US 2003OO94384A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0094384 A1 Vreeke et al. (43) Pub. Date: May 22, 2003

(54) REAGENTS AND METHODS FOR Related U.S. Application Data DETECTING ANALYTES, AND DEVICES COMPRISING REAGENTS FOR DETECTING (60) Provisional application No. 60/318,716, filed on Sep. ANALYTES 14, 2001. Publication Classification (76) Inventors: Mark S. Vreeke, Houston, TX (US); Mary Ellen Warchal-Windham, (51) Int. Cl...... G01N 27/327 Osceola, IN (US); Christina Blaschke, (52) U.S. Cl...... 205/777.5; 204/403.01 White Pigeon, MI (US); Barbara J. Mikel, Mishawaka, IN (US); Howard A. Cooper, Elkhart, IN (US) (57) ABSTRACT Reagents for detecting an analyte are described. A reagent Correspondence Address: comprises (a) an Selected from the group consisting Jerome L. Jeffers, Esq. of a flavoprotein, a quinoprotein, and a combination thereof; Bayer Corporation and (b) a mediator Selected from the group consisting of a P.O. BOX 40 phenothiazine, a phenoxazine, and a combination thereof. In Elkhart, IN 46515-0040 (US) addition, reagents having good Stability to radiation Steril ization are described. Electrochemical Sensors and Sampling devices comprising Such reagents, methods of producing a (21) Appl. No.: 10/231,539 Sterilized device including Such reagents, and methods for detecting an analyte which utilize Such reagents are (22) Filed: Sep. 3, 2002 described as well.

Layer 1 Layer 2 Layer3

Laminated Structure Patent Application Publication May 22, 2003. Sheet 1 of 6 US 2003/0094384 A1

FIG. 1

Layer 1 Layer 2 Layer3

Laminated Structure Patent Application Publication May 22, 2003. Sheet 2 of 6 US 2003/0094384 A1 FG. 2

Patent Application Publication May 22, 2003. Sheet 3 of 6 US 2003/0094384 A1 FIG. 3 Background Current After Irradiation 1400 1200 --F25 000 ... N g : : N a COntrol 200Gill : NN -f- N o50kGyis looköy NAD-MB PQQ-Ferri PQQ-MLB Chemistry

600mg/dL Response After irradiation 18000 16000 14000 12000 %. : Š a 10000 s % .. N C 80006000 N 3. i.. N SRS, 2013. N 3. 1 N sity NAD-MLB PQQ-Ferri PQQ-MLB Chemistry

PQQ-GDH/MLB Dose Response Curve 20000 15000 g -0- 5 SeC. 5 10000 -- 10 SeC. as 5000 -A- 15 SeC. -O - 20 SeC. O O 200 400 600 mg/dL Aqueous Glucose Patent Application Publication May 22, 2003 Sheet 4 of 6 US 2003/0094384 A1

MLB 92 with 3 25 a 20 S 15 CN y = 0.0423x + 0.53 5 R4 = 0.9934 92 S O 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 5 10000 A RT 4Wk 40%RH is 5000 nodes O Linear (RT 4wk) O 200 400 600 Glucose (mg/dL)

PQQ-GDHChemistry irradiation Stability Study Formulation 2

S 10000: -0-Control (S) 8 8000 --25 kGy Sis 40006999 -A-50 kG y 3 2000 -o- 100 kGy CC O C O 200 400 600 mg/dL Aqueous Glucose Patent Application Publication May 22, 2003 Sheet 5 of 6 US 2003/0094384 A1

PQQ-GDHChemistry irradiation Stability Study Formulation 3: 20000 9 16000 e É 12000 8000 -o-Control d 4000 --25kGy g O 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose

PQQ-GDH Chemistry Irradiation Stability Study O Formulation 3 8 20000 S 16000 9, 12000 or 8000 -o-Control d 4000 --25kGy CC O 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose

PQQ-GDHChemistry irradiation Stability Study O Formulation IV 3 8 20000 S 16000 9 is 12000 1. s --Control O -H25kGy 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose Patent Application Publication May 22, 2003. Sheet 6 of 6 US 2003/0094384 A1 FIG. 12

PQQ-GDHChemistry irradiation Stability Formulation V CD 3 20000 Se 16000 9, 12000 4. --Control O O --25kGy 0 1 00 200 300 400 500 600 mg/dL Aqueous Glucose US 2003/0094384 A1 May 22, 2003

REAGENTS AND METHODS FOR DETECTING would be desirable to provide biosensor reagents that are ANALYTES, AND DEVICES COMPRISING Stable to the radiation conditions commonly employed in REAGENTS FOR DETECTING ANALYTES lancet Sterilization. Reagents stable to Such radiation Steril BACKGROUND ization could be incorporated into highly user-convenient units in which lancet and biosensor are combined. 0001. The present invention relates to reagents, methods and devices for measurement of analytes and, more particu 0008. The present invention is directed to electron trans larly, to reagents, methods and devices for the measurement fer mediators for use in flavoprotein- and quinoprotein of glucose in the blood. based biosensor reagents, which exhibit improved Stability to both environmental interferents and to radiation Steriliza 0002 The monitoring of certain analyte concentrations in tion. the body enables early detection of health risks, and iden tifies the need for the introduction of therapeutic measures. SUMMARY One of the most commonly monitored analytes is glucose, the blood concentration of which is important in determining 0009. The scope of the present invention is defined solely the appropriate dosages of insulin for diabetics. Various by the appended claims, and is not affected to any degree by methods have been developed for monitoring glucose levels the Statements within this Summary. By way of introduction, in the blood, including the use of electrochemical biosen the presently preferred embodiments described herein are Sors. Electrochemical biosensors are based on enzyme directed towards remedying the aforementioned Stability catalyzed chemical reactions involving the analyte of inter problems of electron transfer mediators and enzyme biosen est. In the case of glucose monitoring, the relevant chemical SOS. reaction is the oxidation of glucose to gluconolactone. This 0010 Briefly stated, a composition aspect of the present oxidation is catalyzed by a variety of , Some of invention is directed to a reagent for detecting an analyte, which may contain a bound coenzyme Such as nicotinamide comprising (a) an enzyme selected from the group consist adenine dinucleotide (phosphate) (NAD(P)), while others ing of a flavoprotein, a quinoprotein, and a combination may contain a bound Such as flavin adenine thereof, and (b) a mediator Selected from the group consist dinucleotide (FAD) or pyrroloquinolinequinone (PQQ). ing of a phenothiazine, a phenoxazine, and a combination 0003. In biosensor applications, the redox equivalents thereof. generated in the course of the oxidation of glucose are 0011. A first apparatus aspect of the present invention is transported to the Surface of an electrode whereby an elec directed to an electrochemical Sensor comprising: (a) a trical signal is generated. The magnitude of the electrical working electrode having a Surface; and (b) a second elec Signal is then correlated with concentration of glucose. The trode coupled to the working electrode. The Surface of the transfer of redox equivalents from the Site of chemical working electrode is coated with a Solution or mixture of a reaction in the enzyme to the Surface of the electrode is reagent comprising an enzyme Selected from the group accomplished with the use of electron transfer mediators. consisting of a flavoprotein, a quinoprotein, and a combi 0004. A significant problem with the use of electron nation thereof, and a mediator Selected from the group transfer mediators in biosensors is the instability of these consisting of a phenothiazine, a phenoxazine, and a combi compounds upon exposure to common environmental con nation thereof. ditions Such as temperature and moisture. As a result, the 0012 A Second apparatus aspect of the present invention number of mediators Suitable for use in glucose biosensors is directed to a device for measuring an analyte, comprising is quite limited. (a) a lancet; and (b) a sampling chamber connected to the 0005 U.S. Pat. No. 5,520,786 (786) to Bloczynski et al. lancet. The Sampling chamber comprises a reagent compris describes families of phenothiazine and phenoxazine com ing an enzyme Selected from the group consisting of PQQ pounds Suitable for use as electron transfer mediators with glucose dehydrogenase, FAD-glucose oxidase, and a com the enzymes dihydronicotinamide adenine dinucleotide bination thereof, and (b) a mediator Selected from the group (NADH), NADPH, and analogs thereof. Cofactor based consisting of a phenothiazine, a phenoxazine, and a combi enzymes Such as FAD-glucose oxidase and PQQ-glucose nation thereof. dehydrogenase have Several advantages over NAD-based 0013 A first method aspect of the present invention is enzymes, including lower cost, higher enzyme activity, directed to a method of producing a Sterilized device for increased Stability, and bound as opposed to readily disso measuring an analyte, comprising (a) providing a device in ciable cofactor. accordance with the present invention, and (b) irradiating the 0006 Electron transfer mediators previously used with device with E-beam or gamma ray radiation. FAD-glucose oxidase and POO-glucose dehydrogenase 0014) A second method aspect of the present invention is include quinones, phenzine methoSulfate, dichlorophenolin directed to a method for detecting an analyte which under dophenol and ferricyanide. Unfortunately, these compounds goes a , the method comprising (a) pro have proven to be highly Susceptible to the environmental viding an electrode Surface; (b) catalyzing the chemical agents described above, and result in biosensor reagents of reaction with an enzyme Selected from the group consisting low stability. Thus, mediators are needed which exhibit good of a flavoprotein, a quinoprotein, and a combination thereof; Stability upon exposure to commonly-encountered environ (c) generating a redox equivalent by the chemical reaction; mental agents, and which can be used in flavoprotein- and and (d) transferring the redox equivalent to the electrode quinoprotein-based Systems. Surface using a mediator Selected from the group consisting 0007. In addition to the need for biosensor reagents that of a phenothiazine, a phenoxazine, and a combination are stable to the environmental agents described above, it thereof. US 2003/0094384 A1 May 22, 2003

0.015 The presently preferred embodiments discussed tion of an enzyme that catalyzes a reaction of an analyte, and herein may possess one or more advantages relative to other a phenothiazine and/or phenoxazine mediator. The term flavoprotein- and quinoprotein-based biosensor reagents, “bioburden” refers to the population of viable microorgan which can include but are but not limited to: improved isms on a determined immediately prior to irradia biosensor reagent Stability; enhanced electron transfer capa bility of mediators; ability to tune mediators for optimum tion. electrode operation; reduced Susceptibility of media 0025. A biosensor reagent for detecting an analyte in tors, increased thermal Stability of mediators, increased stability of mediators to ambient humidity; lower redox accord with the present invention includes (1) 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, and (2) a mediator Sterilization conditions. Selected from the group consisting of a phenothiazine, a phenoxazine, and a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS 0026. The nature of the analyte monitored in accord with 0016 FIG. 1 shows a schematic illustration of a device the present invention is unrestricted, provided the analyte for measuring an analyte that embodies features of the undergoes a chemical reaction that is catalyzed by an present invention. enzyme Selected from the group consisting of a flavoprotein, 0017 FIG. 2 shows a perspective view of an integrated a quinoprotein, and a combination thereof. Preferred ana lancet/biosensor device for use in accordance with the lytes include but are not limited to glucose, lactate, D-amino present invention. acids, ascorbate, alcohol, cholesterol, choline, and acetyl 0.018 FIG. 3 shows a graph of background currents for choline. 3 formulations of biosensor reagents exposed to increasing levels of radiation. 0027 Flavoproteins in accord with the present invention include FAD-glucose oxidase (Enzyme Classification No. 0.019 FIG. 4 shows a graph of the current response of 1.1.3.4), Flavin-hexose oxidase (EC No. 1.1.3.5) and FAD radiation Sterilized biosensor reagents upon exposure to glucose dehydrogenase (EC No. 1.1.99.10) For information glucose. relating to these flavoproteins, See: Adriaan Joseph Jan 0020 FIG. 5 shows a plot of current vs. glucose con Olsthoorn, “Structural and Mechanistic Aspects of Soluble centration at increasing time intervals for a PQQ-glucose Quinoprotein Glucose Dehydrogenase from Acinetobacter dehydrogenase/phenothiazine biosensor. calcoaceticus,” Ph.D. dissertation, Delft University of Tech 0021 FIG. 6 shows a plot of current vs. glucose con nology, The Netherlands, 1999. Additional oxidase enzymes centration for a FAD-glucose oxidase/phenothiazine bio for use in accord with the present invention include but are SCSO. not limited to lactate oxidase, cholesterol oxidase, alcohol 0022 FIG. 7 shows a plot of current vs. glucose con oxidase (e.g., methanol oxidase), d-aminoacid oxidase, cho centration for a PQQ-glucose dehydrogenase/phenothiazine line oxidase, and FAD derivatives thereof. A preferred biosensor reagent Subjected to heat StreSS and humidity flavoprotein for use in accord with the present invention is StreSS. FAD-glucose oxidase. 0023 FIGS. 8-12 show plots of current vs. glucose 0028 Quinoproteins in accord with the present invention concentration for 5 formulations of PQQ-glucose dehydro include but are not limited to membrane bound and soluble genase/phenothiazine biosensors exposed to varying levels PQQ-glucose dehydrogenase (EC No. 1.1.99.17). Informa of radiation. tion relating to PQQ-glucose dehydrogenase can be found in DETAILED DESCRIPTION OF THE the Olsthoorn reference cited above. Additional quinopro PRESENTLY PREFERRED EMBODIMENTS tein enzymes for use in accord with the present invention include but are not limited to , alde 0024. Throughout this description and in the appended hyde dehydrogenase, methylamine dehydrogenase, alcohol claims, the following definitions are to be understood: The dehydrogenase (e.g., methanol dehydrogenase), and PQQ term “analyte' refers to one or a plurality of Species having derivatives thereof. A preferred quinoprotein for use in a concentration of interest. The term “flavoprotein” refers to accord with the present invention is PQQ-glucose dehydro enzymes containing flavin cofactors. The term "quinopro tein’ refers to enzymes containing PQQ or Similar cofactors. genase. The phrase “redox equivalent” refers to one or a plurality of 0029 Mediators in accord with the present invention charged species (e.g., electrons) produced in electrochemi include phenothiazines having the formula cal reactions involving the analyte. The phrase "E-beam irradiation' or “electron beam irradiation” refers to a proceSS of exposure to a concentrated, high-current Stream of elec trons. The terms “alkyl,”“alkenyl,”“alkynyl,”“aryl,”“het eroaryl,”“cyclic,”“heterocyclic,”“halo,”“haloalkyl,”“car boxy,”“carboxyalkyl,”“alkoxycarbonyl,”“aryloxycarbonyl, “aromatic keto,”“aliphatic keto,”“alkoxy,”“aryloxy, g nitro,”“dialkylamino,”“aminoalkyl,”“sulfo, “dihydroxyboron,” and the like refer to substituents well known in the art, which may be branched or unbranched and may themselves be substituted with one or more substitu ents. The phrase “biosensor reagent” refers to the combina US 2003/0094384 A1 May 22, 2003

0030) and phenoxazines having the formula oxadiazoyl)phenylimino)-3H-phenothiazine, aminoethyl-phenylimino)-3H-phenothiazine, ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H R6 R1 phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy R O N R2 ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- 21 ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, 2 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- R8 N R5 R3, dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe R9 R4 nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H phenothiazinedisulfonic acid, and 3-(3-phenylimino)-3H 0031) wherein R', R. R. R. R. R. R7, R, and Rare phenothiazineSulfonic acid. the same or different, and are independently Selected from 0035 More preferably, the mediator used in accord with the group consisting of hydrogen, alkyl, alkenyl, alkynyl, the present invention is Selected from the group consisting of aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, car boxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aro matic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialky Mediator I lamino, aminoalkyl, Sulfo, dihydroxyboron, and S N CO2H combinations thereof. 2

0032. In contrast to the single electron transfer carrying 2 capability of K-Fe(CN), mediators in accord with the N present invention have the ability to carry two redox equiva lents, and are therefore well Suited for use in FAD and CO2H quinoprotein oxidation/reduction processes, which generally and involve the transfer of two electrons. Moreover, the potential Mediator II of mediators of the present invention can be tuned to the S N optimum potential (i.e., the potential where the Signal con 2 tribution from interferences is minimized) for a specific Sample matrix by varying the Substitution on the aromatic N SOH. rings. Electron-donating Substituents (e.g., alkyl, alkoxy, amine, hydroxy, etc.) result in decreased redox potentials, while electron-withdrawing Substituents (e.g., carboxylic 0036 Relative to ferricyanide, phenothiazine media acid, ester, aldehyde, ketone, nitrile, nitro, Sulfonic acid, tors-in particular mediator I-are leSS Susceptible to oxy trifluromethyl, etc.) result in increased redox potentials. For gen degradation, more thermally Stable, and more stable to blood or plasma Samples, the ideal potential usually lies ambient humidity. In addition, mediator I works at a lower between about -200 and about 100 mV versus an Ag/AgCl redox potential than ferricyanide. For example, E for reference. mediator I is approximately 0 mV versus an Ag/AgCl 0033. The substituents on the aromatic rings, in addition reference, whereas Eo for ferricyanide is approximately 250 to their utility in tuning the redox potentials of the mediators, mV verSusan. Ag/AgCl reference. The lower redox potential can also be used to enhance mediator Solubility. For of phenothiazine mediators is advantageous in that there is example, the introduction of a Substituent having the capac a region around 0 mV verSuSanAg/AgCl reference in which ity for hydrogen bonding can be expected to render the the amount of electrochemical interferences are minimized. mediator more water Soluble than a mediator lacking Such Thus, the impact from chemical interferents in the blood can Substitution. In addition, these Substituents can Serve as be minimized by using these mediators. functional groups for immobilizing the mediators to a Sup 0037 Reagents embodying features of the present inven port (e.g., the electrode Surface or, alternatively, a chemical tion can be incorporated into a variety of biosensor devices, matrix Such as a polymer backbone, which is Suitable for including but not limited to the ones described in U.S. Pat. application to the electrode Surface). No. 5,120,420 and No. 5,798,031, the entire contents of both 0034 Preferably, mediators used in biosensor reagents of which are incorporated herein by reference, except that in according to the present invention include 3-(4-chloro the event of any inconsistent disclosure or definition from phenylimino)-3H-phenothiazine, 3-(4'-diethylamino-phe the present application, the disclosure or definition herein nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H shall be deemed to prevail. phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H 0038 Turning now to the drawings, FIG. 1 shows a phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H representative electrochemical Sensor in accordance with the phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, present invention. The electrochemical Sensor 2 is com 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- prised of an insulating base 4 upon which is printed (typi (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri cally by Screen printing techniques) an electrode pattern (6 fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe and 8) and a reagent layer 10 that contains a reagent nothiazine, 3-(4'-(o-carboxy-n-butyl-phenylimino)-3H embodying features of the present invention. The two parts phenothiazine, 3-(4-aminomethyl-phenylimino)-3H of the electrode print, 6 and 8, provide the working and phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- reference electrodes necessary for the electrochemical deter US 2003/0094384 A1 May 22, 2003

mination. A lancet element 12 can be incorporated into the resulted in a background Signal which constituted a signifi electrochemical Sensor (e.g., interposed between layers 1 cant amount of the glucose signal. While formulation (2) and 2), as is described more fully hereinbelow. The three exhibited good tolerance to the radiation process, the activ layers shown in FIG. 1 can be joined by means of an ity of the extracted enzyme was lower than the correspond adhesive (e.g., pressure Sensitive, hot melt, etc.) or by Sonic ing activity of the enzyme extracted from formulation (3). welding, depending on the identity of the materials. FIG. 4 shows a graph of current response when these 0039. It has been found that biosensor reagents compris radiation-Sterilized Sensors were exposed to 600 mg/dL ing PQQ-glucose dehydrogenase and certain phenothiazine glucose. mediators exhibit high Stability to radiation Sterilization. A 0044) The manner in which a device embodying features preferred application of radiation Stable biosensor reagents of the present invention is made, and the process by which in accord with the present invention is for the development Such a device is used for monitoring an analyte, will be of integrated lancet/biosensor devices. An example of Such abundantly clear to one of ordinary skill in the art based an integrated device is illustrated in FIG. 2 and described in upon joint consideration of both the preceding description, U.S. Pat. No. 5,801,057, the entire contents of which are and the following representative procedures. It is to be incorporated herein by reference, except that in the event of understood that many variations in the presently preferred any inconsistent disclosure or definition from the present embodiments illustrated herein will be obvious to one of application, the disclosure or definition herein shall be ordinary skill in the art, and remain within the Scope of the deemed to prevail. appended claims and their equivalents. 0040. As shown in FIG. 2, the integrated lancet/biosen 0045 For example, the working electrode employed in Sor device 14 has a finely bored needle 16 connected to a electrochemical Sensors according to the present invention sampling chamber 18. Sampling chamber 18 has at least one can be varied, with Suitable electrodes including but not optical window 20 and a vent 22 through which air can be limited to carbon electrodes, platinum electrodes, palladium displaced when the chamber 18 fills with blood or other electrodes, gold electrodes, and the like. Similarly, the fluids. Preferably, sampling chamber 18 comprises a bio reference electrode can be varied, with Suitable electrodes Sensor reagent comprising PQQ-glucose dehydrogenase and including but not limited to silver-silver chloride electrodes, a phenothiazine and/or phenoxazine mediator. Preferably, calomel electrodes, Saturated calomel electrodes, and the the mediator is a phenothiazine. More preferably, the media like. Alternatively, a quasi-reference electrode (e.g., a large tor has a Structure represented by mediator I or mediator II Surface area platinum electrode) of the type commonly used above. Once sampling chamber 18 has been loaded with in non-aqueous electrochemical experiments (i.e., an elec biosensor reagent, the entire device 14 can be Subjected to trode which does not have a specific redox Species to which radiation sterilization. Preferably, the method of sterilization its potential is referenced) can be used in accord with the involves electron beam (E-beam) irradiation or gamma present invention. The Surface areas of all electrodes irradiation. employed in accordance with the present invention are 0041 Asset forth in the Association for the Advancement likewise subject to variation. Preferably, the working elec of Medical Instrumentation document ANSI/AAMI/ISO trode has dimensions of about 0.6 mmx1.2 mm. 11137-1994, products that penetrate the skin and come into 0046. Furthermore, the compositions and pH of the buffer contact with the blood must have a Sterility assurance level Solutions employed, and the enzyme activities and concen (SAL) of 10, which corresponds to a one in a million trations of components of the biosensor reagents, are Subject probability of a viable microorganism being present on a to wide variation. Suitable buffer Solutions include but are product unit after Sterilization. The Sterilization dose needed not limited to HEPES (i.e., N-2-hydroxyethylpiperazine-N'- to achieve a 10 SAL depends on the bioburden of the 2-ethanesulfonic acid), MOPS (i.e., 3-(N-morpholino)pro Sample. For example, a Sample with a bioburden of 1,021 panesulfonic acid), TES (i.e., N-tris(hydroxymethyl)methyl requires a sterilization dose of 24.9 kGy to achieve a 10 2-aminoethaneSulfonic acid), 2-(2-hydrox-1,1- SAL. bis(hydroxymethyl)-ethylamino)ethanesulfonic acid), 0042. In the examples described hereinbelow, electron PIPES (i.e., piperazine-N,N'-bis(2-ethanesulfonic acid)), beam (E-beam) irradiation was employed as the method of 1,4-piperazinediethaneSulfonic acid), ACES (i.e., N-(car Sterilization. The biosensor reagents Subjected to the elec bamoylmethyl)-2-aminoethaneSulfonic acid), N-(2-acetami tron beam absorb energy from the electrons. The energy that dol)-2-aminoethanesulfonic acid, BES (i.e., N,N-bis(2-hy is absorbed per unit mass of material is referred to as the droxyethyl)-2-aminoethaneSulfonic acid, and Dulbecco's absorbed dose, and it is this absorption of energy-or dose buffer (i.e., 0.008M sodium phosphate, 0.002M potassium delivery-that destroys the reproductive cells and DNA phosphate, 0.14M sodium chloride, 0.01M potassium chlo chains of microorganisms, thereby rendering a product Ster ride, pH 7.4). ile. E-beam doses of 25, 50 and 100 kGy were used because 0047 The manner in which reagents and devices the bioburden of the biosensor reagents was unknown. embodying features of the present invention are made, and the methods by which these reagents and devices are used 0.043 FIG. 3 shows a graph of the background currents for monitoring an analyte, will be abundantly clear to one of observed for three formulations of biosensor reagents exposed to increasing levels of radiation: (1) NAD-glucose ordinary skill in the art based upon joint consideration of dehydrogenase with Mediator I, (2) PQQ-glucose dehydro both the preceding description, and the following represen genase with Ferricyanide, and (3) PQQ-glucose dehydroge tative procedures. nase with Mediator I. The POO formulations tolerated the 0048 While the examples provided hereinbelow relate to irradiation extremely well. In contrast, the NAD formulation in vitro applications of the biosensor reagents in accord with exhibited poor tolerance to the Sterilization conditions, and the present invention, it is contemplated that these reagents US 2003/0094384 A1 May 22, 2003 can also be adapted for in Vivo analyte monitoring by mediator I in 100 mM phosphate buffer (pH 7.4), and of the chemically immobilizing the phenoxazine and/or phenothi enzyme PQQ-glucose dehydrogenase (10 U/ul ) was depos azine mediators (e.g., by chemical reaction at one or more of ited on the Surface of the working electrode and allowed to the Substituent groups on the aromatic rings), and incorpo dry at room temperature for 5 minutes prior to desiccation. rating the immobilized mediators into a device which can be The electrodes were assembled into a format having a Small implanted Subcutaneously into a patient. capillary gap, which allowed inoculation of the Sensors with Sample Solutions. EXAMPLES 0057. In subsequent tests, the sensors were subjected to the following environmental conditions prior to testing: 1) 0049 Preparation of Biosensor and Glucose Dose-Re 50° C. for 2, 4, and 8 weeks; and 2) room temperature with Sponse 40% relative humidity. The sensors were poised at a poten 0050. A liquid chemistry reagent was prepared to be 20 tial of 150 mV relative to the Ag/AgCl reference electrode UnitS/ull pyrolloquinolinequinone-glucose dehydrogenase and the resulting current was measured. This mediator/ (PQQ-GDH) and 24 mM mediator I in 100 mM Sodium enzyme combination is quite Stable to both heat StreSS and Phosphate, pH 7.4. The first component of the reagent was humidity stress as shown in FIG. 7. made by dissolving the mediator in 100 mM phosphate pH 0.058 Sterilization of Biosensors and Radiation Stability 7.4, adjusting the pH back to 7.4, and filtering the Solution Data by forcing it through a Whatman 0.45 micron PTFE Syringe filter. The reagent was completed by adding lyophilized 0059 Five formulations of biosensor reagents (Table 1) PQQ-GDH (Toyobo Product No. GLD-321) to an activity of were prepared and Subjected to E-beam irradiation using SureBeam(R) sterilization technology at Titan Scan Tech 20 Uful. nologies (San Diego, Calif.). Formulation I was irradiated at 0051. The chemistry formulation was deposited onto 25 kGy, 50 kGy, and 100 kGy, whereas each of Formulations electrodes, which had been produced using a 3-pass Screen II-V was irradiated at 25 kGy only. In the two rightmost printing process by Conductive Technologies, Inc. During column headings of Table 1, the abbreviation CMC refers to this process, the silver/silver chloride (DuPont 5870 ink) carboxymethylcellulose, and the abbreviation PEO refers to leads and reference electrode were printed first onto poly polyethylene oxide. carbonate base material. The second pass of Dupont 7102T carbon-graphite working electrode was printed on top of TABLE 1. this. A final pass of Norcote RDMSK4954-A2 dielectric For- Enzyme defined the working electrode area to be 0.0113 cm. mula- Concentration Concentration Polymer Polymer tion POO-GDH Mediator I Concentration Concentration 0.052 The chemistry was deposited over the working # Units mM CMC 9% PEO % electrode with the use of an Asymtek Automove(R) 402 Dispensing System. The System was programmed to per I 2O 12 O O II 2O 12 O O form the transfer by dipping a 62 mL StainleSS Steel pin into III 2O 12 1. O a 1.5 mL Eppendorf vial filled with reagent. Polycarbonate IV 2O 12 2 O lid material was laminated to the Sensors creating a capillary V 2O 12 O 2 area over the working and reference electrodes capable of holding approximately 3 till of test Solution. The capillary area, which defines the Sample Volume, is first formed in the 0060 FIGS. 8-12 show glucose dose response curves for polycarbonate lid material by a coining or Stamping process. each of the five formulations both before and after irradia tion. The stability of the five formulations is high, as is 0053 As shown in FIG. 5, reactivity of the chemistry clearly shown by the near overlapping of the glucose was analyzed by generating a glucose dose-response curve response generated before and after irradiation. with buffered (100 mM phosphate, 100 mM sodium chlo ride, pH 7.4) samples containing a range of glucose con 0061 Table 2 shows the results of enzyme assays con centrations from 0 to 600 mg/dL. Current generated at each ducted on the five formulations both before and after irra of the glucose concentrations was measured using a poten diation. Enzyme activity following irradiation remains high tiostat programmed to apply 150 mV potential with trigger in all instances. level Set to 100 nA, and timing programmed to record the current at 5, 10, 15, and 20 seconds. The trigger level refers TABLE 2 to a threshold level above which timing and recording are initiated. Formulation # kGy Level Enzyme Activity I O 4.67 0054 Sensors formulated with 20 U Glucose Oxidase/ 25 4.32 sensor and 6 mM mediator I were deposited onto electrode 50 4.2O sensors as above. The dose response plot shown in FIG. 6 1OO 4.24 was obtained. II O 3.31 25 3.34 III O 4.93 0.055 Preparation of Electrochemical Biosensor and 25 4.87 Heat/Humidity Stability IV O 4.96 25 4.86 0056 Electrochemical biosensors were constructed using V O 3.63 a Screen-printing process. Sensors were comprised of a 25 4.05 carbon working electrode and a Silver/silver chloride refer ence electrode. A solution (150 to 800 nl) containing 12 mM US 2003/0094384 A1 May 22, 2003

0062) The foregoing detailed description and examples phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H have been provided by way of explanation and illustration, phenothiazineSulfonic acid, and combinations thereof. and are not intended to limit the Scope of the appended 4. The reagent of claim 1, wherein the mediator comprises claims. Many variations in the presently preferred embodi S N CO2H. ments illustrated herein will be obvious to one of ordinary 21 skill in the art, and remain within the Scope of the appended claims and their equivalents. 2 N 1. A reagent for detecting an analyte, comprising: COH an enzyme Selected from the group consisting of a fla 5. The reagent of claim 1, wherein the mediator comprises voprotein, a quinoprotein, and a combination thereof; S N and 21 a mediator Selected from the group consisting of a phe 2 nothiazine, a phenoxazine, and a combination thereof. SOH. 2. The reagent of claim 1, wherein the mediator is Selected from the group consisting of 6. The reagent of claim 1, wherein the flavoprotein is Selected from the group consisting of FAD-glucose oxidase, R6 R1 flavin-hexose oxidase, FAD-glucose dehydrogenase, FAD lactate oxidase, FAD-cholesterol oxidase, FAD-alcohol R. 2' R2 oxidase, FAD-d-aminoacid oxidase, FAD-choline oxi dase, and combinations thereof. 2 7. The reagent of claim 1, wherein the quinoprotein is R8 N R5 R3, Selected from the group consisting of PQQ-membrane bound glucose dehydrogenase, POO-Soluble glucose dehy R9 R4 drogenase, PQQ-lactate dehydrogenase, PQQ-aldehyde R6 R1 dehydrogenase, PQQ-methylamine dehydrogenase, PQQ-, and combinations thereof. R t e R2 8. The reagent of claim 1, wherein the enzyme is Selected from the group consisting of FAD-glucose oxidase, PQQ 2 glucose dehydrogenase, and a combination thereof. R8 N R5 R3, 9. The reagent of claim 8, wherein the mediator is selected R9 R4 from the group consisting of and combinations thereof, wherein R, R R,R,R,R,R, R6 R1 R, and Rare the same or different, and are independently Selected from the group consisting of hydrogen, alkyl, R. e R2 alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy 2 carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, R8 N R5 R3, nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and combinations thereof. R9 R4 3. The reagent of claim 1, wherein the mediator is Selected R6 R1 from the group consisting of 3-(4-chloro-phenylimino)-3H phenothiazine, 3-(4-diethylamino-phenylimino)-3H-phe R 2 R2 nothiazine, 3-(4'ethyl-phenylimino)-3H-phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H-phenothiazine, 2 3-(4-methoxycarbonyl-phenylimino)-3H-phenothiazine, R8 N R5 R3, 3-(4-nitro-phenylimino)-3H-phenothiazine, 3-(4-methoxy phenylimino)-3H-phenothiazine, 7-acetyl-3-(4-methoxy R9 R4 carbonylphenylimino)-3H-phenothiazine, 7-trifluorom and combinations thereof, wherein R, R R,R,R,R,R, ethyl-3-(4-methoxycarbonylphenylimino)-3H phenothiazine, 3-(4-(o-carboxy-n-butyl-phenylimino)-3H R, and Rare the same or different, and are independently phenothiazine, 3-(4-aminomethyl-phenylimino)-3H Selected from the group consisting of hydrogen, alkyl, phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4-3- haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy combinations thereof. ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy 10. The reagent of claim 8, wherein the mediator is ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- Selected from the group consisting of 3-(4-chloro-phe ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, US 2003/0094384 A1 May 22, 2003

3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- 17. The reagent of claim 14, further comprising a polymer (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri Selected from the group consisting of carboxymethylcellu fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe lose, polyethylene oxide, and combinations thereof. nothiazine, 3-(4'-(o-carboxy-n-butyl-phenylimino)-3H 18. A reagent for detecting an analyte, comprising: phenothiazine, 3-(4-aminomethyl-phenylimino)-3H phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- an enzyme Selected from the group consisting of PQQ oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4-3- glucose dehydrogenase, FAD-glucose oxidase, and a aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- combination thereof; ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H a mediator Selected from the group consisting of a phe phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy nothiazine, a phenoxazine, and combinations thereof. ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy 19. The reagent of claim 18, wherein the mediator com ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- prises ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- S N CO2H. dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy 21 phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H N phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H CroCO2H phenothiazineSulfonic acid, and combinations thereof. 11. The reagent of claim 8, wherein the mediator com 20. The reagent of claim 18, wherein the mediator com prises prises

S N S N 21 2

2 2 N SOH. t SOH. 12. The reagent of claim 8, wherein the mediator com 21. An electrochemical Sensor comprising: prises a working electrode having a Surface; and S 2 CO2H. a second electrode coupled to the working electrode, wherein the Surface of the working electrode is coated with a N Solution of a reagent comprising CroCOH an enzyme Selected from the group consisting of a 13. The reagent of claim 1, further comprising a polymer flavoprotein, a quinoprotein, and a combination Selected from the group consisting of carboxymethylcellu thereof; and lose, polyethylene oxide, and combinations thereof. a mediator Selected from the group consisting of a 14. A reagent for detecting glucose, comprising: phenothiazine, a phenoxazine, and a combination thereof. PQQ-glucose dehydrogenase in an activity of about 20 22. The electrochemical sensor of claim 21, wherein the Units/ul, working electrode is Selected from the group consisting of a a buffer having a concentration between about 0.1 mM carbon electrode, a platinum electrode, a palladium elec and about 100 mM, and a pH between about 4.5 and trode, and a gold electrode. about 9.5; and 23. The electrochemical sensor of claim 21, wherein the Second electrode is Selected from the group consisting of a a mediator having a structure reference electrode and a quasi-reference electrode. 24. The electrochemical sensor of claim 21, wherein the S N COH, 21 Second electrode is a Silver/silver chloride reference elec trode. 2 25. The electrochemical sensor of claim 21, wherein the N Surface of the working electrode has an area of about COH 0.00113 cm’. 26. An electrochemical Sensor comprising: wherein the mediator has a concentration in the buffer a working electrode having a Surface; and between about 0.1 mM and about 30 mM. a reference electrode coupled to the working electrode, 15. The reagent of claim 14, wherein the buffer is selected wherein from the group consisting of Sodium phosphate, potassium phosphate, Hepes, MOPS, TES, Pipes, ACES, BES, Dul the Surface of the working electrode is coated with a becco's, and combinations thereof. Solution of a reagent comprising 16. The reagent of claim 14, wherein the buffer comprises PQQ-glucose dehydrogenase in an activity of about Sodium phosphate. 1 Units/ull to about 100 Units/ull; US 2003/0094384 A1 May 22, 2003

a buffer having a concentration of about 100 mM and nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe a pH of about 7.4; and nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H phenothiazine, 3-(4'-trifluoromethyl-phenylimino)-3H a mediator having a structure phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H S N COH, phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, 2 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri 2 fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe N nothiazine, 3-(4'-(o-carboxy-n-butyl-phenylimino)-3H phenothiazine, 3-(4-aminomethyl-phenylimino)-3H CO2H phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4- oxadiazoyl)phenylimino)-3H-phenothiazine, 3-(4-3- wherein the mediator has a concentration in the aminoethyl-phenylimino)-3H-phenothiazine, 6-(4'- buffer of about 1 mM to about 100 mM. ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H 27. The electrochemical sensor of claim 26, wherein the phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy activity of the PQQ-glucose dehydrogenase is about 20 ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy UnitS/ull, and wherein the concentration of the mediator in ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- the buffer is about 24 mM. ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, 28. The electrochemical sensor of claim 26, wherein the 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- activity of the PQQ-glucose dehydrogenase is about 10 dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy UnitS/ull, and wherein the concentration of the mediator in phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe the buffer is about 12 mM. nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H 29. The reagent of claim 26, further comprising a polymer phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H Selected from the group consisting of carboxymethylcellu phenothiazineSulfonic acid, and combinations thereof. lose, polyethylene oxide, and combinations thereof. 33. The device of claim 30, wherein the mediator com 30. A device for measuring an analyte, comprising: prises S N CO2H. a lancet; and 2 a Sampling chamber connected to the lancet, comprising: 2 a reagent comprising: N an enzyme Selected from the group consisting of COH PQQ-glucose dehydrogenase, FAD-glucose oxi dase, and a combination thereof, and 34. The device of claim 30, wherein the mediator com prises a mediator Selected from the group consisting of a S N phenothiazine, a phenoxazine, and a combination 2 thereof. 31. The device of claim 30, wherein the mediator is 2 Selected from the group consisting of N SOH. R6 R1 35. A method of producing a sterilized device for mea Suring an analyte, comprising: R. t 2 R2 providing a device comprising: 2 a reagent, comprising: R8 N R5 R3, an enzyme Selected from the group consisting of R9 R4 PQQ-glucose dehydrogenase, FAD-glucose oxi R6 R1 dase, and a combination thereof, and a mediator Selected from the group consisting of a R O 2' R2 phenothiazine, a phenoxazine, and a combination thereof; and R8 N R5 R3, irradiating the device with E-beam or gamma ray irradia tion. R9 R4 36. A method of producing a Sterilized device for mea Suring an analyte, comprising: and combinations thereof, wherein R, RR,R,R,R,R", providing a device comprising: R, and R are the same or different, and are independently Selected from the group consisting of hydrogen, alkyl, a lancet; and alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, a Sampling chamber connected to the lancet, compris haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy ing: carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and a reagent, comprising: combinations thereof. an enzyme Selected from the group consisting of 32. The device of claim 30, wherein the mediator is PQQ-glucose dehydrogenase, FAD-glucose Selected from the group consisting of 3-(4-chloro-phe oxidase, and a combination thereof, and US 2003/0094384 A1 May 22, 2003

a mediator Selected from the group consisting of a oxadiazoyl)phenylimino)-3H-phenothiazine, phenothiazine, a phenoxazine, and a combina aminoethyl-phenylimino)-3H-phenothiazine, tion thereof, and ethylphenyl)amino-3-(4-ethyl-phenylimino)-3H irradiating the device with E-beam or gamma ray irradia phenothiazine, 6-(4-2-(2-ethanoloxy)ethoxy tion. ethoxyphenyl)amino-3-(4-2-(2-ethanoloxy)ethoxy 37. A method for detecting an analyte which undergoes a ethoxy-phenylimino-3H-phenothiazine, 3-(4-2-(2- chemical reaction, the method comprising: ethanoloxy)ethoxyethoxy-phenylimino-3H-phenothiazine, 3-(4'-phenylimino)-3H-phenothiazineboronic acid, (3-(3',5'- providing an electrode Surface; dicarboxy-phenylimino)-3H-phenothiazine, 3-(4-carboxy catalyzing the chemical reaction with an enzyme Selected phenylimino)-3H-phenothiazine, 3-(3',5'-dicarboxy-phe from the group consisting of a flavoprotein, a quino nylimino)-3H-phenoxazine, 3-(3',5'-phenylimino)-3H protein, and a combination thereof; phenothiazinedisulfonic acid, 3-(3-phenylimino)-3H generating a redox equivalent by the chemical reaction; phenothiazineSulfonic acid, and combinations thereof. 41. The method of claim 37, wherein the mediator com and prises transferring the redox equivalent to the electrode Surface using a mediator Selected from the group consisting of a phenothiazine, a phenoxazine, and a combination CO2H. 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 COH

R6 R1 42. The method of claim 37, wherein the flavoprotein is Selected from the group consisting of FAD-glucose oxidase, FAD-glucose dehydrogenase, lactate oxidase, cholesterol R. t e R2 oxidase, , d-aminoacid oxidase, choline oxi 1 dase, and combinations thereof. R8 N R5 R3, 43. The method of claim 37, wherein the quinoprotein is R9 R4 Selected from the group consisting of PQQ-glucose dehy R6 R1 drogenase, lactate dehydrogenase, aldehyde dehydrogenase, methylamine dehydrogenase, alcohol dehydrogenase, and combinations thereof. R t 2' R2 44. The method of claim 37, wherein the enzyme is 2 Selected from the group consisting of FAD-glucose oxidase, R8 N R5 R3, PQQ-glucose dehydrogenase, and a combination thereof. R9 R4 45. The method of claim 44, wherein the mediator com prises and combinations thereof, wherein R, R R,R,R,R,R, R, and Rare the same or different, and are independently S N COH, Selected from the group consisting of hydrogen, alkyl, 21 alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo, 2 haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxy N carbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, Sulfo, dihydroxyboron, and COH combinations thereof. 40. The method of claim 37, wherein the mediator is Selected from the group consisting of 3-(4-chloro-phe 46. The method of claim 44, wherein the mediator com nylimino)-3H-phenothiazine, 3-(4-diethylamino-phe prises nylimino)-3H-phenothiazine, 3-(4'ethyl-phenylimino)-3H phenothiazine, 3-(4-trifluoromethyl-phenylimino)-3H phenothiazine, 3-(4-methoxycarbonyl-phenylimino)-3H S N phenothiazine, 3-(4-nitro-phenylimino)-3H-phenothiazine, 21 3-(4-methoxy-phenylimino)-3H-phenothiazine, 7-acetyl-3- 2 (4'-methoxycarbonylphenylimino)-3H-phenothiazine, 7-tri N SOH. fluoromethyl-3-(4-methoxycarbonylphenylimino)-3H-phe nothiazine, 3-(4'-(o-carboxy-n-butyl-phenylimino)-3H phenothiazine, 3-(4-aminomethyl-phenylimino)-3H phenothiazine, 3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-