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(12) Patent Application Publication (10) Pub. No.: US 2003/0094384 A1 Vreeke Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2003/0094384 A1 Vreeke Et Al 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 enzyme 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 Glucose Response After irradiation 18000 16000 14000 12000 %. : Š a 10000 s % .. N C 60008000 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 Glucose Oxidase 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 enzymes, 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 cofactor 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 chemical reaction, 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.
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