WO2019180660A1

WO2019180660A1 - Composite membrane-coated electrodes

Info

Publication number: WO2019180660A1
Application number: PCT/IB2019/052312
Authority: WO
Inventors: Wesley Langdon STORM; Jonathan Edward MCDUNN; Micah Daniel BROWN; Mark Schoenfisch
Original assignee: Clinical Sensors, Inc.; The University Of North Carolina At Chapel Hill
Priority date: 2018-03-22
Filing date: 2019-03-21
Publication date: 2019-09-26

Abstract

An analyte-selective electrode is described herein, which includes an electrode having a surface, and a composite membrane on at least a part of the surface, the composite membrane including, e.g., a first component including a permselective, electropolymerized material and a second component including a sol-gel-derived material, wherein these materials can be in different configurations with respect to one another. The coated electrodes can be incorporated within sensors. The disclosure also provides methods of making such electrodes, methods of incorporating such electrodes within sensors, and methods of detecting one or more analytes of interest.

Description

COMPOSITE MEMBRANE-COATED ELECTRODES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number All 12064 awarded by The National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and devices for the detection and quantification of various analytes, e.g., nitric oxide (NO).

BACKGROUND OF THE INVENTION

Nitric oxide (NO) is an endogenous, physiologically active metabolite. Within the human body, nitric oxide is produced, e.g., by oxidation of the guanidine group of L-arginine, facilitated by various nitric oxide synthase (NOS) enzymes. Human NOS enzymes include endothelial (eNOS or NOS-1) enzymes, inducible (iNOS or NOS-2) enzymes, and neuronal (nNOS or NOS-3) enzymes. Based on differential tissue expression and regulation of these enzymes, the NO produced within the body regulates diverse physiologic processes, including, but not limited to, vascular tone, immunologic response to inflammatory and infectious stimuli, and neurophysiology. Wound healing, vasodilation, angiogenesis, platelet aggregation, long-term memory potentiation, and inflammation all depend on NO in its role as an intercellular gasotransmitter to initiate and mediate these processes.

NO can react with other compounds to generate diverse storage forms, including, but not limited to, inorganic compounds such as nitrite, nitrate, and nitrosopersulfide (SSNO); small molecule organic compounds such as S-nitrosocysteine, S-nitrosoglutathione, nitrosamines; S-nitrosopenacillamine, and post- translationally modified proteins such as proteins with S-nitroso, N-nitroso, nitrotyrosine, and metal- centered nitrosyl modifications. These storage forms have longer half-lives than free NO, readily traffic through the body, and release NO through both spontaneous and catalytic processes, therefore impacting systemic and local levels of NO. With these properties, NO and/or its storage forms are potential biomarkers for cardiovascular disease, neovascularization (for example, tumor-induced angiogenesis), infection and inflammation (including autoimmune disease), and neurologic disease. Recent studies have shown that some pathogenic microbes exploit NO physiology and, therefore, NO may also have biomarker properties in gastrointestinal and digestive diseases. The quantification of NO and NO storage forms is thus of considerable importance in helping to anticipate and understand a range of diseases.

The high reactivity and lipopermeability of NO present challenges to in situ detection and quantification. The lifetime of NO in biological milieu is usually limited to a few seconds because it can be easily scavenged by thiols, oxygen, free metal ions, and

heme-containing proteins. Indirect detection methods alternatively measure NO’s stable oxidative byproducts, nitrite and nitrate (or collectively, NO_x). Although the effects of scavenging are largely circumvented, indirect methods fail to capture NO dynamisms in real-time and often require extensive sample preparation (e.g., the Griess assay). Direct and highly sensitive detection can be achieved under chemiluminescence and electron paramagnetic resonance, but these techniques demand complex instrumentation and are not well suited to real-time analysis in complex media. Currently, electrochemical techniques offer the highest spatial and temporal resolution for in situ, real-time monitoring of NO in biological media.

However, accurate electrochemical detection and quantification of nitric oxide (NO) from biological media is commonly frustrated, due to the presence of electroactive interferent species and proteins. The former contribute erroneously to the current response, demanding that selective barriers be used to modify the electrode surface. Likewise, protein biofouling on the sensor surface may degrade analytical performance and must be mitigated as much as possible. It would be useful to provide materials and methods for efficient analyte (e.g., NO) detection and measurement in the presence of electroactive interferent species and proteins.

SUMMARY OF THE INVENTION

The present disclosure provides composite membranes, electrodes comprising such composite membranes, and sensors incorporating at least one electrode comprising a composite membrane. The disclosure further provides methods of making composite membrane-coated electrodes and sensors incorporating such electrodes and to methods of using the electrodes and sensors to detect and/or quantify certain analytes in a given sample.

One aspect of the invention is directed to an analyte-selective electrode, comprising: an electrode having a surface, and a composite membrane on at least a part of the surface, the composite membrane comprising: a first component comprising a permselective, electropolymerized material; and a second component comprising a sol-gel-derived material. These materials can be in various configurations with respect to one another. For example, in some embodiments, the first component is directly associated with at least a portion of the electrode surface and the second component is directly associated with at least a portion of the first component. In some embodiments, the second component is directly associated with at least a portion of the electrode surface and the first component is directly associated with at least a portion of the second component. In some embodiments, the composite membrane comprises a mixture of the first and second components.

The analyte for which the disclosed electrode is sensitive can vary and, in various embodiments, can be selected from the group consisting of nitric oxide (NO), nitrite, ascorbic acid, acetaminophen, uric acid, cysteine, xanthine, hypoxathine, vanillylmandelic acid, glutathione (reduced and/or oxidized), xanthosine, methoxy-hydroxyphenylglycol, homogentisic acid, 7-methylxanthine, methionine, norepinephrine, guanosine, L-dopa, guanine, 3-hydroxykynurenine, epinephrine, tyrosine, dihydroxyphenylacetic acid, 4- hydroxyphenyllactic acid, 3 -hydroxy anthranilic acid, 1,7-dimethylxanthine, 5 -hydroxy tryptophan, 1,3- dimethylxanthine, 4-hydroxybenzoic acid, 3-o-methyldopa, 5 -hydroxy indole acetic acid, kynureinine, normetanephrine, dopamine, metanephrin, acetylserotonin, homovanillic acid, 4-hydroxyphenylacetic acid, try amine, 2-hydroxyphenylacetic acid, 5 -serotonin, 3-methoxytyramine, methylserotonin, tryptophan, melatonin, tryptophol, indole-3 -acetic acid, indole-3 -propionic acid, (+)-a-tocopherol, (+)-6-tocopherol, and (+)-y-tocopherol. In certain specific embodiments, the electrode is selective for NO. In other specific embodiments, the electrode is selective for nitrite or ascorbate.

The composition of the permselective, electropolymerized material of the disclosed composite membrane can vary. In some embodiments, the permselective, electropolymerized material comprises a polymer with monomeric units selected from the group consisting of: optionally functionalized alcohols; optionally functionalized amines; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof. In certain embodiments, the monomeric units are selected from the group consisting of: phenol; eugenol; naphthol; phenylenediamine; aniline; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof. In more specific embodiments, the monomeric units are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4- (trifluoromethyl)phenol, 3,5-bis(trifluoromethyl)phenol, and combinations thereof. As one specific example, the permselective, electropolymerized material can comprise poly-5-amino-l-naphthol (5A1N). In some embodiments, the permselective, electropolymerized material is in the form of a film with an average thickness of about 3 nm to about 10 pm, e.g., an average thickness of about 3 nm to about 15 nm.

The composition of the sol-gel-derived material of the disclosed composite membrane can also vary. In some embodiments, the sol-gel-derived material comprises a xerogel. Exemplary xerogels include, but are not limited to, fluorinated alkoxysilane xerogels.

In some embodiments, the electrode comprises platinum. In some embodiments, the electrode is a radial disk electrode or planar electrode.

Various additional components can be associated with the disclosed analyte-selective electrodes.

For example, in some embodiments, the analyte-selective electrode further comprises a third component comprising a material selected from the group consisting of poly(tetrafluoroethylene), Nafion, cellulose acetate, polyurethanes, and combinations and copolymers thereof, which can be associated with one or both of the first component and the second component.

The disclosure also provides electrochemical analyte sensing devices, comprising: a substrate; an analyte-selective electrode as disclosed herein; and an analyte detecting element. The disclosure further provides a process for preparing an analyte-selective electrode as disclosed herein, comprising: providing the electrode; associating the composite membrane with at least a portion of the surface, wherein the associating comprises: electropolymerizing one or more monomers to give the first component; and applying the second component.

One aspect of the invention is directed to a process for preparing an analyte-selective electrode, comprising: providing an electrode having a surface; associating a composite membrane with at least a portion of the surface, wherein the associating comprises: electropolymerizing one or more monomers to give a first component comprising a permselective, electropolymerized material; and applying a second component comprising a sol-gel-derived material. The order of the electropolymerizing and applying steps is not limited. As such, in some embodiments, the electropolymerizing step is conducted before the applying step. In other embodiments, the electropolymerizing step is conducted after the applying step. In certain embodiments, the electropolymerizing comprises electrooxidation or radical coupling. The electropolymerizing can comprise, for example, a potential cycling or potentio static process. In some embodiments, the electropolymerizing comprises electropolymerizing the one or more monomers from an electrolyte-containing solution of the one or more monomers. The electrolyte-containing solution and the monomer(s) employed in this process can vary. In some embodiments, the electrolyte-containing solution comprises phosphate buffered saline (PBS). The one or more monomers can, in certain embodiments, have a concentration in the electrolyte-containing solution of about 10 to about 100 mM.

The one or more monomers can be, for example, selected from the group consisting of optionally functionalized alcohols, optionally functionalized amines, and combinations thereof. In certain embodiments, the one or more monomers are selected from the group consisting of: phenol; eugenol;

naphthol; phenylenediamine; aniline; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof. In specific embodiments, the one or more monomers are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-(trifluoromethyl)phenol, 3,5- bis(trifluoromethyl)phenol, and combinations thereof. In one particular embodiment, the one or more monomers comprise 5-amino-l -naphthol (5A1N).

In some embodiments, the applying comprises a sol gel process. For example, in certain embodiments, the applying comprises spray coating a sol onto the first component and drying the sol.

The present disclosure includes, without limitation, the following embodiments:

Embodiment 1 : An analyte-selective electrode, comprising: an electrode having a surface, and a composite membrane on at least a part of the surface, the composite membrane comprising: a first component comprising a permselective, electropolymerized material; and a second component comprising a sol-gel- derived material.

Embodiment 2: The analyte-selective electrode of Embodiment 1, wherein the first component is directly associated with the electrode surface and the second component is directly associated with the first component.

Embodiment 3: The analyte-selective electrode of Embodiment 1, wherein the second component is directly associated with the electrode surface and the first component is directly associated with the second component.

Embodiment 4: The analyte-selective electrode of Embodiment 1, wherein the composite membrane comprises a mixture of the first and second components.

Embodiment 5: The analyte-selective electrode of any preceding embodiment, wherein the electrode is selective for an analyte selected from the group consisting of nitric oxide (NO), nitrite, ascorbic acid, acetaminophen, uric acid, cysteine, xanthine, hypoxathine, vanillylmandelic acid, glutathione (reduced and/or oxidized), xanthosine, methoxy-hydroxyphenylglycol, homogentisic acid, 7-methylxanthine, methionine, norepinephrine, guanosine, L-dopa, guanine, 3-hydroxykynurenine, epinephrine, tyrosine, dihydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 3 -hydroxy anthranilic acid, 1,7-dimethylxanthine,

5 -hydroxy tryptophan, 1,3-dimethylxanthine, 4-hydroxybenzoic acid, 3-o-methyldopa, 5 -hydroxy indole acetic acid, kynureinine, normetanephrine, dopamine, metanephrin, acetylserotonin, homovanillic acid, 4- hydroxyphenylacetic acid, tryamine, 2-hydro xyphenylacetic acid, 5-serotonin, 3-methoxytyramine, methylserotonin, tryptophan, melatonin, tryptophol, indole-3 -acetic acid, indole-3 -propionic acid, (+)-<x- tocopherol, (+)-6-tocopherol, and (+)-y-tocopherol.

Embodiment 6: The analyte-selective electrode of any preceding embodiment, wherein the electrode is selective for NO.

Embodiment 7: The analyte-selective electrode of any preceding embodiment, wherein the electrode is selective for nitrite or ascorbate.

Embodiment 8: The analyte-selective electrode of any preceding embodiment, wherein the permselective, electropolymerized material comprises a polymer with monomeric units selected from the group consisting of: optionally functionalized alcohols; optionally functionalized amines; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof.

Embodiment 9: The analyte-selective electrode of the preceding embodiment, wherein the monomeric units are selected from the group consisting of: phenol; eugenol; naphthol; phenylenediamine; aniline;

heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof.

Embodiment 10: The analyte-selective electrode of Embodiment 8, wherein the monomeric units are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4- (trifluoromethyl)phenol, 3,5-bis(trifluoromethyl)phenol, and combinations thereof.

Embodiment 11: The analyte-selective electrode of any of Embodiments 1-7, wherein the permselective, electropolymerized material comprises poly-5-amino-l-naphthol (5A1N).

Embodiment 12: The analyte-selective electrode of any preceding embodiment, wherein the permselective, electropolymerized material is in the form of a film with an average thickness of about 3 nm to about 10 pm. Embodiment 13: The analyte-selective electrode of any preceding embodiment, wherein the permselective, electropolymerized material is in the form of a film with an average thickness of about 3 nm to about 15 nm. Embodiment 14: The analyte-selective electrode of any preceding embodiment, wherein the second component comprises a xerogel.

Embodiment 15: The analyte-selective electrode of the preceding embodiment, wherein the xerogel is a fluorinated alkoxysilane xerogel.

Embodiment 16: The analyte-selective electrode of any preceding embodiment, wherein the electrode comprises platinum.

Embodiment 17: The analyte-selective electrode of any preceding embodiment, wherein the electrode is a radial disk electrode or planar electrode.

Embodiment 18: The analyte-selective electrode of any preceding embodiment, further comprising a third component comprising a material selected from the group consisting of poly(tetrafluoroethylene), Nafion, cellulose acetate, polyurethanes, and combinations and copolymers thereof associated with one or both of the first component and the second component.

Embodiment 19: An electrochemical analyte sensing device, comprising: a substrate; the analyte-selective electrode of any preceding embodiment; and an analyte detecting element. Embodiment 20: A process for preparing the analyte-selective electrode of any preceding embodiment, comprising: providing the electrode; associating the composite membrane with at least a portion of the surface, wherein the associating comprises: electropolymerizing one or more monomers to give the first component; and applying the second component.

Embodiment 21 : A process for preparing an analyte-selective electrode, comprising: providing an electrode having a surface; associating a composite membrane with at least a portion of the surface, wherein the associating comprises: electropolymerizing one or more monomers to give a first component comprising a permselective, electropolymerized material; and applying a second component comprising a sol-gel-derived material.

Embodiment 22: The process of any preceding embodiment, wherein the electropolymerizing step is conducted before the applying step.

Embodiment 23: The process of Embodiment 20 or 21, wherein the electropolymerizing step is conducted after the applying step.

Embodiment 24: The process of any preceding embodiment, wherein the electropolymerizing comprises electrooxidation or radical coupling.

Embodiment 25: The process of any of Embodiments 20-23, wherein the electropolymerizing comprises a potential cycling or potentio static process.

Embodiment 26: The process of any preceding embodiment, wherein the electropolymerizing comprises electropolymerizing the one or more monomers from an electrolyte-containing solution of the one or more monomers.

Embodiment 27: The process of the preceding embodiment, wherein the electrolyte-containing solution comprises phosphate buffered saline (PBS).

Embodiment 28: The process of Embodiment 26 or 27, wherein the one or more monomers have a concentration in the electrolyte-containing solution of about 10 to about 100 mM.

Embodiment 29: The process of any preceding embodiment, wherein the one or more monomers are selected from the group consisting of optionally functionalized alcohols, optionally functionalized amines, and combinations thereof.

Embodiment 30: The process of any preceding embodiment, wherein the one or more monomers are selected from the group consisting of: phenol; eugenol; naphthol; phenylenediamine; aniline;

Embodiment 31 : The process of any preceding embodiment, wherein the one or more monomers are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-

(trifluoromethyl)phenol, 3,5-bis(trifluoromethyl)phenol, and combinations thereof

Embodiment 32: The process of any preceding embodiment, wherein the one or more monomers comprise

5-aminoindole.

Embodiment 33: The process of any preceding embodiment, wherein the applying comprises a sol gel process. Embodiment 34: The process of any preceding embodiment, wherein the applying comprises spray coating a sol onto the first component and drying the sol.

These and other features, aspects, and advantages of the disclosure will be apparent from a reading of the following detailed description together with the accompanying drawings, which are briefly described below. The invention includes any combination of two, three, four, or more of the above-noted embodiments as well as combinations of any two, three, four, or more features or elements set forth in this disclosure, regardless of whether such features or elements are expressly combined in a specific embodiment description herein. This disclosure is intended to be read holistically such that any separable features or elements of the disclosed invention, in any of its various aspects and embodiments, should be viewed as intended to be combinable unless the context clearly dictates otherwise. Other aspects and advantages of the present invention will become apparent from the following.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to provide an understanding of embodiments of the invention, reference is made to the appended drawings, which are not necessarily drawn to scale, and in which reference numerals refer to components of exemplary embodiments of the invention. The drawings are exemplary only, and should not be construed as limiting the invention.

FIG. 1 A is a schematic illustration of a longitudinal cross-sectional view of an electrode comprising a composite membrane-coated sensing tip;

FIG. IB is a schematic illustration of a top view of the sensing end of the electrode shown in FIG.

1 A, shown in the absence of any electrode coating;

FIG. 2 is a graph of selectivity coefficients of bare electrodes and electrodes coated with the composite membranes disclosed herein for NO against various common interferents;

FIG. 3 is a graph of NO sensitivity retention for bare electrodes and electrodes coated with the composite membranes disclosed herein;

FIGs. 4A and 4B are plots of NO-release profiles of stimulated (A) and unstimulated (B) microphages, measured using a sensor comprising a composite membrane -coated electrode as disclosed herein;

FIG. 5 is a representation of macrophage-to-sensor separation distance dependence of an amount of NO measured electrochemically upon stimulation;

FIG. 6 is a plot of NO sensitivity of a sensor comprising a composite membrane-coated electrode as disclosed herein after exposure to human blood;

FIG. 7 is a plot of relative standard deviation of a blank sample analyzed by a sensor comprising a composite membrane-coated electrode as disclosed herein between injections of human blood;

FIG. 8 is a plot comparing plasma response to bare electrode and electrode coated with the composite membrane disclosed herein; and

FIG. 9 is a plot comparing NO sensitivity of bare electrode and electrode coated with the composite membrane disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. As used in this specification and the claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Reference to“dry weight percent” or“dry weight basis” refers to weight on the basis of dry ingredients (/.<?.. all ingredients except water).

The present disclosure provides an analyte-selective electrode for electrochemical

detection/measurement, comprising an electrode with a composite membrane on at least a portion of the electrode surface, referred to generally herein as a“composite membrane-coated electrode.” The composite membrane can incorporate various membrane components (e.g., two or more membrane components) with different functions, rendering the disclosed analyte-selective electrodes suitable for electrochemical analysis of a range of analytes in a range of different environments. Advantageously, the principles disclosed herein are tunable (with at least two separate components associated with the composite membrane) such that, based on the present disclosure, analyte-selective electrodes for various applications can be appreciated. For example, based on the principles disclosed herein, composite membrane-coated electrodes can be developed to offer selectivity to different analytes and can be developed to provide such selectivity in the context of various samples (understood to commonly include components that typically interfere with such selectivities in traditional measurement techniques).

“Analyte” or“molecular species” as used herein is a metabolite with a molecular weight of less than 200 Da. Analytes that can be sensed and/or quantified using the electrodes and sensors described herein include, but are not limited to, nitric oxide (NO), nitrite, ascorbic acid, acetaminophen, uric acid, cysteine, xanthine, hypoxathine, vanillylmandelic acid, glutathione (reduced and/or oxidized), xanthosine, methoxy- hydroxyphenylglycol, homogentisic acid, 7-methylxanthine, methionine, norepinephrine, guanosine, L- dopa, guanine, 3 -hydroxy kynurenine, epinephrine, tyrosine, dihydroxyphenylacetic acid, 4- hydroxyphenyllactic acid, 3 -hydroxy anthranilic acid, 1,7-dimethylxanthine, 5 -hydroxy tryptophan, 1,3- dimethylxanthine, 4-hydroxybenzoic acid, 3-o-methyldopa, 5 -hydroxy indole acetic acid, kynureinine, normetanephrine, dopamine, metanephrin, acetylserotonin, homovanillic acid, 4-hydroxyphenylacetic acid, try amine, 2-hydroxyphenylacetic acid, 5 -serotonin, 3-methoxytyramine, methylserotonin, tryptophan, melatonin, tryptophol, indole-3 -acetic acid, indole-3 -propionic acid, (+)-a-tocopherol, (+)-6-tocopherol, and/or (+)-y-tocopherol.

The disclosed electrodes can be used in various electrochemical techniques. Potentiodynamic techniques (e.g., cyclic voltammetry, differential pulse voltammetry) offer higher selectivity, but typically at the expense of temporal resolution and limit of detection (LOD). Far more common are potentiostatic techniques (e.g., amperometry, chronocoulometry) for their simplicity and high temporal resolution.

In addition to the exact electrochemical technique/process in which the disclosed composite membrane-coated electrodes are employed, the electrode size, geometry, and material can affect sensor function and must be chosen with the particular system under observation (e.g., the analyte to be detected and/or the likely contaminants) in mind. The type of electrode to which the disclosed composite membranes can be applied are not particularly limited and these membranes may be employed in the context of electrodes of all sizes and geometries. The electrodes can be, e.g., micro-scale electrodes, ultramicro-scale electrodes, or macro-scale electrodes. In some embodiments, the electrodes are macrodisc electrodes, macrocylinder electrodes, macroring electrodes, microdisc electrodes, microcylinder electrodes, and microring electrodes. Suitable electrode materials include any electrically conductive metals and other materials such as, but not limited to, platinum, palladium, rhodium, ruthenium, osmium, iridium, tungsten, nickel, copper, gold, silver, and carbon and carbon fibers, as well as, oxides, dioxides, combinations, or alloys thereof. In some embodiments, the electrically conductive material is selected from carbon (including glassy carbon), carbon fibers, platinum (including platinum black), tungsten, silver, silver/silver chloride, gold, copper, indium tin oxide, iridium oxide, nickel and combinations thereof.

The composite membrane disclosed herein to be associated with at least a portion of the electrode generally includes at least two components. As will be described in further detail, the two components of the composite membrane include an electropolymerized material (referred to herein as a“first component”) and a sol-gel-derived material (referred to herein as a“second component”). The reference to“first” and “second” components is not necessarily indicative of the configuration of the two components or the order in which the two components are applied to the electrode, as will be clear based on a reading of the application in its entirety. This combination of the first and second component to give a composite membrane provides a unique synergy in the context of a coated electrode, with respect to the detection and quantification of various analytes with enhanced selectivity and sensitivity. In some embodiments, the composite membrane provides for selective detection maintained over long-term use in complex (e.g., proteinaceous) media without deterioration.

The composite membrane is generally provided such that it is directly associated with at least a portion of the surface of the electrode. By“directly associated with” is meant that at least a portion of the composite membrane is in direct contact with at least a portion of the electrode or is in a working relationship with at least a portion of the electrode, but spaced apart therefrom. In some embodiments, a hydrogel or other material may separate the composite membrane from the surface of the electrode.

The two components of the composite membrane can be configured in various manners with respect to one another. In some embodiments, the two components are in a substantially layered configuration. The first component, in certain embodiments, is directly associated with the electrode surface, and the second component is directly associated with at least a portion of the first component (i.e., as a top“layer”). In other embodiments, the second component is directly associated with the electrode surface, and the first component is directly associated with at least a portion of the second component (i.e., as a top“layer”). Although, in such embodiments, these components may be largely described as being in independent layers (or as comprising discrete“membranes”), such a strictly layered structure is not required according to the present disclosure. In some embodiments, the two components in these substantially layered configurations may not exist as discrete layers, and there may be, in some embodiments, no strictly defined boundary between the first component“layer” and the second component“layer.” In other embodiments, the two components of the composite membrane are mixed. The mixing can be homogeneous or non-homogeneous.

As referenced, the first component of the composite membrane is an electropolymerized material. Electropolymerized materials, in some embodiments, rely on size-exclusion and hydrophobicity to allow differential permeation of certain analytes while blocking bulkier species. Electropolymerized materials also are advantageous for their facile and highly reproducible depositions on electroactive surfaces.

Such electropolymerized materials are commonly and advantageously in the form of a selectively permeable membrane. Selectively permeable materials, e.g., membranes, are also referred to as “permselective.“ Thus, a permselective or selectively permeable membrane allows some molecules to pass through the membrane, while other molecules cannot pass through the membrane. In some embodiments, the term“permselective” as used herein refers to a material/membrane that selectively allows small, nonpolar gaseous molecules to pass through, while being impermeable to larger or more polar molecules. In some embodiments, the permselectivity is enhanced by closer and more orderly packing of oligomer chains in the first component and/or by the hydrophobicity of such oligomers.

In some embodiments, this first permselective or selectively permeable material or membrane is selectively permeable to nitric oxide and oxygen, while not being permeable to compounds such as nitrite (N0₂ ^~), ascorbic acid, uric acid, acetaminophen, dopamine, and aqueous liquids (where detection of nitric oxide and/or oxygen is desirable). In other embodiments, the first permselective or selectively permeable material or membrane is selectively permeable to other analytes, while not being permeable to compounds likely to present in a sample containing such analytes. In one particular embodiment, the first component is selected so as to afford a good NO/nitrite selectivity coefficient or a good N0/H₂0₂ selectivity. In some embodiment, the first component is selected so as to afford a good selectivity coefficient for NO over uric acid, 5-hydroxytryptamine, ascorbic acid, dopamine, serotonin, glucose, 1-arginine, N0₂ , NH₃, C0₂, H₂0₂, and/or CO.

The properties of the first component can vary and can be specifically designed for a given application. For example, the first component can be conducting (“non-passivating”) or non-conducting (“ self-passivating”) .

The monomer(s) electropolymerized to form the first component can vary. In some embodiments, the first component is prepared from monomers such as optionally functionalized alcohols, optionally functionalized amines, and combinations thereof. In certain embodiments, the first component is prepared from an electroactive aromatic small molecule capable of polymerization under oxidizing conditions.

Exemplary monomers include, but are not limited to: phenol; eugenol, naphthol, phenylenediamines (o- phenylenediamine, m-phenylenediamine, /? -p h c n\ 1 c nc d i a m i nc ) . aniline, 2,3-diaminonaphthalene (2,3-DAN), heteroaromatic, deuterated, and fluorinated analogues thereof, and combinations of two or more of the foregoing. Certain fluorinated analogues that are employed as monomers in certain embodiments include, but are not limited to, 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-(trifluoromethyl)phenol, 3,5- bis(trifluoromethyl)phenol, and combinations thereof. In one particular embodiment, the second component is an electropolymerized 5-amino-l-naphthol (5A1N) film (comprising poly-5-amino-l-naphthol, p-5AlN) and in another particular embodiment, the first component is an electropolymerized 5-aminoindole (comprising poly-5 -amino -indole).

Where the first component is a substantially discrete layer, this component typically has a relatively low thickness (which can be tuned, e.g., by the particular application technique employed). In some embodiments, the average thickness of the first component is about 2 to about 50 nm, e.g., about 2 to about 20 nm, or about 3 to 15 nm. In other embodiments, e.g., where the deposition/formation of the first component results in 3D polymer growth (extending orthogonal to the electrode surface), larger thicknesses may be obtained. In some embodiments, the average thickness of the first component is about 2 nm to about 20 microns, e.g., about 3 nm to about 10 microns. Such thicknesses can be obtained, e.g., based on electrochemical quartz crystal microbalance measurements or from cross-sectional scanning electron microscopy (SEM). In some embodiments, thicknesses are estimated from the total charge passed during electropolymerization.

The second component, i.e., the second selectively permeable component (e.g., membrane), as referenced above, generally comprises a sol-gel-derived material/membrane. In some embodiments, the second component comprises a xerogel, e.g., in the form of a xerogel-based membrane. In some embodiments, the second component is selectively permeable to nitric oxide and oxygen, while not being permeable to compounds such as nitrite (N0₂ ^~), ascorbic acid, uric acid, acetaminophen, dopamine, and aqueous liquids. In other embodiments, the second component is selectively permeable to other analytes, while not being permeable to compounds likely to present in a sample containing such analytes. In certain embodiments, the second component can further improve selectivity of the composite membrane and/or serves to mitigate protein adsorption on the composite membrane surface. In particular embodiments, the second component is specifically designed to provide better selectivity against certain interferents, e.g., those interferents that are not effectively blocked by the first component.

A xerogel is generally a polymeric networked, formed via a sol-gel process. In particular, the term “xerogel” can be used to refer to polysiloxane networks formed from the co-condensation of solutions containing silane mixtures. Generally, xerogels are formed upon the acid-catalyzed hydrolysis and co condensation of alkoxysilane precursors, followed by an extensive drying period, providing a material that is commonly rather rigid.

The terms“silane” (and“silyl”) refer to chemical groups and compounds comprising silicon atoms (Si). The term“polysiloxane” refers to a polymeric material comprising a backbone of silicon-oxygen bonds (i.e.,— Si— O— Si— O— Si— ) having the formula R_nSiX_yO_m, wherein each R is an H, alkyl, aryl, aralkyl, or substituted alkyl group and each X is an alkoxy, aryloxy, aralkoxy, hydroxyl or halo group. In some embodiments, each silicon atom is covalently bonded to one R group, for example one alkyl or fluorinated alkyl group. Each silicon atom is also crosslinked to one, two, or three other silicon atoms via silicon-oxygen bonds and bonded to zero, one, or two X groups, such as ethoxy, methoxy, hydroxyl, or chloro. Thus, in some embodiments, the higher the level of crosslinking in the polysiloxane, the fewer X groups are present. The terms“polysiloxane” and“silicone” can be used interchangeably. Exemplary xerogels suitable for use in the disclosed composite membranes are disclosed in Hunter et al, Anal. Chem. 2013, 85, 6066-6072 and U.S. Patent No. 8,551,322 to Schoenfisch et al., which are incorporated herein by reference in their entireties. The xerogel can generally comprise a polysiloxane network comprising both alkyl and fluorinated alkyl groups. The term“alkyl” refers to C1-C20 inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.“Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary branched alkyl groups include, but are not limited to, isopropyl, isobutyl, tert-butyl, “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a Ci_₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.“Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments,“alkyl” refers, in particular, to Ci_₈ straight-chain alkyls. In other embodiments,“alkyl” refers, in particular, to Ci_₈branched- chain alkyls.

Alkyl groups can optionally be substituted (a“substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term“alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as“alkylaminoalkyl”), or aryl. Thus, as used herein, the term“substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. In some embodiments, the substituted alkyl group is a fluorinated alkyl group. The term“fluorinated alkyl” refers to an alkyl group (i.e., C1-C20 linear, branched, or cyclic alkyl) wherein one or more of the C— H bonds are replaced by C— F bonds. In some embodiments, the entire length or a portion of the entire length (i.e., several consecutive carbon atoms) of the alkyl group is perfluorinated (i.e., each of the C— H bonds is replaced by a C— F bond). Fluorinated xerogels, in particular, have been previously demonstrated to have NO permselective characteristics based on size-exclusion and hydrophobicity and thus, in some embodiments, may be particularly useful for use in composite coatings to give NO-selective electrodes.

One or more further components, e.g., membranes, can optionally be included in association with the electrode in addition to the composite membranes disclosed herein. For example, other membranes including catalysts, redox-mediators, or high electroactive surface area particles can be incorporated to enhance sensitivity toward certain analytes of interest. Protective and biofouling-resistant membranes can be incorporated to help prevent performance degradation with extended use and/or placement in proteinaceous media. Exemplary additional membranes include, but are not limited to, poly(tetrafluoroethylene) (PTFE) membranes, Nafion membranes, cellulose acetate membranes, and/or polyurethane membranes. The particular application will dictate which type of membrane(s) are most suitable to ensure accurate NO measurement.

One or more of the composite membrane-coated electrodes disclosed herein is generally included within a sensing device or“sensor.” A sensor generally includes, in addition to the composite membrane- coated electrode(s) and, optionally, one or more additional electrodes (comprising an“electrode assembly”), a detector for measuring current at an electrode. The electrode assembly can comprise one, two, three, or more electrodes. In some embodiments, the electrode assembly comprises one electrode (i.e., a working electrode). In some embodiments, the sensor includes a two- or a three-electrode configuration. Thus, in some embodiments, the electrode assembly comprises a working electrode and a reference electrode. In some embodiments, the electrode assembly comprises a working electrode, a counter electrode, and a reference electrode.

The electrode assembly can further include one or more insulating materials or components to physically contain at least a portion of the electrode or electrodes, or to insulate electrodes from one another. In some embodiments, the electrode assembly can comprise a coating to protect the electrode or electrodes from the environment and/or to enhance the biocompatibility of the electrode assembly. For example, the electrode assembly can comprise a biocompatible polymeric coating covering those portions of the assembly not covered by the composite membrane disclosed herein, so long as such coating does not interfere with the ability of the sensor to detect the gaseous species.

FIG. 1 A shows a schematic illustration of a longitudinal cross view of a representative Clark-type sensor 100, which comprises a composite coating as disclosed herein. The shaft of sensor 100 comprises electrode assembly 102, which includes both working electrode 104 and reference electrode 106. The electrodes can be of any suitable electrode material and can have any suitable dimensions to correspond to the desired dimensions of the electrode assembly and/or sensor as a whole. In some embodiments, the electrodes can have outer diameters ranging from between a few mm and a few tenths of a micrometer. Working electrode 104 can comprise, for example, platinized Pt having an outer diameter of 127 pm.

Reference electrode 106 can comprise Ag/AgCl, having an outer diameter of 250 pm. Thus, electrode assembly 102 can have an outer diameter of, for example, 1.5 mm. Electrodes 104 and 106 are surrounded by insulating material 108 (e.g., borosilicate glass), which insulates electrodes 104 and 106 from one another. End 110 of electrode assembly 102 is covered by the disclosed composite membrane, comprising the first component (e.g., electropolymerized membrane) 112 and the second component (e.g., sol-gel- derived membrane) 116. Thus, to reach electrodes 104 and 106, any species from a sample that comes into contact with sensor 100 must first diffuse through the composite membrane, i.e., through components 116 and 112. It is noted that these two components are shown in FIG. 1A in a layered configuration; however, as noted above, these components may not be present as discrete layers as depicted. Further, as described above, components 116 and 112 may, in some embodiments, be mixed such that the composite membrane does not comprise two separate components as depicted. The other end of electrode assembly 102 (not shown) can be attached to the detector. Sandwiched between electrodes 104 and 106 and the composite coating is hydrogel 114, which is optional.

FIG. IB is a schematic illustration showing the view looking down on electrode assembly 102 at the surface of electrode assembly end 110. End 104' of working electrode 104 and end 106' of working electrode 106 are not covered by insulating material 108 at the surface of end 110.

The disclosed composite membranes are typically prepared in situ, i.e., by direct deposition or formation on at least a portion of the electrode surface. The first and second components referenced herein above (and any additional components, e.g., further membranes) are typically applied to the electrode surface sequentially. In certain embodiments, the first component is applied first and the second component is applied second. In other embodiments, the second component is applied first and the first component is applied second. It is noted that the order of application is not necessarily indicative of the final composite membrane configuration. For example, in some embodiments, the second component is applied before the first component is applied, but in the composite membrane-coated electrode, the first component is directly associated with the electrode. The following describes the preparation of a composite membrane wherein the first component is applied first; however, it is to be understood that the method is not limited thereto and that the order of application of the two components to the electrode can be the inverse of that described below. In some embodiments, application of the first and second components with the first component applied first and the second component applied second leads to a material that functions substantially the same as a material wherein the second component is applied first and the first component is applied second.

In certain embodiments, to prepare a first component on the electrode, the electrode is brought into contact with a solution of the desired monomer. The concentration of monomer can vary, e.g., on the specific monomer(s) chosen and the properties thereof. Exemplary amounts of monomer in solution include, but are not limited to, about 1 mM to about 100 mM, e.g., about 5 mM to about 50 mM. The solution selected can vary and can generally be any solution sufficient to at least mostly dissolve the specific monomer(s) chosen. In some embodiments, the solution is aqueous. One exemplary solution for the solution of monomer is phosphate buffered saline (PBS). The pH of the solution selected can also vary, but is typically not strongly acidic or basic and is, e.g., within the range of about 5 to about 10, e.g., about 6 to about 8.

After the electrode is brought into contact with the solution, deposition of the monomer from solution is effected. Electropolymerization to form the first component can be conducted by various techniques. Exemplary such techniques include, but are not limited to the steps of electrooxidation

(monomeric radical formation), radical coupling (oligomer formation) and precipitation on a surface (film formation). The specific electropolymerization technique may, in some embodiments, involve potential cycling and/or potentio static deposition. In one embodiment, deposition of the first component is initiated by application of a cyclic voltammetry wave form.

Such processes can advantageously use various electrolytes, monomer concentrations, applied potential and duration, potential range, scan rate, and number of cycles. One of skill in the art is aware of the variables that can be adjusted to obtain an electropolymerized material as the first component in the hybrid membranes disclosed herein. Suitable deposition techniques can be electrochemically irreversible or can be quasi-reversible. The voltage, number of scans, and sweep rate can be varied to achieve the desired deposition. In certain embodiments, slower formation of the electropolymerized material as the first component leads to more orderly packing of oligomers and, correspondingly, better permselectivity of the first component for certain monomers. However, for other monomers, the inverse has been observed, and faster formation leads to more orderly packing and better permselectivity. In certain embodiments, decreasing monomer concentration leads to more orderly packing of oligomers and, correspondingly, better permselectivity of the first component.

In certain embodiments, depositions by cyclic voltametry, as opposed to depositions by constant potential amperometry afford better NO selectivity against most interferents of interest except nitrite. It is noted that, in some embodiments, a specific deposition procedure may be chosen to protect against a particular interferent.

In this method, the second component is then applied. Generally, the electropolymerized material- coated electrode is contacted with a colloidal solution (“sol”). This contact can occur, e.g., by spray coating the electropolymerized material-coated electrode with the sol, spin coating the electropolymerized material- coated electrode with the sol, dipping the electropolymerized material-coated electrode with the sol, or otherwise associating the electropolymerized material-coated electrode with the sol. The sol, in some embodiments, can be an aqueous colloidal solution or can be, e.g., a colloidal solution in ethanol.

The disclosure further provides methods of assembling sensors and devices comprising such sensors. Sensors (e.g., NO sensors) can be assembled largely using known processes, as disclosed, e.g., in U.S. Patent No. 8,551,322 to Schoenfisch et ak, which is incorporated by reference in its entirety, modified so as to associate, e.g., the disclosed composite membrane with one or more of the electrodes of such sensors.

The principles generally disclosed herein can be applied in the context of various types of devices and various configurations of such devices. For example, these principles can be applied in the context of handheld analyzers, benchtop analyzers, etc. Advantageously, in some embodiments, a sensor is provided which can be directly implemented within known analyzers. Advantageously, the principles and materials disclosed herein are applicable in the context of analyzing complex fluids, including biological fluids, physiological fluids, and clinical fluids. Exemplary materials that can be analyzed according to the disclosed method include, but are not limited to, whole blood, cell culture supernatant, wound fluid/exudate, plasma, serum, cerebrospinal fluid, interstitial fluid, bone marrow aspirate, bronchoalveolar lavage fluid, endotracheal aspirate, saliva, lymph extracts, sweat, and urine and, as such, the composite membrane-coated electrodes provided herein are suitable, in some embodiments, for use within biosensors.

As such, the methods and sensors disclosed herein provide for the direct analysis of analyte levels in a sample, e.g., from a human patient. The disclosure thus provides methods of analyzing various analytes from a range of sample sources, including, but not limited to, sources such as blood, cell culture supernatant, wound exudate, plasma, and urine. Such methods generally comprise contacting the sample with a sensor comprising an electrode assembly comprising at least one composite membrane-coated electrode as disclosed herein, which comprises, in addition to the electrode(s) disclosed herein, a detector for measuring current at each of the electrodes. The method further comprises evaluating the current at each of the electrodes and correlating the current with analyte content to evaluate the amount of analyte present within the sample. In some embodiments, the disclosed composite membranes reduce the signal from common interferents in complex fluids, e.g., biologically important media (including, but not limited to, plasma).

Advantageously, the composite membranes disclosed herein exhibit synergy, as, in some embodiments, such membrane show a greater than additive effect of the electropolymerized component and the sol-gel-derived component. For example, an electrode coated with the composite membrane disclosed herein can, in some embodiments, provide for greater selectivity and/or sensitivity for a given analyte than would be expected based on results obtained for a comparable electrode coated with just an

electropolymerized component and for a comparable electrode coated with just a sol-gel-derived component. In some embodiments, the electrodes and sensors incorporating such electrodes can be used for analysis of complex mixtures, are suitable for extended use, e.g., in proteinaceous media.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

EXPERIMENTAL

Example 1

Materials and reagents

Trimethoxymethylsilane (MTMOS), 5-amino-l-naphthol (5A1N), hydrochloric acid (HC1), sodium nitrite, sodium nitrite standard (0.1 M), L-ascorbic acid (AA), acetaminophen (AP), dopamine hydrochloride (DA), lipopolysaccharide (LPS), fetal bovine serum (FBS), \ -( 1 - naphthyl)ethylenediamine dihydrochloride (NED), and sulfanilamide were purchased from Sigma (St. Louis, MO). (Heptadecafluoro-l,l,2,2-tetrahydrodecyl)trimethoxysilane (17FTMS) was purchased from Gelest (Morrisvile, PA). Ethanol (200 proof), ammonium hydroxide, sodium hydroxide (NaOH), sodium chloride, sulfuric acid, phosphoric acid, and hydrogen peroxide (30 wt.% H202) were obtained from Fisher Scientific (Hampton, NH). Dulbecco’s modification of Eagle’s medium (DMEM; 4.5 g/mL glucose; no phenol or L-glutamine) was purchased from Coming (Coming, NY). Interferon-gamma (IFN- g) was obtained from BioLegend (San Diego, CA). Murine RAW 264.7 macrophages were obtained from the American Type Culture Collection (Manassas, VA). Nitric oxide (99.5%), carbon monoxide (99.3%), nitrogen (N2), and argon (Ar) gases were purchased from National Welders Supply (Raleigh, NC). Other solvents and chemicals were analytical-reagent grade and used as received without further purification.

A Millipore Milli-Q UV Gradient A10 System (Bedford, MA) was used to purify distilled water to a resistivity of 18.2 MW-cm and a total organic content of <6 ppb. Saturated NO solutions (1.9 mM) were made by purging ~20 mL of phosphate buffered saline (PBS; 0.01 M, pH 7.4) sequentially with Ar for 25 min and NO gas for 25 min over ice and were used the day of preparation. Electrochemical experiments were carried out using an 8-channel CH Instruments 1030C Electrochemical Analyzer (Austin, TX). In total, 8 inlaid 2 mm dia. polycrystalline platinum (Pt) disc working electrodes sealed in Kel-F (6 mm total diameter; CH Instruments), a silver-silver chloride (Ag|AgCl) reference electrode (3.0 M KC1; CH

Instruments), and a coiled Pt wire counter electrode were employed in the electrochemical arrangement. All reported potentials herein are versus the Ag|AgCl reference electrode.

Preparation ofPt/p-5AlN/XG sensors

Platinum working electrodes were mechanically polished with deagglomerated alumina slurries down to 0.05 pm in particle size (Buehler; Lake Bluff, IL) and ultrasonicated in ethanol to remove residual alumina. Electrodes were then electrochemically polished in 1 N hydrosulfuric acid by cycling between -0.4 and +1.8 V for 40 cycles (500 mV s ¹). Electrodes were then rinsed with water and transferred to a monomer solution of 5A1N. To improve solubility, 10 mM 5A1N was dissolved in 10 mM NaCl solution titrated to pH 1 with HC1. Poly -5 AIN films were electropolymerized via cyclic voltammetry (CV), sweeping the potential from 0 to +1.0 V (20 cycles; positive direction initial sweep) at a scan rate of 10 mV s ¹. Film- modified electrodes were then rinsed copiously with distilled water to remove unbound oligomer and allowed to dry in ambient for >1 h before spray -coating.

A fluoroalkoxysilane sol solution was prepared via acid-catalyzed hydrolysis and co-condensation of MTMOS and 17FTMS precursors (30% 17FTMS, v/v balance MTMOS). Ordered additions of 3.600 mL EtOH, 630 pL MTMOS, 270 pL 17FTMS, 960 pL H20, and 60 pL 0.5 M HC1 constituted the sol solution, which was then stirred vigorously for 1 h. The sol was spray-coated onto Pt disc electrodes (both bare and electropolymerized film-modified) with an airbrush gun (Iwata HP-BC1 Plus; Yokohama, Japan) pressurized at 42 psi with a nozzle-target separation of 50 cm and 5 s of continuous dispersal. The sol-coated electrodes were then dried in ambient for >48 h to allow condensation of the xerogel matrix before further testing.

Steady-state amperometric measurements

Bare and modified electrodes were polarized at +0.8 V in deoxygenated PBS for 10 min to achieve a constant background current prior to measurements. Sensitivity for NO was determined with consecutive injections (475 nM each) of saturated NO solution into deoxygenated PBS. Calibrations were also done in FBS -supplemented DMEM to determine sensitivity retention relative to PBS trials. Limits of detection (LOD) were calculated with respect to 3 standard deviations of the noise ( S/N= 3). The current responses to injections of sodium nitrite (1 mM), L-ascorbate (1 mM), acetaminophen (50 pM), dopamine hydrochloride (100 pM), carbon monoxide (1 pM), ammonium hydroxide (1 mM), and hydrogen peroxide (1 mM) were measured and compared to the NO sensitivity. Selectivity coefficients were calculated using Eq. 1, where logLYO,/ is the selectivity coefficient for NO over interferent /. A/} is the current response to interferent /. C, is the concentration of interferent /. and S_N0 is the NO sensitivity of the sensor.

Sensor performance with extended use and in proteinaceous media

The long-term stabilities of Pt/p-5AlN/XG, Pt/p-5AlN, Pt/XG, and bare Pt electrodes were evaluated in PBS with 12 h of a continuously applied working potential (+0.8 V). Sensitivity towards NO, selectivity against nitrite, and the LOD were re-measured at the 12 h time-point and compared to initial measurements to assess performance maintenance. All sensor designs were also tested for sensitivity retention in DMEM. The Pt/p-5AlN/XG sensors were evaluated for long-term stability in FBS- supplemented DMEM with 24 h of continuously applied working potential (+0.8 V). Again, analytical metrics were re-measured at the 24 h time-point and compared to initial measurements to assess performance maintenance.

Measurements from RA W 264.7 macrophages

The RAW 264.7 macrophages were seeded into a 12-well plate at a density of 1.0 ^c 10⁶ cells/well and given 4 hours to adhere to the plate. Each well contained 2.0 mL of phenol-free, FBS-supplemented DMEM. A custom well plate cover was designed to support a Pt/p-5AlN/XG working electrode and reference/counter electrodes in each individual well while the plate remained in an incubator. Sensors were positioned such that the transducer surface was approximately ~100 pm above the well bottom.

Electrodes were given ~8 h to polarize (amperometry, +0.8 V) in solution before pro- inflammatory stimulants were injected into the cell (20 ng/mL LPS and 10 ng/mL IFN-g). Continuous NO measurements were collected for 24 h post-stimulation. An exponential fit of the first 8 h prestimulation was used to background subtract non-Faradaic polarization current from the entire trace. The current measured at t = 2 h post-stimulation (i.e., t = 10 h overall) was also background subtracted from all traces as a reference point where no NO evolution from the cells was anticipated. Data collection was repeated for unstimulated macrophage cells with all other parameters and data processing kept the same.

After electrochemical experiments were completed, supernatant solutions were collected and stored at -20 °C. Endpoint Griess analysis was carried out on samples to quantitate nitrite totals from both stimulated and unstimulated cell runs as a measure of total NO production. Briefly, sulphanilic acid was reacted with nitrite in the sample to form a diazonium salt before reaction with NED to yield the characteristic pink azo dye detectable via absorbance spectroscopy at l = 540 nm. Nitrite calibration standards were prepared in FBS-supplemented DMEM (the same matrix as the samples).

Statistical analysis

The NO sensitivity, sensitivity retention, selectivity coefficients, and LOD of the bare and modified electrodes are presented as the mean ± standard error of the mean. Comparisons between groups were performed using a two-tailed /-test with p <0.05 considered to be statistically significant.

Sensor preparation

Electropolymerization of the p-5AlN was carried out using a deposition procedure previously optimized for NO permselectivity against nitrite and AA.²⁷ As the potential is swept positively, 5A1N monomers are oxidized via the amine group to radical cations, which then dimerize, oligomerize, and ultimately precipitate out of solution, onto the electroactive surface. As the film grows with successive cycles, fewer and fewer monomers are able to reach the underlying electrode to generate new radical cations. As a result, the process is self-terminating and results in thin (66-98 nm), reproducible (>5% relative error in total charge passed) films overall. After electrodeposition, a prepared sol solution was spray -coated onto the electrode and allowed to dry/condense for >48 h. The presence of amines is known to catalyze the condensation of alkoxysilane precursors in sol-gel systems; thus, the presence of uncoupled amines in p- 5A1N films may improve xerogel rigidity /adherence. Confirmation of xerogel formation was achieved with contact angle measurements increasing from 62° ± 3° to >90° post-deposition.

Sensor selectivity

Composite Pt/p-5AlN/XG sensors were then evaluated for selectivity for NO against common biological interferents (FIG. 2). FIG. 2 provides a graph of selectivity coefficients of bare electrodes (“Pt”) and electrodes modified with the coatings of the present example (Pt/p-5AlN/XG). The electrodes were tested against common biological interferents, including nitrite (N0₂ ), L-ascorbic acid (AA), acetaminophen (AP), dopamine (DA), carbon monoxide (CO), hydrogen peroxide (H2O2), and ammonia (NH₃; n=8).

The selectivity against nearly all interferents studied was markedly improved with the addition of both the p-5AlN and XG membranes, both of which confer selectivity through size-exclusion and hydrophobic interactions with NO. Most notable perhaps was the improvement in selectivity against nitrite to a coefficient of nearly -5, signifying 100,000-fold greater sensitivity towards NO over nitrite. This merit is important, as poor sensor selectivity against nitrite will result in a continually increasing signal response if a NO-releasing system is under observation (e.g., a therapeutic or cell), falsely suggesting an increase in the NO flux.

Selectivity coefficients against larger interferents AA, AP, and DA fell between -3 and -4, highlighting size-exclusion as a key sieving mechanism. Poorer selectivity coefficients were observed for smaller interferents H202, NH3, and particularly CO, for which there was no improvement over bare Pt electrodes. Nitric oxide and CO share a similar size, neutral charge, and lipophilicity— all traits that facilitate their roles as intercellular gasotransmitters. Therefore, their permeability characteristics were expected to be similar.

Sensor performance with extended use and in proteinaceous media

In order to continuously measure the NO release off of stimulated macrophages accurately, the longterm sensor performance needed to be evaluated. As such, bare Pt, Pt/p-5AlN, Pt/XG, and composite Pt/p- 5A1N/XG sensors were tested for NO sensitivity, LOD, and selectivity versus nitrite. All sensors were then polarized for 12 h at a continuously applied potential of +0.8 V in the absence of NO. Endpoint measurements of relevant analytical performance metrics are compared in Table 1. The Pt/p-5AlN sensors, though bearing high selectivity against nitrite initially, were unable to maintain high selectivity throughout the experiment. Coupled with an increase in NO sensitivity (and error), the reduced selectivity was likely the result of p-5AlN membrane damage or partial delamination under the rigors of such a high operating potential. The Pt/XG sensors did not have as high a selectivity coefficient versus nitrite compared to Pt/p- 5A1N, but were able to maintain all performance metrics within error over the course of the experiment. The XG membrane is conclusively more stable and capable of withstanding continuously applied high working potentials. The combination of p-5AlN and XG membranes yielded a sensor with markedly higher selectivity against nitrite than either component evaluated individually. Not only does this improvement suggest that XG deposition does not disrupt the p-5AlN film, but rather that their layering results in a significant synergism to sieving NO. After 12 h of continuous polarization, Pt/p-5AlN/XG sensors experienced no change in LOD or nitrite selectivity. The overall composite design is able to benefit from both the high selectivity of the p-5AlN film and the performance stability of the XG membrane.

A critical challenge to in situ monitoring of NO is biofouling, specifically the accumulation of proteins at the transducer surface. The effects of protein buildup typically manifest as reduced sensor sensitivity, increased response time, and/or membrane damage (with concomitant reductions in selectivity). Sensors were therefore evaluated for NO sensitivity retention in FBS-supplemented DMEM relative to calibrations carried out in PBS. See FIG. 3. FIG. 3 provides a graph of data with respect to retention of NO sensitivity of bare and modified Pt electrodes in FBS-supplemented DMEM relative to calibrations carried out in deoxygenated PBS (n = 8).

Without any kind of membrane modification, Pt electrodes maintained <20% of their original NO sensitivity when transferred to proteinaceous media. It should be noted that sensitivity reduction also occurs due to protein scavenging of free NO used for calibration, irrespective of any protein-surface interaction. The single-layered Pt/p-5 AIN and Pt/XG sensors saw improved sensitivity retention compared to bare Pt, but remained <70%. Composite Pt/p-5 A 1N/XG sensors demonstrated ~80% sensitivity retention, possibly due to their higher hydrophobicity. To account for the ~20% reduction in sensitivity, sensors should be calibrated in the matrix of their application, ideally after polarization pre-treatment. Finally, Pt/p-5AlN/XG sensors were evaluated for long-term performance in FBS-supplemented DMEM with 24 h of continuously applied working potential (Table 1). Due to the reduction in NO sensitivity in DMEM, the nitrite selectivity was observed to drop slightly compared to PBS trials. Endpoint comparisons, however, reveal that the LOD and selectivity were maintained over the course of the experiment in DMEM. The sensitivity was seen to increase slightly, which, without a coupled reduction in nitrite selectivity, may be attributed to hydration of the membrane-protein layer (i.e., the formation of water channels towards the electrode surface).

Table 1. Analytical performance metrics of bare and modified Pt electrodes with extended use (n = 81.

t Sensitivity LOD

Electrode Medium lo kNO,NQ₂- h A/ M M

Measurements from RA W 264.7 macrophages

Given its superior preservation of performance in proteinaceous media, the Pt/p-5 A1N/XG sensor design was deemed suitable for long-term measurements of NO from cultured macrophages. Sensors were first polarized for 2 h and calibrated in FBS-supplemented DMEM before use to circumvent protein-related sensitivity reductions. The macrodisc sensors were then positioned approximately 100 pm above the well bottom. After 8 h of continuous measurement, stimulated trials were co-injected with 20 ng/mL LPS and 10 ng/mL IFN-g (FIG. 4A). The former stimulant is recognized by the macrophages as a structural element from the cell walls of E. coli, and the latter is an inflammatory cytokine; the combination is known to elicit a robust inflammation response. As can be seen in FIG. 4, the traces begin to increase and plateau approximately ~8 h post-stimulation, in agreement with the literature. Approximately ~14 h post-stimulation, the traces begin to fall, indicative of L-arginine exhaustion. Griess measurements revealed that the total nitrite content in the supernatant was 56 ± 3 pM, compared to 0.69 pM in unstimulated trials (approaching the LOD). The nitrite concentrations are therefore a stoichiometric quantifier of total NO release from the cells. The small relative error of the nitrite totals (5.4%) indicates that the cells were prepared and stimulated in a uniform fashion. The question then remains why electrochemical traces were observed to plateau at different values, which can in part be ascribed to non-uniform electrode placement. Nitric oxide diffuses radially from cells, and the equilibrium between NO supply and solution scavengers results in a unique [NO] gradient surrounding the cell. When expanded to a plane of near-confluent cells, the gradient extends linearly rather than radially (FIG. 4, providing a representation of the macrophage-to-sensor separation distance-dependence (depicted along the z-axis) of the amount of NO measured

electrochemically upon stimulation). The [NO] measured is therefore dependent on this gradient and subject to change with imprecise sensor placement. Future scanning electrochemical microscope (SECM) studies will seek to profile this gradient as a sensor approaches stimulated cells from the bulk solution.

Unstimulated macrophages were also continuously monitored for NO release (FIG. 4B). The lack of any release profile indicates that no NO was evolved from the cells, as corroborated by Griess assay measurements (0.69 mM nitrite). The comparison between stimulated and unstimulated trials verifies that NO was truly detected in the former trial and that the background subtraction methods employed did not artificially create/enhance the release profiles measured.

Biocompatibility Analysis

The sensitivity to nitric oxide before and after n=20 independent exposures to blood was determined. Sensitivity was determined from the slope of a 4-point standard calibration (0-950 nM nitric oxide), and results are shown in FIG. 6. The relative standard deviation of n=4 measurements of a“blank” sample (phosphate buffered saline, pH 7.4) analyzed in between injections of human blood, was determined, as provided in FIG. 7. Each blank injection was performed following five independent exposures of human blood to the sensor.

Example 2

A three-electrode system consisting of a Pt working electrode, Ag/AgCl reference electrode, and Pt counter electrode was prepared by evaporating platinum and silver onto a planar electrode. The Pt working electrode was either left bare, or coated with a composite xerogel/electropolymerized o-phenylenediamine membrane by electropolymerizing o-phenylenediamine via cyclic voltammetry and spraycoating a 30 mol% (heptadecafluoro- 1 , 1 ,2,2-tetrahydrodecyl)trimethoxy silane balanced with a 70 mol%

methyltrimethoxysilane backbone. The planar electrode system was incorporated within a microfluidic assembly, polarized for 2 hours at 0.6 V vs. Ag/AgCl. Human plasma was injected (500 uL) within the microfluidic chamber and the average current was recorded over a period of 10 s, providing a representation of total signal from interfering electroactive compounds endogenous to the plasma sample. As shown in FIG. 8, the composite membrane effectively reduced the signal from electro-oxidation of interferents in plasma. Electrodes coated with the composite membrane exhibited an average oxidative current of 17 nanoamperes (nA) while bare electrodes exhibited an average current of l.OlxlO³ nA. The reduced response to the endogenous interferent pool did not come at the expense of loss in NO sensitivity, as sensors coated with the composite membrane retained their sensitivity to nitric oxide (FIG. 9), demonstrating both the improved selectivity over interferents and anti-fouling properties of this composite membrane.

Claims

CLAIMS What is claimed is:

1. An analyte-selective electrode, comprising:

an electrode having a surface, and

a composite membrane on at least a part of the surface, the composite membrane comprising:

a first component comprising a permselective, electropolymerized material; and a second component comprising a sol-gel-derived material.

2. The analyte-selective electrode of claim 1, wherein the first component is directly associated with the electrode surface and the second component is directly associated with the first component.

3. The analyte-selective electrode of claim 1, wherein the second component is directly associated with the electrode surface and the first component is directly associated with the second component.

4. The analyte-selective electrode of claim 1, wherein the composite membrane comprises a mixture of the first and second components.

5. The analyte-selective electrode of any of claims 1-4, wherein the electrode is selective for an analyte selected from the group consisting of nitric oxide (NO), nitrite, ascorbic acid, acetaminophen, uric acid, cysteine, xanthine, hypoxathine, vanillylmandelic acid, glutathione (reduced and/or oxidized), xanthosine, methoxy -hydro xyphenylgly col, homogentisic acid, 7-methylxanthine, methionine, norepinephrine, guanosine, L-dopa, guanine, 3 -hydroxy kynurenine, epinephrine, tyrosine, dihydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 3 -hydroxy anthranilic acid, 1,7- dimethylxanthine, 5 -hydroxy tryptophan, 1,3-dimethylxanthine, 4-hydroxybenzoic acid, 3-o- methyldopa, 5 -hydroxy indole acetic acid, kynureinine, normetanephrine, dopamine, metanephrin, acetylserotonin, homovanillic acid, 4-hydroxyphenylacetic acid, tryamine, 2-hydroxyphenylacetic acid, 5-serotonin, 3-methoxytyramine, methylserotonin, tryptophan, melatonin, tryptophol, indole-3- acetic acid, indole-3 -propionic acid, (+)-a-tocopherol, (+)-6-tocopherol, and (+)-y-tocopherol.

6. The analyte-selective electrode of claim 5, wherein the electrode is selective for NO.

7. The analyte-selective electrode of claim 5, wherein the electrode is selective for nitrite or ascorbate.

8. The analyte-selective electrode of any of claims 1-7, wherein the permselective, electropolymerized material comprises a polymer with monomeric units selected from the group consisting of: optionally functionalized alcohols; optionally functionalized amines; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof.

9. The analyte-selective electrode of claim 8, wherein the monomeric units are selected from the group consisting of: phenol; eugenol; naphthol; phenylenediamine; aniline; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof.

10. The analyte-selective electrode of claim 8, wherein the monomeric units are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-(trifluoromethyl)phenol, 3,5- bis(trifluoromethyl)phenol, and combinations thereof.

11. The analyte-selective electrode of any of claims 1-7, wherein the permselective, electropolymerized material comprises poly-5-amino-l-naphthol (5A1N).

12. The analyte-selective electrode of any of claims 1-3 or 5-11, wherein the permselective,

electropolymerized material is in the form of a film with an average thickness of about 3 nm to about 10 pm.

13. The analyte-selective electrode of claim 12, wherein the permselective, electropolymerized material is in the form of a fdm with an average thickness of about 3 nm to about 15 nm.

14. The analyte-selective electrode of any of claims 1-13, wherein the second component comprises a xerogel.

15. The analyte-selective electrode of claim 14, wherein the xerogel is a fluorinated alkoxysilane

xerogel.

16. The analyte-selective electrode of any of claims 1-15, wherein the electrode comprises platinum.

17. The analyte-selective electrode of any of claims 1-16, wherein the electrode is a radial disk electrode or planar electrode.

18. The analyte-selective electrode of any of claims 1-17, further comprising a third component

comprising a material selected from the group consisting of poly(tetrafluoroethylene), Nafion, cellulose acetate, polyurethanes, and combinations and copolymers thereof associated with one or both of the first component and the second component.

19. An electrochemical analyte sensing device, comprising: a substrate; the analyte-selective electrode of any of claims 1-18; and an analyte detecting element.

20. A process for preparing the analyte-selective electrode of any of claims 1-18, comprising:

providing the electrode;

associating the composite membrane with at least a portion of the surface, wherein the associating comprises:

electropolymerizing one or more monomers to give the first component; and applying the second component.

21. A process for preparing an analyte-selective electrode, comprising:

providing an electrode having a surface;

associating a composite membrane with at least a portion of the surface, wherein the associating comprises:

electropolymerizing one or more monomers to give a first component comprising a permselective, electropolymerized material; and

applying a second component comprising a sol-gel-derived material.

22. The process of claim 21, wherein the electropolymerizing step is conducted before the applying step.

23. The process of claim 21, wherein the electropolymerizing step is conducted after the applying step.

24. The process of any of claims 21-23, wherein the electropolymerizing comprises electrooxidation or radical coupling.

25. The process of any of claims 21-23, wherein the electropolymerizing comprises a potential cycling or potentiostatic process.

26. The process of any of claims 21-25, wherein the electropolymerizing comprises electropolymerizing the one or more monomers from an electrolyte-containing solution of the one or more monomers.

27. The process of claim 26, wherein the electrolyte-containing solution comprises phosphate buffered saline (PBS).

28. The process of claim 26, wherein the one or more monomers have a concentration in the electrolyte- containing solution of about 10 to about 100 mM.

29. The process of any of claims 21-28, wherein the one or more monomers are selected from the group consisting of optionally functionalized alcohols, optionally functionalized amines, and combinations thereof.

30. The process of any of claims 21-28, wherein the one or more monomers are selected from the group consisting of: phenol; eugenol; naphthol; phenylenediamine; aniline; heteroaromatic, deuterated, and fluorinated analogues thereof; and combinations thereof.

31. The process of any of claims 21-28, wherein the one or more monomers are selected from the group consisting of 2-(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-(trifluoromethyl)phenol, 3,5- bis(trifluoromethyl)phenol, and combinations thereof

32. The process of any of claims 21-28, wherein the one or more monomers comprise 5-aminoindole.

33. The process of any of claims 21-32, wherein the applying comprises a sol gel process.

34. The process of any of claims 21-33, wherein the applying comprises spray coating a sol onto the first component and drying the sol.