USOO71636.16B2

(12) United States Patent (10) Patent No.: US 7,163,616 B2 Vreelke et al. (45) Date of Patent: Jan. 16, 2007

(54) REAGENTS AND METHODS FOR (56) References Cited DETECTING ANALYTES, AND DEVICES U.S. PATENT DOCUMENTS COMPRISING REAGENTS FOR DETECTING ANALYTES 4,545,382 A 10/1985 Higgins et al...... 128,635 4,711,245 A 12/1987 Higgins et al...... 128,635 4,863,016 A 9/1989 Fong et al...... 206,210 4.941,308 A 7, 1990 Grabenkort et al...... 53.425 (75) Inventors: Mark S. Vreeke, Houston, TX (US); 5, 120,420 A 6/1992 Nankai et al...... 204,403 Mary Ellen Warchal-Windham, 5,206,147 A 4, 1993 Hoenes ...... 435/25 Osceola, IN (US); Christina Blaschke, 5,212,092 A 5/1993 Jackson et al...... 436/11 White Pigeon, MI (US); Barbara J. 5,236,567 A 8/1993 Nanba et al...... 204,403 Mikel, Mishawaka, IN (US); Howard (Continued) A. Cooper, Elkhart, IN (US) FOREIGN PATENT DOCUMENTS (73) Assignee: Bayer Corporation, Elkhart, IN (US) EP O 330 517 A 8, 1989 (Continued) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 102 days. Taylor, C.; Kenausis, G.; Katakis, I.; Heller, A.; “Wiring” of oxidase within a hydrogel made with polyvinyl imidazole complexed with Ox-4,4'-dimethoxy-2,2'-bipyridin)CI+2+, Jour (21) Appl. No.: 10/231,539 nal of Electroanalytical Chemistry, 1995, vol. 396, pp. 511-515.* (Continued) (22) Filed: Sep. 3, 2002 Primary Examiner Kaj K. Olsen (74) Attorney, Agent, or Firm Jenkens & Gilchrist (65) Prior Publication Data US 2003 FOO94384 A1 May 22, 2003 (57) ABSTRACT Reagents for detecting an analyte are described. A reagent Related U.S. Application Data comprises (a) an selected from the group consisting (60) Provisional application No. 60/318,716, filed on Sep. of a flavoprotein, a quinoprotein, and a combination thereof. 14, 2001. and (b) a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof. In (51) Int. C. addition, reagents having good stability to radiation steril GOIN 27/327 (2006.01) ization are described. Electrochemical sensors and sampling (52) U.S. Cl...... 205/777.5; 204/403.14 devices comprising Such reagents, methods of producing a (58) Field of Classification Search ...... 204/403, sterilized device including Such reagents, and methods for 204/409, 412,403.14; 205/775, 777.5; 435/14, detecting an analyte which utilize Such reagents are 435/25, 26,810, 817: 436/806, 808, 149, described as well. 436/151 See application file for complete search history. 40 Claims, 6 Drawing Sheets

12 Layer 1 Layer 2 Layer3

Laminated Structure US 7,163,616 B2 Page 2

U.S. PATENT DOCUMENTS calcoaceticus'; Ph.D. Dissertation, Delft University of Technology, The Netherlands; 1999: pp. i-vi. 1-69. 5,298,144. A 3/1994 Spokane ...... 204,403 5,334.508 A 8, 1994 Hoenes ...... 435/25 Dewanti, Asteriani Ratih; "Enzymology of Quinoprotein Glucose 5,393,615 A 2, 1995 Corey et al...... 429.43 Dehydrogenases from Acinetobacter calcoaceticus'; Ph.D. Disser 5,411,647 A 5/1995 Johnson et al...... 205/777.5 tation, Delft University of Technology, The Netherlands; 2000; pp. 5,520,786 A 5/1996 Bloczynski et al. ... 204/403.14 i-viii. 1-107. 5,798,031 A 8, 1998 Charlton et al...... 204,403 Abstract of JP6114041A2 (issued Apr. 26, 1994): Jackson, Jeffrey 5,801,057 A 9, 1998 Smart et al...... 436/68 6,057,120 A 5, 2000 Heindl et al...... 435/25 T.; Hui, Henry K. “Storage and Calibration Solution for Blood Gas 6,299,757 B1 * 10/2001 Feldman et al...... 205/775 Sensor Device of Blood in Multi-Charateristic Value Blood Vessel': 6,773,564 B1* 8/2004 Yugawa et al...... 204,403.14 pp. 1-2. Abstract of JP3180750A2 (issued Aug. 6, 1991): Yamaguchi, FOREIGN PATENT DOCUMENTS Hideichiro. "Cell for Chemical Sensor and Chemical Sensor Having EP 1 130 111 A 9, 2001 This Cell'; pp. 1-2. WO WO92 O7263 A 4f1992 Abstract of JP7083870A2 (issued Mar. 31, 1995): Kojima, Naomi; WO WO92 O7953. A 5, 1992 Sugama, Akio; Suzuki, Hiroaki. “Sterilization Method for Enzyme OTHER PUBLICATIONS Film”: pp. 1-2. Abstracts from ISI Current Contents, 1992-1999; 306 records. Olsthoorn, Arjen. "Structural and Mechanistic Aspects of Soluble Quinoprotein Glucose Dehydrogenase from Acinetobacter * cited by examiner U.S. Patent Jan. 16, 2007 Sheet 1 of 6 US 7,163,616 B2

12 Layer 1 Layer 2 Layer 3

Laminated Structure U.S. Patent Jan. 16, 2007 Sheet 2 of 6 US 7,163,616 B2 FG. 2

U.S. Patent Jan. 16, 2007 Sheet 3 of 6 US 7,163,616 B2

FIG. 3 Background Current After Irradiation 1400 1200 --Fas 1000 N S 800600 N a Control d 400 N :25kGy 200 .. N o50kGy O : N N 100 kGy NAD-MLB PQQ-Ferri PQQ-MLB Chemistry

600mg/dL Glucose Response After irradiation 18000 16000 S 1400012000 Že s SS %: a Control

° 33. NES 2666%.61%. f N 333. N D50is 100kéy kGy NAD-MLB POQ-Ferri PQQ-MLB Chemistry

PQQ-GDH/MLB Dose Response Curve 20000 15000 g -0- 5 SeC. 5 10000 -H 10 SeC. as 5000 -A- 15 SeC. O O - 20 Sec. O 200 400 600 mg/dL Aqueous Glucose U.S. Patent Jan. 16, 2007 Sheet 4 of 6 US 7,163,616 B2

MLB 92 with 3 25 a 20 S 15 CN y = 0.0423x + 0.53 (e) 105 R42 = 0.9934 9 S 0 C 0 100 200 300 400 500 600 glucose (mg/dL)

GDH-PQQ With MLB 92 20000 0 initial 5 15000 4 wks at 50C E 10000 A RT 4Wk 40%RH 5 5000 no des O Linear (RT 4wk) O 200 400 600 Glucose (mg/dL)

PQQ-GDHChemistry irradiation Stability Study Formulation 9.

S 10000: -0- Control (S) 8 8000 --25kGy Sis 40006999 -A-50 kG y 3 2000 -o-100kGy g O O 200 400 600 mg/dL Aqueous Glucose U.S. Patent Jan. 16, 2007 Sheet 5 of 6 US 7,163,616 B2

PQQ-GDHChemistry irradiation Stability Study Formulation 20000 9 16000 (e) E. 12000 8000 -o-Control d 4000 --25kGy g O 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose

PQQ-GDHChemistry irradiation Stability Study Os Formulation ill 3 3 20000 S 16000 9 oris 120008000 -o-Control d 4000 --25kGy KC O 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose

PQQ-GDHChemistry irradiation Stability Study 2 Formulation IV 3 3 20000 S 16000 9 is 12000 5is de 80004000 --Control CDg O --25kGy 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose U.S. Patent Jan. 16, 2007 Sheet 6 of 6 US 7,163,616 B2

FIG. 12

PQQ-GDHChemistry irradiation Stability Formulation W O 93 20000 S 16000 s 12000 s: -o-Control O O --25kGy 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose US 7,163,616 B2 1. 2 REAGENTS AND METHODS FOR the radiation conditions commonly employed in lancet ster DETECTING ANALYTES, AND DEVICES ilization. Reagents stable to Such radiation sterilization COMPRISING REAGENTS FOR DETECTING could be incorporated into highly user-convenient units in ANALYTES which lancet and biosensor are combined. The present invention is directed to electron transfer This application claims benefit to provisional application mediators for use in flavoprotein- and quinoprotein-based 60/318,716, filed on Sep. 14, 2001. biosensor reagents, which exhibit improved stability to both environmental interferents and to radiation sterilization. BACKGROUND 10 SUMMARY The present invention relates to reagents, methods and devices for measurement of analytes and, more particularly, The scope of the present invention is defined solely by the to reagents, methods and devices for the measurement of appended claims, and is not affected to any degree by the glucose in the blood. statements within this Summary. By way of introduction, the The monitoring of certain analyte concentrations in the 15 presently preferred embodiments described herein are body enables early detection of health risks, and identifies directed towards remedying the aforementioned stability the need for the introduction of therapeutic measures. One of problems of electron transfer mediators and enzyme biosen the most commonly monitored analytes is glucose, the blood SOS. concentration of which is important in determining the Briefly stated, a composition aspect of the present inven appropriate dosages of insulin for diabetics. Various meth tion is directed to a reagent for detecting an analyte, com ods have been developed for monitoring glucose levels in prising (a) an enzyme selected from the group consisting of the blood, including the use of electrochemical biosensors. a flavoprotein, a quinoprotein, and a combination thereof. Electrochemical biosensors are based on enzyme-catalyzed and (b) a mediator selected from the group consisting of a chemical reactions involving the analyte of interest. In the phenothiazine, a phenoxazine, and a combination thereof. case of glucose monitoring, the relevant is 25 A first apparatus aspect of the present invention is directed the oxidation of glucose to gluconolactone. This oxidation is to an electrochemical sensor comprising: (a) a working catalyzed by a variety of , some of which may electrode having a Surface; and (b) a second electrode contain a bound coenzyme Such as nicotinamide adenine coupled to the working electrode. The surface of the work dinucleotide (phosphate) (NAD(P)), while others may con ing electrode is coated with a solution or mixture of a tain a bound Such as flavin adenine dinucleotide 30 reagent comprising an enzyme selected from the group (FAD) or pyrroloquinolinequinone (PQQ). consisting of a flavoprotein, a quinoprotein, and a combi In biosensor applications, the redox equivalents generated nation thereof, and a mediator selected from the group in the course of the oxidation of glucose are transported to consisting of a phenothiazine, a phenoxazine, and a combi the surface of an electrode whereby an electrical signal is nation thereof. generated. The magnitude of the electrical signal is then 35 A second apparatus aspect of the present invention is correlated with concentration of glucose. The transfer of directed to a device for measuring an analyte, comprising (a) redox equivalents from the site of chemical reaction in the a lancet; and (b) a sampling chamber connected to the lancet. enzyme to the surface of the electrode is accomplished with The sampling chamber comprises a reagent comprising an the use of electron transfer mediators. enzyme selected from the group consisting of PQQ-glucose A significant problem with the use of electron transfer 40 dehydrogenase, FAD-glucose oxidase, and a combination mediators in biosensors is the instability of these compounds thereof, and (b) a mediator selected from the group consist upon exposure to common environmental conditions such as ing of a phenothiazine, a phenoxazine, and a combination temperature and moisture. As a result, the number of media thereof. tors Suitable for use in glucose biosensors is quite limited. A first method aspect of the present invention is directed U.S. Pat. No. 5,520,786 (786) to Bloczynski et al. 45 to a method of producing a sterilized device for measuring describes families of phenothiazine and phenoxazine com an analyte, comprising (a) providing a device in accordance pounds suitable for use as electron transfer mediators with with the present invention, and (b) irradiating the device the enzymes dihydronicotinamide adenine dinucleotide with E-beam or gamma ray radiation. (NADH), NADPH, and analogs thereof. Cofactor based A second method aspect of the present invention is enzymes such as FAD-glucose oxidase and PQQ-glucose 50 directed to a method for detecting an analyte which under dehydrogenase have several advantages over NAD-based goes a chemical reaction, the method comprising (a) pro enzymes, including lower cost, higher enzyme activity, viding an electrode Surface; (b) catalyzing the chemical increased stability, and bound as opposed to readily disso reaction with an enzyme selected from the group consisting ciable cofactor. of a flavoprotein, a quinoprotein, and a combination thereof. Electron transfer mediators previously used with FAD 55 (c) generating a redox equivalent by the chemical reaction; glucose oxidase and PQQ-glucose dehydrogenase include and (d) transferring the redox equivalent to the electrode quinones, phenzine methosulfate, dichlorophenolindophe Surface using a mediator selected from the group consisting nol and ferricyanide. Unfortunately, these compounds have of a phenothiazine, a phenoxazine, and a combination proven to be highly Susceptible to the environmental agents thereof. described above, and result in biosensor reagents of low 60 The presently preferred embodiments discussed herein stability. Thus, mediators are needed which exhibit good may possess one or more advantages relative to other stability upon exposure to commonly-encountered environ flavoprotein- and quinoprotein-based biosensor reagents, mental agents, and which can be used in flavoprotein- and which can include but are but not limited to: improved quinoprotein-based systems. biosensor reagent stability; enhanced electron transfer capa In addition to the need for biosensor reagents that are 65 bility of mediators; ability to tune mediators for optimum stable to the environmental agents described above, it would electrode operation; reduced Susceptibility of media be desirable to provide biosensor reagents that are stable to tors; increased thermal stability of mediators; increased US 7,163,616 B2 3 4 stability of mediators to ambient humidity; lower redox goes a chemical reaction that is catalyzed by an enzyme potential of mediators; reduced susceptibility to interferents selected from the group consisting of a flavoprotein, a in blood; and stability of biosensor reagents to radiation quinoprotein, and a combination thereof. Preferred analytes sterilization conditions. include but are not limited to glucose, lactate, D-amino BRIEF DESCRIPTION OF THE DRAWINGS acids, ascorbate, alcohol, cholesterol, , and acetyl choline. FIG. 1 shows a schematic illustration of a device for Flavoproteins in accord with the present invention include measuring an analyte that embodies features of the present FAD-glucose oxidase (Enzyme Classification No. 1.1.3.4), invention. 10 Flavin-hexose oxidase (EC No. 1.1.3.5) and FAD-glucose FIG. 2 shows a perspective view of an integrated lancet/ dehydrogenase (EC No. 1.1.99.10) For information relating biosensor device for use in accordance with the present to these flavoproteins, see: Adriaan Joseph Jan Olsthoorn, invention. “Structural and Mechanistic Aspects of Soluble Quinopro FIG. 3 shows a graph of background currents for 3 tein Glucose Dehydrogenase from Acinetobacter calcoace formulations of biosensor reagents exposed to increasing 15 levels of radiation. ticus.” Ph.D. dissertation, Delft University of Technology, FIG. 4 shows a graph of the current response of radiation The Netherlands, 1999. Additional oxidase enzymes for use sterilized biosensor reagents upon exposure to glucose. in accord with the present invention include but are not FIG. 5 shows a plot of current vs. glucose concentration limited to lactate oxidase, cholesterol oxidase, alcohol oxi at increasing time intervals for a POQ-glucose dehydroge dase (e.g., methanol oxidase), d-aminoacid oxidase, choline nase/phenothiazine biosensor. oxidase, and FAD derivatives thereof. A preferred flavopro FIG. 6 shows a plot of current vs. glucose concentration tein for use in accord with the present invention is FAD for a FAD-glucose oxidase/phenothiazine biosensor. glucose oxidase. FIG. 7 shows a plot of current vs. glucose concentration for a POQ-glucose dehydrogenase/phenothiazine biosensor 25 Quinoproteins in accord with the present invention reagent Subjected to heat stress and humidity stress. include but are not limited to membrane bound and soluble FIGS. 8–12 show plots of current vs. glucose concentra PQQ-glucose dehydrogenase (EC No. 1.1.99.17). Informa tion for 5 formulations of PQQ-glucose dehydrogenase/ tion relating to POO-glucose dehydrogenase can be found in phenothiazine biosensors exposed to varying levels of radia the Olsthoorn reference cited above. Additional quinopro tion. 30 tein enzymes for use in accord with the present invention include but are not limited to , alde DETAILED DESCRIPTION OF THE hyde dehydrogenase, methylamine dehydrogenase, alcohol PRESENTLY PREFERRED EMBODIMENTS dehydrogenase (e.g., methanol dehydrogenase), and PQQ derivatives thereof. A preferred quinoprotein for use in Throughout this description and in the appended claims, 35 the following definitions are to be understood: The term accord with the present invention is PQQ-glucose dehydro “analyte' refers to one or a plurality of species having a genase. concentration of interest. The term “flavoprotein’ refers to Mediators in accord with the present invention include enzymes containing flavin cofactors. The term "quinopro phenothiazines having the formula tein’ refers to enzymes containing PQQ or similar cofactors. 40 The phrase “redox equivalent” refers to one or a plurality of charged species (e.g., electrons) produced in electrochemi cal reactions involving the analyte. The phrase "E-beam R6 RI irradiation' or “electron beam irradiation” refers to a process R S N R2 of exposure to a concentrated, high-current stream of elec 45 trons. The terms “alkyl,” “alkenyl,” “alkynyl,” “aryl,” “het 2 eroaryl,” “cyclic,” “heterocyclic,” “halo,” “haloalkyl,” “car R8 N O R5 R3, boxy.” “carboxyalkyl.” “alkoxycarbonyl.” “aryloxycarbonyl,” “aromatic keto,” “aliphatic keto.” R9 R4 “alkoxy.” “aryloxy.” “nitro,” “dialkylamino,” “aminoalkyl.” 50 “sulfo,” “dihydroxyboron, and the like refer to substituents well known in the art, which may be branched or and phenoxazines having the formula unbranched and may themselves be substituted with one or more substituents. The phrase “biosensor reagent” refers to the combination of an enzyme that catalyzes a reaction of an 55 analyte, and a phenothiazine and/or phenoxazine mediator. R6 RI The term “bioburden” refers to the population of viable R O N R2 microorganisms on a determined immediately prior to irradiation. 2 A biosensor reagent for detecting an analyte in accord 60 R8 N O R5 R3, with the present invention includes (1) an enzyme selected from the group consisting of a flavoprotein, a quinoprotein, R9 R4 and a combination thereof, and (2) a mediator selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof. 65 The nature of the analyte monitored in accord with the present invention is unrestricted, provided the analyte under US 7,163,616 B2 5 wherein R. R. R. R. R. R. R. R. and Rare the same or different, and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, het Mediator I eroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, car S N CO2H boxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto, 21 aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, ami 2 noalkyl, Sulfo, dihydroxyboron, and combinations thereof. N In contrast to the single electron transfer carrying capa CO2H bility of KFe(CN), mediators in accord with the present 10 and invention have the ability to carry two redox equivalents, Mediator II and are therefore well suited for use in FAD and quinopro S N tein oxidation/reduction processes, which generally involve 21 the transfer of two electrons. Moreover, the potential of mediators of the present invention can be tuned to the 15 2 optimum potential (i.e., the potential where the signal con C SOH. tribution from interferences is minimized) for a specific sample matrix by varying the Substitution on the aromatic Relative to ferricyanide, phenothiazine mediators—in rings. Electron-donating Substituents (e.g., alkyl, alkoxy, particular mediator I—are less Susceptible to oxygen deg amine, hydroxy, etc.) result in decreased redox potentials, while electron-withdrawing Substituents (e.g., carboxylic radation, more thermally stable, and more stable to ambient acid, ester, aldehyde, ketone, nitrile, nitro, Sulfonic acid, humidity. In addition, mediator I works at a lower redox trifluromethyl, etc.) result in increased redox potentials. For potential than ferricyanide. For example, E for mediator I is blood or plasma samples, the ideal potential usually lies approximately 0 mV versus an Ag/AgCl reference, whereas 25 Eo for ferricyanide is approximately 250 mV versus an. between about -200 and about 100 mV versus an Ag/AgCl Ag/AgCl reference. The lower redox potential of phenothi reference. azine mediators is advantageous in that there is a region The Substituents on the aromatic rings, in addition to their around 0 mV versus an Ag/AgCl reference in which the utility in tuning the redox potentials of the mediators, can amount of electrochemical interferences are minimized. also be used to enhance mediator solubility. For example, the 30 Thus, the impact from chemical interferents in the blood can introduction of a Substituent having the capacity for hydro be minimized by using these mediators. gen bonding can be expected to render the mediator more Reagents embodying features of the present invention can water Soluble than a mediator lacking Such substitution. In be incorporated into a variety of biosensor devices, includ addition, these Substituents can serve as functional groups ing but not limited to the ones described in U.S. Pat. No. for immobilizing the mediators to a Support (e.g., the elec 35 5,120,420 and U.S. Pat. No. 5,798,031, the entire contents of trode Surface or, alternatively, a chemical matrix Such as a both of which are incorporated herein by reference, except polymer backbone, which is suitable for application to the that in the event of any inconsistent disclosure or definition electrode surface). from the present application, the disclosure or definition Preferably, mediators used in biosensor reagents accord 40 herein shall be deemed to prevail. ing to the present invention include 3-(4-chloro-phe Turning now to the drawings, FIG. 1 shows a represen nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe tative electrochemical sensor in accordance with the present nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H invention. The electrochemical sensor 2 is comprised of an phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H insulating base 4 upon which is printed (typically by Screen phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H 45 printing techniques) an electrode pattern (6 and 8) and a phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, reagent layer 10 that contains a reagent embodying features 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- of the present invention. The two parts of the electrode print, (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri 6 and 8, provide the working and reference electrodes fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe necessary for the electrochemical determination. A lancet nothiazine, 3-(4-(o-carboxy-n-butyl-phenylimino)-3H 50 element 12 can be incorporated into the electrochemical phenothiazine, 3-(4-aminomethyl-phenylimino)-3H sensor (e.g., interposed between layers 1 and 2), as is phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- described more fully hereinbelow. The three layers shown in oxadiaZoyl)phenylimino)-3H-phenothiazine, 3-(4-B- FIG. 1 can be joined by means of an adhesive (e.g., pressure aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- sensitive, hot melt, etc.) or by Sonic welding, depending on ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H 55 the identity of the materials. phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy It has been found that biosensor reagents comprising ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy PQQ-glucose dehydrogenase and certain phenothiazine ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- mediators exhibit high stability to radiation sterilization. A ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, preferred application of radiation stable biosensor reagents 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- 60 in accord with the present invention is for the development dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy of integrated lancet/biosensor devices. An example of Such phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe an integrated device is illustrated in FIG. 2 and described in nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H U.S. Pat. No. 5,801,057, the entire contents of which are phenothiazinedisulfonic acid, and 3-(3-phenylimino)-3H incorporated herein by reference, except that in the event of phenothiazinesulfonic acid. 65 any inconsistent disclosure or definition from the present More preferably, the mediator used in accord with the application, the disclosure or definition herein shall be present invention is selected from the group consisting of deemed to prevail. US 7,163,616 B2 7 8 As shown in FIG. 2, the integrated lancet/biosensor but not limited to silver—silver chloride electrodes, calomel device 14 has a finely bored needle 16 connected to a electrodes, saturated calomel electrodes, and the like. Alter sampling chamber 18. Sampling chamber 18 has at least one natively, a quasi-reference electrode (e.g., a large surface optical window 20 and a vent 22 through which air can be area platinum electrode) of the type commonly used in displaced when the chamber 18 fills with blood or other non-aqueous electrochemical experiments (i.e., an electrode fluids. Preferably, sampling chamber 18 comprises a bio which does not have a specific redox species to which its sensor reagent comprising PQQ-glucose dehydrogenase and potential is referenced) can be used in accord with the a phenothiazine and/or phenoxazine mediator. Preferably, present invention. The surface areas of all electrodes the mediator is a phenothiazine. More preferably, the media employed in accordance with the present invention are tor has a structure represented by mediator I or mediator II 10 likewise subject to variation. Preferably, the working elec above. Once sampling chamber 18 has been loaded with trode has dimensions of about 0.6 mmx1.2 mm. biosensor reagent, the entire device 14 can be subjected to Furthermore, the compositions and pH of the buffer radiation sterilization. Preferably, the method of sterilization Solutions employed, and the enzyme activities and concen involves electron beam (E-beam) irradiation or gamma trations of components of the biosensor reagents, are subject irradiation. 15 to wide variation. Suitable buffer solutions include but are As set forth in the Association for the Advancement of not limited to HEPES (i.e., N-2-hydroxyethylpiperazine-N'- Medical Instrumentation document ANSI/AAMI/ISO 2-ethanesulfonic acid), MOPS (i.e., 3-(N-morpholino)pro 11137-1994, products that penetrate the skin and come into panesulfonic acid), TES (i.e., N-tris(hydroxymethyl)methyl contact with the blood must have a sterility assurance level 2-aminoethanesulfonic acid), 2-(2-hydrox-1,1-bis (SAL) of 10, which corresponds to a one in a million (hydroxymethyl)-ethylamino)ethanesulfonic acid), PIPES probability of a viable microorganism being present on a (i.e., piperazine-N,N'-bis(2-ethanesulfonic acid)), 1,4-pip product unit after sterilization. The sterilization dose needed erazinediethanesulfonic acid), ACES (i.e., N-(carbamoylm to achieve a 10 SAL depends on the bioburden of the ethyl)-2-aminoethanesulfonic acid), N-(2-acetamidol)-2- sample. For example, a sample with a bioburden of 1,021 aminoethanesulfonic acid, BES (i.e., N,N-bis(2- requires a sterilization dose of 24.9 kGy to achieve a 10 25 hydroxyethyl)-2-aminoethanesulfonic acid, and Dulbecco's SAL. buffer (i.e., 0.008M sodium phosphate, 0.002M potassium In the examples described hereinbelow, electron beam phosphate, 0.14M sodium chloride, 0.01M potassium chlo (E-beam) irradiation was employed as the method of ster ride, pH 7.4). ilization. The biosensor reagents subjected to the electron The manner in which reagents and devices embodying beam absorb energy from the electrons. The energy that is 30 features of the present invention are made, and the methods absorbed per unit mass of material is referred to as the by which these reagents and devices are used for monitoring absorbed dose, and it is this absorption of energy—or dose an analyte, will be abundantly clear to one of ordinary skill delivery—that destroys the reproductive cells and DNA in the art based upon joint consideration of both the pre chains of microorganisms, thereby rendering a product ster ceding description, and the following representative proce ille. E-beam doses of 25, 50 and 100 kGy were used because 35 dures. the bioburden of the biosensor reagents was unknown. While the examples provided hereinbelow relate to in FIG.3 shows a graph of the background currents observed vitro applications of the biosensor reagents in accord with for three formulations of biosensor reagents exposed to the present invention, it is contemplated that these reagents increasing levels of radiation: (1) NAD-glucose dehydroge can also be adapted for in vivo analyte monitoring by nase with Mediator I, (2) PQQ-glucose dehydrogenase with 40 chemically immobilizing the phenoxazine and/or phenothi Ferricyanide, and (3) PQQ-glucose dehydrogenase with azine mediators (e.g., by chemical reaction at one or more of Mediator I. The PQQ formulations tolerated the irradiation the Substituent groups on the aromatic rings), and incorpo extremely well. In contrast, the NAD formulation exhibited rating the immobilized mediators into a device which can be poor tolerance to the sterilization conditions, and resulted in implanted Subcutaneously into a patient. a background signal which constituted a significant amount 45 of the glucose signal. While formulation (2) exhibited good EXAMPLES tolerance to the radiation process, the activity of the extracted enzyme was lower than the corresponding activity Preparation of Biosensor and Glucose Dose-Response of the enzyme extracted from formulation (3). FIG. 4 shows A liquid chemistry reagent was prepared to be 20 Units/uI. a graph of current response when these radiation-sterilized 50 pyrolloquinolinequinone-glucose dehydrogenase (POO sensors were exposed to 600 mg/dL glucose. GDH) and 24 mM mediator I in 100 mMSodium Phosphate, The manner in which a device embodying features of the pH 7.4. The first component of the reagent was made by present invention is made, and the process by which Such a dissolving the mediator in 100 mM phosphate pH 7.4, device is used for monitoring an analyte, will be abundantly adjusting the pH back to 7.4, and filtering the solution by clear to one of ordinary skill in the art based upon joint 55 forcing it through a Whatman 0.45 micron PTFE syringe consideration of both the preceding description, and the filter. The reagent was completed by adding lyophilized following representative procedures. It is to be understood PQQ-GDH (Toyobo Product No. GLD-321) to an activity of that many variations in the presently preferred embodiments 20 U/uL. illustrated herein will be obvious to one of ordinary skill in The chemistry formulation was deposited onto electrodes, the art, and remain within the scope of the appended claims 60 which had been produced using a 3-pass screen-printing and their equivalents. process by Conductive Technologies, Inc. During this pro For example, the working electrode employed in electro cess, the silver/silver chloride (DuPont 5870 ink) leads and chemical sensors according to the present invention can be reference electrode were printed first onto polycarbonate varied, with suitable electrodes including but not limited to base material. The second pass of Dupont 7102T carbon carbon electrodes, platinum electrodes, palladium elec 65 graphite working electrode was printed on top of this. A final trodes, gold electrodes, and the like. Similarly, the reference pass of Norcote RDMSK4954-A2 dielectric defined the electrode can be varied, with suitable electrodes including working electrode area to be 0.0113 cm. US 7,163,616 B2

The chemistry was deposited over the working electrode with the use of an Asymtek Automove R 402 Dispensing TABLE 1-continued System. The system was programmed to perform the trans For- Enzyme fer by dipping a 62 mL stainless steel pin into a 1.5 mL mula- Concentration Concentration Polymer Polymer Eppendorf vial filled with reagent. Polycarbonate lid mate tion PQQ-GDH Mediator I Concentration Concentration rial was laminated to the sensors creating a capillary area i Units mM CMC 9/o PEO 9 over the working and reference electrodes capable of hold III 2O 12 1 O ing approximately 3 uIl of test solution. The capillary area, IV 2O 12 2 O which defines the sample volume, is first formed in the V 2O 12 O 2 polycarbonate lid material by a coining or stamping process. 10 As shown in FIG. 5, reactivity of the chemistry was FIGS. 8–12 show glucose dose response curves for each analyzed by generating a glucose dose-response curve with of the five formulations both before and after irradiation. buffered (100 mM phosphate, 100 mM sodium chloride, pH The stability of the five formulations is high, as clearly 7.4) samples containing a range of glucose concentrations shown by the near overlapping of the glucose response from 0 to 600 mg/dL. Current generated at each of the 15 glucose concentrations was measured using a potentiostat generated before and after irradiation. programmed to apply 150 mV potential with trigger level set Table 2 shows the results of enzyme assays conducted on to 100 nA, and timing programmed to record the current at the five formulations both before and after irradiation. 5, 10, 15, and 20 seconds. The trigger level refers to a Enzyme activity following irradiation remains high in all threshold level above which timing and recording are initi instances. ated. Sensors formulated with 20U Glucose Oxidase?sensor TABLE 2 and 6 mM mediator I were deposited onto electrode sensors Formulation if kGy Level Enzyme Activity as above. The dose response plot shown in FIG. 6 was obtained. 25 I O 4.67 25 4.32 50 4.20 Preparation of Electrochemical Biosensor and Heat/Humid 100 4.24 ity Stability II O 3.31 Electrochemical biosensors were constructed using a 25 3.34 III O 4.93 screen-printing process. Sensors were comprised of a carbon 30 25 4.87 working electrode and a silver/silver chloride reference IV O 4.96 electrode. A solution (150 to 800 ml) containing 12 mM 25 4.86 mediator I in 100 mM phosphate buffer (pH 7.4), and of the V O 3.63 enzyme POO-glucose dehydrogenase (10U/LL) was depos 25 4.OS ited on the surface of the working electrode and allowed to 35 dry at room temperature for 5 minutes prior to desiccation. The foregoing detailed description and examples have The electrodes were assembled into a format having a small been provided by way of explanation and illustration, and capillary gap, which allowed inoculation of the sensors with are not intended to limit the Scope of the appended claims. sample solutions. Many variations in the presently preferred embodiments In Subsequent tests, the sensors were subjected to the 40 illustrated herein be obvious to one of ordinary skill in the following environmental conditions prior to testing: 1) 50° art, and remain within the scope of the appended claims and C. for 2, 4, and 8 weeks; and 2) room temperature with 40% their equivalents. relative humidity. The sensors were poised at a potential of The invention claimed is: 150 mV relative to the Ag/AgCl reference electrode and the 1. A reagent for detecting an analyte, comprising: resulting current was measured. This mediator/enzyme com 45 bination is quite stable to both heat stress and humidity stress an enzyme selected from the group consisting of a fla as shown in FIG. 7. voprotein, a quinoprotein, and a combination thereof; and Sterilization of Biosensors and Radiation Stability Data a mediator selected from the group Five formulations of biosensor reagents (Table 1) were prepared and Subjected to E-beam irradiation using Sure 50 BeamR sterilization technology at Titan Scan Technologies R6 RI (San Diego, Calif.). Formulation I was irradiated at 25 kGy. 50 kGy, and 100 kGy, whereas each of Formulations II-V R S 2 R2 was irradiated at 25 kGy only. In the two rightmost column 55 headings of Table 1, the abbreviation CMC refers to car 2 boxymethylcellulose, and the abbreviation PEO refers to R8 N R5 R3, polyethylene oxide. R9 R4 R6 RI TABLE 1. 60 R O 2. R2 For- Enzyme mula- Concentration Concentration Polymer Polymer tion PQQ-GDH Mediator I Concentration Concentration 2 i Units mM CMC 9/o PEO 9% R8 N R5 R3, I 2O 12 O O 65 R9 R4 II 2O 12 O O US 7,163,616 B2 11 12 and combinations thereof, dehydrogenase, PQQ-methylamine dehydrogenase, wherein R. R. R. R. R. R. R7, R, and R are the PQQ-, and combinations thereof. same or different, and are independently selected from 7. The reagent of claim 1, wherein the enzyme is selected the group consisting of hydrogen, alkyl, alkenyl, alky from the group consisting of FAD-glucose oxidase, POQ nyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, ary glucose dehydrogenase, and a combination thereof. loxycarbonyl, aromatic keto, aliphatic keto, alkoxy, 8. The reagent of claim 7, wherein the mediator is selected aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihy from the group consisting of 3-(4-chloro-phenylimino)-3H droxyboron, and combinations thereof. phenothiazine, 3-(4-diethylamino-phenylimino)-3H-phe 2. The reagent of claim 1, wherein the mediator is selected 10 nothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, from the group consisting of 3-(4-chloro-phenylimino)-3H 3-(4-trifluoromethyl-phenylimino)-3H-phenothiazine, phenothiazine, 3-(4-diethylamino-phenylimino)-3H-phe 3-(4-methoxycarbonyl-phenylimino)-3H-phenothiazine, nothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, 3-(4-methoxy 3-(4-trifluoromethyl-phenylimino)-3H-phenothiazine, 15 phenylimino)-3H-phenothiazine, 7-acetyl-3-(4-methoxy 3-(4-methoxycarbonyl-phenylimino)-3H-phenothiazine, carbonylphenylimino)-3H-phenothiazine, 7-trifluorom 3-(4-nitro-phenylimino)-3H-phenothiazine, 3-(4-methoxy ethyl-3-(4-methoxycarbonylphenylimino)-3H phenylimino)-3H-phenothiazine, 7-acetyl-3-(4-methoxy phenothiazine, 3-(4'-.omega.-carboxy-n-butyl carbonylphenylimino)-3H-phenothiazine, 7-trifluorom phenylimino)-3H-phenothiazine, 3-(4-aminomethyl ethyl-3-(4-methoxycarbonylphenylimino)-3H phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminoph phenothiazine, 3-(4'-.omega.-carboxy-n-butyl enyl)-1,3,4-oxadiaZoyl)phenylimino)-3H-phenothiazine, phenylimino)-3H-phenothiazine, 3-(4-aminomethyl phenylimino)-3H-phenothiazine, 3-(4'-(2"-(5"-(p-aminoph 3-(4-beta.-aminoethyl-phenylimino)-3H-phenothiazine, enyl)-1,3,4-oxadiaZoyl)phenylimino)-3H-phenothiazine, 6-(4-ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H-phe 3-(4-beta.-aminoethyl-phenylimino)-3H-phenothiazine, 25 nothiazine, 6-(4-2-(2-ethanoloxy)ethoxyethoxyphenyl) 6-(4-ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H-phe amino-3-(4-2-(2-ethanoloxy)e-thoxyethoxy-phenylimino nothiazine, 6-(4-2-(2-ethanoloxy)ethoxyethoxyphenyl) 3H-phenothiazine, 3-(4-2-(2-ethanoloxy)ethoxyethoxy amino-3-(4-2-(2-ethanoloxy)e-thoxyethoxy-phenylimino phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H 3H-phenothiazine, 3-(4-2-(2-ethanoloxy)ethoxye-thoxy 30 phenothiazineboronic acid, (3-(3',5'-dicarboxy phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H phenylimino)-3H-phenothiazine, 3-(4-carboxy phenothiazinebor-onic acid, (3-(3',5'-dicarboxy phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy phenylimino)-3H-phenothiazine, 3-(4-carboxy phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H 35 phenothiazinesulfonic acid, and combinations thereof. phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H 9. The reagent of claim 7, wherein the mediator comprises phenothiazinesulfonic acid, and combinations thereof. 3. The reagent of claim 1, wherein the mediator comprises S N 40 21 S N CO2H. 2 2 C SOH.

N 45 10. The reagent of claim 8 wherein the mediator com CroCO2H prises

4. The reagent of claim 1, wherein the mediator comprises S N CO2H. 50 21

S N 21 N

2 CO2H YSOH. 55 11. The reagent of claim 1, further comprising a polymer 5. The reagent of claim 1, wherein the flavoprotein is selected from the group consisting of carboxymethylcellu selected from the group consisting of FAD-glucose oxidase, lose, polyethylene oxide, and combinations thereof. flavin-hexose oxidase, FAD-glucose dehydrogenase, FAD 60 12. A reagent for detecting glucose, comprising: lactate oxidase, FAD-cholesterol oxidase, FAD-, FAD-d-aminoacid oxidase, FAD-choline oxi PQQ-glucose dehydrogenase in an activity of about 20 dase, and combinations thereof. Units/LL; 6. The reagent of claim 1, wherein the quinoprotein is a buffer having a concentration between about 0.1 mM selected from the group consisting of PQQ-membrane 65 and about 100 mM, and a pH between about 4.5 and bound glucose dehydrogenase, POQ-soluble glucose dehy about 9.5; and drogenase, PQQ-lactate dehydrogenase, PQQ-aldehyde a mediator having a structure US 7,163,616 B2 13 14

S N COH, S N

2 5 2 N N SOH. CO2H 19. An electrochemical sensor comprising: 10 a working electrode having a Surface; and wherein the mediator has a concentration in the buffer a second electrode coupled to the working electrode, between about 0.1 mM and about 30 mM. wherein the surface of the working electrode is coated 13. The reagent of claim 12, wherein the buffer is selected with a solution of a reagent comprising an enzyme from the group consisting of Sodium phosphate, potassium Selected from the group consisting of a flavoprotein, a phosphate, Hepes, MOPS, TES, Pipes, ACES, BES, Dul 15 quinoprotein, and a combination thereof, and a media becco's, and combinations thereof. tor selected from 14. The reagent of claim 12, wherein the buffer comprises Sodium phosphate. and combinations thereof, 15. The reagent of claim 12, further comprising a polymer selected from the group consisting of carboxymethylcellu R6 RI lose, polyethylene oxide, and combinations thereof. 16. A reagent for detecting an analyte, comprising: R S N R2 an enzyme selected from the group consisting of PQQ glucose dehydrogenase, FAD-glucose oxidase, and a 2 combination thereof, and 25 R8 N O R5 R3, a mediator selected from the group consisting of R9 R4 R6 RI R6 R1 R O N R2 30 R S N R2 2 r R8 N RS R3, 2 R8 N O R5 R3, R9 R4 R9 R4 35 R6 R1 wherein R', R. R. R. R. R. R. R., and R are the same or different, and are independently selected from R O N R2 the group consisting of hydrogen, alkyl, alkenyl, alky nyl, aryl, heteroaryl, cyclic, heterocyclic, halo, 40 2 haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, ary R8 N O R5 R3, loxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihy R9 R4 droxyboron, and combinations thereof. 20. The electrochemical sensor of claim 19, wherein the and combinations thereof, 45 working electrode is selected from the group consisting of a wherein R', R. R. R. R. R. R. R., and R are the carbon electrode, a platinum electrode, a palladium elec same or different, and are independently selected from trode, and a gold electrode. the group consisting of hydrogen, alkyl, alkenyl, alky 21. The electrochemical sensor of claim 19, wherein the nyl, aryl, heteroaryl, cyclic, heterocyclic, halo, second electrode is selected from the group consisting of a haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, ary 50 reference electrode and a quasi-reference electrode. loxycarbonyl, aromatic keto, aliphatic keto, alkoxy, 22. The electrochemical sensor of claim 19, wherein the aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihy second electrode is a silver/silver chloride reference elec droxyboron, and combinations thereof. trode. 17. The reagent of claim 16, wherein the mediator com 23. The electrochemical sensor of claim 19, wherein the prises 55 Surface of the working electrode has an area of about 0.00113 cm. 24. An electrochemical sensor comprising: CO2H. a working electrode having a Surface; and 60 a reference electrode coupled to the working electrode, wherein the surface of the working electrode is coated with a solution of a reagent comprising PQQ-glucose dehydrogenase in an activity of about 1 CO2H Units/uIL to about 100 Units/uIL; 65 a buffer having a concentration of about 100 mM and a pH 18. The reagent of claim 16, wherein the mediator com of about 7.4; and prises a mediator having a structure US 7,163,616 B2 15 16 carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and a combination thereof. S N CO2H 5 29. The device of claim 28, wherein the mediator is selected from the group consisting of 3-(4-chloro-phe 2 N nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H CO2H phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H 10 phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H wherein the mediator has a concentration in the buffer of phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, about 1 mM to about 100 mM. 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- 25. The electrochemical sensor of claim 24, wherein the (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri activity of the PQQ-glucose dehydrogenase is about 20 15 Units/ul, and wherein the concentration of the mediator in fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe the buffer is about 24 mM. nothiazine, 3-(4-.omega.-carboxy-n-butyl-phenylimino)- 3H-phenothiazine, 3-(4-aminomethyl-phenylimino)-3H phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- S N CO2H. oxadiaZoyl)phenylimino)-3H-phenothiazine, 3-(4-beta.- aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- 2 N ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy CroCO2H 25 ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- 26. The electrochemical sensor of claim 24, wherein the ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, activity of the PQQ-glucose dehydrogenase is about 10 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- Units/ul, and wherein the concentration of the mediator in 30 dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy the buffer is about 12 mM. phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe 27. The reagent of claim 24, further comprising a polymer nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H selected from the group consisting of carboxymethylcellu phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H lose, polyethylene oxide, and combinations thereof. phenothiazinesulfonic acid, and combinations thereof. 28. A device for measuring an analyte, comprising: 35 a lancet; and 30. The device of claim 28, wherein the mediator com a sampling chamber connected to the lancet, comprising: prises a reagent comprising: an enzyme selected from the group consisting of 40 PQQ-glucose dehydrogenase, FAD-glucose oxi dase, and a combination thereof, and S 2 CO2H. a mediator selected from N 45 R6 RI CrcCO2H R. S N R2 21 31. The device of claim 28, wherein the mediator com 2 50 prises R8 N R5 R3,

R9 R4 S N R6 RI 2

R O N R2 55 2 21 N SOH. 2 R8 N R5 R3, 32. A method for detecting an analyte which undergoes a R9 R4 60 chemical reaction, the method comprising: providing an electrode Surface; and combinations thereof, wherein R. R. R. R. R. R. catalyzing the chemical reaction with an enzyme selected R. R. and R are the same or different, and are indepen from the group consisting of a flavoprotein, a quino dently selected from the group consisting of hydrogen, alkyl, 65 protein, and a combination thereof; alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, generating a redox equivalent by the chemical reaction; haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy and US 7,163,616 B2 17 18 transferring the redox equivalent to the electrode surface 35. The method of claim 32, wherein the mediator com using a mediator selected from prises

R6 RI 5 S N CO2H. R. S e R2 2 N

R8 N O R5 R3, and 10 CO2H R9 R4 R6 R1 36. The method of claim 32, wherein the flavoprotein is selected from the group R. O 2 R2 15 S N CO2H R8 N O R5 R3, 2 R9 R4 N CrcCO2H and a combination thereof, wherein R. R. R. R. R. R. R. R. and R are the same or different, and are indepen dently selected from the group consisting of hydrogen, alkyl, consisting of FAD-glucose oxidase, FAD-glucose dehydro alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, 25 genase, lactate oxidase, cholesterol oxidase, alcohol oxi dase, d-aminoacid oxidase, choline oxidase, and combina haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy tions thereof. carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, 37. The method of claim 32, wherein the quinoprotein is nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and selected from the group consisting of PQQ-glucose dehy combinations thereof. drogenase, lactate dehydrogenase, aldehyde dehydrogenase, 33. The method of claim 32, wherein the analyte is 30 methylamine dehydrogenase, alcohol dehydrogenase, and glucose. combinations thereof. 34. The method of claim 32, wherein the mediator is 38. The method of claim 32, wherein the enzyme is selected from the group consisting of 3-(4-chloro-phe selected from the group consisting of FAD-glucose oxidase, nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe 35 PQQ-glucose dehydrogenase, and a combination thereof. nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H 39. The method of claim 38, wherein the mediator com phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H prises phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- S N CO2H. (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri 40 fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe 2 nothiazine, 3-(4'-.omega.-carboxy-n-butyl-phenylimino)- N 3H-phenothiazine, 3-(4-aminomethyl-phenylimino)-3H phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- CO2H oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4-beta.- 45 aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- 40. The method of claim 38, wherein the mediator com ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H prises phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy 50 ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2-etha S N noloxy)ethoxy ethoxy-phenylimino-3H-phenothiazine, 2 3-(4'-phenylimino)-3H-phenothiazinebor-onic acid, (3-(3', 5'-dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-car 2 boxy-phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy SOH. phenylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H 55 phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H phenothiazinesulfonic acid, and combinations thereof.