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(12) Patent Application Publication (10) Pub. No.: US 2009/0045056A1 Berberich Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0045056A1 Berberich Et Al US 20090045056A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0045056A1 Berberich et al. (43) Pub. Date: Feb. 19, 2009 (54) STABLE THREE ENZYME CREATININE (52) U.S. Cl. .................................... 204/403.14; 435/183 BOSENSOR (76) Inventors: Jason Berberich, Pittsburg, PA (US); Mark W. Boden, Harrisville, RI (US); Andy D. C. Chan, (57) ABSTRACT Frenklin, MA (US); Alan Russell, Gibsonia, PA (US) The invention provides methods for preparing a stable, mul tiple-use three enzyme biosensor for the amperometric deter Correspondence Address: mination of creatinine in biological liquids that has a useful SEMENS CORPORATION lifetime that extends significantly beyond that of presently INTELLECTUAL PROPERTY DEPARTMENT available amperometric biosensors. The biosensor prepared 17O WOOD AVENUE SOUTH by the methods of the invention encompasses a plurality of ISELIN, NJ 08830 (US) immobilized enzymes that are applied to the biosensor as an enzyme-polymer composition. The enzymes, which can (21) Appl. No.: 11/664,859 include creatinine amidohydrolase, creatine amidinohydro lase and sarcosine oxidase, are immobilized into the enzyme (22) PCT Filed: Oct. 5, 2005 polymer composition simultaneously as well as applied to the (86). PCT No.: PCT/US05/35959 biosensor simultaneously. Prior to being immobilized, the enzymes can be chemically modified by attaching one or S371 (c)(1), more polyethylene glycol (PEG) chains per enzyme mono (2), (4) Date: Apr. 5, 2007 mer. The polymer component can be provided by a polyure thane membrane. The invention also provides a method of Related U.S. Application Data preparing a biosensor that limits the diffusion of silver ions emanating the reference electrode, thereby preventing, con (60) Provisional application No. 60/616,149, filed on Oct. tact between the silver ions and the enzymes. Related meth 5, 2004. ods of preparing an enzyme-polymer composition for incor poration into a multiple use three enzyme biosensor for the Publication Classification amperometric determination of creatinine in biological liq (51) Int. Cl. uids also are provided. The invention also provides multiple GOIN 27/26 (2006.01) use biosensors and enzyme-polymer compositions prepared CI2N 9/00 (2006.01) by the methods disclosed. Patent Application Publication Feb. 19, 2009 Sheet 1 of 14 US 2009/0045056A1 20 s =- Average Number of PEG Chains Attached Figure 1 120 100 20 25 Time (days) Figure 2 Patent Application Publication Feb. 19, 2009 Sheet 2 of 14 US 2009/004505.6 A1 20 O 8 6 4. 2 SO OO 150 Enzyme Concentration in Hydroge? (U/g) Figure 3 2O OO Assay Number Figure 4 Patent Application Publication Feb. 19, 2009 Sheet 3 of 14 US 2009/004505.6 A1 O 20 40 60 80 Time (days) Figure 5 0.001 0.0 0. l O OO 000 AgNO3 (M) Figure 6 Patent Application Publication Feb. 19, 2009 Sheet 4 of 14 US 2009/004505.6 A1 100 s 80 2 's 60 't 40 a 20 0 1 2 3 4 5 6 7 8 9 O l l 12 Time (days) Figure 7 Patent Application Publication Feb. 19, 2009 Sheet 5 of 14 US 2009/0045056A1 100 4.68 OOO 2 0 O 10 20 30 40 Time (days) Figure 9 Time (days) Figure 10 Patent Application Publication Feb. 19, 2009 Sheet 6 of 14 US 2009/004505.6 A1 s 8O 2 is 60 ed 40 O is 20 O -- -- O 20 40 60 8O 100 120. AgNO3 (M) Figure 11 2O 100 R s A 80 9 A. go Enzyme only 5 60 (a O EDTA A EGTA s Q X Cysteine X Mercaptoethanol 40 ODTT Omidazole 20 6 A PE O ----é-i-S O 20 40 60 80 OO AgNO3 (LM) Figure 12 Patent Application Publication Feb. 19, 2009 Sheet 7 of 14 US 2009/004505.6 A1 's S O 4. O 1 O - O SO OO 150 2OO 250 3OO 350 4OO Residue index Figure 13a Figure 13b Patent Application Publication Feb. 19, 2009 Sheet 8 of 14 US 2009/004505.6 A1 Figure 13c monomer A monomer B O 1OO 2OO 3OO 4OO 500 60of 700 t 800 Residue Index Figure 14 Patent Application Publication Feb. 19, 2009 Sheet 9 of 14 US 2009/004505.6 A1 Road s r re m p. P als n 2. A ad N < W 2 VA. : W W N s PQ.--C 2 2 Patent Application Publication Feb. 19, 2009 Sheet 10 of 14 US 2009/004505.6 A1 (a) 0.09 5 26.” S. C297 0.08 E90 E90 0.07 O.O6 0.05 S 73 O.04 0.03 0.02 0.01 O 100 200 300 4OO 500 600 700 8OO Residue index Figure 16a (b) Figure 16b Patent Application Publication Feb. 19, 2009 Sheet 11 of 14 US 2009/0045056A1 20 e 100 u 1 Š - is 80 - 1 O P t ? P s an 60 - 1 2 ? t 2. 40 P O y Of 1. 20 A) O. O O O 20 40 60 8O 100 Native Protein (%) Figure 17 100 Š w S S SS - 80 Sg Ne' 60 C t e 40 .2 20 Š S. S. Š SS O S. Š No inhibitor Pyrole-2- (Methylthio)acetic carboxylic acid acid Figure 18 Patent Application Publication Feb. 19, 2009 Sheet 12 of 14 US 2009/0045056A1 20 00 8 O 6 O 40 2O O Time (days) Figure 19 O 50 100 50 200 Enzyme Concentration (Units/g polymer) Figure 20 Patent Application Publication Feb. 19, 2009 Sheet 13 of 14 US 2009/004505.6 A1 20 00 Assay Number Figure 21 (9%)ysonœuuÁzu?I Time (days) Figure 22 Patent Application Publication Feb. 19, 2009 Sheet 14 of 14 US 2009/004505.6 A1 S O 10 20 30 40 50 60 Time (days) Figure 23 20 100 2 O. O 0.00 O.O. 0. O OO OOO AgNO3 (M) Figure 24 US 2009/004505.6 A1 Feb. 19, 2009 STABLE THREE ENZYME CREATINNE more polyethylene glycol (PEG) chains per enzyme mono BOSENSOR mer. The polymer component can be provided by a polyure thane membrane. The invention also provides a method of FIELD OF THE INVENTION preparing a biosensor that limits the diffusion of silver ions emanating the reference electrode, thereby preventing con 0001. The present invention relates generally to the field of tact between the silver ions and the enzymes. Related meth diagnostic medicine and, more specifically, to methods for ods of preparing an enzyme-polymer composition for incor producing a multiple-use biosensor for amperometric creati poration into a multiple-use three enzyme biosensor for the nine determination that includes a biopolymer of immobi amperometric determination of creatinine in biological liq lized enzymes. uids also are provided. The invention also provides multiple BACKGROUND OF THE INVENTION use biosensors and enzyme-polymer compositions prepared by the methods disclosed. 0002 The determination of creatinine levels in biological fluids is an increasingly important clinical necessity. BRIEF DESCRIPTION OF THE DRAWINGS Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to ampero 0008 FIG. 1 shows activity retention of PEGylated metrically measurable hydrogen peroxide. Due to the com MSOX modified using PEG-NCO at pH 7.5 (closed circles) plexity of the three-enzyme system, development of these and pH 8.5 (closed squares). biosensors has been slow. 0009 FIG. 2 shows activity retention of MSOX after 0003 Incorporation of enzymes into polymer networks PEGylation in the presence of inhibitors (10:1 (solid) and through multi-point attachment is a rapid and effective gen 10:1 (white) isocyanate to amineratio). The inhibitor concen eral strategy for enhancing the stability of enzymes, while tration was 50 mM. retaining activity. This strategy involves the production of 0010 FIG. 3 shows stability of sarcosine oxidase as a bioplastics in a single step, employing oligomers capable of function of number of PEGs attached at 37°C. Native enzyme chemical reaction with specific functionalities on the enzyme (open Squares); one PEG attached (closed squares); two Surface. PEGs attached (closed circles). 0004. The utility of enzymes in biosensors is limited by 0011 FIG. 4 shows relative activity of MSOX containing their stability. Clinical blood analyzers require enzymes to be polyurethanes as a function of enzyme content. used over and over while in contact with whole blood. Many (0012 FIG. 5 shows reusability of MSOX containing poly blood analyzers operate at 37°C. which further limits enzyme urethane hydrogels. stability. A variety of immobilization procedures are 0013 FIG. 6 shows leaching of sarcosine oxidase from described in the literature for use with biosensors. Although polyurethane hydrogels. 1 mg MSOX/g polymer (closed tri most of the procedures described Suggest utility for biosen angles); 2 mg MSOX/g polymer (open Squares). sors, they are often not tested under conditions that would be (0014 FIG. 7 shows stability of MSOX containing poly applicable under real-life conditions, such as room tempera urethane hydrogels stored in buffer at 37° C. Rates were ture or at 37°C. while in contact with fluid. Enzyme immo normalized to the rate of oxygen consumption after the first bilization is especially critical for continuous use biosensors day. Native enzyme (closed circles); immobilized enzyme where enzyme leaching can be a concern. (open circles). 0005 Enzyme immobilization into a “biopolymer by 0015 FIG. 8 shows ion induced irreversible inhibition of Multipoint covalent attachment affords a straightforward and sarcosine oxidase activity. Incubation time: 5 min (closed convenient method for preparation of immobilized enzymes circles); 1 hr (open squares); 3 hr (closed triangles); 5 hr (open for biosensors. Not only does multipoint covalent immobili circles): 21 hr closed squares).
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