# Patent

## Title
Redox polymers for use in analyte monitoring

## Abstract

Polymers for use as redox mediators in electrochemical biosensors are described. The transition metal complexes attached to polymeric backbones can be used as redox mediators in enzyme based electrochemical sensors. In such instances, transition metal complexes accept electrons from, or transfer electrons to, enzymes at a high rate and also exchange electrons rapidly with the sensor. The transition metal complexes include at least one substituted or unsubstituted biimidazole ligand and may further include a second substituted or unsubstituted biimidazole ligand or a substituted or unsubstituted bipyridine or pyridylimidazole ligand.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 11/503,519, filed Aug. 10, 2006, which is a continuation of U.S. patent application Ser. No. 10/639,181, filed Aug. 11, 2003, issued as U.S. Pat. No. 7,090,756, which is a continuation of U.S. patent application Ser. No. 09/712,452, filed Nov. 14, 2000, issued as U.S. Pat. No. 6,605,201, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60/165,565, filed Nov. 15, 1999, which are incorporated herein by reference.
This application is also related to, and claims the benefit of priority of U.S. patent application Ser. No. 11/929,149 filed Oct. 30, 2007, which is pending and which claims the benefit of priority of U.S. patent application Ser. No. 10/975,207 filed Oct. 27, 2004, which issued Nov. 20, 2007 as U.S. Pat. No. 7,299,082, and which claims the benefit of priority of U.S. Patent Application Ser. No. 60/516,599 of Feldman et al. filed Oct. 31, 2003.
This application is also related U.S. patent application Ser. No. 10/819,498, filed Apr. 6, 2004, which is pending and which claims the benefit of priority U.S. patent application Ser. No. 10/775,604 of Feldman et al., which was filed on Feb. 9, 2004, as a continuation-in-part thereof, and is additionally related to U.S. patent application Ser. No. 10/146,518 of Mao et al., which was filed on May 14, 2002, the corresponding U.S. patent application Publication No. U.S. 2003/0042137 A1 of Mao et al., which was published on Mar. 6, 2003, and U.S. Provisional Patent Application No. 60/291,215 of Mao, which was filed on May 15, 2001. Each of the aforementioned applications, publication, and provisional application, is incorporated herein in its entirety and for all purposes by this reference.


FIELD OF THE INVENTION
The invention relates to polymers for use as redox mediators in electrochemical biosensors.
BACKGROUND OF THE INVENTION
Enzyme-based electrochemical sensors are widely used in the detection of analytes in clinical, environmental, agricultural and biotechnological applications. Analytes that can be measured in clinical assays of fluids of the human body include, for example, glucose, lactate, cholesterol, bilirubin and amino acids. Levels of these analytes in biological fluids, such as blood, are important for the diagnosis and the monitoring of diseases.
Enzyme-based electrochemical sensors are devices in which a signal from an analyte-concentration-dependent biochemical reaction is converted into a measurable optical or electrical signal. Amperometric, enzyme-based biosensors typically employ two or three electrodes, including at least one measuring or working electrode and one reference electrode. The measuring or working electrode is composed of a non-corroding carbon or a metal conductor and is connected to the reference electrode via a circuit, such as a potentiostat. In three electrode systems, the third electrode is a counter-electrode. In two electrode systems, the reference electrode also serves as the counter-electrode.
The working electrode typically includes a sensing layer (also referred to herein as a “reagent layer”) in direct contact with the conductive material of the electrode. The sensing layer may include an enzyme, an enzyme stabilizer such as bovine serum albumin (BSA), and a cross-linker that crosslinks the sensing layer components. Alternatively, the sensing layer may include an enzyme, a polymeric mediator, and a cross-linker that crosslinks the sensing layer components, as in a “wired-enzyme” biosensor.
Upon passage of a current through the working electrode, a redox enzyme is electrooxidized or electroreduced. The enzyme is specific to the analyte to be detected, or to a product of the analyte. The turnover rate of the enzyme is typically related (preferably, but not necessarily, linearly) to the concentration of the analyte itself, or to its product, in the test solution.
The electrooxidation or electroreduction of the enzyme is often facilitated by the presence of a redox mediator in the solution or on the electrode. The redox mediator assists in the electrical communication between the working electrode and the enzyme. The redox mediator can be dissolved in the fluid to be analyzed, which is in electrolytic contact with the electrodes, or can be applied within a coating on the working electrode in electrolytic contact with the analyzed solution. The coating is preferably not soluble in water, though it may swell in water. Useful devices can be made, for example, by coating an electrode with a film that includes a redox mediator and an enzyme where the enzyme is catalytically specific to the desired analyte, or its product. In contrast to a coated redox mediator, a diffusional redox mediator, which can be soluble or insoluble in water, functions by shuttling electrons between, for example, the enzyme and the electrode. In any case, when the substrate of the enzyme is electrooxidized, the redox mediator transports electrons from the substrate-reduced enzyme to the electrode; when the substrate is electroreduced, the redox mediator transports electrons from the electrode to the substrate-oxidized enzyme.
Enzyme-based electrochemical sensors have employed a number of different redox mediators such as monomeric ferrocenes, quinoid-compounds including quinines (e.g., benzoquinones), nickel cyclamates, and ruthenium ammines. For the most part, these redox mediators have one or more of the following limitations: the solubility of the redox mediators in the test solutions is low, their chemical, light, thermal, or pH stability is poor, or they do not exchange electrons rapidly enough with the enzyme or the electrode or both. Additionally, the redox potentials of many of these reported redox mediators are so oxidizing that at the potential where the reduced mediator is electrooxidized on the electrode, solution components other than the analyte are also electrooxidized; in other cases they are so reducing that solution components, such as, for example, dissolved oxygen are also rapidly electroreduced. As a result, the sensor utilizing the mediator is not sufficiently specific. Sensors employing the redox polymers of the invention address these deficiencies.
Various biosensors have been designed to operate partially or wholly in a living body. Indeed, clinical use of such biosensors has been a significant step toward helping diabetic patients achieve tight control over their blood glucose levels.
In an example of an amperometric, enzyme-based glucose biosensor, the sensor utilizes glucose oxidase, which catalyzes the oxidation of glucose by oxygen in a sample of body fluid and generates gluconolactone and hydrogen peroxide, whereupon the hydrogen peroxide is electrooxidized and correlated to the concentration of glucose in the sample (Thom-Duret et al., Anal. Chem. 68, 3822 (1996); and U.S. Pat. No. 5,882,494 of Van Antwerp et al., filed on Aug. 28, 1995). In another example of an amperometric, enzyme-based, glucose biosensor, a polymeric redox mediator “wires” the reaction center of glucose oxidase to an electrode and catalyzes the electrooxidation of glucose to gluconolactone. The principle and the operational details of such a “wired-enzyme” biosensor have been described (Csoregi, et al., Anal. Chem. 1994, 66, 3131; Csoregi, et al., Anal. Chem. 1995, 67, 1240; Schmidtke, et al., Anal. Chem. 1996, 68, 2845; Schmidtke, et al., Anal. Chem. 1998, 70, 2149; and Schmidtke, et al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 294).
Enzyme-immobilized mediators allow electron transport between an enzyme active site and an electrode surface by shortening the electron tunneling steps. The term “wired enzyme” refers to enzymes with covalently attached redox mediators. The enzyme is in effect wired by the mediator to an electrode. The wired enzymes are able to transfer redox equivalents from the enzyme's active site through the mediator to an electrode.
The wired-enzyme principle resulted in subsequent development of enzyme-immobilizing redox polymers. These polymers effectively transfer electrons from glucose-reduced GOx flavin sites to polymer-bound redox centers. A series of chain redox reactions within and between polymers transfer the equivalents to an electrode surface. The redox enzyme and wire are immobilized by cross-linking to form three-dimensional redox epoxy hydrogels. A large fraction of enzymes bound in the three-dimensional redox epoxy gel are wired to the electrode.
In in vivo systems employed to monitor glucose, the electrochemical sensor may be inserted into a blood source, such as a vein or other blood vessel, for example, such that the sensor is in continuous contact with blood and can effectively monitor blood glucose levels. Further by way of example, the electrochemical sensor may be placed in substantially continuous contact with bodily fluid other than blood, such as dermal or subcutaneous fluid, for example, for effective monitoring of glucose levels in such bodily fluid. Relative to discrete or periodic monitoring, continuous monitoring is generally more desirable in that it may provide a more comprehensive assessment of glucose levels and more useful information, such as predictive trend information, for example. Subcutaneous continuous glucose monitoring is also desirable for a number of reasons, one being that continuous glucose monitoring in subcutaneous bodily fluid is typically less invasive than continuous glucose monitoring in blood.
The FREESTYLE NAVIGATOR continuous glucose sensor (Abbott Diabetes Care Inc., Alameda, Calif., USA) is a subcutaneous, electrochemical sensor, which operates for three days when implanted at a site in the body. This sensor is based on WIRED ENZYME sensing technology, as described above in which “wired” enzyme electrodes are made by connecting enzymes to electrodes through crosslinked electron-conducting redox hydrogels (“wires”). In FREESTYLE NAVIGATOR, glucose oxidase is “wired” with polyelectrolytes having electron relaying [Os(bpy)2Cl]+/2+ redox centers in their backbones. Hydrogels are formed upon cros slinking the enzyme and its wire on electrodes.
The operation and performance of an amperometric biosensor, such as those just described, may be complicated at high rates of analyte flux. For example, at high rates of glucose flux, an amperometric glucose biosensor may be kinetically overwhelmed, such that the relationship between the concentration of glucose in a sample fluid and the response from the biosensor becomes non-linear. This kinetic problem may be solved by the interposition of an analyte-flux-limiting membrane between the sample fluid and the sensing layer of the biosensor, as described in the above-mentioned U.S. patent Application Publication No. U.S. 2003/0042137 A1 of Mao et al. Still, the development of analyte-flux-limiting membranes, such as glucose-flux-limiting membranes, has not been without its challenges. Many known membranes have proved difficult to manufacture and/or have exhibited properties that limit their practical use, such as practical use in a living body.
The FREESTYLE NAVIGATOR sensor also comprises an analyte-restricting membrane, which may be a glucose-restricting membrane, disposed over the sensing layer. (See, e.g., U.S. Patent Application Publication No. 2003/0042137 A1 of Mao et al. filed May 14, 2002). The membrane is a bio-compatible polymer used to coat the outer surface of the sensing layer.
Further development of manufacturing techniques and methods, as well as analyte-monitoring devices, systems, or kits employing the same, is desirable.
SUMMARY OF THE INVENTION
The present invention is directed to redox polymers that include a polymeric backbone, a cross-linker, and a transition metal complex having the following formula:




wherein M is cobalt, ruthenium, osmium, or vanadium, and L is selected from the group consisting of:





R1, R2, and R′1 are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups. R3, R4, R5, R6, R′3, R′4, Ra, Rb, Rc, and Rd are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. c is an integer selected from −1 to −5 or +1 to +5 indicating a positive or negative charge. X represents at least one counter ion and d is an integer from 1 to 5 representing the number of counter ions, X. L1, L2, L3 and L4 are other ligands where at least one of L, L1, L2, L3 and L4 couples to the polymeric backbone. The polymer is disposed proximate to the working electrode of an electrochemical biosensor.

In one embodiment, M is osmium and the transition complex has the following formula:




wherein R3, R4, R5, R6, R16, R17, R19, R20, R22 and R23 are —H;
R1 and R2 are independently substituted or unsubstituted C1 to C12 alkyls; and
R18 and R21 are independently —H or substituted or unsubstituted C1-C12 alkoxy, C1-C12 alkylthio, C1-C12 alkylamino, C2-C24 dialkylamino, or C1-C12 alkyl.
In a further embodiment, the cross-linker is PEGDGE having the following general structure, wherein n=ca. 10:




In another embodiment, the polymeric backbone is a poly(vinylpyridine) having the following general structure, where n may be 2, n′ may be 17, and n′′ may be 1:




In yet another embodiment, the redox polymer has the following structure:




Embodiments of the claimed subject matter also include a reagent composition for use in a sensing layer of an electrochemical biosensor, comprising a redox polymer, an enzyme, and a cross-linker. In further embodiments, the enzyme is glucose oxidase and the cross-linker is a bi-functional epoxide.



BRIEF DESCRIPTION OF THE DRAWINGS
A detailed description of various aspects, features and embodiments of the present invention is provided herein with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and are not necessarily drawn to scale. The drawings illustrate various aspects or features of the present invention and may illustrate one or more embodiment(s) or example(s) of the present invention in whole or in part. A reference numeral, letter, and/or symbol that is used in one drawing to refer to a particular element or feature maybe used in another drawing to refer to a like element or feature.
FIG. 1 is a schematic illustration of an analyte-measuring or monitoring device, a portion of which is enlarged for illustration purposes that may be employed, according to various aspects of the present invention.
FIG. 2 is a schematic illustration of a sensing layer that is associated with a working electrode of an analyte-measuring or monitoring device, such as that illustrated in FIG. 1.
FIG. 3 is an illustration of the structure of a redox polymer, which is a component of a sensing layer, such as that illustrated in FIG. 2. The redox polymer shown in FIG. 3 is an Osmium-decorated poly(vinylpyridine)-based polymer, referred to herein as “X7”, and comprises a redox mediator of high molecular weight attached to a poly(vinylpyridine)-based polymer backbone. Epoxides, such as the bi-functional short-chain epoxide known as PEGDGE, may function as cross-linkers. The polymeric backbone has the following general formula, where n may be 2, n′ may be 17, and n′′ may be 1.




The cross-linker such as polyethylene glycol diglycidyl ether (PEGDGE) has the following general formula, where




FIG. 4A is a schematic, side-view illustration of a portion of a two-electrode glucose sensor having a working electrode, a combined counter/reference electrode, and a dip-coated membrane that encapsulates both electrodes. FIGS. 4B and 4C are schematic top- and bottom-view illustrations, respectively, of the portion of the glucose sensor of FIG. 4A. Herein, FIGS. 4A, 4B and 4C may be collectively referred to as FIG. 4.
FIGS. 5 and 6, together, depict a transcutaneous electrochemical sensor. FIG. 5 is a perspective view of a fully fabricated sensor as it would be seen partially implanted into the skin, and FIG. 6 is an expanded and cutaway view of a sensor insertion tip, showing a membrane, covering a sensing layer.
FIG. 7A depicts the head of a fully implantable analyte sensing system. FIG. 7B is a schematic cross sectional view of a sensing region of the head. FIGS. 7A and 7B may be collectively referred to as FIG. 7.



DETAILED DESCRIPTION
In the description of the invention herein, it will be understood that a word appearing in the singular encompasses its plural counterpart, and a word appearing in the plural encompasses its singular counterpart, unless implicitly or explicitly understood or stated otherwise. Merely by way of example, reference to “an” or “the” “analyte” encompasses a single analyte, as well as a combination and/or mixture of two or more different analytes, reference to “a” or “the” “concentration value” encompasses a single concentration value, as well as two or more concentration values, and the like, unless implicitly or explicitly understood or stated otherwise. Further, it will be understood that for any given component described herein, any of the possible candidates or alternatives listed for that component, may generally be used individually or in combination with one another, unless implicitly or explicitly understood or stated otherwise. Additionally, it will be understood that any list of such candidates or alternatives, is merely illustrative, not limiting, unless implicitly or explicitly understood or stated otherwise.
Various terms are described below to facilitate an understanding of the invention. It will be understood that a corresponding description of these various terms applies to corresponding linguistic or grammatical variations or forms of these various terms. It will also be understood that the invention is not limited to the terminology used herein, or the descriptions thereof, for the description of particular embodiments. Merely by way of example, the invention is not limited to particular analytes, bodily or tissue fluids, blood or capillary blood, or sensor designs or usages, unless implicitly or explicitly understood or stated otherwise, as such may vary.
The term “alkyl” includes linear or branched, saturated aliphatic hydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, the term “alkyl” includes both alkyl and cycloalkyl groups.
The term “alkoxy” describes an alkyl group joined to the remainder of the structure by an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, and the like. In addition, unless otherwise noted, the term ‘alkoxy’ includes both alkoxy and cycloalkoxy groups.
The term “alkenyl” describes an unsaturated, linear or branched aliphatic hydrocarbon having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, and the like.
The terms “amperometry” and “amperometrically” refer to the measurement of the strength of a current and include steady-state amperometry, chronoamperometry, and Cottrell-type measurements.
A “biological fluid” is any body fluid or body fluid derivative in which the analyte can be measured, for example, blood, interstitial fluid, plasma, dermal fluid, sweat, and tears.
The term “bodily fluid” in the context of the invention encompasses all non-blood bodily fluid that can be found in the soft tissue of an individual's body, such as subcutaneous, dermal, or interstitial tissue, in which an analyte may be measured. By way of example, the term “bodily fluid” encompasses a fluid such as dermal, subcutaneous, or interstitial fluid., including but not limited to blood, interstitial fluid, plasma, dermal fluid, sweat and tears.
The term “blood” in the context of the invention encompasses whole blood and its cell-free components, such as plasma and serum. The term “capillary blood” refers to blood that is associated with any blood-carrying capillary of the body.
The term “concentration” may refer to a signal that is indicative of a concentration of an analyte in a medium, such as a current signal, for example, to a more typical indication of a concentration of an analyte in a medium, such as mass of the analyte per unit volume of the medium, for example, or the like.
“Coulometry” refers to the determination of charge passed or projected to pass during complete or nearly complete electrolysis of a compound, either directly on the electrode or through one or more electron-transfer agents. The charge is determined by measurement of electrical charge passed during partial or nearly complete electrolysis of the compound or, more often, by multiple measurements during the electrolysis of a decaying current over an elapsed period. The decaying current results from the decline in the concentration of the electrolyzed species caused by the electrolysis.
A “counter electrode” refers to one or more electrodes paired with the working electrode, through which passes an electrochemical current equal in magnitude and opposite in sign to the current passed through the working electrode. The term “counter electrode” is meant to include counter electrodes that also function as reference electrodes (i.e., a counter/reference electrode) unless the description provides that a “counter electrode” excludes a reference or counter/reference electrode.
The term “electrochemical biosensor” refers to analyte sensors for use in vivo and in vitro and may be used interchangeably herein with the terms “sensor” and “biosensor”. All of these are devices configured to detect the presence of or measure the concentration or amount of an analyte in a sample via electrochemical oxidation or reduction reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of analyte.
The term “electrolysis” refers to the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron-transfer agents, such as redox mediators and/or enzymes, for example.
An “immobilized” material refers to a material that is entrapped on a surface or chemically bound to a surface.
An “implantable” device refers to a fully implantable device that is implanted fully within a body and/or an at least partially implantable device that is at least partially implanted within a body. An example of an at least partially implantable sensing device is a transcutaneous sensing device, sometimes referred to as a subcutaneous sensing device, that is associated with a portion that lies outside of a body and a portion that penetrates the skin from the outside of the body and thereby enters the inside of the body.
The term “measure,” as in “to measure the concentration,” is used herein in its ordinary sense and refers to the act of obtaining an indicator, such as a signal, that may be associated with a value, such as concentration, for example, and to the act of ascertaining a value, such as a concentration, for example. The term “monitor,” as in “to monitor the concentration,” refers to the act of keeping track of more than one measurement over time, which may be carried out on a systematic, regular, substantially continuous, and/or on-going basis. The terms measure and monitor may be used generally herein, such as alternately, alternatively, or interchangeably, or more specifically, as just described.
The term “measurement” may refer to a signal that is indicative of a concentration of an analyte in a medium, such as a current signal, for example, to a more typical indication of a concentration of an analyte in a medium, such as mass of the analyte per unit volume of the medium, for example, or the like. The term “value” may sometimes be used herein as a term that encompasses the term “measurement.”
The term “membrane” refers to the component represented schematically in FIG. 1 as 320. It may be an analyte-restricting membrane, and specifically a glucose-restricting membrane. It is disposed over the sensing layer. It may comprise a poly(vinylpyridine-co-styrene) copolymer of high molecular weight, that is cross-linked using a tri-functional, short-chain epoxide. It may be about 50 μm thick and reduce glucose diffusion to the sensing layer by a factor of about 50.
The term “patient” refers to a living animal, and thus encompasses a living mammal and a living human, for example. The term “subject” may sometimes be used herein as a term that encompasses the term “patient.”
A “reactive group” is a functional group of a molecule that is capable of reacting with another compound to couple at least a portion of that other compound to the molecule. Reactive groups include carboxy, activated ester, sulfonyl halide, sulfonate ester, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imido ester, maleimide, hydrazide, hydroxy, and photo-reactive azido aryl groups. Activated esters, as understood in the art, generally include esters of succinimidyl, benzotriazolyl, or aryl substituted by electron-withdrawing groups such as sulfo, nitro, cyano, or halo groups; or carboxylic acids activated by carbodiimides.
A “redox mediator” is an electron transfer agent for carrying electrons between an analyte or an analyte-reduced or analyte-oxidized enzyme and an electrode, either directly, or via one or more additional electron transfer agents. “Redox polymers” act as redox mediators and comprise a polymeric backbone, a cross-linker, and a transition metal complex. Redox polymer transfer electrons between the working electrode and an analyte. In some embodiments, the redox polymer is associated with an enzyme to facilitate electron transfer. Redox polymer may transfer electrons between the working electrode and glucose (typically via an enzyme) in an enzyme-catalyzed reaction of glucose. Redox polymers are particularly useful for forming non-leachable coatings on the working electrode. Such a coating, also referred to herein as a “sensing layer”, may be formed for example, by crosslinking a redox polymer on the working electrode, or by cross-linking the redox polymer and the enzyme on the working electrode.
The term “reference electrode” encompasses a reference electrode that also functions as a counter electrode (i.e., a counter/reference electrode), unless the description provides that a “reference electrode” excludes a counter/reference electrode.
The term “sensing layer” (also referred to as the “reagent layer”) refers to the area shown schematically in FIG. 1 as 312. The sensing layer is comprised of a reagent composition, which may be described as the active chemical area of the biosensor or the “wired” enzyme layer, where electrons from glucose are shuttled to a working electrode via one or more electron transfer agents. The sensing layer formulation, which is a cross-linked, glucose-transducing gel, may comprise, among other constituents, a redox polymer mediator of high molecular weight, glucose oxidase (“GOx”), and a bi-functional, short-chain, epoxide cross-linker. In the sensing layer, GOx may be substituted with other redox enzymes to measure other relevant clinical compounds, such as lactate oxidase for the in vivo detection of lactate.
A “substituted” functional group (e.g., substituted alkyl, alkenyl, or alkoxy group) includes at least one substituent selected from the following: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH2, alkylamino, dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido, hydrazino, alkylthio, alkenyl, and reactive groups.
The term “working electrode” refers to an electrode at which a candidate compound is electrooxidized or electroreduced with or without the agency of a redox mediator.
Generally, the present invention relates to redox polymers for use in electrochemical biosensors. Redox polymers of the invention may comprise, inter alia, a transition metal complex, a cross-linker and a polymeric backbone. Such redox polymers are useful as redox mediators in electrochemical biosensors.
Redox polymers of the invention may comprise a transition metal complex of iron, cobalt, ruthenium, osmium, and vanadium having at least one bidentate ligand containing an imidazole ring.
In at least some instances, the transition metal complexes have one or more of the following characteristics: redox potentials in a particular range, the ability to exchange electrons rapidly with electrodes, the ability to rapidly transfer electrons to or rapidly accept electrons from an enzyme to accelerate the kinetics of electrooxidation or electroreduction of an analyte in the presence of an enzyme or another analyte-specific redox catalyst. For example, a redox mediator may accelerate the electrooxidation of glucose in the presence of glucose oxidase or PQQ-glucose dehydrogenase, a process that can be useful for the selective assay of glucose in the presence of other electrochemically oxidizable species. Compounds having the formula 1 are examples of transition metal complexes of the present invention.




M is a transition metal and is typically iron, cobalt, ruthenium, osmium, or vanadium. Ruthenium and osmium are particularly suitable for redox mediators.
L is a bidentate ligand containing at least one imidazole ring. One example of L is a 2,2′-biimidazole having the following structure 2:




R1 and R2 are substituents attached to two of the 2,2′-biimidazole nitrogens and are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups. Generally, R1 and R2 are unsubstituted C1 to C12 alkyls. Typically, R1 and R2 are unsubstituted C1 to C4 alkyls. In some embodiments, both R1 and R2 are methyl.
R3, R4, R5, and R6 are substituents attached to carbon atoms of the 2,2′-biimidazole and are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, R3 and R4 in combination or R5 and R6 in combination independently form a saturated or unsaturated 5- or 6-membered ring. An example of this is a 2,2′-bibenzoimidazole derivative. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, R3, R4, R5, and R6 are independently —H or unsubstituted alkyl groups. Typically, R3, R4, R5, and R6 are —H or unsubstituted C1 to C12 alkyls. In some embodiments, R3, R4, R5, and R6 are all —H.
Another example of L is a 2-(2-pyridyl)imidazole having the following structure 3:




R′1 is a substituted or unsubstituted aryl, alkenyl, or alkyl. Generally, R′1 is a substituted or unsubstituted C1-C12 alkyl. R′1 is typically methyl or a C1-C12 alkyl that is optionally substituted with a reactive group.
R′3, R′4, Ra, Rb, Rc, and Rd are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, alkoxylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, Rc and Rd in combination or R′3 and R′4 in combination can form a saturated or unsaturated 5- or 6-membered ring. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, R′3, R′4, Ra, Rb, Rc and Rd are independently —H or unsubstituted alkyl groups. Typically, Ra and Rc are —H and R′3, R′4, Rb, and Rd are —H or methyl.
c is an integer indicating the charge of the complex. Generally, c is an integer selected from −1 to −5 or +1 to +5 indicating a positive or negative charge. For a number of osmium complexes, c is +2 or +3.
X represents counter ion(s). Examples of suitable counter ions include anions, such as halide (e.g., fluoride, chloride, bromide or iodide), sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, and cations (preferably, monovalent cations), such as lithium, sodium, potassium, tetralkylammonium, and ammonium. Preferably, X is a halide, such as chloride. The counter ions represented by X are not necessarily all the same.
d represents the number of counter ions and is typically from 1 to 5.
L1, L2, L3 and L4 are ligands attached to the transition metal via a coordinative bond. L1, L2, L3 and L4 can be monodentate ligands or, in any combination, bi-, ter-, or tetradentate ligands For example, L1, L2, L3 and L4 can combine to form two bidentate ligands such as, for example, two ligands selected from the group of substituted and unsubstituted 2,2′-biimidazoles, 2-(2-pyridyl)imidizoles, and 2,2′-bipyridines
Examples of other L1, L2, L3 and L4 combinations of the transition metal complex include:

    
    
        (A) L1 is a monodentate ligand and L2, L3 and L4 in combination form a terdentate ligand;
        (B) L1 and L2 in combination are a bidentate ligand, and L3 and L4 are the same or different monodentate ligands;
        (C) L1 and L2 in combination, and L3 and L4 in combination form two independent bidentate ligands which can be the same or different; and
        (D) L1, L2, L3 and L4 in combination form a tetradentate ligand.
    
    


Examples of suitable monodentate ligands include, but are not limited to, —F, —Cl, —Br, —I, —CN, —SCN, —OH, H2O, NH3, alkylamine, dialkylamine, trialkylamine, alkoxy or heterocyclic compounds. The alkyl or aryl portions of any of the ligands are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Any alkyl portions of the monodentate ligands generally contain 1 to 12 carbons. More typically, the alkyl portions contain 1 to 6 carbons. In other embodiments, the monodentate ligands are heterocyclic compounds containing at least one nitrogen, oxygen, or sulfur atom. Examples of suitable heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, pyrazine and derivatives thereof. Suitable heterocyclic monodentate ligands include substituted and unsubstituted imidazole and substituted and unsubstituted pyridine having the following general formulas 4 and 5, respectively:




With regard to formula 4, R7 is generally a substituted or unsubstituted alkyl, alkenyl, or aryl group. Typically, R7 is a substituted or unsubstituted C1 to C12 alkyl or alkenyl. The substitution of inner coordination sphere chloride anions by imidazoles does not typically cause a large shift in the redox potential in the oxidizing direction, differing in this respect from substitution by pyridines, which typically results in a large shift in the redox potential in the oxidizing direction.
R8, R9 and R10 are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, R9 and R10, in combination, form a fused 5 or 6-membered ring that is saturated or unsaturated. The alkyl portions of the substituents generally contain 1 to 12 carbons and typically contain 1 to 6 carbon atoms. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. In some embodiments, R8, R9 and R10 are —H or substituted or unsubstituted alkyl. Preferably, R8, R9 and R10 are —H.
With regard to Formula 5, R11, R12, R13, R14 and R15 are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, R11, R12, R13, R14 and R15 are —H, methyl, C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 lower alkyl substituted with a reactive group.
One example includes R11 and R15 as —H, R12 and R14 as the same and —H or methyl, and R13 as —H, C1 to C12 alkoxy, —NH2, C1 to C12 alkylamino, C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino, hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12 alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
Examples of suitable bidentate ligands include, but are not limited to, amino acids, oxalic acid, acetylacetone, diaminoalkanes, ortho-diaminoarenes, 2,2′-biimidazole, 2,2′-bioxazole, 2,2′-bithiazole, 2-(2-pyridyl)imidazole, and 2,2′-bipyridine and derivatives thereof. Particularly suitable bidentate ligands for redox mediators include substituted and unsubstituted 2,2′-biimidazole, 2-(2-pyridyl)imidazole and 2,2′-bipyridine. The substituted 2,2′ biimidazole and 2-(2-pyridyl)imidazole ligands can have the same substitution patterns described above for the other 2,2′-biimidazole and 2-(2-pyridyl)imidazole ligand. A 2,2′-bipyridine ligand has the following general formula 6:




R16, R17, R18, R19, R20, R21, R22 and R23 are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, or alkyl. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
Specific examples of suitable combinations of R16, R17, R18, R19, R20, R21, R22 and R23 include R16 and R23 as H or methyl; R17 and R22 as the same and —H or methyl; and R19 and R20 as the same and —H or methyl. An alternative combination is where one or more adjacent pairs of substituents R16 and R17, on the one hand, and R22 and R23, on the other hand, independently form a saturated or unsaturated 5- or 6-membered ring. Another combination includes R19 and R20 forming a saturated or unsaturated five or six membered ring.
Another combination includes R16, R17, R19, R20, R22 and R23 as the same and —H and R18 and R21 as independently —H, alkoxy, —NH2, alkylamino, dialkylamino, alkylthio, alkenyl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. As an example, R18 and R21 can be the same or different and are —H, C1-C6 alkyl, C1-C6 amino, C1 to C12 alkylamino, C2 to C12 dialkylamino, C1 to C12 alkylthio, or C1 to C12 alkoxy, the alkyl portions of any of the substituents are optionally substituted by a —F, —Cl, —Br, —I, aryl, C2 to C12 dialkylamino, C3 to C18 trialkylammonium, C1 to C6 alkoxy, C1 to C6 alkylthio or a reactive group.
Examples of suitable terdentate ligands include, but are not limited to, diethylenetriamine, 2,2′,2′′-terpyridine, 2,6-bis(N-pyrazolyl)pyridine, and derivatives of these compounds. 2,2′,2′′-terpyridine and 2,6-bis(N-pyrazolyl)pyridine have the following general formulas 7 and 8 respectively:




With regard to formula 7, R24, R25 and R26 are independently —H or substituted or unsubstituted C1 to C12 alkyl. Typically, R24, R25 and R26 are —H or methyl and, in some embodiments, R24 and R26 are the same and are —H. Other substituents at these or other positions of the compounds of formulas 7 and 8 can be added.
With regard to formula 8, R27, R28 and R29 are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Typically, the alkyl and alkoxy groups are C1 to C12 and, in some embodiments, R27 and R29 are the same and are —H.
Examples of suitable tetradentate ligands include, but are not limited to, triethylenetriamine, ethylenediaminediacetic acid, tetraaza macrocycles and similar compounds as well as derivatives thereof.
Examples of suitable transition metal complexes are illustrated using Formula 9 and 10:




With regard to transition metal complexes of formula 9, the metal osmium is complexed to two substituted 2,2′-biimidazole ligands and one substituted or unsubstituted 2,2′-bipyridine ligand. R1, R2, R3, R4, R5, R6, R16, R17, R18, R19, R20, R21, R22, R23, c, d, and X are the same as described above.
In one embodiment, R1 and R2 are methyl; R3, R4, R5, R6, R16, R17, R19, R20, R22 and R23 are —H; and R18 and R21 are the same and are —H, methyl, or methoxy. Preferably, R18 and R21 are methyl or methoxy.
In another embodiment, R1 and R2 are methyl; R3, R4, R5, R6, R16, R17, R18, R19, R20, R22 and R23 are —H; and R21 is halo, C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. For example, R21 is a C1 to C12 alkylamino or C2 to C24 dialkylamino, the alkyl portion(s) of which are substituted with a reactive group, such as a carboxylic acid, activated ester, or amine. Typically, the alkylamino group has 1 to 6 carbon atoms and the dialkylamino group has 2 to 8 carbon atoms.
With regard to transition metal complexes of formula 10, the metal osmium is complexed to two substituted 2,2′-biimidazole ligands and one substituted or unsubstituted 2-(2-pyridyl)imidazole ligand. R1, R2, R3, R4, R5, R6, R′1, R′3, R′4, Ra, Rb, Rc, Rd, c, d, and X are the same as described above.
In one embodiment, R1 and R2 are methyl; R3, R4, R5, R6, R′3, R′4 and Rd are independently —H or methyl; Ra and Rc are the same and are —H; and Rb is C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portions of any of the substituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
A list of specific examples of preferred transition metal complexes with respective redox potentials is shown in Table 1.







TABLE 1







Redox Potentials of Selected Transition Metal Complexes









Complex
Structure
E1/2(vs Ag/AgCl)/mV*












I




−110


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-




dimethylamino-2,2′-bipyridine)]Cl3



 


II




−100


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-




methylamino-2,2′-bipyridine)]Cl3



 


III




128


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-bromo-




2,2′-bipyridine)]Cl3



 


IV




−86


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-di(2-




methoxyethyl)amino-2,2′-bipyridine)]Cl3



 


V




−97


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(3-




methoxypropyl)amino-2,2′-bipyridine)]Cl3



 


VI




−120


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-




diethylamino-2,2′-bipyridine)]Cl3



 


VII




32


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4,4′-




dimethyl-2,2′-bipyridine)]Cl3



 


VIII




−100


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(6-




hydroxyhexyl)amino-2,2′-bipyridine)]Cl3



 


IX




−93


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(6-




aminohexyl)amino-2,2′-bipyridine)]Cl3



 


X




−125


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-




methoxypyridine)2]Cl3



 


XI




−60


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(N-(4-




carboxy)piperidino)-2,2′-bipyridine)]Cl3



 


XII




−74


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(1-methyl-2-




(2-pyridyl)imidazole)]Cl3



 


XIII




−97


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(1-methyl-2-




(6-methylpyrid-2-yl)imidazole)]Cl3



 


IVX




−81


 



[Os(1,1′-dimethyl-2,2′-biimidazole)2(1-(6-




aminohexyl)-2-(6-methylpyrid-2-




yl)imidazole)]Cl3



 


VX




−230


 



[Os(3,3′-dimethyl-2,2′-biimidazole)3]Cl3





*Redox potentials were estimated by averaging the positions of the reduction wave peaks and the oxidation wave peaks of cyclic voltammograms (CVs) obtained in pH 7 PBS buffer with a glassy carbon working electrode, a graphite counter electrode and a standard Ag/AgCl reference electrode at a sweep rate of 50 mV/s.






The transition metal complexes of Formula 1 also include transition metal complexes that are coupled to a polymeric backbone through one or more of L, L1, L2, L3, and L4. Additional examples of suitable transition metal complexes are described in U.S. patent application Ser. No. 09/712,065, entitled “Polymeric Transition Metal Complexes and Uses Thereof”, incorporated herein by reference. In some embodiments, the polymeric backbone has functional groups that act as ligands of the transitional metal complex. Such polymeric backbones include, for example, poly(4-vinylpyridine) and poly(N-vinylimidazole) in which the pyridine and imidazole groups, respectively, can act as monodentate ligands of the transition metal complex.
An example of a precursor polymer that can be used to form a polymeric transition metal complex is presented as Formula 18. This precursor polymer is poly(4-vinylpyridine) quaternized with an alkyl moiety substituted with a reactive group.




where Ω is the reactive group, m is typically 1 to 18, n and n′ are the average numbers of pyridinium and pyridine subunits respectively in each repeating polymer unit, and n′′ is the number of repeating polymer units.
The polymeric backbone has the following general formula, where n may be 2, n′ may be 17, and n′′ may be 1:




In other embodiments, the transition metal complex of the redox polymers of the invention can be the reaction product between a reactive group on a precursor polymer and a reactive group on a ligand of a precursor transition metal complex (such as a complex of Formula 1 where one of L, L1, L2, L3 and L4 includes a reactive group as described above). Suitable precursor polymers include, for example, poly(acrylic acid) (Formula 11), styrene/maleic anhydride copolymer (Formula 12), methylvinylether/maleic anhydride copolymer (GANTREX polymer) (Formula 13), poly(vinylbenzylchloride) (Formula 14), poly(allylamine) (Formula 15), polylysine (Formula 16), carboxy-poly(vinylpyridine (Formula 17), and poly(sodium 4-styrene sulfonate) (Formula 18).




Alternatively, the transition metal complex can have reactive group(s) for immobilization or conjugation of the complexes to other substrates or carriers, examples of which include, but are not limited to, macromolecules (e.g., enzymes) and surfaces (e.g., electrode surfaces).
For reactive attachment to polymers, substrates, or other carriers, the transition metal complex precursor includes at least one reactive group that reacts with a reactive group on the polymer, substrate, or carrier. Typically, covalent bonds are formed between the two reactive groups to generate a linkage. Examples of such linkages are provided in Table 2, below. Generally, one of the reactive groups is an electrophile and the other reactive group is a nucleophile.







TABLE 2







Examples of Reactive Group Linkages









First Reactive Group
Second Reactive Group
Resulting Linkage





Activated ester*
Amine
Carboxamide


Acrylamide
Thiol
Thioether


Acyl azide
Amine
Carboxamide


Acyl halide
Amine
Carboxamide


Carboxylic acid
Amine
Carboxamide


Aldehyde or ketone
Hydrazine
Hydrazone


Aldehyde or ketone
Hydroxyamine
Oxime


Alkyl halide
Amine
Alkylamine


Alkyl halide
Carboxylic acid
Carboxylic ester


Alkyl halide
Imidazole
Imidazolium


Alkyl halide
Pyridine
Pyridinium


Alkyl halide
Alcohol/phenol
Ether


Alkyl halide
Thiol
Thioether


Alkyl sulfonate
Thiol
Thioether


Alkyl sulfonate
Pyridine
Pyridinium


Alkyl sulfonate
Imidazole
Imidazolium


Alkyl sulfonate
Alcohol/phenol
Ether


Anhydride
Alcohol/phenol
Ester


Anhydride
Amine
Carboxamide


Aziridine
Thiol
Thioether


Aziridine
Amine
Alkylamine


Aziridine
Pyridine
Pyridinium


Epoxide
Thiol
Thioether


Epoxide
Amine
Alkylamine


Epoxide
Pyridine
Pyridinium


Halotriazine
Amine
Aminotriazine


Halotriazine
Alcohol
Triazinyl ether


Imido ester
Amine
Amidine


Isocyanate
Amine
Urea


Isocyanate
Alcohol
Urethane


Isothiocyanate
Amine
Thiourea


Maleimide
Thiol
Thioether


Sulfonyl halide
Amine
Sulfonamide





*Activated esters, as understood in the art, generally include esters of succinimidyl, benzotriazolyl, or aryl substituted by electron-withdrawing groups such as sulfo, nitro, cyano, or halo; or carboxylic acids activated by carbodiimides.






Transition metal complexes used in the redox polymers of the present invention can be soluble in water or other aqueous solutions, or in organic solvents. In general, transition metal complexes can be made soluble in either aqueous or organic solvents by having an appropriate counter ion or ions, X. For example, transition metal complexes with small counter anions, such as F−, Cl−, and Br−, tend to be water soluble. On the other hand, transition metal complexes with bulky counter anions, such as I−, BF4− and PF6−, tend to be soluble in organic solvents. Preferably, the solubility of transition metal complexes of the present invention is greater than about 0.1 M (moles/liter) at 25° C. for a desired solvent.
The use of transition metal complexes as redox mediators is described, for example, in U.S. Pat. Nos. 5,262,035; 5,262,305; 5,320,725; 5,365,786; 5,593,852; 5,665,222; 5,972,199; and 6,143,164 and U.S. patent application Ser. Nos. 09/034,372, (now U.S. Pat. No. 6,134,461); 09/070,677, (now U.S. Pat. No. 6,175,752); 09/295,962, (now U.S. Pat. No. 6,338,790) and 09/434,026, all of which are herein incorporated by reference. The transitional metal complexes described herein can typically be used in place of those discussed in the references listed above.
Redox polymers of the invention may comprise, inter alia, a transition metal complex, a cross-linker and a polymeric backbone. Such redox polymers are useful as redox mediators in electrochemical biosensors for the detection of analytes in bio-fluids, for example.
In general, the redox polymer is disposed on or in proximity to (e.g., in a solution surrounding) a working electrode. The redox polymer transfers electrons between the working electrode and an analyte. In some preferred embodiments, an enzyme is also included to facilitate the transfer. For example, the redox polymer transfers electrons between the working electrode and glucose (typically via an enzyme) in an enzyme-catalyzed reaction of glucose. Redox polymers are particularly useful for forming non-leachable coatings on the working electrode. These can be formed, for example, by crosslinking the redox polymer on the working electrode, or by cross-linking the redox polymer and the enzyme on the working electrode
Transition metal complexes can enable accurate, reproducible and quick or continuous assays. Transition metal complex redox mediators accept electrons from, or transfer electrons to, enzymes or analytes at a high rate and also exchange electrons rapidly with an electrode. Typically, the rate of self exchange, the process in which a reduced redox mediator transfers an electron to an oxidized redox mediator, is rapid. At a defined redox mediator concentration, this provides for more rapid transport of electrons between the enzyme (or analyte) and electrode, and thereby shortens the response time of the sensor. Additionally, the transition metal complex redox mediators disclosed herein are typically stable under ambient light and at the temperatures encountered in use, storage and transportation. Preferably, the transition metal complex redox mediators do not undergo chemical change, other than oxidation and reduction, in the period of use or under the conditions of storage, though the redox mediators can be designed to be activated by reacting, for example, with water or the analyte.
A transition metal complex can be used as a redox mediator in combination with a redox enzyme to electrooxidize or electroreduce the analyte or a compound derived of the analyte, for example by hydrolysis of the analyte. The redox potentials of the redox mediators are generally more positive (i.e. more oxidizing) than the redox potentials of the redox enzymes when the analyte is electrooxidized and more negative when the analyte is electroreduced. For example, the redox potentials of the preferred transition metal complex redox mediators used for electrooxidizing glucose with glucose oxidase or PQQ-glucose dehydrogenase as enzyme is between about −200 mV and +200 mV versus a Ag/AgCl reference electrode, and the most preferred mediators have redox potentials between about −100 mV and about +100 mV versus a Ag/AgCl reference electrode
Crosslinking in Transition Metal Complex Polymers
Electron transport involves an exchange of electrons between segments of the redox polymers (e.g., one or more transition metal complexes coupled to a polymeric backbone, as described above) in a crosslinked film disposed on an electrode. A transition metal complex can be bound to the polymer backbone though covalent, coordinative or ionic bonds, where covalent and coordinative binding are preferred. Electron exchange occurs, for example, through the collision of different segments of the crosslinked redox polymer. Electrons transported through the redox polymer can originate from, for example, electrooxidation or electroreduction of an enzymatic substrate, such as, for example, the oxidation of glucose by glucose oxidase.
The degree of crosslinking of the redox polymer can influence the transport of electrons or ions and thereby the rates of the electrochemical reactions. Excessive crosslinking of the polymer can reduce the mobility of the segments of the redox polymer. A reduction in segment mobility can slow the diffusion of electrons or ions through the redox polymer film. A reduction in the diffusivity of electrons, for example, can require a concomitant reduction in the thickness of the film on the electrode where electrons or electron vacancies are collected or delivered. The degree of crosslinking in a redox polymer film can thus affect the transport of electrons from, for example, an enzyme to the transition metal redox centers of the redox polymer such as, for example, Os2+/3+ metal redox centers; between redox centers of the redox polymer; and from these transition metal redox centers to the electrode.
Inadequate crosslinking of a redox polymer can result in excessive swelling of the redox polymer film and to the leaching of the components of the redox polymer film. Excessive swelling can also result in the migration of the swollen polymer into the analyzed solution, in the softening of the redox polymer film, in the film's susceptibility to removal by shear, or any combination of these effects.
Crosslinking can decrease the leaching of film components and can improve the mechanical stability of the film under shear stress. For example, as disclosed in Binyamin, G. and Heller, A; Stabilization of Wired Glucose Oxidase Anodes Rotating at 1000 rpm at 37° C.; Journal of the Electrochemical Society, 146(8), 2965-2967, 1999, herein incorporated by reference, replacing a difunctional cross-linker, such as polyethylene glycol diglycidyl ether, with a trifunctional cross-linker such as N,N-diglycidyl-4-glycidyloxyaniline, for example, can reduce leaching and shear problems associated with inadequate crosslinking.
Examples of other bifunctional, trifunctional and tetrafunctional cross-linkers are listed below:
Amine-Reactive Bifunctional Crosslinkers




Pyridine- or Imidazole-Reactive Bifunctional Crosslinkers




Pyridine- or Imidazole-Reactive Trifunctional Crosslinker




Pyridine- or Imidazole-Reactive Tetrafunctional Crosslinkers




Alternatively, the number of crosslinking sites can be increased by reducing the number of transition metal complexes attached to the polymeric backbone, thus making more polymer pendant groups available for crosslinking. One important advantage of at least some of the redox polymers is the increased mobility of the pendant transition metal complexes, resulting from the flexibility of the pendant groups. As a result, in at least some embodiments, fewer transition metal complexes per polymer backbone are needed to achieve a desired level of diffusivity of electrons and current density of analyte electrooxidation or electroreduction.
Coordination in Transition Metal Complex Polymers
Transition metal complexes can be directly or indirectly attached to a polymeric backbone, depending on the availability and nature of the reactive groups on the complex and the polymeric backbone. For example, the pyridine groups in poly(4-vinylpyridine) or the imidazole groups in poly(N-vinylimidazole) are capable of acting as monodentate ligands and thus can be attached to a metal center directly. Alternatively, the pyridine groups in poly(4-vinylpyridine) or the imidazole groups in poly(N-vinylimidazole) can be quaternized with a substituted alkyl moiety having a suitable reactive group, such as a carboxylate function, that can be activated to form a covalent bond with a reactive group, such as an amine, of the transition metal complex. (See Table 2 for a list of other examples of reactive groups).
Redox centers such as, for example, Os2+/3+ can be coordinated with five heterocyclic nitrogens and an additional ligand such as, for example, a chloride anion. An example of such a coordination complex includes two bipyridine ligands which form stable coordinative bonds, the pyridine of poly(4-vinylpyridine) which forms a weaker coordinative bond, and a chloride anion which forms the least stable coordinative bond.
Alternatively, redox centers, such as Os2+/3+, can be coordinated with six heterocyclic nitrogen atoms in its inner coordination sphere. The six coordinating atoms are preferably paired in the ligands, for example, each ligand is composed of at least two rings. Pairing of the coordinating atoms can influence the potential of an electrode used in conjunction with redox polymers of the present invention.
Typically, for analysis of glucose, the potential at which the working electrode, coated with the redox polymer, is poised is negative of about +250 mV vs. SCE (standard calomel electrode). Preferably, the electrode is poised negative of about +150 mV vs. SCE. Poising the electrode at these potentials reduces the interfering electrooxidation of constituents of biological solutions such as, for example, urate, ascorbate and acetaminophen. The potential can be modified by altering the ligand structure of the complex.
The redox potential of a redox polymer, as described herein, is related to the potential at which the electrode is poised. Selection of a redox polymer with a desired redox potential allows tuning of the potential at which the electrode is best poised. The redox potentials of a number of the redox polymers described herein are negative of about +150 mV vs. SCE and can be negative of about +50 mV vs. SCE to allow the poising of the electrode potentials negative of about +250 mV vs. SCE and preferably negative of about +150 mV vs. SCE.
The strength of the coordination bond can influence the potential of the redox centers in the redox polymers. Typically, the stronger the coordinative bond, the more positive the redox potential. A shift in the potential of a redox center resulting from a change in the coordination sphere of the transition metal can produce a labile transition metal complex. For example, when the redox potential of an Os2+/3+ complex is downshifted by changing the coordination sphere, the complex becomes labile. Such a labile transition metal complex may be undesirable when fashioning a metal complex polymer for use as a redox mediator and can be avoided through the use of weakly coordinating multidentate or chelating heterocyclics as ligands.
Electrode Interference
Transition metal complexes used as redox mediators in electrodes can be affected by the presence of transition metals in the analyzed sample including, for example, Fe3+ or Zn2+. The addition of a transition metal cation to a buffer used to test an electrode results in a decline in the current produced. The degree of current decline depends on the presence of anions in the buffer which precipitate the transition metal cations. The lesser the residual concentration of transition metal cations in the sample solution, the more stable the current. Anions which aid in the precipitation of transition metal cations include, for example, phosphate. It has been found that a decline in current upon the addition of transition metal cations is most pronounced in non-phosphate buffers. If an electrode is transferred from a buffer containing a transition metal cation to a buffer substantially free of the transition metal cation, the original current is restored.
The decline in current is thought to be due to additional crosslinking of a pyridine-containing polymer backbone produced by the transition metal cations. The transition metal cations can coordinate nitrogen atoms of different chains and chain segments of the polymers. Coordinative cros slinking of nitrogen atoms of different chain segments by transition metal cations can reduce the diffusivity of electrons.
Serum and other physiological fluids contain traces of transition metal ions, which can diffuse into the films of electrodes made with the redox polymers of the present invention, lowering the diffusivity of electrons and thereby the highest current reached at high analyte concentration. In addition, transition metal ions like iron and copper can bind to proteins of enzymes and to the reaction centers or channels of enzymes, reducing their turnover rate. The resulting decrease in sensitivity can be remedied through the use of anions which complex with interfering transition metal ions, for example, in a buffer employed during the production of the transition metal complex. A non-cyclic polyphosphate such as, for example, pyrophosphate or triphosphate, can be used. For example, sodium or potassium non-cyclic polyphosphate buffers can be used to exchange phosphate anions for those anions in the transition metal complex which do not precipitate transition metal ions. The use of linear phosphates can alleviate the decrease in sensitivity by forming strong complexes with the damaging transition metal ions, assuring that their activity will be low. Other complexing agents can also be used as long as they are not electrooxidized or electroreduced at the potential at which the electrode is poised.
Enzyme Damage and its Alleviation
Glucose oxidase is a flavoprotein enzyme that catalyzes the oxidation by dioxygen of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide. Reduced transition metal cations such as, for example, Fe2+, and some transition metal complexes, can react with hydrogen peroxide. These reactions form destructive OH radicals and the corresponding oxidized cations. The presence of these newly formed transition metal cations can inhibit the enzyme and react with the metal complex. Also, the oxidized transition metal cation can be reduced by the FADH2 centers of an enzyme, or by the transition metal complex.
Inhibition of the active site of an enzyme or a transition metal complex by a transition metal cation, as well as damaging reactions with OH radicals can be alleviated, thus increasing the sensitivity and functionality of the electrodes by incorporating non-cyclic polyphosphates, as discussed above. Because the polyphosphate/metal cation complex typically has a high (oxidizing) redox potential, its rate of oxidation by hydrogen peroxide is usually slow. Alternatively, an enzyme such as, for example, catalase, can be employed to degrade hydrogen peroxide.
EXAMPLES
Unless indicated otherwise, all of the chemical reagents are available from Aldrich Chemical Co. (Milwaukee, Wis.) or other sources. Additional examples are provided in U.S. Pat. No. 6,605,200 entitled “Polymeric Transition Metal Complexes and Uses Thereof”, incorporated herein by reference. For purposes of illustration, the synthesis of several transition metal complex ligands are shown below:
Example 1
Synthesis of 4-(5-carboxypentyl)amino-2,2′-bipyridyl
This example illustrates how a carboxy reactive group is introduced onto a 2.2′-bipyridyl derivative.
Synthesis of Compound D:
To compound C (formed from A and B according to




Wenkert, D.; Woodward, R. B. J. Org. chem. 48, 283 (1983)) (5 g) dissolved in 30 mL acetic acid in a 100 ml round bottom flask was added 16 mL acetyl bromide. The yellow mixture was refluxed for 1.5 h and then rotovaporated to dryness. The resulting light yellow solid of D was sufficiently pure enough for the next step without further purification. Yield: 95%

Synthesis of Compound E:
To a stirred suspension of compound D in 60 mL CHCl3 was added 12 mL PCl3 at room temperature. The mixture was refluxed for 2 h under N2 and then cooled to room temperature. The reaction mixture was poured into 100 mL ice/water. The aqueous layer was separated and saved. The CHCl3 layer was extracted three times with H2O (3×60 mL) and then discarded. The combined aqueous solution was neutralized with NaHCO3 powder to about pH 7 to 8. The resulting white precipitate was collected by suction filtration, washed with H2O (30 mL) and then dried under vacuum at 50° C. for 24 h. Yield: 85%.
Synthesis of Compound F:
Compound F was synthesized from compound E (5 g) and 6-aminocaproic acid methyl ester (6 g) using the palladium-catalyzed amination method of aryl bromides described by Hartwig et al. (Hartwig, J. F., et al. J. Org. Chem. 64, 5575 (1999)). Yield: 90%.
Synthesis of Compound G:
Compound F (3 g) dissolved in 20 mL MeOH was added to a solution of NaOH (0.6 g) in 30 mL H2O. The resulting solution was stirred at room temperature for 24 h and then neutralized to pH 7 with dilute HCl. The solution was saturated with NaCl and then extracted with CHCl3. The CHCl3 extract was evaporated to dryness and then purified by a silica gel column eluted with 10% H2O/CH3CN. Yield: 70%.
Example 2
Synthesis of a 4-((6-Aminohexyl)amino)-2,2′-bipyridine




This example illustrates the general synthesis of a 2,2′-bipyridyl with an amine reactive group.
Synthesis of Compound H:
A mixture of compound E (2.5 g) and 1,6-diaminohexane (15 g) in a 250 mL round bottom flask was heated under N2 at 140° C. in an oil bath for 4-5 h. Excess 1,6-diaminohexane was removed by high vacuum distillation at 90-120° C. The product was purified by a silica gel column, eluting with 5% NH4OH in isopropyl alcohol. Yield: 70%.
Example 3
Synthesis of 1,1′-dimethyl-2,2′-biimidazole
This example illustrates the synthesis of 2,2′-biimidazole derivatives.




The alkylation step can be carried out stepwise so two different alkyl groups can be introduced. For example:




Synthesis of Compound K:
To a stirred solution of compound J (formed from I according to Fieselmann, B. F., et al. Inorg. Chem. 17, 2078 (1978)) (4.6 g, 34.3 mmoles) in 100 mL dry DMF in a 250 ml round bottom flask cooled in an ice/water bath was added in portions NaH(60% in mineral oil, 2.7 g, 68.6 mmoles). After the solution was stirred at 0° C. for one more hour under N2, methyl toluenesulfonate (10.3 mL, 68.6 mmoles) was added in small portions using a syringe over 30 min. The stiffing of the solution in the ice/water bath was continued for 1 h and then at room temperature for 3 h. The solvent was removed by vacuum distillation. The dark residue was triturated with ether and then suction filtered and dried under vacuum. The product was purified by sublimation. Yield: 80%.
Synthesis of Compound L:
Compound L was prepared using the method described for the synthesis of compound K except that only one equivalent each of compound J, NaH and methyl toluenesulfonate was used. The product was purified by sublimation.
Synthesis of Compound M:
To a stirred solution of compound L (1 g, 6.8 mmoles) in 20 mL dry DMF in a 50 ml round bottom flask cooled in a ice/water bath is added in portions NaH(60% in mineral oil, 0.27 g, 6.8 mmoles). After the solution is stirred at 0° C. for one more hour under N2, ethyl bromoacetate (0.75 mL, 6.8 mmoles) is added in small portions via a syringe over 15 min. The stiffing of the solution is continued in the ice/water bath for 1 h and then at room temperature for 3 h. The solvent is removed by vacuum distillation. The product is purified by a silica gel column using 10% MeOH/CHCl3 as the eluent.
Synthesis of Compound N:
Compound M (1 g) is hydrolyzed using the method described for the synthesis of compound G. The product is purified by a silica gel column using 10% H2O/CH3CN as the eluent.
Example 4
Synthesis of 2-(2-Pyridyl)imidazole Heterobidentate Ligands
This example illustrates a general synthesis of heterobidentate ligands containing an imidazole ring.




Synthesis of Compound O:
A solution of 6-methylpyridine-2-carboxaldehyde (26 g, 0.21 mole) and glyoxal (40%, 30 mL) in 50 mL EtOH in a three-necked 250 mL round bottom flask fitted with a thermometer and an addition funnel was stirred in a NaCl/ice bath. When the solution was cooled to below 5° C., conc. NH4OH was added dropwise through the addition funnel. The rate of the addition was controlled so that the temperature of the solution was maintained at below 5° C. After the addition, the stirring of the yellow solution was continued in the ice bath for 1 h and then at room temperature overnight. The light yellow crystals were collected by suction filtration and washed with H2O (20 mL). The crystals were resuspended in H2O (200 mL) and boiled briefly, followed by suction filtration, to collect the product which was dried under high vacuum. Yield: 35%.
Synthesis of Compound P:
Sodium t-butoxide (2 g, 20.8 mmoles) was added in one portion to a stirred solution of compound O (3 g, 18.9 mmoles) in 50 mL dry DMF. After all of the sodium t-butoxide was dissolved, iodomethane (1.3 mL) was added dropwise using a syringe. The stirring of the solution was continued at room temperature for 2 h and then the solution was poured into H2O (150 mL). The product was extracted with EtOAc, and the extract was dried with anhydrous Na2SO4 and then evaporated to give crude compound P. The product was purified by separation on a silica gel column using 10% MeOH/CHCl3 as the eluent. Yield: 70%.
Example 5
Synthesis of Transition Metal Complexes with Multiple Identical Ligands
Transition metal complexes containing multiple identical bidentate or tridentate ligands can be synthesized in one step from a metal halide salt and the ligand. This example illustrates the synthesis of an osmium complex with three identical 2,2′-biimidazole bidentate ligands.




Synthesis of Compound Q:
Ammonium hexachloroosmate (200 mg, 0.46 mmoles) and compound K (221 mg, 1.37 mmoles) were mixed in 15 mL ethylene glycol in a 100 mL three-necked round bottom flask fitted with a reflux condenser. The mixture was degassed with N2 for 15 min and then stirred under N2 at 200-210° C. for 24 hrs. The solvent was removed by high vacuum distillation at 90-100° C. The green colored crude product was dissolved in 15 mL H2O and stirred in air to be fully oxidized to the dark blue colored Os(III) oxidation state (about 24 h). The product was purified on a LH-20 reverse phase column using H2O as the eluent. Yield: 50%.
Example 6
Synthesis of Transition Metal Complexes with Mixed Ligands
Transition metal complexes containing multiple types of ligands can be synthesized stepwise. First, a transition metal complex intermediate that contains one desired type of ligand and halide ligand(s), for example, chloride, is synthesized. Then the intermediate is subjected to a ligand substitution reaction to displace the halide ligand(s) with another desired type of ligand. The preparation of the following osmium complex illustrates the general synthetic scheme.




Synthesis of Compound U:
Potassium hexachloroosmate (1 g, 2.08 mmoles), compound K (0.67 g, 4.16 mmoles) and LiCl (1 g, 23.8 mmoles) were suspended in 40 mL ethylene glycol in a 250 mL three-necked round bottom flask fitted with a reflux condenser. The suspension was degassed with N2 for 15 min and then stirred under N2 at 170° C. in an oil bath for 7-8 h, resulting in a dark brown solution. The solvent was removed by high vacuum distillation at 90-100° C. bath temperature. The gummy solid was triturated with acetone twice (2×50 mL) and then with H2O once (50 mL). The product was dried at 50° C. under high vacuum for 24 h.
Synthesis of Compound W:
A suspension of compound U (119 mg, 0.192 mmole) and 4-(4-carboxypiperidino)amino-2,2′-bipyridyl (prepared from compound E and ethyl isonipecotate using the synthetic methods for compounds F and G) was made in 10 mL ethylene glycol in a 100 mL three-necked round bottom flask equipped with a reflux condenser. The suspension was degassed with N2 for 15 min and then stirred under N2 at 130° C. in an oil bath for 24 h. The dark brown solution was cooled to room temperature and then poured into EtOAc (50 mL). The precipitate was collected by suction filtration. The dark brown solid thus obtained was compound W with osmium in a 2+ oxidation state. For ease of purification, the osmium 2+ complex was oxidized to an osmium 3+ complex by dissolving the dark brown solid in 20 mL H2O and stiffing the solution in open air for 24 h. The resulting dark green solution was poured into a stirred solution of NH4 PF6 (1 g) in 20 mL H2O. The resulting dark green precipitate of [Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(4-carboxypiperidino)amino-2,2′-bipyridyl)]3+3 PF6− was collected by suction filtration and washed with 5 mL H2O and then dried at 40° C. under high vacuum for 48 h. The counter anion PF6− of [Os(1,1′-dimethyl-2,2′-biimidazole)2(4-(4-carboxypiperidino)amino-2,2′-bipyridyl)]3+3 PF6− was exchanged to the more water soluble chloride anion. A suspension of the PF6− salt of compound W (150 mg) and Cl− resin (10 mL) in H2O (20 mL) was stirred for 24 h, at the end of which period all of osmium complex was dissolved. The dark green solution was separated by suction filtration and then lyophilized to give compound W.
A suitable sensor may operate as now described. The sensor is placed, transcutaneously, for example, into a subcutaneous site such that subcutaneous fluid of the site comes into contact with the sensor. The sensor operates to electrolyze an analyte of interest in the subcutaneous fluid such that a current is generated between the working electrode and the counter electrode. A value for the current associated with the working electrode is determined periodically. If multiple working electrodes are used, current values from each of the working electrodes may be determined periodically. A microprocessor may be used to collect these periodically determined current values or to further process these values.
If an analyte concentration is successfully determined, it may be displayed, stored, and/or otherwise processed to provide useful information. By way of example, analyte concentrations may be used as a basis for determining a rate of change in analyte concentration, which should not change at a rate greater than a predetermined threshold amount. If the rate of change of analyte concentration exceeds the predefined threshold, an indication maybe displayed or otherwise transmitted to indicate this fact.
As demonstrated herein, the redox polymers of the present invention are particularly useful in connection with a device that is used to measure or monitor a glucose analyte, such as any such device described herein. These redox polymers may also be used in connection with a device that is used to measure or monitor another analyte, such as oxygen, carbon dioxide, proteins, drugs, or another moiety of interest, for example, or any combination thereof, found in bodily fluid, such as subcutaneous fluid, dermal fluid (sweat, tears, and the like), interstitial fluid, or other bodily fluid of interest, for example, or any combination thereof. Preferably, the device is in good contact, such as thorough and substantially continuous contact, with the bodily fluid.
According to a preferred embodiment of the present invention, the measurement sensor is one suited for electrochemical measurement of analyte concentration, and preferably, glucose concentration, in a bodily fluid. In this embodiment, the measurement sensor comprises at least a working electrode and a counter electrode. It may further comprise a reference electrode, although this is optional. The working electrode typically comprises a glucose-responsive enzyme and a redox mediator, both of which are agents or tools in the transduction of the analyte, and preferably, glucose. Preferably, the redox mediator is non-leachable relative to the working electrode. Merely by way of example, the redox mediator may be, and preferably is, immobilized on the working electrode.
According to a most preferred embodiment of the present invention, the measurement sensor is one suited for in vivo, continuous, electrochemical measurement or monitoring of analyte concentration, and preferably, glucose concentration, in a bodily fluid. In this embodiment, the measurement sensor is sufficiently biocompatible for its partial or full implantation within the body. By way of explanation, when an unnatural device is intended for use, particularly long-term use, within the body of an individual, protective mechanisms of the body attempt to shield the body from the device. (See co-pending U.S. application Ser. No. 10/819,498 of Feldman et al. filed Apr. 6, 2004). That is, such an unnatural device or portion thereof is more or less perceived by the body as an unwanted, foreign object.
Protective mechanisms of the body may encompass encapsulation of the device or a portion thereof, growth of tissue that isolate the device or a portion thereof, formation of an analyte-impermeable barrier on and around the device or a portion thereof, and the like, merely by way of example. Encapsulation and barrier formation around all or part of the implantable sensor may compromise, significantly reduce, or substantially or completely eliminate, the functionality of the device. Preferably, the measurement sensor is sufficiently biocompatible to reduce, minimize, forestall, or avoid any such protective mechanism or its effects on the sensor functionality, or is associated with or adapted to incorporate a material suitable for promoting biocompatibility. Preferably, the measurement sensor is sufficiently biocompatible over the desired, intended, or useful life of the sensor.
Evidence suggests that improved glycemic control can minimize many of the complications associated with Type 1 diabetes. (See, Diabetes Control and Complications Trial Research Group: The Effect of Intensive Treatment of Diabetes on the Development and Progression of Long-Term Complications in Insulin-Dependent Diabetes Mellitus, N. Engl. J. Med., 329, pp. 977-986 (1993)). Frequent self-monitoring of blood glucose, in concert with intensive insulin therapy, greatly improves glycemic control.
Continuous glucose sensing provides all of the advantages of high-frequency, discrete testing. It also provides advantages of its own. By way of example, continuous glucose sensing may provide valuable information about the rate and direction of changes in glucose levels, which information may be used predictively or diagnostically. Further by way of example, as continuous glucose sensing occurs at times when discrete testing does not usually occur, such as post-prandially or during sleep, for example, continuous glucose sensing may provide sensitive alarms for hyperglycemia and hypoglycemia that may be associated with post-prandial or resting conditions.
The above-mentioned FREESTYLE NAVIGATOR continuous glucose sensor is a subcutaneous, electrochemical sensor, which operates for three days when implanted at a site in the body. This sensor is based on the above-mentioned WIRED ENZYME sensing technology, a mediated glucose-sensing technology that offers a number of advantages over conventional oxygen-dependent, electrochemical, glucose-sensing technologies, which utilize hydrogen peroxide (H2O2) detection at high applied potential ( ̃500 mV vs. a silver/silver chloride (Ag/AgCl) reference electrode). (See, Csoregi, E., Schmidtke, D. W., and Heller, A., Design and Optimization of a Selective Subcutaneously Implantable Glucose Electrode Based on “Wired” Glucose Oxidase, Anal. Chem., 67, pp. 1240-1244 (1995)).
WIRED ENZYME technology works at a relatively gentle oxidizing potential of +40 mV, using an osmium (Os)-based mediator molecule specifically designed for low potential operation and stably anchored in a polymeric film for in vivo use. The sensing element is a redox active gel that comprises Os-based mediator molecules, attached by stable bidentate anchors to a polymeric backbone film, and glucose oxidase (GOx) enzyme molecules, permanently coupled together via chemical cross-linking. This redox active gel is a glucose-sensing gel, which accurately transduces glucose concentrations to a measured current over a glucose range of 20-500 mg/dL.
WIRED ENZYME sensing technology offers three primary advantages over conventional H2O2-based detection systems, which rely on oxygen for signal generation. One advantage is that this WIRED ENZYME technology affords electrochemical responses that are extremely stable. This is not the case with many other implanted, or in vivo, glucose sensors, which have been associated with drifts in sensitivity (output per unit glucose concentration) over their lifetimes. (See: Roe, J. N., and Smoller, B. R., Bloodless Glucose Measurements, Crit. Rev. Ther. Drug Carrier Syst., 15, pp. 199-241 (1998); and Wisniewsky, N., Moussy, F., and Reichert, W. M., Characterization of Implantable Biosensor Membrane Biofouling, Fresenius J. Anal. Chem., 366, pp. 611-621 (2000)). Because of these drifts, many other implanted glucose sensors require frequent and/or retrospective calibration. By contrast, after an initial break-in period, WIRED ENZYME implanted glucose sensors have extremely stable in vivo sensitivities, typically losing no more than 0.1% sensitivity per hour.
Another advantage is that WIRED ENZYME technology does not rely on oxygen for signal generation. Although oxygen can compete for electrons with the Os-based mediator molecules, and thereby modestly reduce the sensor output, the overall effect is much smaller than exists in conventional H2O2-measuring systems, which can generate no signal in the absence of oxygen. This reduced oxygen dependency results in minimal sensitivity to in vivo oxygen variations and good linearity at high glucose concentrations. Yet another advantage is that WIRED ENZYME implanted glucose sensors operate at an applied potential of only +40 mV, which is much gentler than the  ̃500 mV required by H2O2-sensing systems. Oxidation of many interferents (acetaminophen, uric acid, etc.) and subsequent.
Sensor Description
A continuous glucose sensor 300 is schematically shown in FIG. 1. This continuous glucose sensor 300 is the FREESTYLE NAVIGATOR continuous glucose monitoring device that is based on WIRED ENZYME technology, as described above. The sensor 300 is an amperometric sensor that comprises three electrodes, a working electrode 302, a reference electrode 304, and a counter electrode 306, contacts of which are shown in FIG. 1. Each of the working electrode 302 and the counter electrode 306 is fabricated from carbon. The reference electrode 304 is an Ag/AgCl electrode. The sensor 300 has a subcutaneous portion 308 having dimensions of about 5 mm in length, 0.6 mm in width, and 0.25 mm in thickness, as further detailed in the enlarged portion of FIG. 1.
Wired Enzyme/Sensing Layer
The working electrode 302 has an active area 310 of about 0.15 mm2. This active area 310 is coated with the WIRED ENZYME sensing layer 312, which is a cross-linked, glucose-transducing gel. As this sensing layer or gel 312 has a relatively hydrophilic interior, glucose molecules surrounding the subcutaneous portion 308 of the sensor 300 are free to diffuse into and within this glucose-transducing gel. The gel 312 is effective in the capture of electrons from these glucose molecules and the transportation of these electrons to the working electrode 302. A schematic illustration of the WIRED ENZYME sensing layer 312, showing various of its components (as further described below), as well as the path of electron flow in the direction depicted by arrows 314, from the glucose to the working electrode 302, is shown in FIG. 2.
The sensing layer or gel 312 comprises a redox polymer mediator 316 of high molecular weight, glucose oxidase (“GOx”) 318, and a bi-functional, short-chain, epoxide cross-linker (not shown), the former two of which are shown in FIG. 2. The redox polymer is an Osmium-decorated poly(vinylpyridine)-based polymer. The sensing layer 312 has a mass of approximately 300 ng (at a dry thickness of about 2 μm) and comprises about 35% by weight redox polymer 316, 40% by weight GOx enzyme 318, and 25% by weight cross-linker.
The redox polymer 316, the structure of which is illustrated in FIG. 3, comprises a modified poly(vinylpyridine) backbone, which is loaded with poly(bi-imidizyl) Os complexes that are securely anchored to the backbone via bidentate linkage. (See: U.S. Patent Application Ser. No. 60/165,565 of Mao et al. filed Nov. 15, 1999; U.S. Pat. Nos. 6,605,200 and 6,605,201 of Mao et al. filed Nov. 14, 2000; U.S. Patent Application Publication No. 2004/0040840 A1 of Mao et al. filed Aug. 11, 2003; U.S. Pat. No. 6,676,816 of Heller et al. filed May 9, 2002; and U.S. Patent Application Publication No. 2004/0099529 A1 of Heller et al. filed Nov. 14, 2003). This polymer 316 is an effective mediator or facilitator of electron transport in the sensing layer.
The sensing layer may be described as the active chemical area of the biosensor or the “wired” enzyme layer, where electrons from glucose are shuttled to a working electrode via one or more electron transfer agents. In the sensing layer, GOx may be substituted with other redox enzymes to measure other relevant clinical compounds, such as lactate oxidase for the in vivo detection of lactate, which may be important in determining if an organ is receiving sufficient oxygen through the blood, for example. (See, e.g., Heller et al. U.S. Pat. No. 6,284,478, hereby incorporated by reference in its entirety).
Useful redox polymers and methods for producing the sensing layer are described herein as well as in U.S. Pat. Nos. 5,264,104; 5,356,786; 5,262,035, and 5,320,725. Additional redox polymers include, for example, poly(1-vinyl imidazole); poly(4-vinyl pyridine); or copolymers of 1-vinyl imidazole such as poly(acrylamide co-1-vinyl imidazole) where the imidazole or pyridine complexes with [Os (bpy)2Cl]+/2+; [Os (4,4′-dimethyl bipyridine)2 Cl]+/2+; [Os (4,4′-dimethyl phenanthroline)2Cl]+/2+; [Os (4,4′-dimethyoxy phenanthroline)2Cl]+/2+; and [Os (4,4′-dimethoxy bipyridine)2Cl]+/2+; to imidazole rings.
Membrane/Barrier Layer
As shown in FIG. 1, the sensor 300 also comprises an analyte-restricting membrane 320, here, a glucose-restricting membrane, disposed over the sensing layer 312. (See: U.S. Patent Application Publication No. 2003/0042137 A1 of Mao et al. filed May 14, 2002). The membrane 320 comprises a poly(vinylpyridine-co-styrene) copolymer of high molecular weight, that is cross-linked using a tri-functional, short-chain epoxide. The membrane 320, which is about 50 μm thick, serves to reduce glucose diffusion to the active sensing layer 312 by a factor of about 50. The hydrophilic membrane 320 provides a surface that is biocompatible, such that bodily irritation from the subcutaneous portion 308 of the sensor 300 is reduced.
The sensor 300 is associated with an in vivo sensitivity of about 0.1 nA/(mg/dL) and a linear response over a glucose concentration range 20-500 mg/dL. Additionally, in terms of response to an instantaneous change in glucose concentration, the sensor 300 is associated with a response time of about three minutes.
The membrane polymer preparation may comprise 16 mg/mL of a formulation called 10Q5, as depicted below (wherein x=0.85, y=0.1, z=0.05, n=9, m=1, and p=about 10), 8 mg/ml triglycidyl glycerol (the cross-linker), and optionally 7.5 mg/ml manganese 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride (MnTPyP), a compound possessing both superoxide dismutase and catalase activity. 10Q5 is based on a poly(vinylpyridine-co-styrene) copolymer, which has been further derivitized by the addition of propylsulfonate and poly(ethyleneoxide) moieties.




In other embodiments, the membrane may have the following general structure:




Such membranes may be employed in a variety of sensors, such as the two- or three-electrode sensors described previously in detail in U.S. Patent Application Publication No. US 2003/0042137A1 of Mao et al., published on Mar. 6, 2003, which is incorporated in its entirety herein by this reference. By way of example, the membrane may be used in a two-electrode amperometric glucose sensor, as shown in FIGS. 4A-4C (collectively FIG. 4). The amperometric glucose sensor 10a of FIG. 4 comprises a substrate 13 disposed between a working electrode 29a that is typically carbon-based, and an Ag/AgCl counter/reference electrode 29b. A sensor or sensing layer 18a is disposed on the working electrode. A membrane or membrane layer 30a encapsulates the entire glucose sensor 10a, including the Ag/AgCl counter/reference electrode. The sensing layer 18a of the glucose sensor 10a consists of crosslinked glucose oxidase and a low potential polymeric osmium complex mediator, as disclosed in the above-mentioned Published PCT Application, International Publication No. WO 01/36660 A2. The enzyme- and mediator-containing formulation that can be used in the sensing layer, and methods for applying them to an electrode system, are known in the art, for example, from the above-mentioned U.S. Pat. No. 6,134,461 of Say et al. According to the present invention, the membrane overcoat was formed by thrice dipping the sensor into a membrane solution comprising 4 mg/mL poly(ethylene glycol)diglycidyl ether (molecular weight of about 200) and 64 mg/mL of a polymer of Formula 1 above, wherein [n/(n+l+p)]×100% ̃10%; [l/(n+l+p)]×100% ̃80%; and [p/(n+l+p)]×100%=10%, and curing the thrice-dipped sensor at ambient temperature and normal humidity for at least 24 hours, such as for about one to about two days. The q value for such a membrane overcoat may be greater than or equal to about 950, where n is 1, l is 8, and p is 1.
Sensor Configuration
The subcutaneous portion 308 of the sensor may be placed into the subcutaneous tissue of the upper arm or the abdomen of a subject or patient using a spring-actuated insertion mechanism. (See: U.S. Patent Application Ser. No. 60/424,099 of Funderburk et al. filed Nov. 5, 2002; and U.S. Patent Application Publication No. 2004/0133164 A1 of Funderburk et al. filed Nov. 5, 2003). The sensor 300 may be connected via a cord (not shown) to a portable, potentiostat-data logger device (not shown), which may be used to maintain the glucose-sensing working electrode 302 at a potential of +40 mV versus the Ag/AgCl reference electrode 304, while obtaining and storing instantaneous current values at 10-second intervals.
FIGS. 5 and 6, together, illustrate a fully fabricated sensor, as the sensor would be seen placed on the skin, with a portion of the sensor transcutaneously inserted into the subcutaneous space. FIG. 5 provides a perspective view of a sensor 10a, the major portion of which is above the surface of the skin 50, with an insertion tip 11 penetrating through the skin and into the subcutaneous space 52, where it is bathed in biofluid 40. Contact portions of a working electrode 29aa, a reference electrode 29bb, and a counter electrode 29cc can be seen on the portion of the sensor 10a situated above the skin surface. Working electrode 29a, a reference electrode 29b, and a counter electrode 29c can be seen at the end of the insertion tip 11. FIG. 6 provides an expanded and cutaway view of sensor insertion tip 11. The working electrode 29a is shown resting on top of a plastic substrate 13, a wired enzyme sensing layer 18a rests on top of a portion of the working electrode 29a. Overlaying the sensing layer and a portion of the electrode, depicted transparently, is an interfacing membrane 30a, and associated with and dispersed throughout the membrane is a optionally a catalytic agent 32, the membrane covering the sensing layer 18a of the enzyme-based electrochemical sensor. The tip 11 is in the subcutaneous space 52 (as seen in FIG. 5) and is consequently bathed in the surrounding biofluid 40. The catalytic agent may be dispersed in the membrane by admixing into the membrane solution used in the synthesis of the membrane, a bulk loading procedure, as described in U.S. patent application Ser. No. 10/819,498 of Feldman et al., filed on Apr. 6, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/775,604 of Feldman et al., filed on Feb. 9, 2004. This procedure is a modification of a membrane synthesis procedure described earlier in the U.S. Patent Application Publication No. US 2003/0042137A1 of Mao et al., published on Mar. 6, 2003.
FIGS. 7A and 7B provide an illustration of a portion of an implantable sensor, with a protective membrane between the biofluid and the transducing apparatus, and optionally with a catalytic agent 32 associated with the membrane. FIG. 7A is a cutaway perspective view of a portion of an electrochemical sensor 10e; in particular a head portion which is attached to a body portion (not shown) that includes a housing that encloses other parts that contribute to the sensor function, such as a circuit board and microprocessor for processing electrochemical input into an informative signal, a battery for power, and an antenna for sending signal to an external device. Three electrodes, a working electrode 29a, a reference electrode 29b, and a counter electrode 29c are partially exposed in the figure, and can be seen each to be surrounded for part of their length within the ceramic head portion of the sensor 10e, with one end of each penetrating the surface of the head portion, and the other end of each extending into the interior of the body of the sensor. The most distal portion of the head portion of the sensor 10e, where the electrodes terminate is the sensing region 17, and is shown in an enlarged side view in FIG. 7B. There, the end portions of electrodes 29a, 29b, and 29c can be seen terminating in such a way as to be contiguous with the surface of the head of sensor 10e. Covering the sensing region 17 is a transducer, including a sensing membrane 18a that includes glucose oxidase, which recognizes glucose and initiates the first step in transduction of the glucose concentration into an informative signal. The external portion of the sensing region 17 is exposed to the surrounding biofluid 50. The sensing membrane 18a and its function is analogous to the sensing membrane 18 of the transcutaneous sensor shown in FIG. 6. Covering this sensing layer 18a is a second layer, an interfacing membrane 30, which may include a catalytic agent 32.
In fully implantable sensors, such as those described above, and as in the example illustrated in FIG. 7, it can be appreciated that the sensing surface represents but a small fraction of the total surface of the implanted sensor. A biological response to the sensor, such as foreign body response that would be mounted by the immune system, could thus be directed to the sensor as a whole, and such response to the entirety or a portion of the surface could contribute to diminishing biocompatibility or performance of the sensor through its sensing surface. Accordingly, biocompatibility-promoting agents could be disposed over or associated with portions of the sensor other than immediately over the sensing region.
Method for Making Multi-Layered Biosensors
Insulated non-corroding metal or carbon wires that have been etched as described above to contain a recess at the tip, are placed in a block that serves as an X-Y positioner. The wires vertically traverse the block and are held in place, e.g., by pressure. The blocks with the wires can be formed of elements, each element having multiple half-cylinder grooves running vertically. The wires are placed in these grooves and the elements are assembled into the block using screws. For example, the block may be formed of aluminum having equally spaced holes, (900 for a 30×30 array of wires), each hole to contain one wire. The block is positioned under a fixed micronozzle that ejects a fluid in to the recess of the insulated wire.
To reduce the requirement of precision in the positioning of the block and the micronozzle, the nozzle is electrically charged, with the wire having an opposite charge, or the wire being grounded or at least having a potential such that there is a potential difference between the nozzle and the wire. Because the nozzle is charged, the microdroplets it ejects are also charged with the same type of charge (positive or negative) as the nozzle. The higher the potential on the nozzle (e.g., versus ground potential), the higher the charge on the ejected microdroplets. If the tip of the wire to be coated is at ground potential or has a charge of the opposite type, the charged microdroplets are guided into the recess to deposit on the electrode, even if the jet of microdroplets is not vertical, i.e., even if the micronozzle is not precisely aligned above the wire's tip. This coating method is useful in making any small biosensor, not only those in recessed zones.
Clinical Use of Biosensors
The biosensors of the present invention have sufficient sensitivity and stability to be used as very small, subcutaneous biosensors for the measurement of clinically relevant compounds such as glucose and lactate. The electrodes accurately measure glucose in the range of about 2-30 μM and lactate in the range of about 0.5-10 mM. One function of the implanted biosensor is to sound an alarm when, for example, a patient's glucose concentration is too low or too high. When pairs of implanted electrodes are used, there are three situations in which an alarm is triggered: low glucose concentration, high glucose concentration; sensor malfunction as determined by a discrepancy between paired readings of the two sensors. A discrepancy sufficient to trigger the alarm may be, for example more than two or three times the standard deviation persisting for a defined period, e.g., not less than ten minutes. Such a system may be useful in sleeping patients, and also in emergency and intensive care hospital rooms, where vital functions are continuously monitored.
Another function of the biosensors described herein is to assist diabetics in maintaining their blood glucose levels near normal. Many diabetics now maintain higher than normal blood glucose levels because of danger of coma and death in severe hypoglycemia. However, maintaining blood glucose levels substantially, e.g., approximately 40% or more above normal leads to retinopathy and blindness as well as to kidney failure. Use of the subcutaneous biosensors to frequently, if not continuously, monitor glucose concentrations is desirable so that glucose concentrations can be maintained closer to an optimum level.
The subcutaneous biosensors can be used to measure the rate of rise and decline of glucose concentrations after a meal or the administration of glucose (e.g., a glucose tolerance test). The sensors are also useful in feedback loops for automatic or manually controlled maintenance of glucose concentrations within a defined range. For example, when used in conjunction with an insulin pump, a specified amount of insulin is delivered from the pump if the sensor glucose reading is above a set value.
In all of these applications, the ability to promptly confirm that the implanted sensor reading is accurate is essential. Prompt confirmation and rapid recalibration are possible only when one-point calibration is valid. Generally, even if a sensor's response is linear through the relevant concentration range, calibration requires at least two blood or fluid samples, withdrawn from the patient at times when the glucose concentration differs. It usually takes several hours for the glucose concentration to change sufficiently to validate proper functioning by two-point calibration. The ability to confirm and recalibrate using only one point is thus a highly desirable feature of the present invention.
It is preferred that the biosensors be implanted in subcutaneous tissue so as to make the sensor relatively unobtrusive, and at a site where they would not be easily dislodged, e.g., with turning or movement. It is also preferred, when readings are not corrected for temperature (which they generally are) that the sensors be implanted where they are likely to be at body temperature, e.g., near 37° C., and preferably covered by clothing. Convenient sites include the abdomen, inner thigh and arm.
Although we describe here continuous current measurement for assaying glucose, the electrical measurement by which the glucose concentration is monitored can be continuous or pulsed. It can be a current measurement, a potential measurement or a measurement of charge. It can be a steady state measurement, where a current or potential that does not substantially change during the measurement is monitored, or it can be a dynamic measurement, e.g., one in which the rate of current or potential change in a given time period is monitored. These measurements require at least one electrode in addition to the sensing electrode. This second electrode can be placed on the skin or can be implanted, e.g., subcutaneously. When a current is measured it is useful to have a potentiostat in the circuit connecting the implanted sensing electrode and the second electrode, that can be a reference electrode, such as an Ag/AgCl electrode. When a current is measured the reference electrode may serve also as the counter electrode. The counter electrode can also be a separate, third electrode, such as a platinum, carbon, palladium or gold electrode.
In addition to implanting the sensing electrode in the body, fluid from the body, particularly fluid from the subcutaneous region, can be routed to an external sensor. It is preferred in this case to implant in the subcutaneous region a microfiltration giver and pull fluid to an evacuated container, the fluid traversing a cell containing the sensing electrode. Preferably this cell also contains a second electrode, e.g., a reference electrode which may serve also as a counter electrode. Alternatively, the reference and counter electrodes may be separate electrodes. In coulometric measurements only two electrodes, the sensing electrode and the counter electrode are required. The flow of body fluid may be pulsed or continuous. Other than an implanted microfiltration fiber, also a microdialysis fiber may be used, preferably in conjunction with a pump.
The present invention is applicable to corded or cabled glucose-sensing systems, as described above, as well as other analyte-sensing or glucose-sensing systems. For example, it is contemplated that suitable results, along the lines of those described herein, may be obtained using a wireless glucose-sensing system that comprises a pager-sized, hand-held, informational display module, such as a FREESTYLE NAVIGATOR wireless glucose-sensing system. The FREESTYLE NAVIGATOR system employed herein is capable of providing real-time glucose information at 1-minute intervals and information regarding rates and trends associated with changes in glucose levels. This system is further capable of providing a visual indication of glucose level rates, allowing users to discriminate among glucose rate changes of less than 1 mg/dL per minute, 1-2 mg/dL per minute (moderate change), and greater than 2 mg/dL per minute (rapid change). It is contemplated that sensors having features such as these will be advantageous in bringing information of predictive or diagnostic utility to users. The FREESTYLE NAVIGATOR system is also designed to provide hypoglycemic and hyperglycemic alarms with user-settable thresholds.
Each of the various references, presentations, publications, provisional and/or non-provisional U.S. patent applications, U.S. patents, non-U.S. patent applications, and/or non-U.S. patents that have been identified herein, is incorporated herein in its entirety by this reference.
Although various aspects and features of the present invention may have been described largely with respect to applications, or more specifically, medical applications, involving diabetic humans, it will be understood that such aspects and features also relate to any of a variety of applications involving non-diabetic humans and any and all other animals. Further, although various aspects and features of the present invention may have been described largely with respect to applications involving partially implanted sensors, such as transcutaneous or subcutaneous sensors, it will be understood that such aspects and features also relate to any of a variety of sensors that are suitable for use in connection with the body of an animal or a human, such as those suitable for use as fully implanted in the body of an animal or a human. Finally, although the various aspects and features of the present invention have been described with respect to various embodiments and specific examples herein, all of which may be made or carried out conventionally, it will be understood that the invention is entitled to protection within the full scope of the appended claims.

## Claims

1. A redox polymer comprising
(a) a polymeric backbone;
(b) a cross-linker; and
(c) a transition metal complex having the following formula:




wherein
(i) M is osmium; and
(ii) L is selected from the group consisting of:





wherein:
R1, R2, and R′1 are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups;
R3, R4, R5, R6, R′3, R′4, Ra, Rb, Rc, and Rd are independently —H, —F, —Cl, —Br, —I, —NO2, —CN, —CO2H, —SO3H, —NHNH2, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl;
c is an integer selected from −1 to −5 or +1 to +5 indicating a positive or negative charge;
X represents at least one counter ion;
d is an integer from 1 to 5 representing the number of counter ions; and
L1, L2, L3 and L4 are ligands,
wherein L1 comprises a heterocyclic compound coupled to the polymeric backbone; and
wherein L1 and L2 in combination form a first bidentate ligand.

2. The redox polymer of claim 1, wherein M is osmium and the transition complex has the following formula:



wherein R3, R4, R5, R6, Ra, Rb, Rc, Rd, R′3 and R′4 are —H;
R1 and R2 are independently substituted or unsubstituted C1 to C12 alkyls; and
R′1 is independently —H or substituted or unsubstituted C1-C12 alkoxy, C1-12 alkylthio, C1-C12 alkylamino, C2-C24 dialkylamino, or C1-C12 alkyl.

3. The redox polymer of claim 2, wherein at least one of R1, R2, and R′1 comprises a reactive group selected from the group consisting of carboxy, activated ester, sulfonyl halide, sulfonate ester, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxyamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imido ester, maleimide, hydrazide, hydroxy, and photo-reactive azido aryl groups.

4. The redox polymer of claim 3, wherein at least one of R1, R2, and R′1 is coupled to a polymeric backbone.

5. The redox polymer of claim 1, wherein the cross-linker is PEGDGE.

6. The redox polymer of claim 1, having the following structure:

7. The redox polymer of claim 1, wherein the polymeric backbone is a poly(vinylpyridine) having the structure:



wherein m is 1 to 18, n and n′ are the average number of pyridinium and pyridine subunits and n′′ is the number of repeating polymer units.