WO2004060297A2

WO2004060297A2 - Hydrophilic cross-linking agents for use in enzymatic sensors

Info

Publication number: WO2004060297A2
Application number: PCT/US2003/041060
Authority: WO
Inventors: William P. Van Antwerp
Original assignee: Medtronic Minimed, Inc.
Priority date: 2002-12-31
Filing date: 2003-12-19
Publication date: 2004-07-22
Also published as: EP1587546A2; AU2003297503A8; WO2004060297A3; EP1587546A4; AU2003297503A1; CA2511549A1

Abstract

An enzymatic sensor comprising an enzyme that generates a signal upon contact with an analyte; a substrate; and a cross-linking agent that comprises one or more hydrophilic moieties. The enzyme is immobilized in the substrate via the hydrophilic cross-linking agent. A biocompatible membrane, comprising the enzyme, substrate and the hydrophilic cross-linking agent, is applied to a sensor to produce an enzymatic sensor. The enzymatic sensor provides enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensor. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as H202 and the analyte of interest. The membrane and enzymatic sensor can be used in a method for detecting an analyte, such as glucose.

Description

HYDROPHILIC CROSS-LINKING AGENTS FOR USE IN ENZYMATIC

SENSORS

TECHNICAL FIELD OF THE INVENTION

[0001] This invention relates generally to cross-linking agents for use with enzymatic sensors and to methods of making and using such materials. The cross-linking agents are hydrophilic and suitable for use with enzymatic sensors for detecting analytes, such as glucose.

BACKGROUND OF THE INVENTION

[0002] Biosensors are small devices that use biological recognition properties for selective detection of various analytes or biomolecules. Typically, the sensor will produce a signal that is quantitatively related to the concentration of the analyte. To achieve a quantitative signal, a recognition molecule or combination of molecules is often -mrnobiltzed at a suitable transducer, which converts the biological recognition event into a quantitative response.

[0003] The need for the continuous monitoring of biological markers (analytes) in medicine has sparked a tremendous interest in the study of biosensors in recent years. Without question, the greatest interest has been geared toward the development of sensors to detect glucose. In particular, enzymatic (amperometric) glucose electrodes have been studied in more detail than any other biosensors. Electroenzymatic biosensors use enzymes to convert a concentration of analyte to an electrical signal. For a review of some of the operating principles of biosensors, see Bergveld, et al., Advances in Biosensors, Supplement 1, p. 31-91, Turner ed., and Collison, et al., Anal. Chem. 62:425-437 (1990). Typically, the enzyme is immobilized onto the sensor via use of a cross-linking agent, such as glutaraldehyde. [0004] An additional commercial application of this technology focuses on sensors that can be used to monitor fermentation reactions in the biotechnology industry. From a scientific and commercial standpoint, interest has grown beyond glucose to other analytes for the diagnosis of numerous medical conditions other than diabetes. One example of another analyte detectable via enzymatic sensors is lactate.

[0005] A typical glucose sensor works by a reaction in which glucose reacts with oxygen in the presence of glucose oxidase (GOd) to form gluconolactone and hydrogen peroxide. The gluconolactone further reacts with water to hydrolyze the lactone ring and produce gluconic acid. The H₂O₂ formed is electrochemically oxidized at an electrode as shown below (Equation 1):

H₂O₂ → 0₂ +2e- +2H⁺ (I)

[0006] The current measured by the sensor/potentiostat (+0.5 to +0.7 v oxidation at Pt black electrode) is the result of the two electrons generated by the oxidation of the H₂O₂. Alternatively, one can measure the decrease in the oxygen by amperometric measurement (-0.5 to -1 N reduction at a Pt black electrode).

[0007] The stoichiometry of the GOd reaction points to a challenge of developing a reliable glucose sensor. If oxygen and glucose are present in equi olar concentrations, then the H₂O₂ is stoichiometricaUy related to the amount of glucose that reacts at the enzyme. In this case, the ultimate current is also proportional to the amount of glucose that reacts with the enzyme. If there is insufficient oxygen for all of the glucose to react with the enzyme, then the current will be proportional to the oxygen concentration, not the glucose concentration. For the sensor to be a true glucose sensor, glucose must be the limiting reagent, i.e. the 0₂ concentration must be in excess for all potential glucose concentrations.

[0008] For example, the glucose concentration in the body of a diabetic patient can vary from 2 to 30 mM (rrii.limoles per liter or 36 to 540 mg/dl), whereas the typical oxygen concentration in the tissue is 0.02 to 0.2 mM (see, Fisher, et al., Biomed. Biochem. Acta. 48:965-971 (1989). This ratio in the body means that the sensor would be running in the Michaelis Menten limited regime and would be very insensitive to small changes in the glucose concentration. This problem has been called the "oxygen deficit problem". Accordingly, a method or system must be devised to either increase the 0₂ in the GOd enzyme layer, decrease the glucose concentration, or devise a sensor that does not use 0₂.

[0009] There is a need for an enzymatic sensor with enhanced sensing capabilities and that overcomes difficulties associated with conventional cross-linking agents such as glutaraldehyde. The present invention fulfills these needs and provides other related advantages.

SUMMARY OF THE INVENTION

[0010] The invention provides a biocompatible membrane comprising an enzyme and a hyc-Jrophilic cross-linking agent, which membrane can be applied to a sensor. Also provided is an enzymatic sensor comprising an enzyme that generates a signal upon contact with an analyte; a substrate; and a cross-linking agent that comprises one or more hy<-kopkilic moieties. The enzyme is immobilized onto the substrate via the hydrophilic cross-linking agent. The enzymatic sensor provides enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensor. The incorporation of hydropliilic moieties into the cross-liriking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as H₂0₂ and the analyte of interest.

[0011] In one embodiment, the one or more hydrophilic moieties comprise a polyol selected from the group consisting of polyethylene glycol, polypropylene glycol and a copolymer of polypropylene glycol and polyethylene glycol, and the cross-linking agent comprises an aldehyde, diimrnide, cyanate, isocyanate, or diisocyanate. A typical cross-linking agent of the invention comprises: PEG

R-(C-C-C-C)_N-R;

PEG; or PEG

R-(C=C-C-C)_N-R;

PEG; or

wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0012] Examples of enzymes for use with the invention include, but are not limited to, glucose oxidase, α-hydroxy oxidase, lactate oxidase, urease, creatine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, and fructose dehydrogenase. [0013] The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method comprises introducing an enzymatic sensor of the invention into the tissue of the subject, and detecting the signal generated by die enzyme. The amount of signal corresponds to the amount of analyte. Preferably, the analyte is glucose and the enzyme is glucose oxidase. DETAILED DESCRIPTION

[0014] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0015] As used herein, "adhered to" or "adhered thereto" means stuck to or fused with such that a substance adhered to a surface remains substantially attached to or closely associated with the surface.

[0016] As used herein, "a" or "an" means at least one, and unless clearly indicated otherwise, includes a plurality.

Overview

[0017] The invention is based on the discovery that hy<-bophi-ic cross-linking agents can be used in enzymatic sensors, resulting in sensors with improved hydration and enhanced sensitivity. The invention provides a biocompatible membrane comprising an enzyme that generates a signal upon contact with an analyte, a substrate, and a hydrophilic cross-linking agent. The enzyme is immobilized in the substrate via the hyt-kophilic cross-linking agent. The cross-linking agent comprises one or more hy<-hOphilic moieties. The membrane can be applied to a sensor to produce an enzymatic sensor.

[0018] The enzymatic sensors of the invention provide enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensors. The incorporation of hy<-bophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hy<- Opbilic channels or pathways for reactive species, such as H₂O and the analyte of interest. The increased hydration of the sensor environment may also reduce the formation of a skin over the sensor membrane, as occurs with use of the conventional cross-linking agent, glutar aldehyde. [0019] In addition, the cross-linking agents of the invention avoid or diminish negative consequences of e-beam sterilization of sensors that occurs with conventional cross-linking agents. For example, glutaraldehyde and other conventional cross-linking agents can leave by-products that damage the sensor during e-beam sterilization.

Biocompatible Membranes

[0020] A glucose sensor intended for in vivo use requires that the supply of oxygen in the vicinity of the sensing element not be depleted. Additionally, the glucose should diffuse to the sensor at a controlled rate. This diffusion of the analyte to the sensor occurs through a membrane adhered to the surface of the sensor. Overall, the membrane should control the relative rates of diffusion of oxygen and glucose to the sensor so that the local concentration of oxygen is not depleted. Additionally, glucose sensors intended for in vivo use must also be biocompatible with the body. Thus, the enzyme(s) used in such sensors must be protected from degradation or denaturation, while the elements of such sensors must be protected from molecules that would foul the sensors or their accuracy will decrease over time.

[0021] In one aspect, the present invention provides a biocompatible membrane comprising an enzyme immobilized in a substrate by a hydrophilic cross- linking agent. The substrate is typically a polymer matrix. Suitable polymeric compositions are known in the art (see, e.g., U.S. Patent Nos. 5,777,060 and 5,786,439, both of which are incorporated herein by reference). The hydrophilic cross-linking agent comprises a cross-linking agent that includes one or more hydi-ophilic moieties. Examples of cross-linldng agents having hydrophilic moieties are described below.

[0022] In another aspect of the invention, the homogeneity of the membrane can be further enhanced by introducing hydrophilic moieties into other proteins present in the membrane. For example, pegylation of albumin, glucose oxidase, or other enzyme present in the membrane would create an even more homogeneous structure. HydiOphilic Cross-linking Agents

[0023] The biocompatible membrane of the invention comprises a polymeric composition that contains an enzyme that generates a signal upon contact with an analyte of interest. Typically, the enzyme is immobilized in the composition or membrane that covers the sensor via a cross-linldng agent. Examples of cross-linking agents suitable for use with the invention include, but are not limited to, molecules that comprise aldehydes, diimmides, cyanates, isocyanates, and diisocyanates, into which hydrophilic moieties are incorporated. Examples of hydrophilic moieties include, but are not limited to: polyols, such as polyethylene glycol, polypropylene glycol and copolymers of polypropylene glycol and polyethylene glycol; and other non-ionic surfactants, including Tweens 20-80.

[0024] Representative embodiments of hydrophilic cross-linldng agents include:

PEG

R-(C-C-C-C)N-R; PEG;

PEG

R-(C=C-C-C)_N-R; PEG; and

wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Biosensor

[0025] Biosensors typically include a transducer, or enzyme, that generates a signal upon contact with an analyte of interest, and is adhered to a detector, such as an electrode. For example, glucose sensors suitable for in vivo use can be prepared by depositing a membrane comprising a glucose sensitive enzyme, such as glucose oxidase, onto an electrode via an electromotive plating process. The membrane can be applied by immersion of the sensor in a bath comprising glucose oxidase, a stabilizing protein, a surfactant and a buffer for conductivity and stability of the protein solution, and the enzyme is then deposited onto the electrode potentiometrically. Alternatively, the membrane can be applied using a microelectrogravimetric plating method, such as is described in U.S. Patent No. 6,340,021, issued January 22, 2002.

[0026] The invention provides a sensor for measuring an analyte of interest in biological tissue, the sensor having a coating comprising a biocompatible membrane of the invention that includes an enzyme serving as a transducer that generates a signal upon contact with the analyte. The sensor can be used in vitro, or is suitable for use as an implantable biosensor or other in vivo applications. In a preferred embodiment, the analyte is glucose and the transducer is glucose oxidase. Other enzymes can serve as transducers as appropriate for the analyte of interest and examples of such enzymes include, but are not limited to, α-hydroxy oxidase, lactate oxidase, urease, creatine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, and fructose dehydrogenase.

Methods

[0027] The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method comprises introducing an enzymatic sensor of the invention into the tissue of the subject, and detecting the signal generated by the enzyme. The amount of signal generated corresponds to the amount of analyte. Preferably, the analyte is glucose and the enzyme is glucose oxidase. As described herein, other analytes and other corresponding enzymes can be used. The sensor is typically introduced into the tissue in vivo, via subcutaneous implantation, although those skilled in the art will appreciate other means for introducing the sensor into the tissue.

[0028] The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example a variety of solvents, membrane formation methods, and other materials may be used without departing from the scope of the invention. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

[0029] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

WHAT IS CLAIMED IS:

1. A biocompatible membrane comprising:

(a) an enzyme that generates a signal upon contact with an analyte;

(b) a substrate; and

(c) a cross-linldng agent that comprises one or more hydiOpl-ilic moieties,

wherein the enzyme is iα-rnobilized in the substrate via the hydrophilic cross- linldng agent.

2. The membrane of claim 1, wherein the one or more hydrophilic moieties comprise a polyol selected from the group consisting of polyethylene glycol, polypropylene glycol and a copolymer of polypropylene glycol and polyethylene glycol.

3. The membrane of claim 1, wherein the cross-linking agent comprises an aldehyde, di-j mide, cyanate, isocyanate, or diisocyanate.

4. The membrane of claim 1, wherein the cross-linldng agent comprises

PEG

R-(C-C-C-C)_N-R;

PEG wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate, and N is an integer from 1 to about 10.

5. The membrane of claim 4, wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6. The membrane of claim 1, wherein the cross-linking agent comprises

PEG

PEG wherein R is an aldehyde, diim -nide, cyanate, isocyanate, or diisocyanate, N is an integer from 1 to about 10.

7. The membrane of claim 6, wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

8. The membrane of claim 1, wherein the cross-linking agent comprises

wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate.

9. The membrane of claim 1, wherein the enzyme is glucose oxidase, -hydroxy oxidase, lactate oxidase, urease, creatine amidohydrolase, creatine an dinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, or fructose dehydrogenase.

10. The membrane of claim 1, wherein the analyte is glucose and the enzyme is glucose oxidase.

11. The membrane of claim 1, wherein the substrate comprises a polymer matrix.

12. An enzymatic sensor comprising an electrode and the membrane of claim 1 adhered to the electrode.

13. The sensor of claim 12, wherein the analyte is glucose and the enzyme is glucose oxidase.

14. A method of measuring an analyte in a tissue of a subject, the method comprising introducing a sensor of claim 12 into the tissue of the subject and detecting the signal generated by the enzyme, wherein the amount of signal corresponds to the amount of analyte.

15. The method of claim 14, wherein the enzyme is glucose oxidase, α-hydroxy oxidase, lactate oxidase, urease, creatine amidohydrolase, creatine amidinohydiOlase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, or fructose dehydrogenase.

16. The method of claim 14, wherein die analyte is glucose and the enzyme is glucose oxidase.