WO2019180662A1

WO2019180662A1 - Sensing devices and methods of production

Info

Publication number: WO2019180662A1
Application number: PCT/IB2019/052316
Authority: WO
Inventors: Wesley Langdon STORM; Elizabeth Dora EASTER; Grace Elizabeth WRIGHT
Original assignee: Clinical Sensors, Inc.
Priority date: 2018-03-22
Filing date: 2019-03-21
Publication date: 2019-09-26

Abstract

Methods of preparing an electrode assembly and an electrochemical sensor body including such an electrode assembly are provided herein. The method generally involves providing a sensor base comprising a working electrode and a silver electrode; and conducting three or more of the following steps: applying a mask to a portion of the sensor base to shield the silver electrode, keeping the working electrode exposed; cleaning the working electrode; removing the mask from the sensor base; contacting the sensor base with a chloride-containing component to convert the silver electrode to an Ag/AgCl pseudoreference electrode; and depositing one or more membranes on the working electrode. The disclosure also provides electrode assemblies and electrochemical sensor bodies prepared by such methods.

Description

SENSING DEVICES AND METHODS OF PRODUCTION

FIELD OF THE INVENTION

The present invention relates to sensors (e g., microfluidic devices) and components thereof for the detection and quantification of various analytes.

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, nitrosammes; S-mtrosopenacillamine, 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 an electrochemical sensor comprising a fluidic assembly for introducing and removing samples to be analyzed; a pseudoreference electrode; and a working electrode with controlled surface chemistry. Advantageously, the working electrode is free from unwanted surface contamination and, in some embodiments, can be modified with one or more membranes associated with at least a portion of the electrode to provide additional functionality (e.g., including, but not limited to, enhanced sensitivity, enhanced selectivity, or mitigation of fouling).

In one aspect, the disclosure provides a method of preparing an electrochemical sensor body, comprising: providing a sensor base comprising a working electrode and a silver electrode; and conducting three or more of steps a) to e): a) applying a mask to a portion of the sensor base to shield the silver electrode, keeping the working electrode exposed; b) cleaning the working electrode; c) removing the mask from the sensor base; d) contacting the sensor base with a chloride-containing component to convert the silver electrode to an Ag/AgCl pseudoreference electrode; and e) depositing one or more membranes on the working electrode.

The specific steps employed can vary. In some embodiments, step a) is required. In some embodiments, step b) is required. In certain exemplary embodiments, the method comprises steps a), b), c), and d). In certain exemplary embodiments, the method comprises steps a), b), and e). in certain exemplary embodiments, the method comprises steps b), d), and e).

In some embodiments, the method further comprises attaching a fluidic cell to the electrochemical sensor body. In some embodiments, the providing step comprises photolithography.

In certain embodiments, the silver electrode has a thickness of at least about 1 micron. In some embodiments, the working electrode comprises a platinum electrode. In some embodiments, the working electrode comprises a gold electrode, a carbon fiber electrode, or a palladium electrode.

The disclosed method, in some embodiments, comprises step a), wherein the mask in step a) is adhesive. The disclosed method, in some embodiments, comprises step b), wherein the cleaning in step b) comprises electrochemical cleaning. The disclosed method, in some embodiments, comprises step b), wherein the cleaning in step b) comprises chemical cleaning, physical cleaning, electrolytic cleaning, or a combination of two or more. The disclosed method, in some embodiments, comprises step b) and the cleaning in step b) comprises contacting the working electrode with an acid. Exemplary acids include, but are not limited to, a sulfuric acid solution, a nitric acid solution, a hydrochloric acid solution or a combination of two or more. For example, in certain embodiments, the acid is a sulfuric acid solution.

In certain embodiments, the disclosed method comprises step b) and the cleaning comprises (e g., in addition to the types of cleaning referenced above), applying a potential to the working electrode. In some embodiments, the potential is cycled from about -0.4 to 2.0 V versus an independent reference electrode and an independent counter electrode. In various embodiments, the disclosed method comprises step d) and the contacting in step d) comprises anodization. In various embodiments, the disclosed method comprises step d) and the chloride -containing component in step d) is a solution of ferric chloride.

In some embodiments, the disclosed method comprises step e) and the depositing in step e) is electrochemical depositing. The electrochemical depositing can, in certain embodiments, result in the deposition of one or more of a membrane to enhance selectivity over interferents, a membrane to enhance sensitivity to analytes of interest, and a membrane to mitigate electrode fouling. Advantageously, in some embodiments, the working electrode is substantially free of surface contamination at certain steps of the process, e.g., prior to step e).

In another aspect, the disclosure provides an electrochemical sensor body prepared according to any of the methods disclosed herein. The disclosure further provides, in an additional aspect, a microfluidic device for measuring an amount of an analyte, comprising the referenced electrochemical sensor body.

The present disclosure provides, as a further aspect, a microfluidic device for measuring an amount of an analyte, comprising: a substantially planar body; an electrode assembly comprising a working electrode and reference electrode, wherein the reference electrode comprises silver and wherein the reference electrode is prepared from silver metal; and at least one gas-permeable membrane disposed on and coating at least a portion of the working electrode, wherein the working electrode is substantially free of adhered organic or inorganic non-electrode contaminants prior to membrane deposition.

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

Embodiment 1: A method of preparing an electrochemical sensor body, comprising: providing a sensor base comprising a working electrode and a silver electrode; and conducting three or more of steps a) to e): a) applying a mask to a portion of the sensor base to shield the silver electrode, keeping the working electrode exposed; b) cleaning the working electrode; c) removing the mask from the sensor base; d) contacting the sensor base with a chloride -containing component to convert the silver electrode to an Ag/AgCl

pseudoreference electrode; and e) depositing one or more membranes on the working electrode.

Embodiment 2: The method of the preceding embodiment, comprising steps a), b), c), and d)

Embodiment 3: The method of Embodiment 1, comprising steps a), b), and e).

Embodiment 4: The method of Embodiment 1, comprising steps b), d), and e).

Embodiment 5: The method of any preceding embodiment, further comprising attaching a fluidic cell to the electrochemical sensor body.

Embodiment 6: The method of any preceding embodiment, wherein the providing step comprises

photolithography . Embodiment 7: The method of any preceding embodiment, wherein the silver electrode has a thickness of at least about 1 micron.

Embodiment 8: The method of any preceding embodiment, wherein the working electrode comprises a platinum electrode

Embodiment 9: The method of any preceding embodiment, wherein the working electrode comprises a gold electrode, a carbon fiber electrode, or a palladium electrode.

Embodiment 10: The method of any preceding embodiment, wherein the mask in step a) is adhesive.

Embodiment 11 : The method of any preceding embodiment, wherein the cleaning in step b) comprises electrochemical cleaning.

Embodiment 12: The method of any preceding embodiment, wherein the cleaning in step b) comprises chemical cleaning, physical cleaning, electrolytic cleaning, or a combination of two or more.

Embodiment 13: The method of any preceding embodiment, wherein the cleaning in step b) comprises contacting the working electrode with an acid.

Embodiment 14: The method of the preceding embodiment, wherein the acid comprises a sulfuric acid solution, a nitric acid solution, a hydrochloric acid solution or a combination of two or more.

Embodiment 15 The method of the preceding embodiment, wherein the acid is a sulfuric acid solution.

Embodiment 16: The method of any preceding embodiment, wherein the cleaning further comprises applying a potential to the working electrode.

Embodiment 17: The method of the preceding embodiment, wherein the potential is cycled from about -0.4 to 2.0 V versus an independent reference electrode and an independent counter electrode.

Embodiment 18: The method of any preceding embodiment, wherein the contacting in step d) comprises anodization.

Embodiment 19: The method of any preceding embodiment, wherein the chloride-containing component in step d) is a solution of ferric chloride.

Embodiment 20: The method of any preceding embodiment, wherein the depositing in step e) is

electrochemical depositing.

Embodiment 21 : The method of any preceding embodiment, wherein the one or more membranes deposited in step e) comprise one or more of a membrane to enhance selectivity over interferents, a membrane to enhance sensitivity to analytes of interest, and a membrane to mitigate electrode fouling.

Embodiment 22: The method of any preceding embodiment, wherein the working electrode is substantially free of surface contamination prior to step e).

Embodiment 23 : An electrochemical sensor body prepared according to the method of any preceding embodiment.

Embodiment 24: A microfluidic device for measuring an amount of an analyte, comprising the electrochemical sensor body of the preceding embodiment.

Embodiment 25 : A microfluidic device for measuring an amount of an analyte, comprising: a substantially planar body; an electrode assembly comprising a working electrode and reference electrode, wherein the reference electrode comprises silver and wherein the reference electrode is prepared from silver metal; and at least one gas-permeable membrane disposed on and coating at least a portion of the working electrode, wherein the working electrode is substantially free of adhered organic or inorganic non-electrode contaminants prior to membrane deposition.

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 is an electrode assembly of a microfluidic device according to one embodiment of the present disclosure;

FIG. 2 is a top view of a portion of the microfluidic device (the electrode layer)) according to one embodiment of the present disclosure;

FIG 3 is a top view of a microfluidic device according to one embodiment of the present disclosure;

FIG. 4 is a schematic flow chart of a method for production of microfluidic devices according to one embodiment of the present disclosure;

FIG. 5 is a plot of current versus voltage traces between two subsequent cyclic voltamraagrams, used to demonstrate a substantially clean electrode according to the present disclosure;

FIG. 6 is a plot of conductance before and after cleaning when a mask is not employed; and

FIG. 7 is a plot of intra-test variance versus Ag/AgCl (MV) when a silver electrode is chlorinated before it is masked (instead of after it is masked)

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 (i.e., all ingredients except water).

The present disclosure generally provides an electrode assembly and electrochemical sensing device (“sensor”) comprising the electrode assembly. In preferred embodiments, the electrochemical sensor is a microfluidic device. Exemplary microfluidic devices to which the described components and methods are applicable are described, for example, in U.S. Patent No. 9,201,037 to Schoenfisch et al., which is incorporated herein by reference in its entirety. The electrode assembly and sensor described herein can advantageously be used in various contexts, for the detection and quantification of a range of molecular species present in a range of samples.

FIGS. 1-3 illustrate one particular microfluidic device 10, which may be referred to generally as a “sensor,” and which includes a body 11 and an electrode assembly 100. Generally, the microfluidic device is configured to detect the presence of the molecular species in a sample and may further quantify the amount of the species therein. The device may use an electrochemical technique, such as a voltammetric or coulometric technique, for such analysis. In some embodiments, the device is an amperometric sensor (i.e., it detects the redox current produced by the oxidation of the molecular species overtime at a fixed voltage potential). In some embodiments, the device includes a potentiostat. In some embodiments, electrochemical methods for measuring the amount of a molecular species in a sample are employed, such as amperometry, cyclic voltammetry, fast scan cyclic voltammetry, pulsed voltammetry, step voltammetry, thin-layer electrochemistry, and chronocoulometry.

The electrode assembly may 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 microfluidic device may comprise a two- or three-electrode configuration. Thus, in some embodiments, the electrode assembly comprises a working electrode and a reference electrode. In some embodiments, the electrode assembly may comprise a working electrode, a reference electrode and a counter electrode. Further, the electrode assembly may comprise at least one integrated electrode having a sensor width and a sensor pitch.

The electrode assembly may 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 may comprise a biocompatible polymeric coating covering those portions not covered by a membrane (referenced herein below), so long as such coating does not interfere with the ability of the device to detect the analyte/molecular species of interest.

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 platinized platinum, tungsten, silver/silver chloride, gold, copper, indium, tin oxide, iridium oxide, nickel, and combinations thereof. In some embodiments, the working electrode comprises a material selected from platinum, platinized platinum, tungsten, gold, carbon, carbon fiber, and combinations thereof In some embodiments, the reference electrode comprises silver/silver chloride. In some embodiments, the counter electrode comprises platinum. In addition to the exact electrochemical technique/process in which the 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(s) within the assemblies disclosed herein are not particularly limited and can include electrodes of all sizes and geometries.

According to one embodiment, the electrode assembly 100 comprises at least one working electrode (102 or 104) that is integrated or otherwise coupled to the body. According to one embodiment, the microfluidic device may have a plurality of electrodes, including counter electrodes (101), reference electrodes (103), or working electrodes (102 and 104), configured to be in electrical communication with a detector, such as a potentiostat, for measuring current at the electrodes.

Further, the microfluidic device generally includes a membrane 120 in association with at least one of the integrated working electrodes (102 or 104), as shown generally in FIG. 3. Membranes, e.g., selectively permeable membranes (also referred to as“permselective” membranes) are commonly employed in association with electrodes, e.g., to allow certain molecules to pass through the membrane, while other molecules cannot pass through the membrane. In some embodiments, suitable membranes selectively allow small, nonpolar gaseous molecules to pass through, while having reduced permeability to larger or more polar molecules. The exact membrane employed can vary and can be any matenal suitable for association with an electrode that provides a benefit with respect to, e.g., selectivity, sensitivity, or decreased fouling of the surface. Protective and biofouling-resistant membranes can be incorporated to help prevent performance degradation with extended use and/or placement in proteinaceous media. Certain membranes that can be useful in association with one or more electrode in the disclosed assemblies 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 suitable.

In some embodiments, a permselective membrane is provided which is selectively permeable to nitric oxide and oxygen, while having reduced permeability 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 permselective 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 embodiments, the permselective membrane 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 membrane 120 can be a single material or, in some embodiments, can comprise a composite membrane comprising two or more components (e.g., two different membrane layers). The membrane may, in some embodiments, comprise a polysiloxane network wherein one or more silicon atoms in the polysiloxane network is covalently attached to an alkyl group and one or more silicon atoms in the polysiloxane network is covalently attached to a fluorinated alkyl group. In some embodiments, the polysiloxane network is a condensation product of a silane mixture comprising an alkylalkoxysilane and a fluorosilane. The chemical structure and the relative amounts of the silanes in the silane mixture can be varied to alter the biocompatibility, surface wettability and porosity characteristics of the polysiloxane network, depending upon the intended use of the device. Certain such materials (xerogels) that are suitable for inclusion within the microfluidic devices provided herein include those disclosed in Hunter el aί, Ahaί Chem. 2013, 85, 6066-6072 and U.S. Patent No. 8,551,322 to Schoenfisch el al., which are incorporated herein by reference in their entireties.

In one embodiment, the microfluidic device 10 includes a body, which may be a substantially planar body comprising a substantially planar substrate 20 and a substantially planar cap layer 30. In one embodiment, the substrate 20 and the cap layer 30 are bonded or otherwise coupled to one another. The substrate 20 and cap layer 30 may, in some embodiments, be disposed generally parallel to one another. In some embodiments, the body may have a rectangular cross section when viewed along its longitudinal axis. The substrate can comprise various materials, e.g., including, but not limited to, glass, paper, cellulose, fabric, polymers (e.g.,

polydimethylsiloxane, polyimide, polystyrene) or other suitable matenals known in the art.

The substrate 20 may further comprise an integrated reference electrode 103. According to one embodiment, the integrated reference electrode comprises silver/silver chloride. The substrate 20 may further comprise at least one integrated working electrode 102 and at least one integrated counter electrode 101. The substrate 20 may further optionally comprise an additional working electrode 104. It is noted that, although four electrodes are shown in device 10, the invention is not limited thereto, and devices and electrode assemblies provided according to the present disclosure can comprise various numbers of electrodes (e.g., from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8). The substrate layer comprising integrated electrodes 101, 102, 103, and 104 is shown independently in FIG. 2. In certain embodiments, the darkened electrodes (102 and 104) comprise a membrane coating, although the disclosure is not limited thereto.

In another embodiment, the cap layer may comprise a plurality of integrated electrodes, at least one of the plurality of integrated electrodes being an integrated working electrode and at least one of the plurality of integrated electrodes being an integrated counter electrode. The integrated working electrode 102 may be selected, e.g., from the group consisting of platinum, platinized platinum, tungsten, gold, carbon, carbon fiber and combinations thereof. The integrated counter electrode 101, according to one embodiment, comprises platinum As previously mentioned, the planar body of the microfluidic device may comprise a substrate 20 and a cap layer 30 and, can also, in some embodiments, include a fluidic well layer 40.

In certain embodiments, one or more microfluidic channels are defined within the body, such as a longitudinal channel defined between the substrate 20 and the cap layer 30. The channel is configured to guide or otherwise house a sample in the device. In one embodiment, the substrate 20, cap layer 30, and fluidic well layer 40 may cooperate to define the channel. In some embodiments, a material is included between the substrate 20 and the cap layer 30 with, e.g., a pair of raised tracks or an opening cut from a polymer of defined thickness disposed on the substrate 20 so as to define a microfluidic channel 25 there between. In the embodiment shown in FIG. 1, the microfluidic channel is directly provided within fluidic well layer 40.

According to one embodiment, the microfluidic channel 25 may have a channel width of about 2 to 3 mm and a channel height (as measured between the substrate and the cover glass) of about 250 to 1000 pm. Other embodiments may include a channel having a channel height and width suitable for measuring a gaseous species, such as NO, from the necessary flow of a sample comprising about 100 pL or less. One skilled in the art will appreciate a number of combinations of channel height, width, and cross sections exist to accomplish the necessary flow of such a sample. For example, the channel may have different cross sections, such as rectangular or a combination of planar and/or curved surfaces, which may depend on the sample to be analyzed.

According to one embodiment shown in FIG. 2, the cap layer may define an inlet aperture 26 and an outlet aperture 27, the inlet aperture being configured to accept a sample reservoir. As such, the microfluidic channel 25, the inlet aperture 26, the outlet aperture 27, and the sample reservoir may be configured to be in fluid communication with one another and the integrated electrode assembly. According to one embodiment, the microfluidic channel 25 may comprise a proximal end 28 and a distal end 29, wherein the fluid flow of the microfluidic sample travels from the proximal end to the distal end. Further, the fluid flow of the microfluidic sample may be oriented in a perpendicular fashion to the longitudinal onentation of the plurality of the integrated electrodes. As such, according to one embodiment, the flow of the microfluidic sample traverses each of the integrated working electrode, integrated reference electrode, and integrated counter electrode. The flow of the microfluidic sample may be encouraged by a number of methods known to those skilled in the art.

According to one embodiment, the sample may be engaged to flow from the inlet aperture to the outlet aperture by positive pressure applied to the sample at the inlet aperture by a peristaltic pump, a syringe pump, a pressure pump, or by gravity. In another embodiment, the flow of the microfluidic sample may be encouraged by applying negative pressure to the outlet aperture.

The present disclosure also provides a method for preparing an electrode assembly for use in such microfluidic devices, as well as in other sensors and, further provides a method for providing an

electrochemical sensor such as the microfluidic devices described herein above. As will be more fully detailed below, the particular order of certain steps of the disclosed method is important in some embodiments to obtain an electrochemical sensor that exhibits mechanical and performance integrity. In certain embodiments, the disclosed method comprises steps as outlined generally in method 200, depicted in FIG. 4. Although, in some embodiments, all such steps shown in method 200 are conducted in sequence, it is noted that in other embodiments, one or more of these steps may be eliminated. The method generally comprises step 210;

however, in various embodiments, the method can include any combination of two or more of steps 220 to 260, any combination of three or more of steps 220 to 260, any combination of four or more of steps 220 to 260, or all of steps 220 to 260. For example, certain embodiments provide a method comprising, in addition to step 210: steps 220 and 230; steps 220, 230, and 240; steps 220, 230, 240, and 250; steps 220, 230, and 250; steps 230, 240 (without“removing the mask,” as no mask is applied in this embodiment), and 250; and steps 230 and 250. All such methods further commonly further comprise step 260, although the disclosure is not limited thereto.

The method 200 first comprises preparing an electrode assembly on a substrate (such as substrate 20) (step 210). The exact construction, geometry, and dimensions of the substrate can vary widely.

Advantageously, in some embodiments, the substrate is substantially planar or planar. The electrode assembly, in some embodiments, comprises a silver electrode and a working electrode (where the working electrode can comprise any material, such as those referenced above).

The silver electrode is commonly in the form of a layer on the substrate and, although the thickness of the silver layer can vary, in some embodiments it may be about 0.5 to about 2 microns thick, e.g., about 1 micron thick. In some embodiments, providing a minimum thickness of the silver layer (e.g., about 0.5 microns or greater, about 0.6 microns or greater, about 0.7 microns or greater, about 0.8 microns or greater, or about 0.9 microns or greater) is important to ensure retention of the silver electrode after subsequent steps (e.g., as thinner silver layers may, in some embodiments, be affected by, e.g., step 240, and in particular, chlorination processes, as described below).

In preferred embodiments, the method further comprises applying a mask (step 220) to the electrode assembly to shield the silver electrode and its leads. The type of mask can vary, but can be, e.g., any photoresist mask sufficient to effectively shield the silver electrode and its leads while providing access to the working electrode(s) and, optionally, other components on the substrate. In certain preferred embodiments, the mask is provided so as to expose only the working electrode(s). In certain embodiments, the mask is an adhesive mask. The mask, in some embodiments, comprises an acid-resistant material and is effective to shield, e.g., the silver electrode from acidic conditions. Adhesive masks are generally known in the art and any material commonly used can be employed in the disclosed method.

Following application of the mask, the working electrode is cleaned (step 230). The exact method of cleaning can vary and can include, for example, physical cleaning, chemical cleaning, electrolytic cleaning, and the like, as well as combinations of two or more such cleaning techniques. In some embodiments, the cleaning comprises contacting the working electrode with an acid. Suitable acids include, but are not limited to, sulfuric acid, nitric acid, hydrochloride acid, and combinations of any two or more such acids. The pH and concentration of the acid solution used in such embodiments can vary. In one particular embodiment, the cleaning comprises contacting the working electrode with a 0.5 M sulfuric acid solution. In some

embodiments, cleaning step 230 comprises applying a potential to the working electrode, e.g., a programmed potential sequence. In one particular embodiment, the potential is cycled from about -0.4 to about 2 0 V versus an independent reference and an independent counter electrode.

Advantageously, cleaning step 230 provides the working electrode in a form that is substantially free of surface contamination. In some embodiments,“substantially free” includes“free” of surface contamination.

By“substantially free” of surface contamination is meant that adhered organics such as residual photoresist, solvents, oils and inorganics are substantially removed from the electrode surface to achieve a more stable surface chemistry as evidenced by overlap in the current vs. voltage traces in two subsequent cyclic voltammograms.

The mask is then removed from the substrate and the electrode assembly is treated to form a pseudoreference electrode (step 240). The pseudoreference electrode is generally formed by modification of the silver electrode, e g., by contacting the silver electrode with a chloride-containing solution to form an Ag/AgCl pseudoelectrode. The chloride-containing solution can vary and may be, for example, a ferric chloride solution or a sodium hypochlorite solution. This step can, in some embodiments, further comprise applying a voltage against the silver electrode. For example, the pseudoelectrode can, in some embodiments, be formed via anodization (wherein a voltage is applied against the silver electrode in a concentrated chloride containing solution.

Following formation of the pseudoreference electrode, one or more membranes can be deposited on at least a portion of the working electrode(s) (step 250). The one or more membranes, as outlined above, can provide various functions and can comprise various compositions. For example, in some embodiments, this step comprises electrodepositing one or more membranes. In some embodiments, this step comprises applying an electropolymerized film or electrodeposited inorganic compounds with metal centers. In certain embodiments, as referenced above, a composite membrane is formed on at least a portion of the working electrode, e.g., to give an electropolymerized component directly associated with (i.e., in direct contact with or in a working relationship with, but spaced apart therefrom) at least a portion of the working electrode and a sol- gel-derived component (e.g., a fluorinated xerogel) associated with at least a portion of the electropolymerized component.

In some embodiments, step 240 may comprise providing a physical mask on at least a portion of the substrate to shield components other than, e.g., the working electrode. In such embodiments, the mask employed is advantageously non-adhesive. Use of a non-adhesive mask at this step, where a mask is required, is important, as the inventors have found more inconsistent reference potentials resulting from use of adhesive masks on the pseudoelectrode during this step. Where a mask is employed in step 240, this step further comprises removing the mask from the electrode assembly after deposition of the one or more membranes on the working electrode.

In preferred embodiments, following deposition of the one or more membranes, the surface of the electrode remains substantially contaminant-free on at least a majority (e.g., all) of its surface (with the exception, in some embodiments, of the referenced one or more membranes associated therewith). Although the one or more membranes may, in some embodiments, be in direct contact with the electrode surface, the interface between the electrode surface and the one or more membranes is advantageously consistent, i.e., with substantially no contaminant(s) present there between.

The resulting finished electrode assembly is then assembled into a sensing device (“sensor”) (step 260) and, in particular, into a microfluidic sensor device. Assembly into such a sensor generally comprises providing a fluidic assembly and associating the fluidic assembly with the finished electrode assembly. Typically, the association is done via an adhesive, and types of adhesives for this purpose are known. See, e.g., Tsao, C.W. and DeVoe, D.L., 2009. Bonding of thermoplastic polymer micro fluidics. Microfluidics and Nanofluidic , 6(1), pp.1-16, which is incorporated herein by reference. The dimensions of the fluidic cell are generally selected so as to shield the leads from each electrode, such that the leads do not come into contact, during use, with a sample to be analyzed.

If the fluidic assembly is in place prior to step 250 (i.e., prior to deposition of the one or more membranes), the polymer comprising the fluidic assembly may absorb a portion of the material used to prepare the one or more membranes. Once absorbed, these materials may, e.g., introduce an electroactive leachate that compromises the sensor’s ability to detect and quantify analytes. As such, assembly after deposition of membranes (if used) is important to ensuring the efficacy of the resulting sensor device.

The resulting microfluidic device can be configured to detect and/or quantify a range of analytes in a sample. Samples of interest can include, but are not limited to, biological samples and environmental samples. “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-hydroxykynurenine, epinephrine, tyrosine, dihydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 3-hydroxyanthranilic acid, 1,7-dimethylxanthine, 5-hydroxytryptophan, 1,3-dimethylxanthine, 4- hydroxybenzoic acaid, 3-o-methyldopa, 5-hydroxymdole acetic acid, kynureinine, normetanephnne, dopamine, metanephrin, acetylserotonin, homovanilhc 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/or (+)-y-tocopherol.

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, as well as environmental 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 electrode assemblies provided herein are suitable, in some embodiments, for use within biosensors.

As such, the methods and sensors disclosed herein provide, e.g., 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 as disclosed herein, which comprises, in addition to the substrate, electrode assembly, and microfluidic channels 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.

Experimentals

Aspects of the present disclosure are more fully illustrated by the following examples, which are set forth to illustrate certain aspects of the present disclosure and are not to be construed as limiting thereof.

Electrochemical sensor bodies were prepared according to the methods outlined herein to evaluate the effect of various method steps disclosed herein. Certain results are disclosed and data is provided in FIGs. 5-7.

The data in FIG. 5 shows current versus voltage traces for two sets of subsequent cyclic

voltammagrams (-0.4 to 1.8 V vs. Ag/AgCl in 1 N H₂S0₄). As referenced above, comparing two subsequent current versus voltage traces taken for a given electrode provides information as to the surface of the electrode (i.e., how clean the electrode surface is). An electrode is considered to be“substantially clean” with stabilized surface chemistry where there is agreement between two traces for two subsequent cyclic voltammograms. FIG. 5A shows a sets of scans (Scan 1 and Scan 2) that are not in agreement (i.e., the electrode tested is not considered to be substantially free of surface contamination). FIG. 5B shows a set of scans (Scan 6 and Scan 7) that are in agreement (i.e., the current versus voltage traces in these scans substantially completely overlap).

The data in FIG. 6 shows a loss of conductivity of the silver electrode following exposure to acid during cleaning when a mask is not employed. This data thus demonstrates the importance of masking the silver electrode prior to contacting the sensor body with acid. Although not intending to be limited by theory, it is believed that the loss of conductivity is due to loss/dissolution of silver metal from the silver electrode upon contact with the acid.

The data in FIG. 7 shows that, when silver is chlorinated before it is masked (pre-mask), as opposed to being chlorinated after it is masked (post-mask), the resulting electrode exhibits a more variable reference electrode potential. This data thus demonstrates the importance of chlorinating the silver electrode after it is masked, rather than before it is masked.

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.

Claims

CLAIMS What is claimed is:

1. A method of preparing an electrochemical sensor body, comprising:

providing a sensor base comprising a working electrode and a silver electrode; and conducting three or more of steps a) to e):

a) applying a mask to a portion of the sensor base to shield the silver electrode, keeping the working electrode exposed;

b) cleaning the working electrode;

c) removing the mask from the sensor base;

d) contacting the sensor base with a chloride-containing component to convert the silver electrode to an Ag/AgCl pseudoreference electrode; and

e) depositing one or more membranes on the working electrode.

2 The method of claim 1, comprising steps a), b), c), and d).

3 The method of claim 1, comprising steps a), b), and e).

4 The method of claim 1, comprising steps b), d), and e).

5 The method of claim 1, further comprising attaching a fluidic cell to the electrochemical sensor body.

6 The method of claim 1, wherein the providing step comprises photolithography.

7 The method of claim 1, wherein the silver electrode has a thickness of at least about 1 micron.

8 The method of claim 1, wherein the working electrode comprises a platinum electrode.

9 The method of claim 1, wherein the working electrode comprises a gold electrode, a carbon fiber electrode, or a palladium electrode.

10 The method of claim 1, wherein the mask in step a) is adhesive.

11. The method of claim 1, wherein the cleaning in step b) comprises electrochemical cleaning.

12. The method of claim 1, wherein the cleaning in step b) comprises chemical cleaning, physical cleaning, electrolytic cleaning, or a combination of two or more.

13. The method of claim 1, wherein the cleaning in step b) comprises contacting the working

electrode with an acid.

14. The method of claim 13, wherein the acid comprises a sulfuric acid solution, a nitric acid

solution, a hydrochloric acid solution or a combination of two or more.

15. The method of claim 13, wherein the acid is a sulfuric acid solution.

16. The method of any of claims 13-15, wherein the cleaning further comprises applying a potential to the working electrode.

17. The method of claim 16, wherein the potential is cycled from about -0.4 to 2.0 V versus an

independent reference electrode and an independent counter electrode.

18. The method of claim 1, wherein the contacting in step d) comprises anodization.

19. The method of claim 1, wherein the chloride-containing component in step d) is a solution of ferric chloride.

20. The method of claim 1, wherein the depositing in step e) is electrochemical depositing.

21. The method of claim 1, wherein the one or more membranes deposited in step e) comprise one or more of a membrane to enhance selectivity over interferents, a membrane to enhance sensitivity to analytes of interest, and a membrane to mitigate electrode fouling.

22. The method of any of claims 1-21, wherein the working electrode is substantially free of surface contamination prior to step e).

23. An electrochemical sensor body prepared according to the method of any of claims 1-22.

24. A microfluidic device for measuring an amount of an analyte, comprising the electrochemical sensor body of claim 23.

25. A microfluidic device for measuring an amount of an analyte, comprising: a substantially planar body;

an electrode assembly comprising a working electrode and reference electrode, wherein the reference electrode comprises silver and

wherein the reference electrode is prepared from silver metal; and at least one gas-permeable membrane disposed on and coating at least a portion of the working electrode,

wherein the working electrode is substantially free of adhered organic or inorganic non-electrode contaminants prior to membrane deposition.