electrode surface; (b) catalyzing the chemical reaction with an enzyme selected from the group consisting of a fla-s voprotein, a quinoprotein, and a combination thereof; (c) generating a redox equivalent by the chemical reaction; and (d) transferring the redox equivalent to the electrode surface using a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
The presently preferred embodiments discussed herein may possess l o one or more advantages relative to other flavoprotein- and quinoprotein-based biosensor reagents, which can include but are but not limited to: im-proved biosensor reagent stability; enhanced electron transfer capability of mediators; ability to tune mediators for optimum electrode operation; reduced oxygen susceptibility of mediators; increased thermal stability of mediators;
15 increased stability of mediators to ambient humidity; lower redox potential of mediators; reduced susceptibility to interferents in blood; and stability of bio-sensor reagents to radiation sterilization conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an exploded perspective view of an electrochemical sen-2 o sor embodying features of the present invention.
FIG. 2 shows a partial view of an electrode surface coated with a rea-gent embodying features of the present invention.
FIG. 3 shows an exploded perspective view of a device for measuring an analyte in accordance with the present invention.
2s FIG. 4 shows a graph of background currents for 3 formulations of bio-sensor reagents exposed to increasing levels of radiation.
FIG. 5 shows a graph of the current response of radiation sterilized biosensor reagents upon exposure to glucose.
FIG. 6 shows a plot of current vs. glucose concentration at increasing 3o time intervals for a PQQ-glucose dehydrogenase/phenothiazine biosensor.
FIG. 7 shows a plot of current vs. glucose concentration for a [FAD]-glucose oxidase/phenothiazine biosensor.
FIG. 8 shows a plot of current vs. glucose concentration for a PQQ-glucose dehydrogenase/phenothiazine biosensor reagent subjected to heat 5 stress and humidity stress.
FIGS. 9-13 show plots of current vs. glucose concentration for 5 for-mulations of PQQ-glucose dehydrogenase/phenothiazine biosensors ex-posed to varying levels of radiation.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EM-io BODIMENTS
Throughout this description and in the appended claims, the following definitions are to be understood: The term "analyte" refers to one or a plural-ity of species having a concentration of interest. The term "flavoprotein" re-fers to enzymes containing flavin cofactors. The term "quinoprotein" refers to enzymes containing PQQ or similar cofactors. The phrase "redox equivalent"
refers to one or a plurality of charged species (e.g., electrons) produced in electrochemical reactions involving the analyte. The phrase "E-beam irradia-tion" or "electron beam irradiation" refers to a process of exposure to a con-centrated, high-current stream of electrons. The terms "alkyl," "alkenyl," "al-e o kynyl," "aryl," "heteroaryl," "cyclic," "heterocyclic," "halo,"
"haloalkyl," "carboxy,"
"carboxyalkyl," "alkoxycarbonyl," "aryloxycarbonyl," "aromatic keto,"
"aliphatic keto," "alkoxy," "aryloxy," "nitro," "dialkylamino," "aminoalkyl," "sulfo,"
"dihy-droxyboron," and the like refer to substituents well known in the art, which may be branched or unbranched and may themselves be substituted with one 2 5 or more substituents. The phrase "biosensor reagent" refers to the combina-tion of an enzyme which catalyzes a reaction of an analyte, and a phenothi-azine and/or phenoxazine mediator. The term "bioburden" refers to the population of viable microorganisms on a product determined immediately prior to irradiation.
The presently preferred embodiments discussed herein may possess l o one or more advantages relative to other flavoprotein- and quinoprotein-based biosensor reagents, which can include but are but not limited to: im-proved biosensor reagent stability; enhanced electron transfer capability of mediators; ability to tune mediators for optimum electrode operation; reduced oxygen susceptibility of mediators; increased thermal stability of mediators;
15 increased stability of mediators to ambient humidity; lower redox potential of mediators; reduced susceptibility to interferents in blood; and stability of bio-sensor reagents to radiation sterilization conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an exploded perspective view of an electrochemical sen-2 o sor embodying features of the present invention.
FIG. 2 shows a partial view of an electrode surface coated with a rea-gent embodying features of the present invention.
FIG. 3 shows an exploded perspective view of a device for measuring an analyte in accordance with the present invention.
2s FIG. 4 shows a graph of background currents for 3 formulations of bio-sensor reagents exposed to increasing levels of radiation.
FIG. 5 shows a graph of the current response of radiation sterilized biosensor reagents upon exposure to glucose.
FIG. 6 shows a plot of current vs. glucose concentration at increasing 3o time intervals for a PQQ-glucose dehydrogenase/phenothiazine biosensor.
FIG. 7 shows a plot of current vs. glucose concentration for a [FAD]-glucose oxidase/phenothiazine biosensor.
FIG. 8 shows a plot of current vs. glucose concentration for a PQQ-glucose dehydrogenase/phenothiazine biosensor reagent subjected to heat 5 stress and humidity stress.
FIGS. 9-13 show plots of current vs. glucose concentration for 5 for-mulations of PQQ-glucose dehydrogenase/phenothiazine biosensors ex-posed to varying levels of radiation.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EM-io BODIMENTS
Throughout this description and in the appended claims, the following definitions are to be understood: The term "analyte" refers to one or a plural-ity of species having a concentration of interest. The term "flavoprotein" re-fers to enzymes containing flavin cofactors. The term "quinoprotein" refers to enzymes containing PQQ or similar cofactors. The phrase "redox equivalent"
refers to one or a plurality of charged species (e.g., electrons) produced in electrochemical reactions involving the analyte. The phrase "E-beam irradia-tion" or "electron beam irradiation" refers to a process of exposure to a con-centrated, high-current stream of electrons. The terms "alkyl," "alkenyl," "al-e o kynyl," "aryl," "heteroaryl," "cyclic," "heterocyclic," "halo,"
"haloalkyl," "carboxy,"
"carboxyalkyl," "alkoxycarbonyl," "aryloxycarbonyl," "aromatic keto,"
"aliphatic keto," "alkoxy," "aryloxy," "nitro," "dialkylamino," "aminoalkyl," "sulfo,"
"dihy-droxyboron," and the like refer to substituents well known in the art, which may be branched or unbranched and may themselves be substituted with one 2 5 or more substituents. The phrase "biosensor reagent" refers to the combina-tion of an enzyme which catalyzes a reaction of an analyte, and a phenothi-azine and/or phenoxazine mediator. The term "bioburden" refers to the population of viable microorganisms on a product determined immediately prior to irradiation.
A biosensor reagent for detecting an analyte in accord with the present invention includes (1) an enzyme selected from the group consisting of a fla-voprotein, a quinoprotein, and a combination thereof; and (2) a mediator se-lected from the group consisting of a phenothiazine, a phenoxazine, and a s combination thereof.
The nature of the analyte monitored in accord with the present inven-tion is unrestricted, provided the analyte undergoes a chemical reaction that is catalyzed by an enzyme selected from the group consisting of a flavopro-tein, a quinoprotein, and a combination thereof. Preferred analytes include to but are not limited to glucose, lactate, D-amino acids, ascorbate, alcohol, cholesterol, choline, and acetylcholine.
Flavoproteins in accord with the present invention include FAD-glucose oxidase (Enzyme Classification No. 1.1.3.4), Flavin-hexose oxidase (EC No.
1.1.3.5) and FAD-glucose dehydrogenase (EC No. 1.1.99.10) For information 15 relating to these flavoproteins, see: Adriaan Joseph Jan Olsthoorn, "Struc-tural and Mechanistic Aspects of Soluble Quinoprotein Glucose Dehydroge-nase from Acinetobacfer calcoaceticus," Ph.D. dissertation, Delft University of Technology, The Netherlands, 1999. Additional oxidase enzymes for use in accord with the present invention include but are not limited to lactate oxi-2 o dase, cholesterol oxidase, alcohol oxidase (e.g., methanol oxidase), d-aminoacid oxidase, choline oxidase, and FAD derivatives thereof. A pre-ferred flavoprotein for use in accord with the present invention is FAD-glucose oxidase.
Quinoproteins in accord with the present invention include but are not 2 s limited to membrane bound and soluble PQQ-glucose dehydrogenase (EC
No. 1.1.99.17). Information relating to PQQ-glucose dehydrogenase can be found in the Olsthoorn reference cited above. Additional quinoprotein en-zymes for use in accord with the present invention include but are not limited to lactate dehydrogenase, aldehyde dehydrogenase, methylamine dehydro-3 o genase, alcohol dehydrogenase (e.g., methanol dehydrogenase), and PQQ
The nature of the analyte monitored in accord with the present inven-tion is unrestricted, provided the analyte undergoes a chemical reaction that is catalyzed by an enzyme selected from the group consisting of a flavopro-tein, a quinoprotein, and a combination thereof. Preferred analytes include to but are not limited to glucose, lactate, D-amino acids, ascorbate, alcohol, cholesterol, choline, and acetylcholine.
Flavoproteins in accord with the present invention include FAD-glucose oxidase (Enzyme Classification No. 1.1.3.4), Flavin-hexose oxidase (EC No.
1.1.3.5) and FAD-glucose dehydrogenase (EC No. 1.1.99.10) For information 15 relating to these flavoproteins, see: Adriaan Joseph Jan Olsthoorn, "Struc-tural and Mechanistic Aspects of Soluble Quinoprotein Glucose Dehydroge-nase from Acinetobacfer calcoaceticus," Ph.D. dissertation, Delft University of Technology, The Netherlands, 1999. Additional oxidase enzymes for use in accord with the present invention include but are not limited to lactate oxi-2 o dase, cholesterol oxidase, alcohol oxidase (e.g., methanol oxidase), d-aminoacid oxidase, choline oxidase, and FAD derivatives thereof. A pre-ferred flavoprotein for use in accord with the present invention is FAD-glucose oxidase.
Quinoproteins in accord with the present invention include but are not 2 s limited to membrane bound and soluble PQQ-glucose dehydrogenase (EC
No. 1.1.99.17). Information relating to PQQ-glucose dehydrogenase can be found in the Olsthoorn reference cited above. Additional quinoprotein en-zymes for use in accord with the present invention include but are not limited to lactate dehydrogenase, aldehyde dehydrogenase, methylamine dehydro-3 o genase, alcohol dehydrogenase (e.g., methanol dehydrogenase), and PQQ
derivatives thereof. A preferred quinoprotein for use in accord with the pres-ent invention is PQQ-glucose dehydrogenase.
Mediators in accord with the present invention include phenothiazines having the formula Rs R' R~ ~ S / N ~ R2 i Ra ~ ~ N ~ 5 ~ ~ Rs R
Rs Ra and phenoxazines having the formula R' R~ N ~ R2 R8 ~5 ~ R3 Ra wherein R', R2 R3, R°, R5, Rs, R', R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, to alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, car-boxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alk-oxy, aryloxy, vitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, and com-binations thereof.
In contrast to the single electron transfer carrying capability of 15 K3Fe(CN)6, mediators in accord with the present invention have the ability to carry two redox equivalents, and are therefore well suited for use in FAD and quinoprotein oxidation/reduction processes, which generally involve the transfer of two electrons. Moreover, the potential of mediators of the present invention can be tuned to the optimum potential (i.e., the potential where the 2o signal contribution from interferences is minimized) for a specific sample ma trix by varying the substitution on the aromatic rings. Electron-donating sub-stituents (e.g., alkyl, alkoxy, amine, hydroxy, etc.) result in decreased redox potentials, while electron-withdrawing substituents (e.g., carboxylic acid, es-ter, aldehyde, ketone, nitrite, vitro, sulfonic acid, trifluromethyl, etc.) result in increased redox potentials. For blood or plasma samples, the ideal potential usually lies between about -200 and about 100 mV versus a Ag/AgCI refer-ence.
The substituents on the aromatic rings, in addition to their utility in tun-s ing the redox potentials of the mediators, can also be used to enhance me-diator solubility. For example, the introduction of a substituent having the ca-pacify for hydrogen bonding can be expected to render the mediator more water soluble than a mediator lacking such substitution. In addition, these substituents can serve as functional groups for immobilizing the mediators to zo a support (e.g., the electrode surface or, alternatively, a chemical matrix such as a polymer backbone, which is suitable for application to the electrode sur-face).
Preferably, mediators used in biosensor reagents according to the pre-sent invention include 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-15 diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-2 o methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-r~-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-[i-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-2 s ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, and 3-(3-phenylimino)-3H-phenothiazinesulfonic acid.
More preferably, the mediator used in accord with the present inven-tion is selected from the group consisting of ~ s ~ ~ I ~ co2H
N
Mediator I
and i so3H
Mediator II
to Relative to ferricyanide, phenothiazine mediators-in particular me-diator I-are less susceptible to oxygen degradation, more thermally stable, and more stable to ambient humidity. In addition, mediator I works at a lower redox potential than ferricyanide. For example, Eo for mediator I is approxi-mately 0 mV versus an Ag/AgCI reference, whereas Ea for ferricyanide is ap-i s proximately 250 mV versus an. Ag/AgCI reference. The lower redox potential of phenothiazine mediators is advantageous in that there is a region around 0 mV versus an Ag/AgCI reference in which the amount of electrochemical in-terferences are minimized. Thus, the impact from chemical interferents in the blood can be minimized by using these mediators.
2 o Reagents embodying features of the present invention can be incorpo-rated into a variety of biosensor devices, including but not limited to the ones described in United States Patent Nos. 5,120,420 and 5,798,031, the entire contents of which are incorporated herein by reference, except that in the event of any inconsistent disclosure or definition from the present application, 2 s the disclosure or definition herein shall be deemed to prevail.
Turning now to the drawings, FIG. 1 shows a representative electro-chemical sensor in accordance with the present invention. The electrochemi-cal sensor 34 is comprised of an insulating base 36 upon which is printed in sequence (typically by screen printing techniques), an electrical conductor s pattern 38, an electrode pattern (39 and 40), an insulating (dielectric) pattern 42 and finally a reagent layer 44, which contains a reagent embodying fea-tures of the present invention. The two parts of the electrode print, 39 and 40, provide the working and reference electrodes necessary for the electro-chemical determination.
to A detailed view of the working electrode of an electrochemical sensor in accordance with the present invention is shown in FIG. 2. A working elec-trode 2 having a surface 4 is coupled to a second electrode 6. The surface 4 is coated with a solution 8 of a reagent in accordance with the present inven-tion.
It has been found that biosensor reagents comprising PQQ-glucose dehydrogenase and certain phenothiazine mediators exhibit high stability to radiation sterilization. A preferred application of radiation stable biosensor reagents in accord with the present invention is for the development of inte-grated lancet/biosensor devices. An example of such an integrated device is 2o described in United States Patent No. 5,801,057, the entire contents of which are incorporated herein by reference, except that in the event of any incon-sistent disclosure or definition from the present application, the disclosure or definition herein shall be deemed to prevail.
FIG. 3 shows an integrated lancet/biosensor devices device 10 in 2 s which a lancet 12 is connected to a sampling chamber 14. Sampling cham-ber 14 comprises a biosensor reagent comprising PQQ-glucose dehydroge-nase and a phenothiazine and/or phenoxazine mediator. Preferably, the me-diator is a phenothiazine. More preferably, the mediator has a structure rep-resented by mediator I or mediator II above. Once sampling chamber 14 has 3 o been loaded with biosensor reagent, the entire device 10 can be subjected to radiation sterilization. Preferably, the method of sterilization involves electron beam (E-beam) irradiation or gamma irradiation.
As set forth in the Association for the Advancement of Medical Instru mentation document ANSI/AAMI/ISO 11137 -1994, products that penetrate s the skin and come into contact with the blood must have a sterility assurance level (SAL) of 10'8, which corresponds to a one in a million probability of a vi-able microorganism being present on a product unit after sterilization. The sterilization dose needed to achieve a 10'8 SAL depends on the bioburden of the sample. For example, a sample with a bioburden of 1,021 requires a ster-eo ilization dose of 24.9 kGy to achieve a 10'8 SAL.
In the examples described hereinbelow, electron beam (E-beam) irra-diation was employed as the method of sterilization. The biosensor reagents subjected to the electron beam absorb energy from the electrons. The en-ergy that is absorbed per unit mass of material is referred to as the absorbed 15 dose, and it is this absorption of energy-or dose delivery-that destroys the reproductive cells and DNA chains of microorganisms, thereby rendering a product sterile. E-beam doses of 25, 50 and 100 kGy were used because the bioburden of the biosensor reagents was unknown.
FIG. 4 shows a graph of the background currents observed for three 2 o formulations of biosensor reagents exposed to increasing levels of radiation:
(1) NAD-glucose dehydrogenase with Mediator I, (2) PQQ-glucose dehydro-genase with Ferricyanide, and (3) PQQ-glucose dehydrogenase with Media-for I. The PQQ formulations tolerated the irradiation extremely well. In con-trast, the NAD formulation exhibited poor tolerance to the sterilization condi-2s tions, and resulted in a background signal which constituted a significant amount of the glucose signal. While formulation (2) exhibited good tolerance to the radiation process, the activity of the extracted enzyme was lower than the corresponding activity of the enzyme extracted from formulation (3). Fig 5 shows a graph of current response when these radiation sterilized sensors 3o were exposed to 600 mg/dL glucose.
The manner in which a device embodying features of the present in-vention is made, and the process by which such a device is used for moni-toring an analyte, will be abundantly clear to one of ordinary skill in the art based upon joint consideration of both the preceding description, and the fol-lowing representative procedures. It is to be understood that many variations in the presently preferred embodiments illustrated herein will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.
For example, the working electrode employed in electrochemical sen-to sors according to the present invention can be varied, with suitable electrodes including but not limited to carbon electrodes, platinum electrodes, palladium electrodes, gold electrodes, and the like. Similarly, the reference electrode can be varied, with suitable electrodes including but not limited to silver-silver chloride electrodes, calomel electrodes, saturated calomel electrodes, and 15 the like. Alternatively, a quasi-reference electrode (e.g., a large surface area platinum electrode ) of the type commonly used in non-aqueous electro-chemical experiements (i.e., an electrode which does not have a specific re-dox species to which its potential is referenced) can be used in accord with the present invention. The surface areas of all electrodes employed in accor-2o dance with~the present invention are likewise subject to variation.
Preferably, the working electrode has dimensions of about 0.6 mm x 1.2 mm.
Furthermore, the compositions and pH of the buffer solutions em-ployed, and the enzyme activities and concentrations of components of the biosensor reagents, are subject to wide variation. Suitable buffer solutions 2s include but are not limited to HEPES (i.e., N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MOPS (i.e., 3-(N-morpholino)propanesulfonic acid), TES
(i.e., N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), 2-([2-hydrox-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid), PIPES (i.e., pipera-zine-N,N'-bis(2-ethanesulfonic acid)), 1,4-piperazinediethanesulfonic acid), 3 o ACES (i.e., N-(carbamoylmethyl)-2-aminoethanesulfonic acid), N-(2-acetamidol)-2-aminoethanesulfonic acid, BES (i.e., N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and Dulbecco's buffer (i.e., 0.008M sodium phos-phate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01 M potas-sium chloride, pH 7.4).
s The manner in which reagents and devices embodying features of the present invention are made, and the methods by which these reagents and devices are used for monitoring an analyte, will be abundantly clear to one of ordinary skill in the art based upon joint consideration of both the preceding description, and the following representative procedures.
to While the examples provided hereinbelow relate to in vitro applications of the biosensor reagents in accord with the present invention, it is contem-plated that these reagents can also be adapted for in vivo analyte monitoring by chemically immobilizing the phenoxazine and/or phenothiazine mediators (e.g., by chemical reaction at one or more of the substituent groups on the 15 aromatic rings), and incorporating the immobilized mediators into a device which can be implanted subcutaneously into a patient.
EXAMPLES
Preparation of Biosensor and Glucose Dose-Response 2 o A liquid chemistry reagent was prepared to be 20 Units/wL pyrolloquin-olinequinone-glucose dehydrogenase (PQQ-GDH) and 24 mM mediator I in 100 mM Sodium Phosphate, ;pH 7.4. The first component of the reagent was made by dissolving the mediator in 100 mM phosphate pH 7.4, adjusting the pH back to 7.4, and filtering the solution by forcing it through a Whatman 0.45 2s micron PTFE syringe filter. The reagent was completed by adding lyophilized PQQ-GDH (Toyobo Product No. GLD-321 ) to an activity of 20 U/~L.
The chemistry formulation was deposited onto electrodes, which had been produced using a 3-pass screen-printing process by Conductive Tech-nologies, Inc. During this process, the silver/silver chloride (DuPont 5870 ink) 3 0 leads and reference electrode were printed first onto polycarbonate base material. The second pass of Dupont 7102T carbon-graphite working elec-trode was printed on top of this. A final pass of Norcote RDMSK4954-A2 di-electric defined the working electrode area to be 0.0113 cm2.
The chemistry was deposited over the working electrode with the use of an Asymtek Automove~402 Dispensing System. The system was pro-grammed to perform the transfer by dipping a 62 mL stainless steel pin into a 1.5 mL Eppendorf vial filled with reagent. Polycarbonate lid material was laminated to the sensors creating a capillary area over the working and refer-ence electrodes capable of holding approximately 3 pL of test solution. The to capillary area, which defines the sample volume, is first formed in the poly-carbonate lid material by a coining or stamping process.
As shown in FIG. 6, reactivity of the chemistry was analyzed by gener-ating a glucose dose-response curve with buffered (100 mM phosphate, 100 mM sodium chloride, pH 7.4) samples containing a range of glucose concen-trations from 0 to 600 mg/dL. Current generated at each of the glucose con-centrations was measured using a potentiostat programmed to apply 150 mV
potential with trigger level set to 100 nA, and timing programmed to record the current at 5,10,15, and 20 seconds. The trigger level refers to a threshold level above which timing and recording are initiated.
2o Sensors formulated with 20U Glucose Oxidase/sensor and 6 mM me-diator I were deposited onto electrode sensors as above. The dose response plot shown in FIG. 7 was obtained.
Preparation of Electrochemical Biosensor and Heat/Humidity Stability Electrochemical biosensors were constructed using a screen-printing 2 s process. Sensors were comprised of a carbon working electrode and a sil-ver/silver chloride reference electrode. A solution (150 to 800n1) containing 12mM mediator I in 100mM phosphate buffer (pH 7.4), and of the enzyme PQQ-glucose dehydrogenase (10 U/oL) was deposited on the surface of the working electrode and allowed to dry at room temperature for 5 minutes prior to desiccation. The electrodes were assembled into a format having a small capillary gap, which allowed inoculation of the sensors with sample solutions.
In subsequent tests, the sensors were subjected to the following envi-ronmental conditions prior to testing: 1 ) 50 °C for 2, 4, and 8 weeks;
and 2) s room temperature with 40% relative humidity. The sensors were poised at a potential of 150mV relative to the Ag/AgCI reference electrode and the re-suiting current was measured. This mediatorlenzyme combination is quite stable to both heat stress and humidity stress as shown in FIG. 8.
Sterilization of Biosensors and Radiation Stability Data 10 . Five formulations of biosensor reagents (Table 1 ) were prepared and subjected to E-beam irradiation using SureBeam~ sterilization technology at Titan Scan Technologies (San Diego, CA). Formulation I was irradiated at 25 kGy, 50 kGy, and 100 kGy, whereas each of Formulations II-V was irradiated at 25 kGy only. In the two rightmost column headings of Table 1, the abbre-15 viation CMC refers to carboxymethylcellulose, and the abbreviation PEO re-fers to polyethylene oxide.
Table 1 FormulationEnzyme Con- ConcentrationPolymer Polymer # centration Mediator ConcentrationConcentration PQQ-GDH I CMC % PEO
Units mM
- -..
--~
FIGS. 9-13 show glucose dose response curves for each of the five 2 o formulations both before and after irradiation. The stability of the five formu-lations is high, as is clearly shown by the near overlapping of the glucose re-sponse generated before and after irradiation.
Table 2 shows the results of enzyme assays conducted on the five formulations both before and after irradiation. Enzyme activity following irra-diction remains high in all instances.
Table 2 Formulation # kGy Level Enzyme Activity I 0 4.67 25 4.32 50 4.20 100 4.24 II 0 3.31 25 3.34 I I I 0 4.93 25 4.87 1V 0 4.96 25 4.86 V 0 3.63 25 4.05 The foregoing detailed description and examples have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodi-ments illustrated herein will be obvious to one of ordinary skill in the art, and to remain within the scope of the appended claims and their equivalents.
Mediators in accord with the present invention include phenothiazines having the formula Rs R' R~ ~ S / N ~ R2 i Ra ~ ~ N ~ 5 ~ ~ Rs R
Rs Ra and phenoxazines having the formula R' R~ N ~ R2 R8 ~5 ~ R3 Ra wherein R', R2 R3, R°, R5, Rs, R', R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, to alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, car-boxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alk-oxy, aryloxy, vitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, and com-binations thereof.
In contrast to the single electron transfer carrying capability of 15 K3Fe(CN)6, mediators in accord with the present invention have the ability to carry two redox equivalents, and are therefore well suited for use in FAD and quinoprotein oxidation/reduction processes, which generally involve the transfer of two electrons. Moreover, the potential of mediators of the present invention can be tuned to the optimum potential (i.e., the potential where the 2o signal contribution from interferences is minimized) for a specific sample ma trix by varying the substitution on the aromatic rings. Electron-donating sub-stituents (e.g., alkyl, alkoxy, amine, hydroxy, etc.) result in decreased redox potentials, while electron-withdrawing substituents (e.g., carboxylic acid, es-ter, aldehyde, ketone, nitrite, vitro, sulfonic acid, trifluromethyl, etc.) result in increased redox potentials. For blood or plasma samples, the ideal potential usually lies between about -200 and about 100 mV versus a Ag/AgCI refer-ence.
The substituents on the aromatic rings, in addition to their utility in tun-s ing the redox potentials of the mediators, can also be used to enhance me-diator solubility. For example, the introduction of a substituent having the ca-pacify for hydrogen bonding can be expected to render the mediator more water soluble than a mediator lacking such substitution. In addition, these substituents can serve as functional groups for immobilizing the mediators to zo a support (e.g., the electrode surface or, alternatively, a chemical matrix such as a polymer backbone, which is suitable for application to the electrode sur-face).
Preferably, mediators used in biosensor reagents according to the pre-sent invention include 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-15 diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-2 o methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-r~-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-[i-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-2 s ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, and 3-(3-phenylimino)-3H-phenothiazinesulfonic acid.
More preferably, the mediator used in accord with the present inven-tion is selected from the group consisting of ~ s ~ ~ I ~ co2H
N
Mediator I
and i so3H
Mediator II
to Relative to ferricyanide, phenothiazine mediators-in particular me-diator I-are less susceptible to oxygen degradation, more thermally stable, and more stable to ambient humidity. In addition, mediator I works at a lower redox potential than ferricyanide. For example, Eo for mediator I is approxi-mately 0 mV versus an Ag/AgCI reference, whereas Ea for ferricyanide is ap-i s proximately 250 mV versus an. Ag/AgCI reference. The lower redox potential of phenothiazine mediators is advantageous in that there is a region around 0 mV versus an Ag/AgCI reference in which the amount of electrochemical in-terferences are minimized. Thus, the impact from chemical interferents in the blood can be minimized by using these mediators.
2 o Reagents embodying features of the present invention can be incorpo-rated into a variety of biosensor devices, including but not limited to the ones described in United States Patent Nos. 5,120,420 and 5,798,031, the entire contents of which are incorporated herein by reference, except that in the event of any inconsistent disclosure or definition from the present application, 2 s the disclosure or definition herein shall be deemed to prevail.
Turning now to the drawings, FIG. 1 shows a representative electro-chemical sensor in accordance with the present invention. The electrochemi-cal sensor 34 is comprised of an insulating base 36 upon which is printed in sequence (typically by screen printing techniques), an electrical conductor s pattern 38, an electrode pattern (39 and 40), an insulating (dielectric) pattern 42 and finally a reagent layer 44, which contains a reagent embodying fea-tures of the present invention. The two parts of the electrode print, 39 and 40, provide the working and reference electrodes necessary for the electro-chemical determination.
to A detailed view of the working electrode of an electrochemical sensor in accordance with the present invention is shown in FIG. 2. A working elec-trode 2 having a surface 4 is coupled to a second electrode 6. The surface 4 is coated with a solution 8 of a reagent in accordance with the present inven-tion.
It has been found that biosensor reagents comprising PQQ-glucose dehydrogenase and certain phenothiazine mediators exhibit high stability to radiation sterilization. A preferred application of radiation stable biosensor reagents in accord with the present invention is for the development of inte-grated lancet/biosensor devices. An example of such an integrated device is 2o described in United States Patent No. 5,801,057, the entire contents of which are incorporated herein by reference, except that in the event of any incon-sistent disclosure or definition from the present application, the disclosure or definition herein shall be deemed to prevail.
FIG. 3 shows an integrated lancet/biosensor devices device 10 in 2 s which a lancet 12 is connected to a sampling chamber 14. Sampling cham-ber 14 comprises a biosensor reagent comprising PQQ-glucose dehydroge-nase and a phenothiazine and/or phenoxazine mediator. Preferably, the me-diator is a phenothiazine. More preferably, the mediator has a structure rep-resented by mediator I or mediator II above. Once sampling chamber 14 has 3 o been loaded with biosensor reagent, the entire device 10 can be subjected to radiation sterilization. Preferably, the method of sterilization involves electron beam (E-beam) irradiation or gamma irradiation.
As set forth in the Association for the Advancement of Medical Instru mentation document ANSI/AAMI/ISO 11137 -1994, products that penetrate s the skin and come into contact with the blood must have a sterility assurance level (SAL) of 10'8, which corresponds to a one in a million probability of a vi-able microorganism being present on a product unit after sterilization. The sterilization dose needed to achieve a 10'8 SAL depends on the bioburden of the sample. For example, a sample with a bioburden of 1,021 requires a ster-eo ilization dose of 24.9 kGy to achieve a 10'8 SAL.
In the examples described hereinbelow, electron beam (E-beam) irra-diation was employed as the method of sterilization. The biosensor reagents subjected to the electron beam absorb energy from the electrons. The en-ergy that is absorbed per unit mass of material is referred to as the absorbed 15 dose, and it is this absorption of energy-or dose delivery-that destroys the reproductive cells and DNA chains of microorganisms, thereby rendering a product sterile. E-beam doses of 25, 50 and 100 kGy were used because the bioburden of the biosensor reagents was unknown.
FIG. 4 shows a graph of the background currents observed for three 2 o formulations of biosensor reagents exposed to increasing levels of radiation:
(1) NAD-glucose dehydrogenase with Mediator I, (2) PQQ-glucose dehydro-genase with Ferricyanide, and (3) PQQ-glucose dehydrogenase with Media-for I. The PQQ formulations tolerated the irradiation extremely well. In con-trast, the NAD formulation exhibited poor tolerance to the sterilization condi-2s tions, and resulted in a background signal which constituted a significant amount of the glucose signal. While formulation (2) exhibited good tolerance to the radiation process, the activity of the extracted enzyme was lower than the corresponding activity of the enzyme extracted from formulation (3). Fig 5 shows a graph of current response when these radiation sterilized sensors 3o were exposed to 600 mg/dL glucose.
The manner in which a device embodying features of the present in-vention is made, and the process by which such a device is used for moni-toring an analyte, will be abundantly clear to one of ordinary skill in the art based upon joint consideration of both the preceding description, and the fol-lowing representative procedures. It is to be understood that many variations in the presently preferred embodiments illustrated herein will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.
For example, the working electrode employed in electrochemical sen-to sors according to the present invention can be varied, with suitable electrodes including but not limited to carbon electrodes, platinum electrodes, palladium electrodes, gold electrodes, and the like. Similarly, the reference electrode can be varied, with suitable electrodes including but not limited to silver-silver chloride electrodes, calomel electrodes, saturated calomel electrodes, and 15 the like. Alternatively, a quasi-reference electrode (e.g., a large surface area platinum electrode ) of the type commonly used in non-aqueous electro-chemical experiements (i.e., an electrode which does not have a specific re-dox species to which its potential is referenced) can be used in accord with the present invention. The surface areas of all electrodes employed in accor-2o dance with~the present invention are likewise subject to variation.
Preferably, the working electrode has dimensions of about 0.6 mm x 1.2 mm.
Furthermore, the compositions and pH of the buffer solutions em-ployed, and the enzyme activities and concentrations of components of the biosensor reagents, are subject to wide variation. Suitable buffer solutions 2s include but are not limited to HEPES (i.e., N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MOPS (i.e., 3-(N-morpholino)propanesulfonic acid), TES
(i.e., N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), 2-([2-hydrox-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid), PIPES (i.e., pipera-zine-N,N'-bis(2-ethanesulfonic acid)), 1,4-piperazinediethanesulfonic acid), 3 o ACES (i.e., N-(carbamoylmethyl)-2-aminoethanesulfonic acid), N-(2-acetamidol)-2-aminoethanesulfonic acid, BES (i.e., N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and Dulbecco's buffer (i.e., 0.008M sodium phos-phate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01 M potas-sium chloride, pH 7.4).
s The manner in which reagents and devices embodying features of the present invention are made, and the methods by which these reagents and devices are used for monitoring an analyte, will be abundantly clear to one of ordinary skill in the art based upon joint consideration of both the preceding description, and the following representative procedures.
to While the examples provided hereinbelow relate to in vitro applications of the biosensor reagents in accord with the present invention, it is contem-plated that these reagents can also be adapted for in vivo analyte monitoring by chemically immobilizing the phenoxazine and/or phenothiazine mediators (e.g., by chemical reaction at one or more of the substituent groups on the 15 aromatic rings), and incorporating the immobilized mediators into a device which can be implanted subcutaneously into a patient.
EXAMPLES
Preparation of Biosensor and Glucose Dose-Response 2 o A liquid chemistry reagent was prepared to be 20 Units/wL pyrolloquin-olinequinone-glucose dehydrogenase (PQQ-GDH) and 24 mM mediator I in 100 mM Sodium Phosphate, ;pH 7.4. The first component of the reagent was made by dissolving the mediator in 100 mM phosphate pH 7.4, adjusting the pH back to 7.4, and filtering the solution by forcing it through a Whatman 0.45 2s micron PTFE syringe filter. The reagent was completed by adding lyophilized PQQ-GDH (Toyobo Product No. GLD-321 ) to an activity of 20 U/~L.
The chemistry formulation was deposited onto electrodes, which had been produced using a 3-pass screen-printing process by Conductive Tech-nologies, Inc. During this process, the silver/silver chloride (DuPont 5870 ink) 3 0 leads and reference electrode were printed first onto polycarbonate base material. The second pass of Dupont 7102T carbon-graphite working elec-trode was printed on top of this. A final pass of Norcote RDMSK4954-A2 di-electric defined the working electrode area to be 0.0113 cm2.
The chemistry was deposited over the working electrode with the use of an Asymtek Automove~402 Dispensing System. The system was pro-grammed to perform the transfer by dipping a 62 mL stainless steel pin into a 1.5 mL Eppendorf vial filled with reagent. Polycarbonate lid material was laminated to the sensors creating a capillary area over the working and refer-ence electrodes capable of holding approximately 3 pL of test solution. The to capillary area, which defines the sample volume, is first formed in the poly-carbonate lid material by a coining or stamping process.
As shown in FIG. 6, reactivity of the chemistry was analyzed by gener-ating a glucose dose-response curve with buffered (100 mM phosphate, 100 mM sodium chloride, pH 7.4) samples containing a range of glucose concen-trations from 0 to 600 mg/dL. Current generated at each of the glucose con-centrations was measured using a potentiostat programmed to apply 150 mV
potential with trigger level set to 100 nA, and timing programmed to record the current at 5,10,15, and 20 seconds. The trigger level refers to a threshold level above which timing and recording are initiated.
2o Sensors formulated with 20U Glucose Oxidase/sensor and 6 mM me-diator I were deposited onto electrode sensors as above. The dose response plot shown in FIG. 7 was obtained.
Preparation of Electrochemical Biosensor and Heat/Humidity Stability Electrochemical biosensors were constructed using a screen-printing 2 s process. Sensors were comprised of a carbon working electrode and a sil-ver/silver chloride reference electrode. A solution (150 to 800n1) containing 12mM mediator I in 100mM phosphate buffer (pH 7.4), and of the enzyme PQQ-glucose dehydrogenase (10 U/oL) was deposited on the surface of the working electrode and allowed to dry at room temperature for 5 minutes prior to desiccation. The electrodes were assembled into a format having a small capillary gap, which allowed inoculation of the sensors with sample solutions.
In subsequent tests, the sensors were subjected to the following envi-ronmental conditions prior to testing: 1 ) 50 °C for 2, 4, and 8 weeks;
and 2) s room temperature with 40% relative humidity. The sensors were poised at a potential of 150mV relative to the Ag/AgCI reference electrode and the re-suiting current was measured. This mediatorlenzyme combination is quite stable to both heat stress and humidity stress as shown in FIG. 8.
Sterilization of Biosensors and Radiation Stability Data 10 . Five formulations of biosensor reagents (Table 1 ) were prepared and subjected to E-beam irradiation using SureBeam~ sterilization technology at Titan Scan Technologies (San Diego, CA). Formulation I was irradiated at 25 kGy, 50 kGy, and 100 kGy, whereas each of Formulations II-V was irradiated at 25 kGy only. In the two rightmost column headings of Table 1, the abbre-15 viation CMC refers to carboxymethylcellulose, and the abbreviation PEO re-fers to polyethylene oxide.
Table 1 FormulationEnzyme Con- ConcentrationPolymer Polymer # centration Mediator ConcentrationConcentration PQQ-GDH I CMC % PEO
Units mM
- -..
--~
FIGS. 9-13 show glucose dose response curves for each of the five 2 o formulations both before and after irradiation. The stability of the five formu-lations is high, as is clearly shown by the near overlapping of the glucose re-sponse generated before and after irradiation.
Table 2 shows the results of enzyme assays conducted on the five formulations both before and after irradiation. Enzyme activity following irra-diction remains high in all instances.
Table 2 Formulation # kGy Level Enzyme Activity I 0 4.67 25 4.32 50 4.20 100 4.24 II 0 3.31 25 3.34 I I I 0 4.93 25 4.87 1V 0 4.96 25 4.86 V 0 3.63 25 4.05 The foregoing detailed description and examples have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodi-ments illustrated herein will be obvious to one of ordinary skill in the art, and to remain within the scope of the appended claims and their equivalents.
Claims (46)
1. A reagent for detecting an analyte, comprising:
an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination thereof; and a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and a combination thereof.
an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination thereof; and a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and a combination thereof.
2. The reagent of claim 1, wherein the mediator is selected from the group consisting of and combinations thereof, wherein R1, R2 R3, R4, R5, R6, R7, R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, vitro, dialkylamino, aminoalkyl, sulfo, di-hydroxyboron, and combinations thereof.
3. The reagent of claim 1, wherein the mediator is selected from the group consisting of 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-[3-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H-phenothiazinesulfonic acid, and combinations thereof.
4. The reagent of claim 1, wherein the mediator comprises
5. The reagent of claim 1, wherein the mediator comprises
6. The reagent of claim 1, wherein the flavoprotein is selected from the group consisting of FAD-glucose oxidase, flavin-hexose oxidase, FAD-glucose dehydrogenase, [FAD]-lactate oxidase, [FAD]-cholesterol oxidase, [FAD]-alcohol oxidase, [FAD]-d-aminoacid oxidase, [FAD]-choline oxidase, and combinations thereof.
7. The reagent of claim 1, wherein the quinoprotein is selected from the group consisting of PQQ-membrane bound glucose dehydrogenase, PQQ-soluble glucose dehydrogenase, [PQQ]-lactate dehydrogenase, [PQQ]-aldehyde dehydrogenase, [PQQ]-methylamine dehydrogenase, [PQQ]-alcohol dehydrogenase, and combinations thereof.
8. The reagent of claim 1, wherein the enzyme is selected from the group consisting of FAD-glucose oxidase, PQQ-glucose dehydrogenase, and a combination thereof.
9.. The reagent of claim 8, wherein the mediator is selected from the group consisting of and combinations thereof, wherein R1, R2 R3, R4, R5, R6, R7, R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, vitro, dialkylamino, aminoalkyl, sulfo, di-hydroxyboron, and combinations thereof.
10. The reagent of claim 8, wherein the mediator is selected from the group consisting of 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2°-(5°-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-.beta.-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H-phenothiazinesulfonic acid, and combinations thereof.
11. The reagent of claim 8, wherein the mediator comprises
12. The reagent of claim 8, wherein the mediator comprises
13. The reagent of claim 1, further comprising a polymer selected from the group consisting of carboxymethylcellulose, polyethylene oxide, and combinations thereof.
14. A reagent for detecting glucose, comprising:
PQQ-glucose dehydrogenase in an activity of about 20 Units/
µL;
a buffer having a concentration between about 0.1 mM and about 100 mM, and a pH between about 4.5 and about 9.5; and a mediator having a structure wherein the mediator has a concentration in the buffer between about 0.1 mM and about 30 mM.
PQQ-glucose dehydrogenase in an activity of about 20 Units/
µL;
a buffer having a concentration between about 0.1 mM and about 100 mM, and a pH between about 4.5 and about 9.5; and a mediator having a structure wherein the mediator has a concentration in the buffer between about 0.1 mM and about 30 mM.
15. The reagent of claim 14, wherein the buffer is selected from the group consisting of sodium phosphate, potassium phosphate, Hepes, MOPS, TES, Pipes, ACES, BES, Dulbecco's, and combinations thereof.
16. The reagent of claim 14, wherein the buffer comprises sodium phosphate.
17. The reagent of claim 14, further comprising a polymer selected from the group consisting of carboxymethylcellulose, polyethylene oxide, and combinations thereof.
18. A reagent for detecting an analyte, comprising:
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof;
a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and combinations thereof.
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof;
a mediator selected from the group consisting of a phenothi-azine, a phenoxazine, and combinations thereof.
19. The reagent of claim 18, wherein the mediator comprises
20. The reagent of claim 18, wherein the mediator comprises
21. An electrochemical sensor comprising:
a working electrode having a surface; and a second electrode coupled to the working electrode; wherein the surface of the working electrode is coated with a so-lution of a reagent comprising an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination thereof;
and
a working electrode having a surface; and a second electrode coupled to the working electrode; wherein the surface of the working electrode is coated with a so-lution of a reagent comprising an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combination thereof;
and
22 a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
22. The electrochemical sensor of claim 21, wherein the working electrode is selected from the group consisting of a carbon electrode, a plati-num electrode, a palladium electrode, and a gold electrode.
22. The electrochemical sensor of claim 21, wherein the working electrode is selected from the group consisting of a carbon electrode, a plati-num electrode, a palladium electrode, and a gold electrode.
23. The electrochemical sensor of claim 21, wherein the second electrode is selected from the group consisting of a reference electrode and a quasi-reference electrode.
24. The electrochemical sensor of claim 21, wherein the second electrode is a silver/silver chloride reference electrode.
25. The electrochemical sensor of claim 21, wherein the surface of the working electrode has an area of about 0.00113 cm2.
26. An electrochemical sensor comprising:
a working electrode having a surface; and a reference electrode coupled to the working electrode; wherein the surface of the working electrode is coated with a so-lution of a reagent comprising PQQ-glucose dehydrogenase in an activity of about 1 Units/ µL to about 100 Units/ µL;
a buffer having a concentration of about 100 mM
and a pH of about 7.4; and a mediator having a structure wherein the mediator has a concentration in the buffer of about 1 mM to about 100 mM.
a working electrode having a surface; and a reference electrode coupled to the working electrode; wherein the surface of the working electrode is coated with a so-lution of a reagent comprising PQQ-glucose dehydrogenase in an activity of about 1 Units/ µL to about 100 Units/ µL;
a buffer having a concentration of about 100 mM
and a pH of about 7.4; and a mediator having a structure wherein the mediator has a concentration in the buffer of about 1 mM to about 100 mM.
27. The electrochemical sensor of claim 26, wherein the activity of the PQQ-glucose dehydrogenase is about 20 Units/ µL, and wherein the con-centration of the mediator in the buffer is about 24 mM.
28. The electrochemical sensor of claim 26, wherein the activity of the PQQ-glucose dehydrogenase is about 10 Units/ µL, and wherein the con-centration of the mediator in the buffer is about 12 mM.
29. The reagent of claim 26, further comprising a polymer selected from the group consisting of carboxymethylcellulose, polyethylene oxide, and combinations thereof.
30. A device for measuring an analyte, comprising:
a lancet; and a sampling chamber connected to the lancet, comprising:
a reagent comprising:
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
a lancet; and a sampling chamber connected to the lancet, comprising:
a reagent comprising:
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
31. The device of claim 30, wherein the mediator is selected from the group consisting of and combinations thereof, wherein R1, R2 R3, R4, R5, R6, R7, R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, di-hydroxyboron, and combinations thereof.
32. The device of claim 30, wherein the mediator is selected from the group consisting of 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-.beta.-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H-phenothiazinesulfonic acid, and combinations thereof.
33. The device of claim 30, wherein the mediator comprises
34. The device of claim 30, wherein the mediator comprises
35. A method of producing a sterilized device for measuring an analyte, comprising:
providing a device comprising:
a reagent, comprising:
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof; and irradiating the device with E-beam or gamma ray irradiation.
providing a device comprising:
a reagent, comprising:
an enzyme selected from the group consisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof; and irradiating the device with E-beam or gamma ray irradiation.
36. A method of producing a sterilized device for measuring an analyte, comprising:
providing a device comprising:
a lancet; and a sampling chamber connected to the lancet, comprising:
a reagent, comprising:
an enzyme selected from the group con-sisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group con-sisting of a phenothiazine, a phenoxazine, and a combination thereof; and irradiating the device with E-beam or gamma ray irradiation.
providing a device comprising:
a lancet; and a sampling chamber connected to the lancet, comprising:
a reagent, comprising:
an enzyme selected from the group con-sisting of PQQ-glucose dehydrogenase, FAD-glucose oxidase, and a combination thereof; and a mediator selected from the group con-sisting of a phenothiazine, a phenoxazine, and a combination thereof; and irradiating the device with E-beam or gamma ray irradiation.
37. A method for detecting an analyte which undergoes a chemical reaction, the method comprising:
providing an electrode surface;
catalyzing the chemical reaction with an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combina-tion thereof;
generating a redox equivalent by the chemical reaction; and transferring the redox equivalent to the electrode surface using a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
providing an electrode surface;
catalyzing the chemical reaction with an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, and a combina-tion thereof;
generating a redox equivalent by the chemical reaction; and transferring the redox equivalent to the electrode surface using a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof.
38. The method of claim 37, wherein the analyte is glucose.
39. The method of claim 37, wherein the mediator is selected from the group consisting of and combinations thereof, wherein R1, R2 R3, R4; R5, R6, R7, R8, and R9 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, hetero-cyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, and combinations thereof.
40. The method of claim 37, wherein the mediator is selected from the group consisting of 3-(4'-chloro-phenylimino)-3H-phenothiazine, 3-(4'-diethylamino-phenylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 3-(4'-nitro-phenylimino)-3H-phenothiazine, 3-(4'-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-trifluoromethyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4'-.beta.-aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'-ethylphenyl)amino-3-(4'-ethyl-phenylimino)-3H-phenothiazine, 6-(4'-[2-(2-ethanoloxy)ethoxy]ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4'-carboxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H-phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H-phenothiazinesulfonic acid, and combinations thereof.
41. The method of claim 37, wherein the mediator comprises
42. The method of claim 37, wherein the flavoprotein is selected from the group consisting of FAD-glucose oxidase, FAD-glucose dehydroge-nase, lactate oxidase, cholesterol oxidase, alcohol oxidase, d-aminoacid oxi-dase, choline oxidase, and combinations thereof.
43. The method of claim 37, wherein the quinoprotein is selected from the group consisting of PQQ-glucose dehydrogenase, lactate dehydro-genase, aldehyde dehydrogenase, methylamine dehydrogenase, alcohol de-hydrogenase, and combinations thereof.
44. The method of claim 37, wherein the enzyme is selected from the group consisting of FAD-glucose oxidase, PQQ-glucose dehydrogenase, and a combination thereof.
45. The method of claim 44, wherein the mediator comprises
46. The method of claim 44, wherein the mediator comprises
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JP5275494B2 (en) | 2013-08-28 |
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EP1293574A2 (en) | 2003-03-19 |
CA2401365C (en) | 2013-12-31 |
DE60223635T2 (en) | 2008-10-30 |
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