Colloid with nanoporous structure, and device and system for non-enzymatic glucose sensing

Abstract

To provide a device and system for sensing non-enzymatic glucose.SOLUTION: This disclosure relates to a colloid composition containing a number of clusters of nanoparticles dispersed in a liquid, a nanoporous layer formed of the colloid, a glucose-oxidation electrode including the nanoporous layer, and a glucose-sensing device, apparatus and system including the glucose-oxidation electrode. This disclosure also relates to a method of producing the colloid composition, nanoporous layer, glucose-oxidation electrode, and glucose-sensing device and system. Further, this disclosure also relates to devices, systems and methods for continuous glucose monitoring (CGM) and blood glucose monitoring (BGM).SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] The present disclosure relates to glucose sensing. [Background technology]

[0002] There is a high level of interest in the healthcare community and industry to improve techniques for sensing and monitoring blood glucose levels. Today, most glucose sensors use electrochemical methods. Most, if not all, electrochemical sensors use enzyme-based electrochemical sensors. Summary of the Invention

[0003] One embodiment of the invention provides a colloidal composition comprising many clusters of nanoparticles dispersed in a liquid, each cluster comprising many nanoparticles clustered together to form irregularly shaped bodies having nano- or micro-sized lengths, each nanoparticle having a separate, generally ovoid or spherical shape with a diameter of about 2 nm to about 5 nm, and interparticular gaps formed between adjacent nanoparticles inside each cluster, with an interparticular gap distance of about 0.5 nm to about 2 nm.

[0004] In the colloid composition, the interparticle gaps may be distributed throughout each cluster. The composition may be substantially free of surfactants. The liquid may include water, and the colloid composition may include a surfactant in an amount of less than 2 parts by weight per 100 parts by weight of nanoparticles contained therein. The nanoparticles contained in the colloid composition may be in an amount of about 0.01 wt% to about 2 wt% based on the total weight of the colloid composition. The nanoparticles contained in the colloid composition may be in an amount of about 0.01 wt% to about 1 wt% based on the total weight of the colloid composition.

[0005] In the colloidal composition, the nanoparticles may be primarily made of at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the foregoing elements. The nanoparticles may be primarily made of platinum (Pt), and the interparticle gaps may be distributed throughout each cluster. The colloidal composition may contain a surfactant in an amount of less than 1 part by weight per 100 parts by weight of nanoparticles contained therein. The amount of nanoparticles contained in the colloidal composition may be about 0.1 wt% to about 1 wt% based on the total weight of the colloidal composition.

[0006] Another aspect of the invention provides a method for producing a nanoporous layer, comprising dispensing the aforementioned colloidal composition onto a substrate; drying the dispensed colloidal composition so that clusters contained in the dispensed composition are deposited on the substrate and stacked on top of each other, thereby providing a nanoporous layer on the substrate, the nanoporous layer comprising irregularly shaped bodies formed from the stacked clusters, the irregularly shaped bodies comprising many nanoparticles clustered together locally and interparticle gaps formed between adjacent ones of the nanoparticles in the irregularly shaped bodies, the irregularly shaped bodies being interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies, and irregularly shaped spaces formed between adjacent portions of the irregularly shaped bodies, which are nano- or micro-sized.

[0007] In the above method, the nanoparticles may be generally oval or spherical in shape with a diameter of about 2 nm to about 5 nm. The interparticle gaps may have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces. The colloidal composition may be dispensed in a predetermined amount to form a nanoporous layer having a roughness factor of about 100 to about 2500. The nanoporous layer may contain a surfactant in an amount of less than 0.5 parts by weight per 100 parts by weight of nanoparticles contained therein.

[0008] Another aspect of the invention provides a method for producing a colloidal composition, comprising: providing a liquid composition comprising metal ions, a surfactant, and a solvent, wherein the surfactant is in a reverse micelle phase defining a hydrophilic space; adding a reducing agent to the liquid composition to cause reduction of the metal ions, thereby forming a first colloid comprising metal nanoparticles and the surfactant, wherein the metal nanoparticles are dispersed in the first colloid together with the reverse micelle phase of the surfactant; and removing the surfactant from the first colloid to provide a second colloid comprising many clusters dispersed in a liquid, each cluster comprising many nanoparticles clustered together to form irregularly shaped bodies having nano- or micro-sized lengths.

[0009] In the aforementioned manufacturing method, no electric potential may be applied to the liquid composition to reduce the metal ions therein. The surfactant may be a nonionic surfactant capable of forming an isotropic reverse micelle phase. The individual nanoparticles may have separate bodies of roughly ovoid or spherical shape with diameters of about 2 nm to about 5 nm, and interparticle gaps may be formed between adjacent nanoparticles inside each cluster, with an interparticle gap distance of about 0.5 nm to about 2 nm. Removing the surfactant removes a significant amount of surfactant from the first colloid, rendering the second colloid substantially free of surfactant. Removing the surfactant removes a significant amount of surfactant from the first colloid, rendering the second colloid containing less than 1 part by weight of surfactant per 100 parts by weight of nanoparticles contained therein.

[0010] In the aforementioned manufacturing method, removing the surfactant may further include centrifuging the first colloid and collecting the bottoms from the centrifuged composition. Removing the surfactant may further include repeating the series of centrifugation and collection multiple times. Removing the surfactant may further include adding an acid or base to the first colloid before centrifugation. Removing the surfactant may further include repeating the series of addition, centrifugation, and collection multiple times. The nanoparticles contained in the second colloid may be in an amount of about 10 wt% to about 40 wt% based on the total weight of the composition. The nanoparticles may be primarily made of at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the aforementioned metals. The nanoparticles may be primarily made of platinum (Pt), the interparticle gaps may be distributed generally throughout each cluster, the composition may contain a surfactant in an amount of less than 2 parts by weight per 100 parts by weight of nanoparticles contained therein, and the nanoparticles contained in the composition may be in an amount of about 0.1 wt % to about 2 wt % based on the total weight of the composition.

[0011] Another aspect of the invention provides a method for producing a nanoporous layer, comprising: applying the aforementioned method for producing a colloidal composition to provide a second colloid; dispensing the second colloid onto a substrate; and drying the dispensed second colloid so that clusters contained in the dispensed composition are deposited on the substrate and stacked one on top of the other, providing a nanoporous layer on the substrate, the nanoporous layer comprising irregularly shaped bodies formed from the stacked clusters, the irregularly shaped bodies comprising many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies. The irregularly shaped bodies are interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies, and the irregularly shaped spaces, nano- or micro-sized, are formed between adjacent portions of the irregularly shaped bodies, and the irregularly shaped spaces are interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces.

[0012] In the aforementioned method for producing a nanoporous layer, the nanoparticles may be generally oval or spherical with a diameter of about 2 nm to about 5 nm, and the interparticle gap distance may be about 0.5 nm to about 2 nm. The colloidal composition may be dispensed in a predetermined amount to form a nanoporous layer having a roughness factor of about 100 to about 2500. The nanoporous layer may contain less than 0.1 parts by weight of surfactant per 100 parts by weight of nanoparticles contained therein.

[0013] Another aspect of the invention provides a nanoporous structure comprising: irregularly shaped bodies comprising a number of nanoparticles clustered together locally and interparticle gaps formed between adjacent ones of the nanoparticles in the irregularly shaped bodies, wherein the nanoparticles can be generally ovoid or spherical with diameters of about 2 nm to about 5 nm, the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm, the irregularly shaped bodies can be interconnected to provide a three-dimensional interconnected network of the irregularly shaped bodies, and the irregularly shaped spaces formed between adjacent portions of the irregularly shaped bodies are nano-sized or micro-sized, and the irregularly shaped spaces are interconnected to provide a three-dimensional interconnected network of the irregularly shaped spaces.

[0014] The nanoporous structure can be substantially free of surfactant molecules. In the nanoporous structure, the interparticle gaps can be substantially free of nanosized organic molecules. The three-dimensional network of irregularly shaped bodies and the three-dimensional network of gaps between irregularly shaped clusters can be complementary, forming a nanoporous structure. The interparticle gaps can be substantially interconnected among themselves and can be further connected to the three-dimensional interconnected network of gaps between irregularly shaped clusters. The nanoporous structure can be formed by dispensing a solid-liquid colloid containing discrete clusters of irregularly shaped bodies dispersed in a liquid and drying the dispensed solid-liquid colloid, allowing the discrete clusters of irregularly shaped bodies to stack, providing a three-dimensional interconnected network of irregularly shaped bodies and a three-dimensional interconnected network of gaps between irregularly shaped clusters. The gaps between the irregularly shaped clusters have an average intercluster gap distance. The nanoparticles may be made from at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the foregoing metals. The nanoporous structure has a roughness factor of about 100 to about 2500.

[0015] Another aspect of the invention provides a device comprising: a substrate comprising a surface; and a nanoporous layer formed on the surface, the nanoporous layer comprising the nanoporous structure described above. Yet another aspect of the invention provides a non-enzymatic glucose-sensing electrode comprising at least one conductive layer comprising a surface; and a nanoporous layer formed on the surface, the nanoporous layer comprising the nanoporous structure described above, wherein the non-enzymatic glucose-sensing electrode does not comprise a glucose-specific enzyme.

[0016] In the aforementioned device or electrode, the at least one conductive layer may include a conductive metal layer and a conductive carbon layer formed on the conductive metal layer. The device or electrode does not include a biocompatible polymer material formed on a nanoporous layer. The device or electrode may include a biocompatible polymer material formed on a nanoporous layer.

[0017] Yet another aspect of the invention provides a single-use glucose sensing device comprising: a reservoir configured to receive and hold a test solution; and the aforementioned electrodes arranged with the reservoir such that the nanoporous layer can contact the test solution when the test solution can be held in the reservoir. In the single-use glucose sensing device, the electrodes do not comprise a biocompatible polymer material formed on the nanoporous layer.

[0018] Yet another aspect of the invention provides a continuous glucose monitoring (CGM) device comprising: a hypodermic needle configured to contact interstitial fluid in a subject's body; and an electrical circuit connected to the hypodermic needle, wherein the hypodermic needle includes the aforementioned electrode and another electrode connected to the electrical circuit.

[0019] Yet another aspect of the invention provides a non-enzymatic glucose sensing device comprising a working electrode comprising a substrate and a nanoporous layer formed on the substrate, wherein the working electrode does not comprise a glucose-specific enzyme, the nanoporous layer may comprise irregularly shaped bodies comprising many nanoparticles clustered together locally, interparticle gaps may be formed between adjacent ones of the nanoparticles in the irregularly shaped bodies, the nanoparticles may be generally ovoid or spherical with diameters of about 2 nm to about 5 nm, the interparticle gaps having an interparticle gap distance of about 0.5 nm to about 2 nm, and the irregularly shaped bodies may be spaced apart from each other. to provide a three-dimensional interconnected network of irregularly shaped bodies extending substantially throughout the nanoporous layer, the irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and may be nano-sized or micro-sized, and the irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces extending substantially throughout the nanoporous layer, and the nanoporous layer may be configured to cause oxidation of glucose molecules therein in the absence of glucose-specific enzymes at a bias voltage applied thereto of about 0.2 V to about 0.45 V.

[0020] In the non-enzymatic glucose sensing device described above, the nanoporous layer can be substantially free of surfactant molecules, and the substrate can include at least one conductive layer comprising a conductive or semiconductive material. The interparticle gaps can be substantially free of nanosized organic molecules. The three-dimensional network of irregularly shaped bodies and the three-dimensional network of irregularly shaped intercluster gaps can be complementary, forming a nanoporous layer. The interparticle gaps can be substantially interconnected with each other and can be further connected to the three-dimensional interconnected network of irregularly shaped intercluster gaps.

[0021] In the non-enzymatic glucose sensing device described above, the nanoporous layer can be formed by dispensing a solid-liquid colloid containing discrete clusters of irregularly shaped bodies dispersed in a liquid and drying the dispensed solid-liquid colloid, allowing the discrete clusters to stack, providing a three-dimensional interconnected network of irregularly shaped bodies and a three-dimensional interconnected network of gaps between the clusters. The nanoparticles can be made from at least one metal selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the foregoing metals. The nanoporous layer has a roughness factor of about 100 to about 2500. The nanoporous electrode may further include a maltose-blocking layer formed on the nanoporous layer and configured to substantially block the passage of maltose contained in the test fluid therethrough and allow the passage of glucose therethrough. The maltose-blocking layer may include poly-phenylenediamine (poly-PD) in a form that allows the passage of glucose molecules therethrough and effectively blocks the passage of maltose molecules therethrough. The bias voltage may be set to be in the range of 0.2 V to 0.45 V.

[0022] Yet another aspect of the invention provides a non-enzymatic glucose sensing system comprising: the non-enzymatic glucose sensing device described above; a counter electrode; and a bias voltage source electrically connected between the working electrode and the counter electrode for providing a bias voltage between the working electrode and the counter electrode.

[0023] Yet another aspect of the invention provides a method for non-enzymatic glucose sensing. The method includes: providing the non-enzymatic glucose sensing device described above; contacting a test fluid with both the working electrode and the counter electrode while applying a bias voltage between the working electrode and the counter electrode, thereby causing oxidation of glucose contained in the test fluid at the nanoporous layer; measuring a current from the working electrode; and processing the current, with or without additional data, to provide a glucose level corresponding to the glucose contained in the test fluid. The bias voltage can be set to be in the range of 0.2 V to 0.45 V.

[0024] Another aspect of the invention provides a glucose-sensing electrode, comprising: a substrate; a nanoporous metal layer formed on the substrate and capable of oxidizing both glucose and maltose without glucose- or maltose-specific enzymes within the glucose-sensing electrode; and a maltose-blocking layer formed on the nanoporous metal layer. In the glucose-sensing electrode, the maltose-blocking layer has a porosity that allows glucose to pass therethrough and blocks maltose from passing therethrough toward the nanoporous metal layer, such that when a bias voltage of 0.2-0.45 V is applied to the nanoporous metal layer relative to a reference electrode, and when the maltose-blocking layer is in contact with a liquid containing glucose at a concentration of 4-20 mM and maltose at a concentration of 4-20 mM, the current caused by the oxidation of glucose alone in the nanoporous metal layer is 10 nA / mMcm. 2 The current induced by the oxidation of maltose alone in the nanoporous metal layer was 5 nA / mMcm 2 It will be lower.

[0025] In the glucose-sensing electrode described above, the nanoporous metal layer can oxidize glucose, so that the current caused by the oxidation of glucose alone is 10 nA / mMcm when it is in contact with a liquid containing glucose at a concentration of 4-20 mM and without a maltose-blocking layer on top, applying a bias voltage of 0.2-0.45 V. 2The nanoporous metal layer can also oxidize maltose, so that the current caused by the oxidation of maltose alone is 10 nA / mMcm when it is in contact with a liquid containing maltose at a concentration of 4-20 mM at a bias voltage of 0.2-0.45 V and without a maltose-blocking layer on top. 2 The maltose-blocking layer may comprise polyphenylenediamine (poly-PD) and have a thickness of 10 to 40 nm. The maltose-blocking layer may consist essentially of polyphenylenediamine (poly-PD) and have a thickness of 10 to 35 nm. The maltose-blocking layer may consist of polyphenylenediamine (poly-PD) and have a thickness of 10 to 40 nm.

[0026] In the glucose sensing electrode described above, the nanoporous metal layer may include irregularly shaped bodies including many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies. Here, the nanoparticles are generally oval or spherical in shape with diameters of about 2 nm to about 5 nm. The interparticle gaps may have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies may be interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies. The irregularly shaped spaces may be nano-sized or micro-sized and may be formed between adjacent portions of the irregularly shaped bodies. The irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces.

[0027] The glucose sensing electrode may further include an electrolyte ion-blocking layer formed on the maltose-blocking layer and a biocompatible layer formed on the electrolyte ion-blocking layer. The electrolyte ion-blocking layer may block Na ions contained in the liquid. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2-from diffusing toward the nanoporous metal layer, so that Na is blocked between above and below the electrolyte ion-blocking layer. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- There is a substantial discontinuity in the total concentration of . The electrolyte ion-blocking layer can facilitate conditioning of the glucose-sensing electrode such that conditioning is completed within 30 minutes of contact with the subject's body fluid using an applied bias voltage of 0.2-0.45 V.

[0028] Another aspect of the invention provides an apparatus including a single, integrated body including a subcutaneous portion and a terminal portion; the subcutaneous portion including the aforementioned glucose sensing electrode and reference electrode, each of which is exposed to contact with interstitial fluid of a first subject when the subcutaneous portion is subcutaneously inserted into the body of the first subject; and the terminal portion is configured to mate with a corresponding device and include a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode.

[0029] Yet another aspect of the invention provides a device including a single, integral body including the glucose sensing electrode and reference electrode described above, the single, integral body further including a reservoir configured to at least temporarily hold a test fluid therein, the glucose sensing electrode and the reference electrode being arranged within the single, integral body such that when the test fluid is held in the reservoir, each of the glucose sensing electrode and the reference electrode is configured to contact the test fluid.

[0030] A further aspect of the invention provides a method for fabricating a glucose-sensing electrode, comprising: providing a nanoporous metal layer capable of oxidizing both glucose and maltose without the presence of glucose- or maltose-specific enzymes within the glucose-sensing electrode; and forming a poly-phenylenediamine (poly-PD) film on a nanoporous platinum layer, the poly-PD film allowing glucose to pass therethrough and blocking maltose from passing therethrough. The poly-PD film has a porosity that allows glucose to pass therethrough and blocks maltose from passing therethrough toward the nanoporous metal layer, such that when a bias voltage of 0.2-0.45 V is applied to the nanoporous metal layer relative to a reference electrode, and when the poly-PD film is in contact with a liquid containing glucose at a concentration of 4-20 mM and maltose at a concentration of 4-20 mM, the current caused by the oxidation of glucose alone at the nanoporous metal layer is 10 nA / mMcm. 2 Furthermore, the current induced by the oxidation of maltose alone in the nanoporous metal layer was 5 nA / mMcm 2 Lower.

[0031] In the aforementioned method of fabricating a glucose-sensing electrode, forming a poly-PD film can include performing electrochemical polymerization using a nanoporous metal layer as an electrode for electrochemical polymerization. Forming a poly-PD film can include providing a polymer layer containing poly-PD, and the polymer layer can have sufficient porosity to allow glucose to pass therethrough, such that the current caused by the oxidation of glucose alone in the nanoporous metal layer is less than 10 nA / mMcm. 2If the porosity of the polymer layer is lower, the method may include adjusting the porosity of the polymer layer. Adjusting the porosity may include subjecting the polymer layer to at least one electric shock while the polymer layer is in contact with an acidic solution. Forming the poly-PD film may include polymerizing poly-PD from a liquid composition including phenylenediamine at a concentration, and if the concentration is higher than a predetermined value, forming the poly-PD film further includes adjusting the porosity of the polymer layer. Adjusting the porosity may include subjecting the polymer layer to at least one electric shock while the polymer layer is in contact with an acidic solution.

[0032] In the aforementioned method of fabricating a glucose-sensing electrode, forming a poly-PD film allows the polymer layer to have sufficient porosity to allow glucose to pass through it, so that the current caused by the oxidation of glucose alone in the nanoporous metal layer is less than 10 nA / mMcm. 2 If the porosity is expected to be higher, the method may include providing a polymer layer comprising poly-PD without further adjusting the porosity of the polymer layer. Forming the poly-PD film may include polymerizing poly-PD from a liquid composition comprising phenylenediamine at a concentration, and if the concentration is lower than a predetermined value, the method does not include adjusting the porosity of the polymer layer to form the poly-PD film.

[0033] One embodiment of the invention provides a glucose-sensing electrode, which includes: a conductive layer; a nanoporous metal layer formed on the conductive layer; an electrolyte ion-blocking layer formed on the nanoporous metal layer; and a biocompatible layer formed on the electrolyte ion-blocking layer. The glucose-sensing electrode does not contain a glucose-specific enzyme. Glucose, Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- When the electrolyte ion-blocking layer comes into contact with a liquid containing Na + , K. + , Ca 2+, Cl - , PO4 3- and CO3 2- from diffusing toward the nanoporous metal layer, so that Na is blocked between above and below the electrolyte ion-blocking layer. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- There is a substantial discontinuity in the total concentration of

[0034] In the glucose sensing electrode described above, when a bias voltage of 0.2-0.45 V relative to a reference electrode is applied to the electrode, the electrode is configured to cause oxidation of glucose in the nanoporous metal layer and generate a current that is the sum of a glucose-oxidation current caused by glucose oxidation alone and a background current caused by other electrochemical interactions between the liquid and the glucose sensing electrode. When the liquid contains glucose at a concentration of 4-20 mM (approximately 72-360 mg / dL), the glucose-oxidation current at steady state is 0.1 μA / mMcm. 2 (10nA / mMcm 2 ) at a higher level.

[0035] In the glucose sensing electrode described above, the total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 10% of the total concentration above the electrolyte ion-blocking layer. The total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 5% of the total concentration above the electrolyte ion-blocking layer. The electrolyte ion-blocking layer prevents the passage of Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- The membrane may include a porous and hydrophobic polymer layer configured to restrict the mobility of the membrane but not the glucose molecules therethrough.

[0036] In the aforementioned glucose sensing electrode, the electrolyte ion-blocking layer can include at least one selected from the group consisting of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). The electrolyte ion-blocking layer can comprise at least one selected from the group consisting of copolymers of methyl methacrylate and butyl methacrylate and polymers obtained from the polymerization of one or more monomers including branched or unbranched C1-C8 alkyl methacrylates, branched or unbranched C1-C8 cycloalkyl methacrylates, branched or unbranched C1-C8 alkyl acrylates, branched or unbranched C1-C8 cycloalkyl acrylates, and branched or unbranched C1-C8 cycloalkyl methacrylates, wherein the one or more monomers are selected from the group consisting of methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, and 2-ethylhexyl acrylate.

[0037] In the glucose sensing electrode described above, the glucose sensing electrode may be a continuous glucose monitoring (CGM) electrode, and the liquid is a body fluid of the subject. The electrolyte ion-blocking layer is configured to promote conditioning of the glucose sensing electrode such that conditioning of the glucose sensing electrode is completed within 30 minutes of contact with the body fluid of the subject using an applied bias voltage of 0.2-0.45 V. Conditioning of the glucose sensing electrode may be considered complete when the rate of decrease in current becomes less than a first predetermined value and / or when the current remains less than a second predetermined value.

[0038] The glucose-sensing electrode can further include a maltose-blocking layer interposed between the nanoporous metal layer and the electrolyte ion-blocking layer, and the maltose-blocking layer can include poly-phenylenediamine (poly-PD). The maltose-blocking layer can be configured to allow glucose to pass therethrough and substantially block maltose from passing therethrough, such that at steady state, the glucose-oxidation current is greater than or equal to 0.1 μA / mMcm. 2 (10nA / mMcm 2 ), whereas the maltose oxidation current caused by maltose oxidation alone was 0.05 μA / mMcm 2 (5nA / mMcm 2 ) lower.

[0039] The reference electrode can be configured to provide a reference level of potential for the bias voltage applied to the glucose sensing electrode, regardless of whether reduction of the chemical component occurs at the reference electrode. In a three-electrode electrochemical cell, in addition to the reference electrode, a counter electrode is provided for reduction of the chemical component therein, while in a two-electrode electrochemical cell, reduction of the chemical component occurs at the reference electrode.

[0040] In the glucose sensing electrode described above, the nanoporous metal layer can include irregularly shaped bodies including many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies, where the nanoparticles are generally ovoid or spherical with diameters of about 2 nm to about 5 nm, and the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies can be interconnected to provide a three-dimensional interconnected network of the irregularly shaped bodies. The irregularly shaped spaces can be nano-sized or micro-sized and can be formed between adjacent portions of the irregularly shaped bodies, where the irregularly shaped spaces are interconnected to provide a three-dimensional interconnected network of the irregularly shaped spaces.

[0041] Another aspect of the invention provides a sensor device that includes: a single, integral body including a subcutaneous portion and a terminal portion; a subcutaneous portion including a glucose sensing electrode and a reference electrode, each of which is exposed to contact with interstitial fluid of a first subject when the subcutaneous portion is subcutaneously inserted into the body of the first subject; and a terminal portion that is coupled to a corresponding device and configured to include a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode. The glucose sensing electrode may include one or more features of the glucose sensing electrodes described above.

[0042] Another aspect of the invention provides a method for continuous glucose monitoring. The method includes: providing a sensor device; subcutaneously inserting a subcutaneous portion of a glucose sensing electrode into a first subject's body so that the glucose sensing electrode and reference electrode contact interstitial fluid in the first subject's body; applying a bias voltage of 0.2-0.45 V to the glucose sensing electrode relative to the reference electrode; measuring a current generated by the glucose sensing electrode; calculating a glucose level using the current value obtained by measuring the current within less than one hour after subcutaneous insertion of the subcutaneous portion and application of the bias voltage; and presenting on a display the calculated glucose level for the first subject within a range of about 4 mM to about 20 mM (approximately about 72 mg / dL to about 360 mg / dL). The glucose sensing electrode can include one or more features of the glucose sensing electrodes described above.

[0043] A further aspect of the invention provides a sensor device that includes: a substrate; a first electrode (or glucose-sensing electrode) including a first conductive layer formed on the substrate and a glucose-oxidation layer formed on the first conductive layer; a first terminal formed on the substrate and electrically connected to the first electrode; a second electrode including a second conductive layer formed on the substrate; a second terminal formed on the substrate and electrically connected to the second electrode; a reference electrode including a third conductive layer formed on the substrate; and a third terminal formed on the substrate and electrically connected to the reference electrode.

[0044] In the sensor device, when the first electrode is contacted with a liquid containing glucose, ascorbic acid, and acetaminophen, and a first bias voltage sufficient to oxidize glucose in the glucose-oxidation layer is applied between the first electrode and the reference electrode, the glucose-oxidation layer of the first electrode is configured to cause oxidation of glucose and at least one of ascorbic acid and acetaminophen therein, and is further configured to generate a first current containing a glucose component caused by glucose oxidation and a first interference component caused by the oxidation of at least one of ascorbic acid and acetaminophen in the glucose-oxidation layer. A second electrode is arranged within the device so that when the first electrode is contacted with the liquid, the second electrode is also contacted with the same liquid. The second electrode does not include a layer configured to cause oxidation of glucose therein, such that application of a second bias voltage between the second electrode and the reference electrode causes oxidation of at least one of ascorbic acid and acetaminophen therein, but not oxidation of glucose therein, and is further configured to generate a second current at the second electrode that includes a second interfering component caused by the oxidation of at least one of ascorbic acid and acetaminophen but not caused by oxidation of glucose. The device is configured to provide a first current at a first terminal and a second current at a second terminal.

[0045] The sensor device described above may be configured to provide a second current associated with a first current when providing the first current. The sensor device may be configured to generate the first current and the second current simultaneously. The sensor device may be configured to provide the first current and the second current together with information indicating the time at which the first current and the second current were generated. The sensor device may be configured to provide the second current together with the first current whenever the first current is provided. In the sensor device described above, the first current may further include a first background current caused by other electrochemical interactions between the liquid and the glucose sensing layer, and the second current may further include a second background current caused by other electrochemical interactions between the liquid and the second electrode.

[0046] In the sensor device described above, when the first bias voltage is 0.2 V to 0.32 V, the glucose-oxidation layer is configured to oxidize glucose and ascorbic acid but not acetaminophen, and the first interfering component is caused by the oxidation of ascorbic acid, not acetaminophen. When the second bias voltage is 0.2 V to 0.32 V, the second electrode is configured to oxidize ascorbic acid but not acetaminophen, and the second interfering component is caused by the oxidation of ascorbic acid, not acetaminophen. In the sensor device described above, when the first bias voltage is 0.34 V to 0.45 V, the glucose-oxidation layer is configured to oxidize glucose, ascorbic acid, and acetaminophen, and the first interfering component is caused by the oxidation of ascorbic acid and acetaminophen. When the second bias voltage is between 0.34 V and 0.45 V, the second electrode is configured to oxidize ascorbic acid and acetaminophen, and the second interfering component is caused by the oxidation of both ascorbic acid and acetaminophen.

[0047] In the sensor device described above, the first electrode may further include a maltose-blocking layer comprising polyphenylenediamine (poly-PD) formed on the glucose-oxidation layer. When contacted with a liquid containing glucose having a concentration of 4-20 mM (approximately 72-360 mg / dL) and when a bias voltage is applied, the maltose-blocking layer is configured to allow glucose to pass therethrough and substantially block maltose from passing therethrough, such that at steady state, the glucose-oxidation current is 0.1 μA / mMcm. 2 (10nA / mMcm 2 ), whereas the maltose oxidation current caused by maltose oxidation alone was 0.05 μA / mMcm 2 (5nA / mMcm 2 ) lower.

[0048] The sensor device may be a continuous glucose monitoring (CGM) electrode module including a subcutaneous portion configured to subcutaneously contact a body fluid of a subject, and the first, second, and reference electrodes are formed within the subcutaneous portion. In the sensor device, the glucose-oxidation layer may include a nanoporous metal layer, and the first electrode may further include an electrolyte ion-blocking layer formed on the nanoporous metal layer and a biocompatible layer formed on the electrolyte ion-blocking layer. The electrolyte ion-blocking layer may be configured to block Na ions contained in the fluid. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- from diffusing toward the nanoporous metal layer, so that Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- There is a substantial discontinuity in the total concentration of

[0049] In the sensor device described above, the electrolyte ion-blocking layer is formed to prevent the passage of Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- The electrolyte ion-blocking layer may comprise a porous and hydrophobic polymer layer configured to restrict the mobility of electrolyte ions but not restrict the mobility of glucose molecules therethrough, and the electrolyte ion-blocking layer may comprise at least one selected from the group consisting of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

[0050] In the above-described sensor device, the electrolyte ion-blocking layer may be configured to facilitate conditioning of the glucose sensing electrode such that conditioning is completed within 30 minutes of contact with the subject's body fluid using an applied bias voltage of 0.2-0.45 V, and conditioning of the glucose sensing electrode is considered complete when the rate of decrease in current becomes less than a first predetermined value and / or when the current remains less than a second predetermined value.

[0051] The sensor device is a blood glucose monitoring (BGM) electrode module including a reservoir configured to receive blood, where the first electrode, the second electrode, and the reference electrode are configured to contact the blood when the blood is received in the reservoir. The first bias voltage is 0.2 V to 0.45 V, and the second bias voltage is the same as or different from the first bias voltage. The glucose oxidation layer can include a nanoporous metal material or a glucose-specific enzyme configured to oxidize glucose. The glucose oxidation layer can include irregularly shaped bodies including many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies, where the nanoparticles are generally ovoid or spherical with diameters of about 2 nm to about 5 nm, and the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies can be interconnected to provide a three-dimensional interconnected network of the irregularly shaped bodies. The irregularly shaped spaces can be formed between adjacent portions of the irregularly shaped bodies and can be nano- or micro-sized, and the irregularly shaped spaces can be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces.

[0052] Yet another aspect of the invention provides a system including the aforementioned sensor device further including a terminal section having an arrangement of first, second, and third terminals; a corresponding device including a first corresponding terminal, a second corresponding terminal, and a third corresponding terminal, a circuit network, and a power source connected to the circuit network, the corresponding device further including a corresponding terminal section configured to couple or engage with the terminal section. Here, the first, second, and third corresponding terminals are arranged within the corresponding terminal section such that, when the terminal section of the sensor device and the corresponding terminal section of the corresponding device are coupled or engaged, the first terminal electrically connects to the first corresponding terminal, the second terminal electrically connects to the second corresponding terminal, and the third terminal electrically connects to the third corresponding terminal. The circuit network of the corresponding device is configured to provide a first bias voltage between the first and third corresponding terminals, and the circuit network of the corresponding device is further configured to provide a second bias voltage between the second and third corresponding terminals.

[0053] In the above-described system, the corresponding device may include a wireless communication module configured to wirelessly communicate with a wirelessly paired computing device, the wireless communication module including at least one processor and at least one memory. The corresponding device may be configured to receive a first current at a first corresponding terminal and a second current at a second corresponding terminal. The corresponding device may be configured to transmit a second current together with or associated with the first current when transmitting the first current. The first current may be transmitted with a first timestamp, and the second current may be transmitted with a second timestamp, the first and second timestamps indicating the same time.

[0054] The above-described system may further include software installed and executable by at least one processor of the wirelessly paired computing device, the software being configured, when executed, to perform a method including: storing, in at least one memory of the computing device, a first current and a second current received together or in association with each other from a corresponding device; processing the first current and the second current to provide a value indicative of glucose oxidation in a glucose-oxidation layer of a first electrode of the sensor device; and presenting the value or corresponding information on a display of the computing device.

[0055] In the above-described system, either or both of the first current and the second current may be in the form of a continuous signal, and processing the first current and the second current may include processing values of the first current and the second current obtained simultaneously. Here, processing the values may include subtracting the second current from the first current. The first current and the second current may be stored in association with each other in at least one memory. The above-described system may further include software installed and executable on the wirelessly paired computing device. When executed, the software is configured to perform data processing using the first current and the second current received from the corresponding device to obtain a glucose level contained in a liquid contacted by the first electrode of the sensor device. Here, the software requires the second current when processing to obtain the glucose level.

[0056] In the above-described system, the compatible device may further include at least one processor, at least one memory, and software stored in the at least one memory and executable by the at least one processor. When executed, the software is configured to implement a method including: causing at least one memory to store a first current and a second current received together or associated with each other from the sensor device; and processing the first current and the second current to provide a value indicative of glucose oxidation in a glucose-oxidation layer of a first electrode of the sensor device. Here, processing may include subtracting the second current from the first current. Either or both of the first current and the second current may be in the form of a continuous signal, and processing the first current and the second current may include processing simultaneously obtained values of the first current and the second current. The compatible device may further include a display, and the method may further include causing the value or corresponding information to be presented on the display. The compatible device may further include a wireless communication module configured to wirelessly pair with the device including the display, and the method may further include communicating the data to the wirelessly paired device for presenting the value or corresponding information on a display of the wirelessly paired device.

[0057] Yet another embodiment of the invention provides a method of electrochemical sensing, comprising: providing a sensor device including a first electrode including a glucose-oxidation layer capable of oxidizing glucose, a second electrode not including a layer capable of oxidizing glucose, and a reference electrode; contacting the first electrode, the second electrode, and the reference electrode with a liquid including glucose, ascorbic acid, and acetaminophen; applying a first bias voltage between the first electrode and the reference electrode sufficient to oxidize glucose at the glucose-oxidation layer, such that glucose and at least one of ascorbic acid and acetaminophen are oxidized at the glucose-oxidation layer; and generating a first current from the first electrode, the first current representing the glucose component caused by glucose oxidation. and a first interfering component caused by oxidation of at least one of ascorbic acid and acetaminophen; applying a second bias voltage between the second electrode and the reference electrode, such that at least one of ascorbic acid and acetaminophen is oxidized at the second electrode, but glucose is not oxidized therein, and causing a second current to be generated from the second electrode, the second current including the second interfering component caused by oxidation of at least one of ascorbic acid and acetaminophen at the second electrode; and providing a first current and a second current for processing, wherein the first current is provided for processing and the second current is also provided in association with the first current.

[0058] In the above-described method, the first and second currents can be generated simultaneously or one after the other within a reasonable period of time during which the glucose level does not change substantially or beyond a predetermined acceptable level. The first current can be provided along with information indicating the time of occurrence of the first current, and the second current can be provided along with information indicating the time of occurrence of the second current. The second current can be provided along with the first current whenever the first current is provided. In the above-described method, the first bias voltage is applied between 0.2 V and 0.32 V, causing the glucose-oxidation layer to oxidize glucose and ascorbic acid but not acetaminophen, in which case the first interfering component is caused by the oxidation of ascorbic acid, not acetaminophen; and the second bias voltage is applied between 0.2 V and 0.32 V, causing the second electrode to oxidize ascorbic acid but not acetaminophen, in which case the second interfering component is caused by the oxidation of ascorbic acid, not acetaminophen. In another case, a first bias voltage of 0.34 V to 0.45 V is applied, causing the glucose-oxidation layer to oxidize glucose, ascorbic acid, and acetaminophen, in which case the first interfering component is caused by the oxidation of ascorbic acid and acetaminophen; a second bias voltage of 0.34 V to 0.45 V is applied, causing the second electrode to oxidize ascorbic acid and acetaminophen, in which case the second interfering component is caused by the oxidation of both ascorbic acid and acetaminophen.

[0059] In the aforementioned method, the sensor device may further include a maltose-blocking layer formed on the glucose-oxidation layer and including poly-phenylenediamine (poly-PD). The sensor device may be a continuous glucose monitoring (CGM) electrode module including a subcutaneous portion configured to subcutaneously contact a body fluid of a subject, the first, second, and reference electrodes being formed within the subcutaneous portion, and contacting the first, second, and reference electrodes with the fluid may include subcutaneously inserting the subcutaneous portion into the body of the subject. The glucose-oxidation layer may include a nanoporous metal layer, and the first electrode may further include: an electrolyte ion-blocking layer formed on the nanoporous metal layer and a biocompatible layer formed on the electrolyte ion-blocking layer. The electrolyte ion-blocking layer may block Na ions contained in the fluid. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- from diffusing towards the nanoporous metal layer, so that Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- There is a substantial discontinuity in the total concentration of

[0060] In the aforementioned method, the sensor device may be a blood glucose monitoring (BGM) electrode module including a reservoir, and contacting the first electrode, the second electrode, and the reference electrode with a liquid may include providing a blood sample into the reservoir. The glucose-oxidation layer may include irregularly shaped bodies including many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies, the nanoparticles being generally ovoid or spherical with diameters of about 2 nm to about 5 nm, and the interparticle gaps having an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies may be interconnected to provide a three-dimensional interconnected network of the irregularly shaped bodies. The irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and may be nano- or micro-sized, and the irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of the irregularly shaped spaces.

[0061] In the above-described method, the sensor device may further include a first terminal electrically connected to the first electrode, a second terminal electrically connected to the second electrode, and a third terminal electrically connected to the reference electrode. The sensor device may further include a terminal section in which the first, second, and third terminals are arranged, and applying the first bias voltage and the second bias voltage may include coupling to a corresponding device including the first corresponding terminal, the second corresponding terminal, and the third corresponding terminal, a circuit network, and power connected to the circuit network. The corresponding device may further include a corresponding terminal section for coupling or engaging with the terminal section of the sensor device. The first, second, and third corresponding terminals are arranged within the corresponding terminal section, and the terminal section of the sensor device and the corresponding terminal section of the corresponding device are coupled or engaged. The first terminal may be arranged to electrically connect to the first corresponding terminal, the second terminal to electrically connect to the second corresponding terminal, and the third terminal to electrically connect to the third corresponding terminal when engaged or mated. The corresponding device circuitry may provide a first bias voltage between the first and third corresponding terminals; and the corresponding device circuitry may provide a second bias voltage between the second and third corresponding terminals.

[0062] Yet another aspect of the invention provides a method for providing or determining a glucose level, the method including: providing software stored in at least one memory and executable by at least one processor provided with the sensor device or another device; executing the software with the at least one processor to process the first current and the second current to provide a value indicative of glucose oxidation in a glucose-oxidation layer of a first electrode of the sensor device; and presenting the value or corresponding information on a display provided with the sensor device, the other device, or yet another device.

[0063] In the aforementioned method, at least one memory and at least one processor are provided in another device. The method may further include: transmitting the first current and the second current to the other device; and, prior to execution, storing the received first current and second current together or in association with each other in at least one memory. In the aforementioned method, the first current is transmitted with a first timestamp, and the second current is transmitted with a second timestamp, the first and second timestamps indicating the same time. In the aforementioned method, either or both of the first current and the second current may be in the form of a continuous signal, and processing the first current and the second current may include processing values of the first current and the second current obtained simultaneously. In the aforementioned method, processing may include subtracting the second current from the first current.

[0064] Yet another aspect of the invention provides a sensor device comprising: a working electrode comprising a nanoporous metal layer; and a reference electrode; and a bias voltage applied between the working electrode and the reference electrode, wherein no glucose-specific enzyme is present at the working electrode.

[0065] In the sensor device, the nanoporous metal layer includes irregularly shaped bodies including many nanoparticles clustered together locally and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped bodies, where the nanoparticles are generally ovoid or spherical with diameters of about 2 nm to about 5 nm, and the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies can be interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies. The irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nano- or micro-sized, and the irregularly shaped spaces are interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces. In the sensor device, the bias voltage is set to be sufficient to cause glucose oxidation in the nanoporous metal layer but not sufficient to cause acetaminophen oxidation in the nanoporous metal layer, and the bias voltage is set within a range of about 0.20 V to about 0.32 V.

[0066] The sensor device may include a continuous glucose monitoring (CGM) electrode module including a subcutaneous portion configured to subcutaneously contact a body fluid of a subject, wherein a working electrode and a reference electrode are formed within the subcutaneous portion. The working electrode may further include an electrolyte ion-blocking layer formed on the nanoporous metal layer; and a biocompatible layer formed on the electrolyte ion-blocking layer. The electrolyte ion-blocking layer may block Na ions contained in the fluid. + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- from diffusing toward the nanoporous metal layer, so that Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2-There is a substantial discontinuity in the total concentration of . The electrolyte ion-blocking layer can be configured to facilitate conditioning of the working electrode such that conditioning of the working electrode is completed within 30 minutes of contact with the subject's body fluid with the application of a bias voltage.

[0067] The sensor device may further include a maltose-blocking layer comprising poly-phenylenediamine (poly-PD) and interposed between the nanoporous metal layer and the electrolyte ion-blocking layer. When contacted with a liquid containing maltose and glucose having a concentration of 4-20 mM (approximately 72-360 mg / dL) and subjected to a bias voltage, the maltose-blocking layer is configured to allow glucose to pass therethrough and substantially block maltose from passing therethrough, such that at steady state, the glucose-oxidation current is 0.1 μA / mMcm. 2 (10nA / mMcm 2 ), whereas the maltose oxidation current caused by maltose oxidation alone was 0.05 μA / mMcm 2 (5nA / mMcm 2 ) lower.

[0068] Yet another aspect of the invention provides a method for glucose sensing, comprising: providing one of the sensor devices described above; and applying a bias voltage between the working electrode (or glucose-sensing electrode) and the reference electrode in the range of about 0.20 V to about 0.32 V, wherein application of the bias voltage causes oxidation of glucose in the nanoporous metal layer, such that the glucose-oxidation current caused by glucose oxidation alone is less than or equal to 0.1 μA / mMcm. 2 (10nA / mMcm 2 ), whereas the application of a bias voltage did not induce sufficient oxidation of acetaminophen in the nanoporous metal layer, so that the acetaminophen oxidation current caused by acetaminophen oxidation in the nanoporous metal layer was 0.05 μA / mMcm 2 (5nA / mMcm 2 ) lower.

[0069] The patent or application file contains drawings executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0070] [Figure 1] 1 illustrates a conceptual electrochemical glucose sensing system according to an embodiment of the invention. [Figure 2] 1 illustrates a working electrode for an enzymatic glucose sensing system according to one embodiment. [Figure 3] 1 shows a working electrode including a nanoporous layer for a non-enzymatic sensing system according to one embodiment. [Figure 4] The top surface and depth of the nanoporous layer are shown. [Figure 5A] 1 illustrates the clustered morphology of a nanoporous layer according to one embodiment. [Figure 5B] 1 is a TEM photographic image of a cluster according to one embodiment. [Figure 5C] FIG. 5B is a zoomed-in image of the TEM photograph of FIG. 5B. [Figure 5D] 1 is a SEM photographic image of a nanoporous layer taken from its top surface, according to one embodiment. [Figure 6A] 1 is a flow chart for fabricating a clustered nanoporous layer according to one embodiment. [Figure 6B] 1 is a flow chart for fabricating a clustered nanoporous layer according to another embodiment. [Figure 7] 1 is an example of a phase diagram of a surfactant showing different phases. [Figure 8] 1 illustrates a reverse micelle phase and a nanoparticle-surfactant colloid according to one embodiment. [Figure 9] 1 includes a TEM photographic image of a nanoparticle cluster according to one embodiment. [Figure 10A] 1 illustrates the non-clustered morphology of a nanoporous layer according to one embodiment. [Figure 10B]1 is a TEM photographic image of the non-clustered morphology of a nanoporous layer formed on a metal surface according to one embodiment. [Figure 11] 1 is a flow chart for fabricating a non-clustered nanoporous layer according to one embodiment. [Figure 12] 1 is a flowchart for fabricating hexagonal nanostructures according to one embodiment. [Figure 13A] 1 illustrates the formation of a hexagonal array according to one embodiment. [Figure 13B] Deposition of metals using hexagonal ordering of liquid crystal phases is shown. [Figure 14] 1 shows particle size distributions for nanoparticle-surfactant colloids prepared according to one embodiment. [Figure 15] 1 shows particle size distributions for cluster colloids prepared according to one embodiment. [Figure 16A] 1A-1C show cross sections of an electrode-based and a non-enzymatic glucose-sensing working electrode, respectively, according to embodiments. [Figure 16B] 1A-1C show cross sections of an electrode-based and a non-enzymatic glucose-sensing working electrode, respectively, according to embodiments. [Figure 17A] 1 is an SEM photograph of a glucose-sensing working electrode according to an embodiment. [Figure 17B] 1 is an SEM photograph of a glucose-sensing working electrode according to an embodiment. [Figure 17C] 1 is an SEM photograph of a glucose-sensing working electrode according to an embodiment. [Figure 18] 10 is a profile of current generated by oxidation of glucose and other materials in PBS, according to an embodiment. [Figure 19] 1 is a profile of current generated by oxidation of glucose and other materials in human serum, according to an embodiment. [Figure 20] This is the structural formula of a maltose molecule. [Figure 21] 1 shows a non-enzymatic working electrode including a maltose-blocking layer according to one embodiment. [Figure 22]1 shows a scan of oxidation potential during cyclic voltammetric electrochemical polymerization of phenylenediamine according to an embodiment. [Figure 23] 1 shows a chronoamperometry setup for an electric shock treatment to adjust the porosity of a porous polymer layer according to one embodiment. [Figure 24] 1 is a flow chart for manufacturing a maltose-blocking layer according to one embodiment. [Figure 25] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 26] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 27] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 28] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 29] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 30] 1 shows the current monitored using a glucose-sensing electrode having a maltose-blocking layer according to an embodiment, where the current signal is presented in color since it is not easily discernible in black and white. [Figure 31] 1 illustrates a CGM working electrode according to one embodiment. [Figure 32]1 illustrates the electrolyte concentration drop across the thickness of an electrolyte ion-blocking layer according to one embodiment. [Figure 33] 1 illustrates a CGM electrode unit according to one embodiment. [Figure 34] 1 is a flowchart for fabricating a CGM electrode unit according to one embodiment. [Figure 35] 35A and 35B show top and cross-sectional views of intermediate products at various stages in fabricating the CGM electrode of FIG. 33, where each cross-section is taken about line 3501 and looking in the direction of the arrows. [Figure 36] 35A and 35B show top and cross-sectional views of intermediate products at various stages in fabricating the CGM electrode of FIG. 33, where each cross-section is taken about line 3501 and looking in the direction of the arrows. [Figure 37] 35A and 35B show top and cross-sectional views of intermediate products at various stages in fabricating the CGM electrode of FIG. 33, where each cross-section is taken about line 3501 and looking in the direction of the arrows. [Figure 38A] 1A and 1B show cross sections of a CGM working electrode with an intermediate product and a functional layer after forming a nanoporous layer according to an embodiment, respectively. [Figure 38B] 1A and 1B show cross sections of a CGM working electrode with an intermediate product and a functional layer after forming a nanoporous layer according to an embodiment, respectively. [Figure 39] 1 illustrates a disposable glucose sensing cartridge according to an embodiment. [Figure 40] 1 illustrates a two-electrode glucose sensing system according to one embodiment. [Figure 41] 1 illustrates a CGM electrode unit for a two-electrode glucose sensing system according to one embodiment. [Figure 42A] 1 is a profile of the current generated by the oxidation of glucose according to one embodiment, where the working electrode does not include an electrolyte ion-blocking layer. [Figure 42B] FIG. 42B is an enlarged view of a portion of the profile of FIG. 42A. [Figure 43]1 is a profile of the current generated by the oxidation of glucose according to one embodiment, where the working electrode includes an electrolyte ion-blocking layer. [Figure 44] 1 is a comparison of the time to condition a working electrode with and without an electrolyte ion-blocking layer. [Figure 45A] 1 is a photograph of a potentiostat according to one embodiment. [Figure 45B] 1 is a photograph of a potentiostat according to one embodiment. [Figure 45C] 1 is a photograph of a potentiostat according to one embodiment. [Figure 46] 10 is a graph showing CGM monitoring of glucose levels in a rat using a non-enzymatic CGM electrode module according to one embodiment. [Figure 47] 1 is a Clarke error grid for a non-enzymatic CGM electrode module according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0071] The presently disclosed subject matter will now be described in more detail with reference to the accompanying drawings in terms of several specific embodiments and examples. Some, but not all, embodiments of the invention are shown herein. Like numerals refer to like elements or parts throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the specific embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter will occur to those skilled in the art to which the presently disclosed subject matter pertains. Therefore, it is to be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0072] Electrochemical glucose sensing system Electrochemical glucose detection Electrochemical glucose sensing measures the concentration of glucose in an electrolyte solution. FIG. 1 conceptually illustrates an electrochemical glucose sensing system 101 for detecting the concentration of glucose in a test fluid or electrolyte solution 102. The system 101 includes a working or sensing electrode 103, a counter electrode 105, and a reference electrode 106, which are connected to a potentiostat 104 and are in contact with the test fluid 102. In an embodiment, the potentiostat includes electrical circuitry for functioning as a voltage source 109 and a current sensor 108. The voltage source 109 provides a bias voltage that drives the redox reaction at the working electrode 103 and the counter electrode 105. The potentiostat further includes electrical circuitry, such as an operational amplifier 107, for maintaining the bias voltage at the working electrode 103 relative to the reference electrode 106. The current sensor 108 detects the current generated by the redox reaction involving glucose contained in the test fluid 102.

[0073] Enzymatic glucose-sensing electrode Most, if not all, electrochemical glucose sensing systems utilize glucose-specific enzymes for the detection of glucose molecules. FIG. 2 shows a working electrode 103E for an enzymatic glucose sensing system, i.e., an enzymatic glucose-sensing electrode. The terms "glucose-sensing electrode" and "working electrode" are used interchangeably in this disclosure. The enzymatic working electrode 103E includes a conductive layer 110 and an enzyme layer 111. Optionally, the enzymatic working electrode 103E can include at least one functional layer 112 on the enzyme layer 111, as in FIG. 2. Alternatively, although not shown, at least one functional layer can be disposed between the enzyme layer 111 and the conductive layer 110. The enzyme layer 111 includes glucose-specific enzyme molecules 115, which are held therein by fixtures 113. When a glucose molecule contacts the glucose-specific enzyme, the enzyme catalyzes the oxidation of glucose to gluconolactone. Electrons from glucose oxidation are ultimately transferred to the conductive layer 110, generating a current in the electrical circuit of the electrochemical sensing system 101.

[0074] glucose oxidase In some enzymatic glucose sensing systems, the enzymatic working electrode 103E contains glucose oxidase (GOx). The glucose oxidase 115 transfers electrons to molecular oxygen that remains near the enzyme, which is reduced to hydrogen peroxide. When the appropriate bias voltage is applied in the system, the conductive layer 110 oxidizes the hydrogen peroxide and extracts electrons from it, thereby generating a current that is indicative of the glucose concentration in the test fluid 102.

[0075] glucose dehydrogenase In other enzymatic glucose sensing systems, the enzymatic working electrode 103E includes glucose dehydrogenase (GDH). Unlike glucose oxidase, glucose dehydrogenase does not use oxygen; instead, it transfers electrons to another adjacent chemical component called an electron mediator, which then transfers electrons from glucose oxidation to the conductive layer 110. The electron mediator can be included in the enzyme layer 111. Alternatively, the electron mediator can be provided in a separate layer (not shown) between the enzyme layer 111 and the conductive layer 110. While glucose dehydrogenase has some advantages over glucose oxidase, such as sensitivity, the enzyme oxidizes maltose as well as glucose, which prevents accurate sensing of glucose concentrations.

[0076] Non-enzymatic glucose-sensing electrodes A non-enzymatic electrochemical glucose sensing system does not use a glucose-specific enzyme or any enzymes to detect glucose. Instead, a non-enzymatic glucose sensing system has a non-enzymatic working electrode that detects glucose without a glucose-specific enzyme. In an embodiment, the non-enzymatic working electrode includes at least one glucose oxidation layer that allows oxidation of glucose molecules at a moderate bias voltage. Generally, the higher the bias voltage, the greater the likelihood that glucose oxidation will occur in the at least one glucose oxidation layer. However, there is a limit to the bias voltage because at high bias voltages, other chemical components are also oxidized. Thus, non-enzymatic electrochemical glucose sensing relies on materials that oxidize glucose at bias voltages that do not cause oxidation of other chemical components contained in the test fluid.

[0077] Nanoporous layers for non-enzymatic glucose-sensing electrodes 3 shows a non-enzymatic working electrode (simply "working electrode") 103NE comprising a conductive layer 110 and a nanoporous glucose oxidation layer (or nanoporous layer) 117. In embodiments, the nanoporous layer 117 comprises a nanoporous interior structure to cause, enable, or promote glucose oxidation at a moderate bias voltage. When glucose oxidation occurs, the conductive layer 110 extracts electrons from the glucose oxidation, and a current is generated in the electrical circuit. The current can be detected by a current sensor 108 and interpreted by the system hardware and software. Optionally, the working electrode 103NE may include at least one functional layer 112 on the nanoporous layer 117 or between the nanoporous layer 117 and the conductive layer 110 (not shown).

[0078] Conductive Layer - Material 2 and 3 extracts electrons from glucose oxidation and transfers them to current sensor 108. In embodiments, conductive layer 110 includes or is fabricated from at least one conductive material and is connected to the electrical circuitry of system 101. In some embodiments, assuming a small-scale conductive layer 110, a semiconductive material may be used instead of a conductive material. Non-limiting examples of conductive layer materials include platinum (Pt), gold (Au), silver (Ag), ruthenium (Ru), stainless steel, silicon (amorphous, polycrystalline, and single crystalline), conductive carbon materials such as graphite, graphene, fluorene, and carbon nanotubes. In embodiments, conductive layer 110 does not include the nanoporous interior structure of glucose oxidation layer 117.

[0079] Conductive layer-structure In embodiments, the conductive layer 110 may be formed from a single layer of homogeneous material. In other cases, the conductive layer 110 may include multiple sublayers made from different materials. In some embodiments, the conductive layer 110 includes a top sublayer and one or more sublayers below the top sublayer. In embodiments, the top sublayer does not include silver, copper, aluminum, or other conductive materials that have a greater tendency to oxidize than silver, copper, or aluminum. The top sublayer may be less conductive than the other sublayer(s). In some embodiments, the conductive layer 110 includes a conductive carbon layer as the top sublayer and a silver layer as another sublayer below the carbon layer. The conductive layer 110 has a thickness that can vary greatly depending on the particular example. In some embodiments, the conductive layer 110 can be omitted, and the nanoporous layer is directly connected to the current sensor via a conductive wire or connection.

[0080] Counter electrode With the application of a bias voltage, reduction of the chemical component occurs at the counter electrode 105. In embodiments, the counter electrode 105 comprises at least one conductive or semiconductive material and is connected to the electrical circuit of the system 101. In embodiments, the counter electrode 105 may be formed from a single layer of homogeneous material or multiple layers made of different materials. The conductive or semiconductive material for the conductive layer 110 may also be used in the counter electrode 105, although in certain systems, non-identical materials are used in the conductive layer 110 and the counter electrode 105. reference electrode

[0081] The reference electrode 106 provides stability in the electrochemical sensing system by maintaining a bias voltage between the sensing electrode 103 and the reference electrode. As a result, glucose oxidation can continue at the sensing electrode 103 even if reduction at the counter electrode 105 is not at the same rate as oxidation at the sensing electrode 103. In some embodiments, the counter electrode 105 can be omitted, and the reference electrode 106 can serve the dual function of counter and reference electrode. In embodiments, the reference electrode 106 can be formed from a single layer of homogeneous material or multiple layers made from different materials. A conductive or semiconductive material for the conductive layer 110 can also be used in the reference electrode 105, although in certain systems, non-identical materials are used in the conductive layer 110 and the reference electrode 106. In some embodiments, the reference electrode 106 can include a salt layer on the conductive or semiconductive material layer. For example, the salt layer can be made of or include silver chloride (AgCl).

[0082] Current Sensor The current sensor 108 measures the current flowing from the working electrode 103. The current sensor 108 may amperometrically detect the current flowing at a particular point in time. Alternatively, the current sensor 108 may be a coulometric charge-measuring device.

[0083] Test fluid In some embodiments, the test fluid is a biological fluid from a human or animal, but is not limited thereto. In some embodiments, the test fluid is a liquid mixture containing a biological fluid and at least one additional substance added to the biological fluid. Examples of biological fluids include, but are not limited to, blood, interstitial fluid, cerebrospinal fluid, lymphatic fluid, or urine. In some embodiments, the test fluid comprises a non-biological liquid prepared for the experiment.

[0084] bias voltage The bias voltage applied between the working electrode 103NE and the reference electrode 106 is at or about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, or 0.46 V. In an embodiment, the applied bias voltage may be within a range formed by selecting any two numbers (two voltage values) listed in the immediately preceding sentence, e.g., about 0.20 V to about 0.30 V, about 0.30 V to about 0.40 V, about 0.28 V to about 0.40 V, about 0.30 V to about 0.38 V, about 0.28 V to about 0.36 V, etc.

[0085] Nanoporous Layer The nanoporous layer 117 for the working electrode 103NE includes nanosized internal structures, such as cavities, spaces, and openings (collectively "nanopores" or "nanopores"). In embodiments, the nanopores in the nanoporous layer 117 allow or facilitate the oxidation of glucose, and the glucose concentration can be measured based on the current caused by glucose oxidation. While no aspect of the invention is bound by any theory or belief, it is believed that glucose oxidation occurs when glucose molecules enter the nanopores and contact the internal surface more frequently and for a longer period of time within the nanoporous layer 117 than on the non-porous surface of the electrode.

[0086] No enzymes and no electron mediators Incorporating the nanoporous layer 117 allows the working electrode 103NE to be free of glucose-specific enzymes, which require a more complex fabrication process and are less stable than the solid material of the nanoporous layer 117. Furthermore, the enzyme-sensing electrode 103NE can operate without an electron mediator to facilitate electron transfer between different materials. In embodiments, the working electrode 103NE does not include an enzyme or an electron mediator.

[0087] Materials for nanoporous layers In some embodiments, the nanoporous layer 117 is made from or includes, but is not limited to, platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), or oxides of the foregoing elements. In other embodiments, the nanoporous layer 117 is made from or includes, but is not limited to, alloy materials of two or more metal elements listed in the previous sentence, including Pt-Ir, Pt-Ru, and Pt-Pd.

[0088] Specified roughness coefficient The roughness factor, or roughness, is the ratio of an object's real surface area to its geometric surface area. Here, geometric surface area refers to the projected area of an object projected onto a plane without considering the internal surfaces within the object. Real surface area refers to the total area of the surface, taking the internal surfaces into account. Referring to FIG. 4, for example, if nanoporous layer 117 is a rectangular block with height or depth 118 and top rectangular surface 119, the projected or geometric surface area of the nanoporous layer is the area of the top rectangular surface that is exposed to the outside. The real surface area of a nanoporous layer can be measured electrochemically, for example, using the well-known cyclic voltammetry technique, which detects a current from proton adsorption on the real surface.

[0089] Roughness coefficient of nanoporous layers The roughness factor value indicates the total amount of internal pores within the nanoporous layer 117. The roughness factor of the nanoporous layer 117 can be related to the sensitivity of the nanoporous layer 117 to glucose oxidation. Generally, the higher the roughness factor, the more glucose oxidation can occur. The roughness factor of the nanoporous layer 117 can be at or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 100, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500. In embodiments, the roughness factor may be within a range formed by selecting any two numbers (two roughness factor values) listed in the immediately preceding sentence, e.g., from about 100 to about 2500, from about 750 to about 1250, or from about 850 to about 1150.

[0090] Nanoporous layer thickness The roughness factor value does not indicate the level of porosity or density of the nanoporous material per unit volume, but the value may indicate the total amount of internal pores. Thus, depending on the level of porosity of the nanoporous material, in embodiments, the thickness of the nanoporous layer may be adjusted to achieve a target value for the roughness factor. In embodiments, the thickness of the nanoporous layer 117 may be about 0.03, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μm. In some embodiments, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the immediately preceding sentence, e.g., about 0.05 μm (50 nm) to about 10 μm, about 0.5 μm to about 8 μm, or about 2 μm to about 7 μm.

[0091] form The nanoporous layer 117 may have a different internal morphology in each specific fabrication. In some embodiments, the nanoporous layer 117 may include or be fabricated from nanoparticles deposited together and forming nanopores between themselves (interparticle nanopores). In other embodiments, the nanoporous layer 117 may include or be fabricated from clusters of nanoparticles deposited together that form interparticle nanopores within the clusters and also spaces between the clusters (intercluster gaps or spaces). In other embodiments, the nanoporous layer 117 may include or be fabricated from repeating nanostructures of a particular shape, such as hexagonal structures, that contain nanopores therein. Also, in each specific fabrication, the nanoporous layer 117 may have different levels of porosity per unit volume and different roughness coefficient values.

[0092] Fabrication of nanoporous layers The nanoporous layer 117 can be prepared using a liquid composition containing metal ions and a surfactant. In embodiments, different morphologies of the nanoporous layer can be formed using different phases of the surfactant. A micellar, reverse micellar, liquid crystalline, or other phase of the surfactant can be used to generate a nanoporous layer with a specific morphology. In these different phases, the metal ions are aligned or locally concentrated adjacent to the hydrophilic portions of the surfactant. The localized metal ions in the liquid composition can be subjected to additional processes for reduction and deposition on the surface to provide a nanoporous layer 117 with a different morphology.

[0093] Clustered nanoporous layer Clustered morphology FIG. 5A shows a vertical cross-section of a nanoporous layer with a clustered morphology 120 on a substrate 129. In nanoscale reality, the top surface of the substrate 129 may not be straight as shown and may be uneven. In the clustered morphology 120, many nanoparticles 121 come together to form irregularly shaped clusters 125. For illustrative purposes, different shading or hatching is used for different clusters 125. These irregularly shaped clusters 125 stack irregularly to form the nanoporous layer. FIG. 5B is a transmission electron microscope (TEM) image of several clusters 125 before they deposit to form the nanoporous layer. FIG. 5C is a zoomed-in image of the circled area in FIG. 5B. FIG. 5D is a scanning electron microscope (SEM) image of the nanoporous layer with a clustered morphology taken from the top surface of the nanoporous layer.

[0094] Clustered morphology of pores and spaces With the irregular stacking of irregularly shaped clusters 125, adjacent clusters form intercluster gaps or spaces 127 between them. These intercluster gaps 127 can be nanosized and microsized. In this disclosure, nanosized means greater than 1 nm and less than 100 nm, and microsized means greater than 100 nm and less than 100 μm. Each cluster 125 includes or is fabricated from roughly spherical or ovoid nanoparticles 121. In each cluster, the individual nanoparticles are generally separated from one another, forming small gaps 123 between them. The small gaps are nanosized and are referred to as interparticle nanopores 123. In embodiments, interparticle nanopores are found throughout the cluster. In embodiments, the interparticle nanopores form interconnected or networked channels within each cluster. Figures 5A and 5D show these interparticle nanopores 123 within each cluster 125.

[0095] Intercluster gap / space formation In an embodiment, to generate a clustered morphology, irregularly shaped clusters 125 are first prepared as a suspension in a liquid. The suspension is then dispensed onto a substrate 129, which is allowed to dry. As the liquid dries, the clusters naturally deposit on the substrate and on one another. No external force can be applied to the clusters during drying. Therefore, the clusters do not pack together as they deposit. As the clusters deposit and stack on top of one another, each cluster may contact the substrate surface or adjacent clusters. After drying is complete, the clusters abut or contact adjacent or neighboring clusters. The deposited clusters are interconnected or integrated through junctions or contacts. Due to the irregular shape of the individual clusters, irregularly shaped gaps and spaces are formed between adjacent clusters, where the gaps and spaces define the irregular shape of the deposited clusters as if the surface and contour of the deposited clusters were surrounded by the irregularly shaped gaps and spaces. The irregularly shaped gaps and spaces are referred to as inter-cluster gaps or spaces 127.

[0096] Distribution of clusters and intercluster gaps In an embodiment, irregularly shaped cluster bodies 125 are distributed throughout the clustered morphology 120 of the nanoporous layer 117. The irregularly shaped cluster bodies 125 are interconnected through junctions, meaning that the cluster bodies contact each other and form a three-dimensional network of cluster bodies generally throughout the nanoporous layer 117. Intercluster gaps 127 define the surfaces of the irregularly shaped cluster bodies, surround them, and interconnect with each other, forming three-dimensional interconnected or networked channels throughout the nanoporous layer 117. The intercluster gaps and spaces 127 are well distributed throughout the nanoporous layer 117 from the top surface (not shown) to the bottom surface (on or just above the substrate 129). The three-dimensional network of irregularly shaped cluster bodies and the three-dimensional network of irregularly shaped gaps are three-dimensionally complementary, forming a highly networked three-dimensional mesh structure. The three-dimensional network of cluster bodies and channels may resemble the three-dimensional internal shape of a sponge, except that the interparticle gaps and spaces are networked together throughout the nanoporous layer 117 .

[0097] Distribution of nanoparticles and interparticle nanopores Assuming that each cluster is formed by many nanoparticles 121 and interparticle nanopores 123, the nanoparticles 121 and interparticle nanopores 123 are distributed generally throughout the nanoporous layer 117. The interparticle nanopores 123 are therefore interconnected within each cluster and with interparticle nanopores of other clusters generally throughout the nanoporous layer 117 via interparticle nanopores in the junctions between clusters and via intercluster gaps 127 that are interconnected throughout the nanoporous layer 117.

[0098] Intercluster gaps / spaces for glucose diffusion In an embodiment, the interconnection of the intercluster gaps 127 provides networked channels for the diffusion of glucose molecules (0.7-0.8 nm in length) within the nanoporous layer 117. It is understood that glucose oxidation occurs primarily within the nanosized interparticle nanopores rather than within the microsized spaces. As the intercluster gaps 127 are networked or interconnected throughout the nanoporous layer 117, glucose molecules can reach almost anywhere within the nanoporous layer 117 through the interparticle spaces, which are large considering the size of glucose molecules. Also, because the intercluster gaps 127 are well interconnected with the interparticle nanopores 123, any interparticle nanopore 123 within the nanoporous layer 117 may be exposed and open for glucose oxidation. Therefore, the three-dimensional interconnected or networked channels of the intercluster gaps can provide more glucose oxidation, i.e., a stronger signal of glucose oxidation (higher current), than a nanoporous layer without such interconnected channels formed in the intercluster gaps.

[0099] Two types of particles and two types of pores As depicted, the clustered morphology 120 includes two different types of particles that define two different types of pores: from the particle perspective, one is a nanoparticle 121, and the other is a cluster 125 made from the nanoparticles 121. From the pore perspective, one is an interparticle nanopore 123 between the nanoparticles 121 within the cluster 125, and the other is an intercluster gap 127 between the clusters 125.

[0100] Clusters of nanoparticles The TEM image in Figure 5B shows irregularly shaped clusters. The number of nanoparticles 121 within each cluster can vary greatly, resulting in correspondingly variable cluster 125 sizes. In the clustered morphology, some clusters 125 are nanosized (smaller than 100 nm) and others are microsized (100 nm to 100 μm). The clusters 125 have lengths or diameters of approximately 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, or 700 nm. In embodiments, the length or diameter of clusters 125 may be within a range formed by selecting any two numbers (two length or diameter values) listed in the immediately preceding sentence, e.g., about 20 nm to about 300 nm, or about 60 nm to about 240 nm. Clusters 125 may have an average diameter or length of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 280, or 300 nm. In embodiments, the average diameter of clusters 125 may be within a range formed by selecting any two numbers (two length or diameter values) listed in the immediately preceding sentence, e.g., about 100 nm to about 220 nm.

[0101] nanoparticles The TEM image in Figure 5C shows nanoparticles within a single cluster. The nanoparticles 121 within the cluster are separate and generally spherical (ball-like) or ovoid (egg-like) in shape, but are not limited thereto. The nanoparticles 121 have a diameter of approximately 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. In embodiments, the diameter may be within a range formed by selecting any two numbers (two diameter values) listed in the immediately preceding sentence, e.g., from about 2 nm to about 5 nm. The nanoparticles 121 may have an average diameter of approximately 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 nm. In embodiments, the average diameter of the nanoparticles 121 may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., about 2.5 nm to about 4.0 nm, about 2.75 nm to about 3.75 nm, or about 2.25 nm to about 3.5 nm. In embodiments, nanoparticles having an average diameter of 2-5 nm are found throughout the nanoporous layer 117.

[0102] Interparticle nanopores The TEM image in Figure 5C also shows interparticle nanopores between nanoparticles within the cluster. The interparticle nanopores are networked and interconnected within the cluster. The interparticle gap or nanopore 123 is the interparticle gap distance between two directly adjacent nanoparticles within the same cluster. The interparticle gap distance is approximately 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 nm. In embodiments, the interparticle gap distance may be within a range formed by selecting any two numbers (two distance values) listed in the immediately preceding sentence, for example, from about 0.5 nm to about 4.5 nm, or from about 1.5 nm to about 4.0 nm. The interparticle nanopores 123 can have an average interparticle gap distance of about 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, or 3.5 nm. In embodiments, the average interparticle gap distance of the nanopores 123 may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., about 0.75 nm to about 1.5 nm, or about 1.0 nm to about 2.5 nm to about 3.0 nm. In embodiments, interparticle nanopores 123 having an average interparticle gap distance of 1-2.5 nm are found throughout the nanoporous layer 117.

[0103] Inter-cluster gaps / spaces The SEM image in Figure 5D shows the opening of the networked intercluster gaps visible from the top surface of the nanoporous layer. While the three-dimensional shape is not clearly visible in the two-dimensional image in Figure 5D, the top surface of the nanoporous layer contains valleys and hills formed by the stacked clusters. Inside the nanoporous layer, the valleys and hills form intercluster gaps. The intercluster gaps or spaces are irregularly shaped. The intercluster gaps 127 are nano- to micro-sized. The intercluster gaps 127 have an intercluster gap distance of approximately 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 nm. In embodiments, the inter-cluster gap distance may be within the range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 100 nm to about 1000 nm. The inter-cluster gaps 127 have an average inter-cluster gap distance of about 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm. In embodiments, the average inter-cluster gap distance may be within the range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 150 nm to about 400 nm.

[0104] Overall process for fabricating clustered nanoporous layers In embodiments, nanoporous layers with clustered morphology can be prepared using an isotropic reverse micelle phase (or "reverse micelle phase") of a surfactant. Referring to FIG. 6A, in step 601, an aqueous liquid composition is prepared using a metal ion source and a surfactant in a reverse micelle phase. The metal ions are locally concentrated within the hydrophilic spaces of individual reverse micelles. Then, in step 603, a reducing agent is added to the reverse micelle phase to form metal nanoparticles dispersed in a liquid composition containing the surfactant (a "nanoparticle colloid" or "nanoparticle-surfactant colloid"). Then, in step 605, the surfactant is removed from the nanoparticle-surfactant colloid, and the nanoparticle clusters dispersed in the liquid (a "cluster colloid" or "cluster-liquid colloid") are collected. Optionally, in step 607, the collected cluster colloids are mixed with a non-surfactant liquid. In step 609, the cluster colloids are distributed onto a surface, for example, by a printing technique, without the use of electroplating. The liquid is then dried in step 611 to form a nanoporous layer 117 on the surface 129 .

[0105] surfactants Surfactants are amphiphilic organic compounds that have a hydrophilic head (or hydrophilic portion) and a hydrophobic tail (hydrophobic portion) within a single molecule. Surfactants can form different structures or phases in water depending on the concentration and temperature. Figure 7 is an example phase diagram for a surfactant showing the different phases, including a micellar phase 131, a hexagonal phase 133, a lamellar phase 135, and a two-micellar phase 137.

[0106] Preparation of isotropic reverse micellar phases In step 601, an isotropic reverse micellar phase is prepared using an aqueous liquid composition containing a surfactant, metal ions, and water. As shown in the conceptual illustration of Figure 8, the reverse micellar phase includes reverse micelles 141 formed by surfactant molecules. Each reverse micelle 141 includes a hydrophilic core 143 surrounded by hydrophobic tails radiating from the hydrophilic core. The hydrophilic core 143 encapsulates the hydrophilic components of the liquid composition, i.e., water and metal ions. Thus, the metal ions are locally concentrated within the hydrophilic core 143 of the reverse micelle.

[0107] Surfactant Examples The surfactant is selected from those that can form an isotropic reverse micelle phase under reasonable processing conditions. In some embodiments, nonionic surfactants are used, but are not limited to these. Non-limiting examples of surfactants include alkyl benzene sulfonate, alkyl polyglycoside, alkyl sulfate, carboxylate, carboxylic acid ester, cetomacrogol 1000™, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, disodium cocoamphodiacetate, ethoxylated fatty alcohols, glycerol monostearate, glycol esters of fatty acids, IGEPAL CA-630™, Isoceteth-20, Lauryl Glucoside, Maltoside, Monolaurin, Mycosubtilin, Naphthalene Sulfonate, Narrow-Range Ethoxylate, Nonidet P-40™, Nonoxynol-9, Nonoxynol, NP-40™, Octaethylene Glycol Monododecyl Ether, N-Octyl β-D-Thioglucopyranoside, Octyl Glucoside, Oleyl Alcohol, PEG-10 Sunflower Glyceride, Pentaethylene Glycol Monododecyl Ether, Polidocanol, Polysorbate Examples of surfactants that may be used include sorbitan, poloxamer 407, polyethoxylated tallow amine, polyethylene glycol esters, polyglycerol polyricinoleate, polyoxyethylene fatty acid amides, polyoxyethylene surfactants, polysorbate, polysorbate 20, polysorbate 80, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, sulfated alkanolamides, sulfonates, Triton X-100™, and Tween 80™. Those skilled in the relevant art will recognize what constitutes reasonable terms.

[0108] Conditions for a reversed micellar phase After selecting a surfactant, its concentration and temperature are adjusted to form an isotropic reverse micelle phase. The surfactant concentration and temperature can be determined in relation to the surfactant phase diagram. If a phase diagram is not available, some experimentation may be required to find the appropriate concentration and temperature using known laboratory techniques and procedures. For example, if Triton X-100™ is used as the surfactant, a concentration of 10-60 wt% and a temperature of 40-80°C can provide a reverse micelle phase.

[0109] Metal ion source One or more metal ions corresponding to the metal or alloy for the nanoporous layer are selected for the liquid composition. The metal ions are added in the form of an ionic metal-containing compound, such as an acid, base, or salt. Non-limiting examples of metal source compounds include HPtCl, HPt(OH), HPtCl(OH), HPt(SO)(OH), PtCl, KPtCl, PdCl, and TiCl.

[0110] Metal ion concentration The concentration of metal ions is also adjusted for best performance: if the concentration is too low, nanoparticles may not form; if the concentration is too high, the formation or stability of the reverse micelle phase of the surfactant may be affected. The concentration of the metal ion is about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024, 0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044, 0.046, 0.048, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095 or 0.1M. In embodiments, the concentration may be within a range formed by selecting any two numbers (two molar concentration values) listed in the immediately preceding sentence, e.g., about 0.01 to about 0.03 M, about 0.02 to 0.03 M, etc. Within the appropriate concentration range, the level of concentration has been observed to affect the rate of nanoparticle formation.

[0111] Different from plating bath The reverse micelle phase prepared in step 601 differs from plating bath compositions for electroplating in that, unlike plating baths, metal chelating agents may not be required.

[0112] Nanoparticle formation In step 603, a reducing agent is mixed into the aqueous liquid composition in the reverse micelle phase. When the reducing agent enters the hydrophilic core 143 of the reverse micelles 141, it reduces metal ions to metal atoms within the hydrophilic core 143. Because the metal ions are locally concentrated within the hydrophilic core 143, initially, the metal atoms remain within the hydrophilic core 143. The metal atoms within each hydrophilic core 143 coagulate together and grow to form metal nanoparticles. Although not limited to this, one metal nanoparticle can grow from one reverse micelle. The resulting metal nanoparticles are generally uncharged, i.e., neutral. However, some nanoparticles may have a slight positive charge on their surface. To date, electricity has not been applied to form metal nanoparticles.

[0113] Nanoparticle Colloid The nanoparticles are dispersed in a liquid to provide a nanoparticle colloid. Figure 8 conceptually illustrates the resulting nanoparticle colloid. During the process of metal ion reduction and nanoparticle growth, some reverse micelles break or collapse, allowing nanoparticles from the collapsed reverse micelles to disperse in the hydrophobic space. Some of these nanoparticles 151 can float freely outside the hydrophilic core of the reverse micelles in the resulting colloidal composition. Some other nanoparticles 153 can be surrounded or bound by the hydrophilic head groups of surfactant molecules outside the hydrophilic core of the reverse micelles. Some nanoparticles 155 remain inside the reverse micelles 141. Overall, in the resulting nanoparticle colloid, solid nanoparticles 151, 153, and 155 are dispersed in a liquid composition containing reverse micelles 141, water, and surfactant molecules. Because the nanoparticles 151, 153, and 155 are significantly separated from each other in the nanoparticle colloidal composition, they are unlikely to aggregate and grow into larger particles.

[0114] reducing agent Reducing agents are chemical moieties that can donate one or more electrons to the metal ions contained within the nanoparticle colloid. Reducing agents are hydrophilic compounds that fit into the hydrophilic core of the reverse micelle. Non-limiting examples of hydrophilic reducing agents include ascorbic acid, acetic acid, formaldehyde, citric acid, hydroxylamine, hypophosphites, etc.

[0115] Amount of reducing agent The hydrophilic reducing agent is added to the nanoparticle colloid in an amount sufficient to reduce the metal ions contained therein. In some embodiments, the reducing agent is added in a substantially greater than stoichiometric excess amount to reduce the total metal ions contained in the nanoparticle colloid, where "substantially greater than" means 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, or 400% greater.

[0116] stirring The mixture may be stirred during and / or after the addition of the reducing agent to promote distribution of the reducing agent. Stirring can promote the reducing agent to enter the hydrophilic space of the reverse micelles. Therefore, the time required to completely reduce the metal ions within the hydrophilic space can be reduced. Stirring can be performed continuously or intermittently. In an embodiment, stirring is performed for a period of 1 hour to 10 hours.

[0117] Removing surfactants and forming clusters In step 605, the surfactant is substantially removed from the nanoparticle colloid composition, forming nanoparticle clusters. In nanoparticle colloids, the surfactant can stabilize individual nanoparticles; therefore, nanoparticles cannot cluster together if a significant amount of surfactant is present. To remove the surfactant from the nanoparticles, the nanoparticle colloid is centrifuged. After centrifugation, most of the nanoparticles settle to the bottom, and surfactant molecules can be present in the supernatant and at the bottom. The supernatant is separated from the bottom, which contains most of the nanoparticles. In embodiments, a liquid can be added to the separated nanoparticles to dilute the surfactant in the collected bottom. The liquid added to the nanoparticles can be water or an aqueous solution, including, but not limited to, an acidic or basic solution. The centrifugation, bottom collection, and liquid addition can be repeated multiple times, and nanoparticles from which the surfactant has been substantially removed are collected.

[0118] Chemical bonding between surfactants and nanoparticles With surfactants, some nanoparticles form strong chemical bonds with the hydrophilic heads of some surfactant molecules. Surfactant molecules with negatively charged hydrophilic heads can form coordinate bonds with the nanoparticle surface. Also, surfactant molecules with electron-rich hydrophilic heads (even if they are uncharged) can form coordinate bonds with the nanoparticle surface. When such surfactants are used, the chemical bonds must be broken to remove the surfactant from the nanoparticle colloid.

[0119] Breaking chemical bonds In some embodiments, an acidic or basic solution is added to the nanoparticle-surfactant colloid after formation in step 604 of FIG. 6B and before centrifugation. The acid or base in the added solution causes a chemical reaction that breaks the coordinate bond between the surfactant and the nanoparticle, freeing the nanoparticle. For example, protons from the acid can bind to negatively charged or electron-rich surfactant head groups, freeing the nanoparticle. Subsequent centrifugation and bottom collection separates the freed nanoparticles from the surfactant molecules. In embodiments, the addition of the acidic or basic solution can be performed at least once before centrifugation. In some embodiments, the addition of the acidic or basic solution can be performed before each centrifugation. In embodiments, the acid and base can be washed out with water or other solvent after centrifugation.

[0120] Acidic or basic solutions In embodiments, the acid or base is selected with consideration given to the surfactant, so that the surfactant molecules are effectively desorbed from the nanoparticles. In embodiments, the acidic solution has a pH value lower than about 3, but is not limited thereto. For example, non-limiting examples of acids for acidic solutions include HCl, HNO3, H2SO4, HClO4, etc. In embodiments, the basic solution has a pH value higher than about 10, but is not limited thereto. For example, non-limiting examples of bases for basic solutions include NaOH, KOH, Ca(OH)2, etc.

[0121] Cluster Colloid After or during the process of removing the surfactant and collecting the nanoparticles, the nanoparticles tend to cluster together or aggregate to form nanoparticle clusters. In the liquid, the clusters are dispersed, forming a cluster colloid. Each cluster contains metal nanoparticles that interact with each other and are then produced to form larger bodies. Individual nanoparticles within a cluster are most likely electrically neutral. While the invention is not bound by any theory or belief, it is believed that protons, hydroxides, and other charged electrolytes can be attached to the nanoparticle surface, and the ionic interactions of these electrolytes with neighboring nanoparticles can hold adjacent nanoparticles together and allow them to form clusters. In fact, although the surfactant molecules have been substantially removed, the liquid of the cluster colloid contains a sufficient amount of electrolytes from the metal ion source and acidic or basic solution used in the previous preparation steps.

[0122] Clusters and Nanoparticles Figure 9 provides TEM photographic images of nanoparticle clusters from a diluted sample of cluster colloids. Two of the images in Figure 9 are also seen in Figures 5B and 5C. In these images, the clusters do not have regular shapes and are approximately 30 to 500 nm long. The nanoparticles 121 within the clusters are separate, roughly spherical or ovoid, and have diameters of approximately 2-3 nm. Between adjacent or neighboring nanoparticles 121, there are interparticle gaps 125 with gap distances of approximately 1-2 nm. These interparticle nanopores 125 are primarily responsible for glucose oxidation in glucose-sensing electrodes with clustered nanoporous layers.

[0123] Centrifugation Centrifugation can be performed at a rotation speed of 3000 to 5000 rpm. Centrifugation can continue for a period of 3 to 15 minutes. After centrifugation, the supernatant is removed and the bottom containing the nanoparticles is collected. Liquid is added to the collected bottom to dilute the surfactant contained therein. Centrifugation, bottom collection, and liquid addition can be repeated multiple times, for example, three or more times.

[0124] Substantially removed surfactant Using multiple centrifugation processes, the surfactant is substantially removed. In the resulting cluster colloid, the surfactant concentration is significantly lower, but it may not be completely removed. Initially, the reverse micellar phase contains about 10 to about 60 wt. of surfactant. The resulting cluster colloid can be completely surfactant-free. In fact, the resulting cluster colloid is substantially surfactant-free. The residual surfactant in the resulting cluster colloid or final bottom collection can be greater than 0.0001 parts by weight per 100 parts by weight of nanoparticles and less than about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.6 parts by weight per 100 parts by weight of nanoparticles. In embodiments, the remaining surfactant may be in an amount of less than about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5 parts by weight per 100 parts by weight of nanoparticles.

[0125] Concentration of nanoparticles in cluster colloids After multiple rounds of centrifugation, the total amount of nanoparticles (as part of clusters and free nanoparticles) in the final bottom collection can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 wt%. In embodiments, the concentration can be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., about 20 to about 30 wt%, about 15 to 25%, etc.

[0126] Preservation of cluster colloids The clusters can be dispersed in the cluster colloid for a long period of time, for example, more than one week or one month, without any processing. After preparation, the cluster colloid can be stored in a container for a period of time before further processing. Once prepared, the cluster colloid can be sold and transported for processing by others or elsewhere. To maintain colloidal properties for a long period of time, the concentration of the nanoparticles can be adjusted after final collection at the bottom. In embodiments, the cluster colloid at the bottom can be stored or transported in a container with or without adjusting the concentration.

[0127] Adjusting the concentration for distribution In step 607, the collected cluster colloid may be stored for a period of time, with or without dilution with a solvent. The dilution may be to adjust the concentration of clusters in the cluster colloid for subsequent processing, e.g., distribution. The solvent may be water or an organic compound. One or more additive compounds may be added. By dilution, the concentration of nanoparticles or clusters is adjusted to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3,2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 wt%. In embodiments, the concentration of nanoparticles or clusters may be within the range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., about 0.5 to about 2 wt%, about 1 to 3 wt%, etc. After dilution, the remaining surfactant may be less than about 0.1, 0.2, 0.4, 0.6, 0.81, 1.2, 1.4, 1.6, 1.8, or 2 wt%.

[0128] Cluster colloid distribution In step 609, the cluster colloids are dispensed onto the substrate 129 to produce a nanoporous layer while maintaining their colloidal properties. Various dispensing techniques can be utilized to dispense the cluster colloids. Dispensing can be controlled to form a certain thickness of dispensed cluster colloids, or to provide an appropriate thickness of the resulting nanoporous layer after subsequent drying. In other cases, dispensing can be controlled to provide an appropriate roughness coefficient value for the resulting nanoporous layer.

[0129] Lower layer substrate The cluster colloids can be applied onto a substrate made of any material. In embodiments for a glucose sensing electrode, the cluster colloids can be applied onto a conductive or semiconductive surface for conductive layer 110, as described above. In some embodiments, the substrate includes two or more conductive layers.

[0130] Drying of liquids to form clustered nanoporous layers In step 611, the dispensed cluster colloid is subjected to conditions to dry the liquid. Once dispensed, the nanoparticle clusters float within the liquid and are free to move horizontally and vertically. As the liquid dries, the height of the cluster colloid decreases. As the liquid continues to dry, the clusters come into contact with neighboring clusters, both vertically and horizontally, between the underlying substrate 129 and the top surface of the cluster colloid. The mobility of the clusters becomes significantly restricted. After a time, the liquid level becomes lower than the clusters located at or near the top surface. Once drying is complete, the nanoparticle clusters are deposited on the substrate 129, forming a nanoporous layer with a clustered morphology 120, as illustrated in FIG. 5A.

[0131] Nanoporous layer thickness The resulting nanoporous layer has a thickness of about 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 μm. In embodiments, the thickness may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 1 μm to about 10 nm.

[0132] No cleaning of the nanoporous layer The resulting nanoporous layer does not require washing with water or other liquids. In embodiments, the resulting nanoporous layer in clustered form is not washed with water or other liquids at all after drying. In embodiments, the nanoporous layer is not subjected to contact with liquids except for subsequent processing to add layers onto the nanoporous layer.

[0133] Yield - Metal Recovery When an excess amount of reducing agent is added to the nanoparticle colloid, most of the metal ions therein are reduced to form metal atoms, which coagulate and form nanoparticles. Subsequent treatment to remove the surfactant also collects most of the nanoparticles in clusters. Thus, most of the metal ions added in the aforementioned process are ultimately collected and deposited in the form of nanoparticle clusters, resulting in the nanoporous layer 117. In embodiments, greater than 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% of the input metal ions are collected in the form of nanoparticle clusters before dispensing.

[0134] mass production The nanoporous layer 117 can be mass-produced by printing the cluster colloids onto the substrate 129. Printing the cluster colloids takes only one or two seconds. Drying of the liquid may take longer, but only requires a large space for drying. In embodiments, many separate substrates are provided, and printing can be performed on each of the separate substrates. Each printed substrate is then dried to form the nanoporous layer. Alternatively, multiple areas can be printed with the cluster colloids onto a single substrate, which can then be cut into multiple pieces, each containing a printed area. The single substrate can be dried before cutting.

[0135] No electroplating or application of electricity Throughout the process, electroplating is not utilized to form the clustered morphology for the nanoporous layer, and furthermore, electricity is not applied to the substrate 129 on which the nanoporous layer is formed.

[0136] Non-clustered nanoporous layer Non-clustered morphology Figure 10A shows a non-clustered morphology 161 for the nanoporous layer 117. Like the clustered morphology 120, the non-clustered morphology 161 includes both nanoparticles 121 and interparticle nanopores 123 formed between adjacent or neighboring nanoparticles 121. The descriptions of nanoparticles 121 and interparticle nanopores 123 generally apply to the non-clustered morphology 161. Figure 10B is a TEM photographic image of the non-clustered morphology of a nanoporous layer formed on a metal surface, in which the dark areas are part of the metal surface. The nanoparticles and interparticle pores in the TEM photographic image are similar to those shown in Figure 10A.

[0137] No clusters and no intercluster gaps Unlike the clustered morphology 120, the non-clustered morphology 161 does not include clusters 123 or inter-cluster gaps 127. To produce the non-clustered morphology, nanoparticles are deposited onto a substrate 129 by electroplating without preparing clusters prior to electroplating. As a result, neither clusters nor inter-cluster gaps are formed in the resulting structure, i.e., the non-clustered morphology 161. Therefore, the non-clustered morphology 161 does not have the properties of the clustered morphology, resulting from the clusters 123 or inter-cluster gaps 127.

[0138] Non-clustered morphology of cavities Although there are no intercluster gaps, the non-clustered morphology 161 may contain internal cavities 133 that are significantly larger than the interparticle nanopores 123. The internal cavities 133 may be formed during the electroplating process because the nanoparticles are not necessarily stacked sequentially on the underlying surface. The internal cavities 133 are irregularly shaped and irregularly sized. The internal cavities 133 may be found throughout the nanoporous layer 117.

[0139] Cavities distinct from intercluster gaps or spaces The cavities 133 in the non-clustered morphology are distinct from the inter-cluster gaps 127 of the clustered morphology 120. The cavities 133 form because electroplating and nanoparticle deposition are not at the same rate on the surface of the substrate 129. The cavities 133 do not surround or define one or more clusters 125 of nanoparticles 121. Rather, each cavity 133 is surrounded or defined by an aggregate or collection of nanoparticles 121. While the cavities 133 may be interconnected by inter-particle nanopores 123, the cavities 133 themselves are not interconnected throughout the entire nanoporous layer 117 or over some substantial portion thereof. Furthermore, the cavities 133 do not occupy as much of the volume of the nanoporous layer 117 (lower roughness factor in the non-clustered morphology) as the inter-cluster gaps 127 (higher roughness factor in the clustered morphology).

[0140] Substrate substantially coated with nanoparticles 10A and 10B, the top surface of the substrate 129 is substantially coated with nanoparticles 121. In some embodiments, including but not limited to, no substantial interior space is formed on or immediately above the substrate 129.

[0141] Comparison of clustered and non-clustered morphologies Overall, the clustered morphology 120 is much less dense than the non-clustered morphology 161. For the same thickness, the clustered morphology 120 has a higher roughness factor than the non-clustered morphology 161, and therefore, to produce the same roughness factor, the clustered morphology 120 can be made thinner than the non-clustered morphology. Also, given the irregular shape of the clusters, the inter-cluster gaps 127 of the clustered morphology 120 are interconnected throughout much of the nanoporous layer 117, whereas the internal cavities 133 of the non-clustered morphology 161 are not connected to the same extent as the inter-cluster gaps 127. Thus, the inter-particle nanopores 125 within the clusters 123 are connected to the network of inter-cluster gaps 127 in the clustered morphology 120, whereas there are no inter-cluster gaps in the non-clustered morphology 161, and the inter-particle nanopores 125 cannot be connected as they are in the clustered morphology 120.

[0142] Fabrication of non-clustered nanoporous layer - the whole electroplating process Nanoporous layers having a non-clustered morphology can be prepared using electroplating. Referring to Figure 11, in step 1101, a plating bath is prepared containing metal ions and a surfactant in a reverse micelle phase. Then, in step 1103, electroplating is performed in the plating bath to deposit a nanoporous layer in a non-clustered morphology. In step 1105, the resulting nanoporous layer is washed to remove the surfactant therefrom.

[0143] Preparation of the plating bath In step 1101, the plating bath is similar to the reverse micelle phase of step 601 in FIG. 6A for producing a clustered nanoporous layer without electroplating. The plating bath includes a surfactant and a metal ion source material in a reverse micelle phase, similar to the production of a clustered nanoporous layer. All descriptions regarding the surfactant and metal ion source material in step 601 in FIG. 6A are applicable to step 1101 in FIG. 11 . However, the plating bath in step 1101 is not identical to the reverse micelle phase of step 601. One important difference may be that the plating bath may require some additional materials in consideration of electroplating in the next step. For many metal source compounds that can be naturally reduced, the plating bath may require a chelating agent to prevent the metal ions from being naturally reduced during and before electroplating. In contrast, such a chelating agent may not be required in the reverse micelle phase of step 601.

[0144] Electroplating In step 1103, electroplating is performed in an aqueous liquid composition in a reverse micelle phase containing metal ions. Cathode and anode electrodes are submerged in a plating bath containing the liquid composition and connected to a power source. When a DC voltage is applied between the cathode and anode electrodes, the cathode electrode supplies electrons to the aqueous liquid composition. The electrons jump from the cathode electrode to the hydrophilic space immediately adjacent to the reverse micelles, where they can reduce positively charged metal ions to metal atoms. The metal atoms combine to form metal particles, which can be deposited on the surface of the cathode electrode. In the process, the reverse micelles can rupture. The electrons supplied to the cathode electrode travel through the deposited nanoparticles and become available on the outer surface of the deposited nanoparticles. The electrons are then available to reduce nearby metal ions, forming metal nanoparticles that are deposited on the previously deposited nanoparticles.

[0145] Time for electroplating Electroplating is carried out for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes to obtain a nanoporous layer having a roughness factor of 100 to 800. In embodiments, the time for electroplating may be within the range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., about 10 to about 30 minutes. In embodiments, the time for electroplating is controlled to obtain a nanoporous layer having a roughness factor of 100 or greater.

[0146] Formation of successive layers and cavities In electroplating reduction, nanoparticles adjacent to the cathode electrode are deposited first on the surface of the cathode. Then, additional nanoparticles are deposited on the previously deposited nanoparticles 121. Thus, nanoparticles generally deposit in successive layers on the cathode electrode. However, because nanoparticle deposition may not occur at the same rate across the cathode surface and the previously deposited layer of nanoparticles, internal cavities 133 may form within the resulting nanoporous layer. The nanoparticle deposition may grow spatially, horizontally, or laterally, where no nanoparticles are deposited and some cavities 133 may be enclosed by nanoparticles formed thereon. Although the cavities 133 may eventually be interconnected via interparticle nanopores 125, microchannels are not formed throughout or in a substantial portion of the nanoporous layer 117 to interconnect the cavities 133.

[0147] Co-deposited surfactants During the electroplating process, the reverse micelles encapsulating these nanoparticles can rupture, and the nanoparticles are deposited on the cathode electrode. A significant amount of surfactant molecules from the ruptured reverse micelles are deposited on the cathode electrode along with the nanoparticles. During the electroplating process, the surfactant molecules can bind to the nanoparticle surface, resulting in the deposition of nanoparticle-surfactant molecule complexes. The surfactant molecules can be intercalated or trapped between the nanoparticles in the resulting nanostructure.

[0148] Remaining surfactants and effects Surfactant molecules deposited along with nanoparticles can occupy the gaps and spaces between nanoparticles, i.e., the interparticle pores. These surfactant molecules can effectively block the nanopores and nanoparticle surfaces involved in glucose oxidation. Furthermore, surfactant molecules can decompose on the metal surface, which can contaminate the nanoparticle surface. Overall, the sensitivity of glucose oxidation can be affected by the surfactant remaining in the nanoporous layer.

[0149] Cleaning In step 1105, the resulting nanoporous layer is washed with water or other liquid to remove surfactant molecules therefrom. However, washing is not effective in substantially removing the surfactant molecules because many surfactant molecules are trapped between adjacent nanoparticles and the washing liquid can only reach a certain level.

[0150] No nanoparticles or colloids In the electroplating method, no reducing agent is added to reduce metal ions and form nanoparticles. During the electroplating process, nanoparticles may be formed within the hydrophilic spaces of reverse micelles adjacent to or near the cathode electrode surface. The nanoparticles may then be deposited on the cathode electrode. However, nanoparticles are not formed in the hydrophilic spaces of reverse micelles throughout the liquid composition. Therefore, nanoparticle colloids are not formed, as illustrated in Figure 8.

[0151] No clusters and no cluster colloids In electroplating, there is no step to remove the surfactant after nanoparticle formation. Rather, the surfactant and nanoparticles are deposited together during the electroplating process. Therefore, no clusters or cluster colloids are formed at any stage of the process.

[0152] Yield - Metal Recovery Upon completion of electroplating, the plating bath contains significant amounts of metal ions, so metal recovery in electroplating methods may not be as high as reduction by adding excess amounts of reducing agent, as in the process for clustered nanoporous layers.

[0153] Fabrication of nanoporous layers using liquid crystal phases Nanoporous metal layers can be fabricated from a liquid crystalline phase of a surfactant. Referring to Figure 12, in step 1201, an aqueous liquid composition is prepared containing metal ions and a surfactant in a liquid crystalline phase, e.g., a hexagonal array. Then, in step 1203, the aqueous liquid composition is subjected to electroplating to deposit a nanoporous layer, where metal atoms are deposited using the liquid crystalline phase as a template. In step 1205, the surfactant is removed from the deposited hexagonal nanostructures. Figure 13A illustrates the formation of a hexagonal array. Figure 13B illustrates the deposition of a metal using a hexagonal array of a liquid crystalline phase.

[0154] Maltose - Blocking Layer Maltose Maltose is a disaccharide consisting of two glucose units, as illustrated in Figure 20. Maltose can be present in human or animal blood or other bodily fluids. The presence of maltose in a test fluid can interfere with accurate sensing of glucose levels in both enzymatic and non-enzymatic glucose sensing systems.

[0155] Maltose interference in enzymatic glucose sensing. Some enzymes used in enzymatic glucose sensing systems oxidize maltose as well as glucose. Therefore, if maltose is present in the test fluid, the enzymatic glucose sensing system may have inaccurate readings of glucose levels due to the maltose. If the inaccurate readings are used to control or adjust insulin infusion, the consequences can be serious.

[0156] Maltose interference in nonenzymatic glucose sensing. The nanoporous layer 117 of the working electrode 103NE can oxidize maltose at the same bias voltage as glucose sensing. As illustrated in FIG. 20, using a length of approximately 1.4-1.6 nm, maltose molecules can enter the interparticle nanopores 123 of the nanoporous layer 117, where they can be oxidized along with glucose. Example 9.11 and FIG. 18 confirm that maltose can be detected in PBS along with glucose and other interfering chemical components. Also, Example 10.9 and FIG. 19 confirm that maltose can be detected in serum along with glucose and other interfering chemical components.

[0157] Non-enzymatic working electrode with a maltose-blocking layer 21 , the working electrode 103NE includes a nanoporous layer 117 and a maltose-blocking or maltose-screening layer 301 on the nanoporous layer 117. In embodiments, the nanoporous layer 117 is capable of oxidizing both maltose and glucose, regardless of whether it includes a clustered or non-clustered form. The maltose-blocking layer 301 can be in contact with the underlying nanoporous layer 117 or can be separated by an intervening layer. The working electrode 103NE can also include an additional functional layer 112 on the maltose-blocking layer 301. In other cases, the additional functional layer 112 can be interposed between the maltose-blocking layer 301 and the nanoporous layer 117.

[0158] Selective blocking of maltose The maltose-blocking layer 301 effectively or substantially blocks or prevents maltose molecules from passing through or penetrating therethrough, while allowing glucose molecules to pass through. With the maltose-blocking layer 301, maltose molecules contained in the test fluid cannot reach the underlying nanoporous layer 117 at all, or in concentrations significant enough to interfere with glucose sensing. Given the selective maltose-blocking effect of the maltose-blocking layer 301, the presence of maltose in the test fluid is unlikely to affect glucose sensing, even if the nanoporous layer 117 were able to oxidize maltose at the same bias voltage as for glucose oxidation. In addition, the maltose-blocking layer 301 effectively blocks or prevents other molecules and components of the test fluid larger than maltose.

[0159] bias voltage In a non-enzymatic glucose sensing system, the addition of the maltose-blocking layer 301 does not require an increase or decrease in the bias voltage for glucose sensing.

[0160] Porous polymer layer In embodiments, maltose-blocking layer 301 is made from or includes a porous polymeric material that allows glucose to pass through but not maltose, and contains at least one poly-phenylenediamine (poly-PD), including poly(m-phenylenediamine) (poly-mPD), poly(o-phenylenediamine) (poly-oPD), and poly(p-phenylenediamine) (poly-pPD).

[0161] Nanosized thickness The maltose-blocking layer 301 has a thickness of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nm or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nm. Throughout the description, the thickness of the maltose-blocking layer refers to the average thickness of the polymer layer, excluding the upper and lower 10% of thickness variations. In embodiments, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the immediately preceding sentence, such as about 15 nm to about 35 nm, about 17 nm to about 33 nm, about 18 nm to about 32 nm, about 20 nm to about 30 nm, about 21 nm to about 29 nm, about 22 nm to about 28 nm, etc.

[0162] Porosity level In embodiments, the maltose-blocking layer 301 has a porosity that allows glucose molecules to pass through its thickness while effectively blocking maltose molecules from passing through. To achieve the goal of allowing glucose to pass through and blocking maltose, the overall porosity of the maltose-blocking layer must be adjusted to a desired level. The overall porosity of the maltose-blocking layer 301 is related to the density (or internal morphology, including pores and channels) and thickness of the layer. The concentration of materials for the maltose-blocking layer and the method of forming the maltose-blocking layer may be related to the density. While some success has been achieved in adjusting the overall porosity using these parameters, it has been found that the level of porosity generally cannot be defined or described using the concentration of materials and the method of forming the layer. The thickness of the maltose-blocking layer is also related to the overall porosity, but it depends on the specific porosity or porosity per volume. Thus, the level of porosity must be defined in a different manner.

[0163] Sensitivity (current density) for glucose and maltose without a maltose-blocking layer For glucose monitoring, at steady state, with an applied bias voltage of 0.2-0.45 V in a test fluid having a glucose concentration of 4-20 mM (typical glucose levels in human body fluids), the nanoporous layer 117 in contact with the test fluid (i.e., without the maltose-blocking layer) exhibits a current of 0.1 μA / mMcm 2 (10nA / mMcm 2 ), it is necessary to generate a glucose-oxidation current (current caused by the oxidation of glucose alone) at a level higher than the minimum current density (sensitivity) for glucose. According to an embodiment, the same nanoporous layer 117 without the maltose-blocking layer generates a similar level of current (i.e., 10 nA / mMcm) at steady state with an applied bias voltage of 0.2-0.45 V in a test fluid containing maltose at a concentration of 4-20 mM (the same as the glucose concentration above). 2 (higher) will be generated.

[0164] Porosity of maltose-blocking layer as a function of current density for glucose and maltose According to an embodiment, the maltose blocking layer 301 has a porosity to allow glucose to migrate therethrough, so that the glucose oxidation current remains higher than the minimum current density for glucose. Thus, when a bias voltage of 0.2-0.45 V is applied in a test fluid having a glucose concentration of 4-20 mM, at steady state, the working electrode 103NE with the maltose blocking layer 301 generates a glucose-oxidation current of 10 nA / mMcm. 2On the other hand, the maltose blocking layer 301 has a porosity that effectively blocks maltose from passing therethrough, so that at steady state, when a bias voltage of 0.2-0.45 V is applied in a test fluid having a maltose concentration of 4-20 mM, the current caused by maltose alone (maltose oxidation current) is 0.05 μA / mMcm 2 (5nA / mMcm 2 ), which is at a level lower than the maximum current density for maltose when a maltose-blocking layer is used.

[0165] Electrochemical polymerization The porous polymer material for the maltose-blocking layer 301 can be formed on the nanoporous layer 117 by electrochemical polymerization (electropolymerization) using a cyclic voltammetry technique. In an embodiment, a working electrode containing a nanoporous layer is immersed in a reaction mixture solution containing a monomer for cyclic voltammetry electrochemical polymerization. By applying a bias voltage between the working electrode and a reference electrode within the oxidation voltage range of the monomer, polymerization occurs and a polymer layer is formed on the nanoporous layer. Further details regarding the polymerization of phenylenediamine are disclosed in "Electropolymerization of O-Phenylenediamine on Pt-Electrode from Aqueous Acidic Solution: Kinetics, Mechanism, Electrochemical Studies and Characterization of the Polymer Obtained," Sayyah et al., Journal of Applied Polymer Science, Vol. 112, No. 6, 3695-3706 (2009), and "Electropolymerization of P-Phenylenediamine on Pt-Electrode from Aqueous Acidic Solution: Kinetics, Mechanism, Electrochemical Studies, and Characterization of the Polymer Obtained," Sayyah et al., Journal of Applied Polymer Science, Vol. 117, No. 2, 943-952 (2010), each of which is hereby incorporated by reference herein.

[0166] Application of oxidation voltage The bias voltage can be varied during cyclic voltammetry. For example, but not limited to, the bias voltage can be gradually increased within the oxidation voltage range during the first time segment and then gradually decreased within the oxidation voltage range during the next time segment. For phenylenediamine, the bias voltage is applied between 0.5 V and 1.0 V. Figure 22 shows an example of scanning the bias voltage during cyclic voltammetric electrochemical polymerization of phenylenediamine.

[0167] Bias voltage scanning rate The bias voltage scan rate between the lower and upper limits of the oxidation voltage range, along with the monomer concentration described below, can be related to the porosity and thickness of the resulting polymer layer. In embodiments, the scan rate is about 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, or 400 mV / sec. In embodiments, the scan rate may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 5 mV / sec to about 200 mV / sec.

[0168] Monomer concentration The concentration of the monomer is about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 or 10 mM. In embodiments, the concentration of the monomer may be within the range formed by selecting any two numbers recited in the immediately preceding sentence, e.g., about 0.05 mM to about 0.8 mM, about 1.0 mM to about 5.0 mM, etc. The foregoing concentrations are applicable to the three phenylenediamines.

[0169] Porosity considering monomer concentration The concentration of the monomer in the reaction mixture solution is related to the porosity of the resulting maltose-blocking layer. In the flowchart for fabricating a maltose-blocking layer of Figure 24, the monomer concentration is first determined in step 2401, and polymerization is carried out in step 2403. In embodiments, a monomer concentration of less than about 0.7 mM, about 0.6 mM, or about 0.5 mM may provide a desirable level of overall porosity for the maltose-blocking layer. In embodiments, when the monomer concentration exceeds about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, or about 1.2 mM, the resulting polymer layer does not have sufficient porosity to allow glucose to pass therethrough, i.e., less than 10 nA / mMcm. 2 , generating a glucose-oxidation current at a level below the minimum current density (sensitivity) for glucose. In step 2405, the resulting polymer layer is subjected to treatment to adjust its porosity in step 2405.

[0170] Electric shock to adjust porosity If the overall porosity of the polymer layer 302 is not at a desired level, the polymer layer can be further processed to adjust its porosity. For example, the polymer layer can be subjected to an electric shock. In an embodiment, an electric shock can be applied to the polymer layer 302 using the chronoamperometry setup shown in FIG. 23, in which the electroshock electrode 309 and the polymer layer 302 formed on the nanoporous layer 117 are submerged in an electrolyte solution 311. A voltage source 305 and a switch 307 are connected between the substrate 303 and the electroshock electrode 309. Operation of the switch 307 causes an electric current to flow through the porous polymer layer 302, causing a morphological change that increases the porosity of the polymer layer 302. As a result, the polymer layer 302 becomes a maltose-blocking layer 301 with a desired level of porosity that allows glucose to pass through its thickness while effectively blocking maltose from passing therethrough.

[0171] acidic solution The electrolyte solution for the electric shock may be, but is not limited to, an acidic solution having a pH of less than about 2, 3, or 4. In some embodiments, the acidic solution may include at least one acid. Non-limiting examples of acids for the acidic solution include phosphoric acid (H3PO4), nitric acid (HNO3), chloric acid (HCl), formic acid, lactic acid, malic acid, citric acid, carbonic acid, sulfonic acid, etc.

[0172] Shock waveform The potential may be applied in a variety of waveforms. In embodiments, the potential is applied in AC or DC. In embodiments, the potential is applied in multiple pulses or a single pulse. In embodiments, the potential may be applied in other shapes of voltage signals.

[0173] Electrical shock potential The potential applied to polymer layer 302 is about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 V. In embodiments, the maximum voltage may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 0.5 to about 2.5 V, from about 1.0 to about 2.0 V, etc.

[0174] Duration for electric shock The duration of application of the potential is about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 seconds. In embodiments, the duration may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, such as about 0.5 to about 2.5 seconds, about 1.0, and about 2.0 seconds.

[0175] Maltose-blocking layer applicable to enzyme sensing In an embodiment, the maltose-blocking layer 301 may be applied to an enzymatic glucose sensing system. Referring back to Figure 2, the maltose-blocking layer 301 may be added as an additional functional layer 112 on the enzyme layer 111, where maltose is blocked and glucose can pass through.

[0176] CGM working electrode CGM system A continuous glucose monitoring (CGM) system includes glucose-sensing electrodes that are in in vivo contact with a subject's biological fluids for measuring glucose levels contained therein. In practice, the CGM electrodes are inserted or implanted into the subject's body for measurements over an extended period of time, such as days, a week, weeks, or months.

[0177] Non-enzymatic CGM working electrode 31 shows a cross section of a non-enzymatic CGM working electrode 501 according to one embodiment. The illustrated CGM working electrode 501 has a laminated structure including a base 503, a conductive layer 110, a nanoporous layer 117, a maltose-blocking layer 301, an electrolyte ion-blocking layer 505, and a biocompatible layer 507.

[0178] Electrode Base The base, base substrate, or electrode base 503 provides support for the laminate structure of the CGM working electrode 501. In embodiments, the base 503 is an electrically insulating layer and may be fabricated from or include materials such as, but not limited to, polyimide, polypropylene, polyethylene glycol, polyhydroxyethyl methacrylate (pHEMA), and other biocompatible polymers. In embodiments, the base 503 may be in the form of a flexible film of an electrically insulating and biocompatible material. The base 503 has a thickness ranging from, but not limited to, about 30 μm to about 200 μm. The base 503 is an optional layer for the CGM sensing electrode 501 and may be omitted in some embodiments.

[0179] Conductive layer The conductive layer 110 may be disposed on the base 503 with or without an intervening layer therebetween. In embodiments, the conductive layer 110 is formed by, but is not limited to, printing or dispensing a conductive or semiconductive material on the base 503. In the CGM working electrode 501, the conductive layer 110 may have a thickness in the range of approximately 100 nm to 100 μm, but is not limited to this. In some embodiments, the conductive layer 119 may include two or more sublayers of conductive or semiconductive material. In embodiments in which the base 503 is omitted, the conductive layer 119 may function as a support for the laminated structure above it.

[0180] Nanoporous Layer The nanoporous layer 117 may be formed on the conductive layer 110. In the CGM working electrode 501, the nanoporous layer 117 has a thickness ranging from about 500 nm to about 10 μm, but is not limited to such. The nanoporous layer 117 may have at least one of a clustered morphology, a non-clustered morphology, a hexagonal nanostructure, or other nanoporous morphology.

[0181] Maltose-blocking layer The maltose-blocking layer 301 can be formed on the nanoporous layer 117 to block maltose molecules from reaching the underlying nanoporous layer 117 while allowing glucose molecules to pass through. In embodiments, the maltose-blocking layer 301 comprises a polymer material, such as poly-PD, with nano-sized pores to allow glucose molecules to pass through but not maltose molecules. The maltose-blocking layer can have a thickness ranging from about 5 nm to about 40 nm, but is not limited thereto. The maltose-blocking layer 301 is an optional layer for the CMG sensing electrode 501 and can be omitted in some embodiments.

[0182] Electrolyte ion-blocking layer (electrode conditioning enhancement / promotion layer) The electrolyte ion-blocking layer 505 blocks small electrolyte ions, such as Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- The electrolyte ion-blocking layer 505 effectively restricts or prevents ions from passing therethrough or diffusing toward the underlying nanoporous layer 117. As described below, the electrolyte ion-blocking layer 505 enhances the conditioning of the CGM working electrode and is also referred to as a working electrode conditioning enhancement or promotion layer. The electrolyte ion-blocking layer 505 is porous so that glucose molecules can pass freely through it. When implemented, the electrolyte ion-blocking layer 505 is hydrophobic so that it does not readily swell by absorbing water contained in the test fluid. The electrolyte ion-blocking layer 505 can have a thickness ranging from about 0.1 μm to about 10 μm, but is not limited thereto.

[0183] Materials for Electrolyte Ion-Blocking Layers The electrolyte ion-blocking layer 505 can include or be made of, for example, at least one of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). The electrolyte ion-blocking layer 505 can also be formed from or additionally include a copolymer of methyl methacrylate and butyl methacrylate, and a polymer resulting from the polymerization of one or more monomers including methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, and 2-ethylhexyl acrylate.

[0184] Biocompatible Layer When the CGM sensor is implanted or inserted into a subject's body, the biocompatible or biologically protective layer 507 contacts the subject's tissues and bodily fluids. The biocompatible layer 507 includes at least one biocompatible material that is not toxic to the subject's tissues and does not cause immune rejection by the subject's body. Additionally, the at least one material of the biocompatible layer 507 must allow bodily fluids to pass therethrough and reach the underlying nanoporous layer 117 so that the presence of the material does not significantly impair glucose concentration sensing. The biocompatible layer 507 can have a thickness ranging from about 5 μm to about 30 μm, but is not limited thereto.

[0185] Materials for the biocompatible layer The biocompatible layer 507 can include or be made of, for example, at least one of poly(vinyl alcohol), poly(ethylene oxide-co-propylene oxide) (PEO-PPO), poly(ethylene oxide) (PEO), poly(sulfone) (PS), poly(ethylene terephthalate) (PET), poly(ether-urethane) (PU), poly(dimethylsiloxane) (PDMS), ethylene-co-vinyl acetate (EVA), poly(methyl methacrylate), poly(tetrafluoroethylene) (PTFE), poly(propylene) (PP), poly(ethylene) (PE), polyethylene glycol, and polyhydroxyethyl methacrylate (pHEMA).

[0186] Modification The CGM working electrode 501 may include one or more additional functional layers, which are not shown in Figure 31. In some embodiments, one or more of the maltose-blocking layer 301, the electrolyte ion-blocking layer 505, and the biocompatible layer 507 may be omitted. In other embodiments, two or more of the maltose-blocking layer 301, the electrolyte ion-blocking layer 505, and the biocompatible layer 507 may be combined into one layer, or their positions may be changed.

[0187] No enzyme layer The CGM working electrode 501 does not contain an enzyme layer containing a glucose-specific enzyme. The CGM working electrode 501 does not contain such an enzyme in any of its layers.

[0188] No oxygen-absorbing layer The CGM working electrode 501 does not include an oxygen-capturing material or layer that would be required to collect and supply molecular oxygen if glucose oxidase were used for the oxidation of glucose.

[0189] No electron carrier The CGM working electrode 501 does not contain an electron transfer material that would be required to transfer electrons if glucose dehydrogenase were used to oxidize glucose.

[0190] Transient signal of the conditioning current of the CGM working electrode or system When an electrochemical cell using a CGM working electrode is constructed with an applied bias voltage, the CGM working electrode generates a current. The current from the CGM working electrode represents the sum of the background noise at the CGM working electrode and the current from glucose oxidation. Initially, the current exhibits transient behavior. As shown in Figures 25-30, initially, the current is very high compared to that caused by glucose oxidation alone and decreases rapidly. Thereafter, the rate of decrease slows. Eventually, the current settles to a certain level, i.e., a steady state, but in vivo, the current may fluctuate slightly within an acceptable range.

[0191] Current for glucose sensing For accurate glucose sensing, the current must be measured when the electrochemical cell and / or CGM working electrode are in a steady state. In other words, the current from the CGM working electrode should not change significantly over time (i.e., settle at a certain level after an initial decrease) when the glucose concentration is unchanged. Furthermore, for accurate glucose sensing, the background current (noise) should not be too high compared to the current caused by glucose oxidation alone. In other words, the total current should not be too high compared to the current resulting from glucose oxidation alone.

[0192] Conditioning the CGM working electrode or electrochemical cell CGM working electrodes require conditioning before glucose sensing. Here, conditioning refers to the process of stabilizing the CGM working electrode for accurate glucose sensing. Once conditioning of the CGM working electrode is complete, the current from it should settle at a certain level and should not be too high compared to the glucose-derived current. To provide accurate glucose levels, the CGM system must use the current measured after conditioning is complete. Conditioning a CGM working electrode can take a long time. Commercially available enzymatic CGM working electrodes require several hours to several days for conditioning.

[0193] Desired rate of change of current Assuming that the current from glucose oxidation in vivo is about tens of nanoamperes, for accurate glucose sensing, the rate of decrease in current from the CGM working electrode must be less than, for example, 20 nA (nanoamperes) per minute. To provide a reference point, the desired rate of current change should be 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nA per minute or less. In embodiments, the rate of current change can be determined over shorter or longer time periods.

[0194] Desired level of current Current from glucose oxidation in vivo is typically tens of nanoamperes. The desired level of total current can vary depending on various factors, including measurement accuracy, signal processing capability, data processing capability, etc. As these factors are further developed, the desired level may increase. Nevertheless, assuming that the current from glucose oxidation in vivo is approximately tens of nanoamperes, for accurate glucose sensing, the current from the CGM working electrode must be less than, for example, 500 nA. To provide a reference point, the desired current should be at or below 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110 or 100 nA.

[0195] Conditioning complete The CGM system determines that conditioning of its CGM working electrode or its electrochemical cell is complete. The CGM system may determine conditioning completion when the rate of current change is at, below, or remains at a predetermined value, such as the desired current change or decrease rate specified above. The CGM system may determine conditioning completion when the total current change remains at or below a predetermined value, such as the desired current level specified above, for a predetermined period of time. The CGM system may determine conditioning completion when the rate of current change is at or below that predetermined value, and further when the total current change remains at or below that predetermined value for a predetermined period of time, such as when the current change rate is less than 5 nA / min and the total current remains below 400 nA for 1 min.

[0196] Conditioning completion notification The CGM system can notify the user that conditioning is complete. Upon forming, or some time after forming, an electrochemical cell for glucose oxidation, the CGM system can begin monitoring the current from the CGM working electrode. When the current meets one or more requirements for conditioning completion, the CGM system can provide a notification to the user to notify them of conditioning completion. The notification can be in any form, including sound, vibration, light, or an information display. Additionally or alternatively, the CGM system may not provide information indicating glucose levels before conditioning is complete.

[0197] Reducing the time for conditioning the CGM working electrode and concentration discontinuities of small electrolyte ions Human body fluids contain significant amounts of Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- In an embodiment, the electrolyte ion-blocking layer 505 contains Na + , K. + , Ca 2+ , Cl - , PO4 3- and CO3 2- The electrolyte ion-blocking layer 505 restricts or prevents small electrolyte ions from passing through it. As a result, the concentration of these electrolyte ions differs significantly between above and below the electrolyte ion-blocking layer 505. FIG. 32 conceptually illustrates the concentration discontinuity on either side of the electrolyte ion-blocking layer 505. With the electrolyte ion-blocking layer 505, the total concentration of small electrolyte ions is significantly less in the nanoporous layer 117 than in the biocompatible layer 507. Without the electrolyte ion-blocking layer 505, the total concentration of small electrolyte ions in the nanoporous layer 117 would be similar to that in the biocompatible layer 507.

[0198] Electrolyte ions - Concentration of small electrolyte ions beneath the blocking layer In embodiments, the total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 is greater than 0% but less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% of the total concentration of the same electrolyte ions above the electrolyte ion-blocking layer 505. The total concentration below the electrolyte ion-blocking layer 505 may be within a range formed by selecting any two numbers (two percentage values) listed in the immediately preceding sentence. As shown in FIG. 32 , for example, the total concentration of electrolyte ions in human interstitial fluid (i.e., above the electrolyte ion-blocking layer 505) is greater than or equal to about 0.1 M; in contrast, the total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 is less than or equal to about 0.01 M. The total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 can be obtained by measuring the double layer capacitance of the nanoporous layer 117 and applying the measurements to the Gouy-Chapman equation, as described in detail in Ionic Strength-Controlled Virtual Area of Mesoporous Platinum Electrode, Boo et al., J. AM. CHEM. Soc. 2004, 126, 4524-4525.

[0199] Acceleration of ionic equilibria in nanoporous layers As described, the ion-blocking layer 505 establishes or creates a substantial discontinuity in the total concentration of small electrolyte ions between above the electrolyte ion-blocking layer 505 and below the same layer. A low concentration of small electrolyte ions is significantly better for conditioning the CGM working electrode 501, particularly the nanoporous layer 117. While no aspect of the invention is bound by any theory or belief, a low concentration of small electrolyte ions can accelerate ionic equilibrium in the nano-sized structures and surfaces of the nanoporous layer 117 that would not occur at larger scales, such as micro-sized structures and surfaces. As ionic equilibrium is accelerated in the nanoporous layer 117, the time to reach ionic equilibrium or steady state inside the nanostructures of the nanoporous layer 117 will be shorter at lower concentrations of electrolyte ions with the presence of the electrolyte ion-blocking layer 505 than at higher concentrations without the electrolyte ion-blocking layer 505.

[0200] Significantly shorter conditioning times By accelerating the ionic equilibrium in the nanoporous layer 117, the electrolyte ion-blocking layer 505 significantly enhances and expedites the conditioning of the non-enzymatic CGM working electrode 501 of Figure 31, i.e., shortens the time to reach a desired current and / or a desired rate of current change, i.e., a steady state. According to embodiments, when using a non-enzymatic CGM working electrode 505 with an electrolyte ion-blocking layer 505, less time is required for completion of conditioning compared to using the same non-enzymatic CGM working electrode without the electrolyte ion-blocking layer 505.

[0201] Conditioning Time If the desired current change rate is 5 nA / min or less, a non-enzymatic CGM working electrode without an electrolyte ion-blocking layer 505 takes about 3 hours in serum containing 0.1 M or more electrolyte ions; in contrast, a non-enzymatic CGM working electrode with an electrolyte ion-blocking layer 505 takes about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, or 30 minutes or less in the same serum. When the desired rate of current change is 3 nA / min or less, a non-enzymatic CGM working electrode without an electrolyte ion-blocking layer 505 takes more than 5 hours in serum containing 0.1 M or more electrolyte ions; in contrast, a non-enzymatic CGM working electrode with an electrolyte ion-blocking layer 505 takes about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 15 minutes, or 10 minutes or less in the same serum. When the desired rate of current change is 2 nA / min or less, a non-enzymatic CGM working electrode without an electrolyte ion-blocking layer 505 takes more than 5 or 10 hours in serum containing 0.1 M or more electrolyte ions; in contrast, a non-enzymatic CGM working electrode with an electrolyte ion-blocking layer 505 takes about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 15 minutes, or 10 minutes or less in the same serum.

[0202] Unexpected results Without proper conditioning, a CGM working electrode cannot provide a current corresponding to an accurate glucose level. Reducing the time for conditioning is a very important practical consideration when developing and manufacturing a CGM working electrode. This is because proper conditioning of a CGM working electrode can take hours, if not tens of minutes, and because people tend to want to know their glucose level immediately after inserting the electrode into their body. With reference to the examples described below, the time for conditioning a CGM working electrode is reduced from approximately 3, 5, or 10 hours to less than 30 minutes, all other conditions remaining the same, simply by including the electrolyte ion-blocking layer 505. This is a very significant improvement and an unexpectedly high achievement.

[0203] Details of the electrolyte ion-blocking layer The electrolyte ion-blocking layer 505 of the non-enzymatic CGM working electrode comprises or is fabricated from at least one porous hydrophobic polymer, including poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). Additional examples of porous hydrophobic polymers include copolymers of methyl methacrylate and butyl methacrylate, and polymers obtained from the polymerization of one or more monomers, including methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, 2-ethylhexyl acrylate, and the like. The average molecular weights for these polymers are about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000, 310,000, 320,000, 330,000, 340,000, 350,000, 360,000, 370,000, 380,000, 390,000, 410,000, 420,000, 430,000, 440,000, 450,000, 460,000, 470,000, 480,000, 490,000, 500,000, 510,000, 520,000, 530,000, 540,000, 550,000, 560,000, 570,00 In an embodiment, the molecular weight may be within a range formed by selecting any two numbers recited in the immediately preceding sentence. The electrolyte ion-blocking layer can have a thickness of about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 μm.In embodiments, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the immediately preceding sentence, e.g., a range of about 2 to about 5 μm, about 1 to about 3 μm, etc.

[0204] Ion concentration drop has no effect on enzymatic glucose-sensing electrodes. In an enzymatic CGM system, the CGM working electrode contains a glucose-specific enzyme for the oxidation of glucose molecules. The enzymatic CGM working electrode may include a functional layer containing a porous hydrophobic material that can effectively lower the concentration of electrolyte ions below the functional layer. However, in an enzymatic CGM system, the concentration drop by the functional layer cannot provide a reduction in the time for conditioning the CGM electrode due to ionic equilibrium at the nanosized surface or structure. This is because enzymatic CGM systems use enzymes to oxidize glucose molecules and do not require a nanoporous layer for glucose oxidation. Therefore, even if a porous hydrophobic layer were included in the enzymatic CGM working electrode, even if such a layer would cause a discontinuity in electrolyte ion concentration across its thickness and even if there was some reduction in the time for conditioning the enzymatic CGM working electrode, such a reduction would not be equal to the reduction in the time for conditioning in a non-enzymatic CGM working electrode 501 that has both an electrolyte ion blocking layer 505 and a nanoporous layer 117.

[0205] CGM subcutaneous electrode module CGM electrode unit In embodiments, the CGM system includes an electrode unit or module that is in subcutaneous contact with the subject's bodily fluids. The electrode unit may include a unitary body that houses one or more electrodes that contact the bodily fluids when inserted into the subject's body. The unitary body may be flexible.

[0206] CGM electrode unit configuration FIG. 33 shows a CGM electrode unit 701 according to one embodiment. The CGM electrode unit 701 includes a subcutaneous portion 703 and a contact terminal portion 705. The subcutaneous portion 703 is for insertion into a subject's body and includes a working electrode 501, a counter electrode 105, and a reference electrode 106 exposed through openings formed through an insulating layer 707 for subcutaneous contact with bodily fluids. The contact terminal portion 705 is for remaining outside the subject's body and for engaging or connecting to a compatible device. The contact terminal portion 703 includes a working electrode terminal 501T, a counter electrode terminal 105T, and a reference electrode terminal 106T electrically connected to the working electrode 501, the counter electrode 105, and the reference electrode 106, respectively, under the insulating layer 707. Here, each of the working electrode 501, the counter electrode 105, and the reference electrode 106 can have, but is not limited to, the features and characteristics described in this disclosure.

[0207] Fabrication of CGM electrode unit FIG. 34 is a flowchart for fabricating a CGM electrode unit 701, according to one embodiment. In step 3401, an electrically insulating, flexible film is provided for the base or electrode base 503 (also shown in FIG. 31). Then, in step 3403, a conductive layer is formed on the base 503 in predetermined shapes 110R, 110W, and 110C, as shown in FIG. 35. Then, in step 3405, an insulating film 707 is applied over the conductive layer, selectively exposing portions or areas of the conductive layer, as in FIG. 36. Then, in step 3407, the intermediate product is cut to provide the shape shown in FIG. 37. In step 3409, a nanoporous layer 117 is formed on the exposed area for the working electrode 501. Then, in 3411, one or more functional layers are formed on the nanoporous layer 117 to provide the laminate configuration of the non-enzymatic CGM working electrode 501, as in FIG. Additionally, a salt layer may be formed on the exposed area for the reference electrode 106. In embodiments, cleavage of the intermediate product in step 3407 may be performed after step 3409 or 3411.

[0208] Conductive layer - multiple conductive elements 35 provides a top view of the intermediate product after step 3403 and its cross section taken along the arrow direction about line 3501 according to one embodiment. As shown, the conductive layer formed on base 503 has three separate elements 110C, 110W, and 110R of predetermined shapes: conductive layer element 110C for the counter electrode, conductive layer element 110W for the working electrode, and conductive layer element 110R for the reference electrode. Each of conductive layer elements 110C, 110W, and 110R includes a conductive portion reserved for a contact terminal (at contact terminal portion 705 in FIG. 33), a conductive portion reserved for an electrode (at subcutaneous portion 703 in FIG. 33), and a conductive connection between the two conductive portions.

[0209] Conductive layer fabrication The conductive layer may be a single layer of conductive material or may be formed from multiple sublayers of different conductive materials. In an embodiment, either or both of the conductive layer element 110C for the counter electrode and the conductive layer element 110W for the working electrode are formed from at least two sublayers, e.g., a silver layer and a conductive carbon layer on the silver layer. In an embodiment, the conductive layer element 110R for the reference electrode is formed from a single layer, e.g., a silver layer. The conductive layer 110 or its sublayers may be formed by printing a conductive ink on or above the base 503 and then drying it. A sublayer formed on another sublayer may also be formed by printing the conductive material for that sublayer. The conductive layer elements 110W, 110C, and 110R in FIG. 35 are all single-layer; however, for purposes of illustrating alternatives, in FIGS. 36-38, the conductive layer elements 110W and 110C have a two-sublayer configuration, i.e., a carbon layer 1605 above a silver layer 1603 (see also FIG. 16A).

[0210] Insulating film FIG. 36 shows an intermediate product after placement of an insulating film according to one embodiment. The insulating film 707 may be pre-cut with openings in the subcutaneous area 703 of FIG. 33 to expose the conductive portions reserved for the counter electrode 105, working electrode 501, and reference electrode 106. The insulating film 707 does not cover the contact terminal portion 705 of FIG. 33, thus exposing the terminal portions of each of the conductive layer elements 110C, 110W, and 110R (representing 105T, 501T, and 106T, respectively). The conductive connections of the conductive layer elements 110C, 110W, and 110R are covered with the insulating film 707. An adhesive layer (not shown) may be inserted between the base film 503 and the insulating film 707. The insulating film 707 may be an adhesive-coated film.

[0211] Disconnect In step 3407, the intermediate product of FIG. 36 is cut, and unnecessary portions of insulating film 707 and base 503 are removed, for example, by die-cutting. FIG. 37 shows the resulting product, in which contact terminal portion 705 (the proximal end portion of CGM electrode unit 701) is wider than subcutaneous portion 703 (the distal end portion of CGM electrode unit 701). In embodiments, the distal portion has a width of approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm in the direction along line 3501. In embodiments, the width may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, for example, from about 1.0 mm to about 1.5 mm. In embodiments, the CGM electrode unit 701 has a length in a direction between its distal and proximal ends of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mm. In embodiments, the length may be within a range formed by selecting any two numbers listed in the immediately preceding sentence, e.g., from about 10 mm to about 20 mm.

[0212] Formation of nanoporous layer In step 3409, a nanoporous layer 117 is formed on the exposed conductive layer element 110W for the working electrode. Figure 38A shows a cross section of the intermediate product in the direction of the arrow about line 3501 after forming the nanoporous layer 117. In embodiments, the nanoporous layer 117 is formed by dispensing a cluster colloid comprising nanoparticle clusters dispersed in a liquid onto the conductive layer 110 and allowing the liquid to dry therefrom. In alternative cases, different forms of the nanoporous layer 117 may be formed using different methods disclosed herein. In some embodiments, the cutting in step 3407 may be performed after forming the nanoporous layer 117.

[0213] Functional layer(s) for the working electrode After the formation of the nanoporous layer 117, one or more functional layers are formed on the nanoporous layer 117 to provide a non-enzymatic CGM working electrode 501, as in FIG. 31 . A maltose blocking layer 301 can be formed on the nanoporous layer 117, but is not limited to such. An electrolyte ion-blocking layer 505 can be formed on the nanoporous layer 117 for improved conditioning of the resulting CGN working electrode 501, but is not limited to such. Additionally, a biocompatible layer 507 can be formed on the nanoporous layer 117, more particularly, on the electrolyte ion-blocking layer 505. FIG. 38B shows a cross-section of a CGM working electrode 501 including the electrolyte ion-blocking layer 505 and the biocompatible layer 507.

[0214] Reference and counter electrodes In embodiments, a salt layer, e.g., AgCl, may be formed on the exposed conductive layer element 110R for the reference electrode 106. The formation of the salt layer may be performed any time after forming the conductive layer element 110R. In embodiments, the counter electrode 105 may not require additional processing on the conductive layer element 110C.

[0215] Subcutaneous insertion of the CGM electrode unit In an embodiment, the subcutaneous portion 703 (distal portion) of the CGM electrode unit 701 is inserted subcutaneously into the subject's body, with or without the use of an insertion tool known in the art or to be developed in the future. With proper subcutaneous insertion, the working electrode 501, reference electrode 106, and counter electrode 105 of the subcutaneous portion 703 contact the subject's interstitial fluid, while the terminal portion 705 of the CGM electrode unit 701 remains outside the subject's body.

[0216] Supported devices In embodiments, terminal portion 705 is then engaged or connected to a compatible device (not shown) that includes compatible ports or terminals corresponding to working electrode terminal 501T, counter electrode terminal 105T, and reference electrode terminal 106T. In embodiments, the compatible device further includes electrical circuitry that completes the electrochemical cell of FIG. 1 together with CGM electrode unit 701 for a continuous monitoring glucose module. In some embodiments, in addition to the electrical circuitry for completing the electrochemical cell, the compatible device may include at least one processor for processing data including the current obtained from the electrochemical cell for conversion to a standardized number representing the glucose level. In some embodiments, the compatible device includes a wireless module for wirelessly transmitting data to another wireless device, such as a smartphone or computing device.

[0217] BGM Disposable Strip Single-Point Device Glucose sensing can be performed in vitro at a single time point. Single-time point glucose sensing systems measure glucose levels in a test fluid, most commonly blood. The systems are therefore called blood glucose monitoring (BGM) systems. BGM systems include single-use disposable cartridges or strips.

[0218] Disposable cartridges 39 shows a BGM disposable cartridge 901 and sensing module 911 for a single-time point glucose sensing system according to an embodiment. The disposable cartridge 901 includes a test fluid reservoir 903, a counter electrode 105, a reference electrode 106, and a cartridge working electrode 905 formed on a base 907 that provides structural support for the electrodes 105, 106, and 905. Electrical connections (not shown) are made between the electrodes and a connector 909 through the base 907.

[0219] Sensing Module In an embodiment, the disposable cartridge 901 is designed to electrically and / or mechanically couple to the sensing module 911 via a connector 909. The sensing module 911 may include electrical circuitry (not shown) for the voltage source 109 and the current sensor 108. When the disposable cartridge 901 is properly connected to the sensing module 911, the electrodes 105, 106, and 905 are connected to the circuitry of the sensing module 911, as in FIG.

[0220] working electrode The working electrode 905, according to one embodiment, includes a conductive layer 110 and a nanoporous layer 117. The working electrode 905 further includes a filter layer 913 to filter and screen cells, lipids, and large molecules contained in the test fluid. In embodiments, the filter layer 913 may be made of or include woven fabric, cotton, or other materials capable of screening out cells, lipids, and other large components of blood, but allowing glucose to pass therethrough.

[0221] Working electrode not included In an embodiment, the working electrode 905 does not contain a glucose-specific enzyme. Furthermore, the working electrode 905 does not contain a surfactant or an electron carrier that may be required in enzymatic glucose sensing. Furthermore, given that the working electrode 905 is an in vitro device, it does not require a biocompatible layer.

[0222] Calibration of the working electrode Current from the working electrode According to embodiments, a non-enzymatic working electrode having a nanoporous glucose-oxidation layer generates a current caused by the oxidation of glucose contained in a test fluid. In practice, the current from the non-enzymatic working electrode includes 1) a current caused only by glucose oxidation (glucose-oxidation current), 2) a current caused by interfering chemical components, if the test fluid contains such interfering chemical components, and 3) a current caused by interactions between the electrochemical cell and other chemical components contained in the test fluid.

[0223] Glucose levels in body fluids Normal glucose levels in healthy individuals are 4.0-6.0 mM (72-108 mg / dL). When considering diabetic patients, glucose levels may range from 4.0-20 mM (72-360 mg / dL).

[0224] Glucose oxidation current In an embodiment, at steady state (after conditioning) in a test fluid containing 4.0-20 mM glucose, when a bias voltage of about 0.2 V to about 0.45 V is applied, the current resulting from glucose oxidation alone (glucose-oxidation current) is 0.1 μA / mMcm 2 (10nA / mMcm 2) above the reference level. In the 4.0-20 mM glucose concentration range, the nanoporous glucose-oxidation layer (and thus the non-enzymatic working electrode) generates a glucose-oxidation current of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 nA for 1 mM glucose in the test fluid. In embodiments, the glucose-oxidation current from 1 mM glucose in the test fluid may be within the range formed by any two numbers in the immediately preceding sentence, e.g., about 1.5 nA to 2.5 nA. Thus, in the 4.0-20 mM glucose concentration range, the glucose-oxidation current from the non-enzymatic working electrode may be about 2.0 nA (4.0 x 0.5) to about 120 nA (20 x 6.0). In embodiments, the glucose-oxidation current can be about 2.0, 4.0, 8.0, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, or 120Na. In embodiments, the glucose-oxidation current from 4.0-20 mM glucose contained in the test fluid may be within the range formed by any two numbers in the immediately preceding sentence, for example, about 1.5 nA to 2.5 nA.

[0225] Current and glucose concentration calibration In embodiments, for the same glucose concentration in a test fluid, the glucose-oxidation current may vary from one nanoporous glucose-oxidation layer to another depending on their particular manufacturing conditions. Also, for a particular nanoporous glucose-oxidation layer, the glucose-oxidation current generally correlates linearly with glucose concentration, but may not be so linear across the entire concentration or current range. In embodiments, for each batch of nanoporous glucose-oxidation layers manufactured using the same conditions, one or more nanoporous glucose-oxidation layers are tested, and a correlation profile between glucose-oxidation current and glucose concentration is determined for the particular batch. Later in the glucose sensing or monitoring process using nanoporous glucose-oxidation layers from the same batch, the correlation profile is used in calculating or determining the glucose level in the test fluid.

[0226] Second Working Electrode Ascorbic Acid Ascorbic acid, also known as vitamin C, plays an important role in the human body. Ascorbic acid is prone to oxidation and is easily oxidized at low oxidation potentials. Ascorbic acid may interfere with glucose sensing from body fluids.

[0227] Currently, there are no layers available to block ascorbic acid Given that ascorbic acid has a negative charge, a negatively charged layer has been proposed to repel ascorbic acid but allow glucose to pass through. However, to date, no commercially available glucose-sensing electrodes have been found that block ascorbic acid.

[0228] Two working electrodes In embodiments, a glucose sensor or sensing system includes at least one additional working electrode in addition to the working electrode 103 of Figure 1. Figure 40 conceptually illustrates a two-working electrode glucose sensing system 4101. In this system, a first working electrode 4103A, a second working electrode 4103B, a counter electrode 105, and a reference electrode 106 are connected to a potentiostat 4104, which includes electrical circuitry to function as operational amplifiers 4107A and 4107B, current sensors 4108A and 4108B, and voltage sources 4109A and 4109B for the two working electrodes 4103A and 4103B.

[0229] Operation of the two-working electrode system In this embodiment, oxidation of both glucose and ascorbic acid occurs at the first working electrode 4103A. Therefore, the current from the first working electrode 4103A represents the total concentration of glucose and ascorbic acid in the test fluid 102. On the other hand, oxidation of ascorbic acid occurs at the second working electrode 4103B, but not oxidation of glucose. Therefore, the current from the second working electrode 4103B represents only the concentration of ascorbic acid in the same test fluid 102. The difference between the two current values represents the concentration or level of glucose contained in the test fluid 102.

[0230] First working electrode (glucose working electrode) In some embodiments, the first working electrode (glucose working electrode) 4103A includes a nanoporous layer 117 on the conductive layer 110, as in Figure 3. The nanoporous layer 117 can include, but is not limited to, a clustered nanoporous structure. In other embodiments, the first working electrode 4103A can include, as in Figure 2, an enzyme layer containing a glucose-specific enzyme for oxidizing glucose, instead of the nanoporous layer 117 of Figure 3. In either embodiment, the first working electrode 4103A does not include a negatively charged membrane or any other membrane to prevent ascorbic acid from passing therethrough.

[0231] Second working electrode (non-glucose working electrode) The second working electrode (non-glucose working electrode) 4103B includes a conductive layer 110 but does not include any layers or features to effectively cause the oxidation of glucose. In embodiments, the second working electrode 4103B does not include a nanoporous layer 117 or a glucose-specific enzyme to oxidize glucose. However, oxidation of ascorbic acid occurs in the conductive layer 110. In embodiments, the conductive layer 110 includes, but is not limited to, a conductive carbon layer formed on a silver layer.

[0232] Same bias voltage for the two electrodes In an embodiment, the same bias voltage is applied to both the first and second working electrodes 4103A and 4103B relative to the reference electrode 106. This is to provide an environment in which approximately the same level of oxidation of ascorbic acid occurs at both the first and second working electrodes 4103A and 4103B. Assuming that the same level of oxidation of ascorbic acid occurs at each of the first and second working electrodes 4103A and 4103B, the difference between the current from the first working electrode 4103A and the current from the second working electrode 4103B should represent the oxidation of glucose at the first working electrode 4103A.

[0233] Addressing additional chemical interferences The two-electrode system 4101 can be used to address interference from more than one chemical component. In an embodiment, by adjusting the bias voltage, the first working electrode 4103A can oxidize not only glucose and ascorbic acid but also an additional interfering chemical component, such as acetaminophen. Similarly, the second working electrode 4103B oxidizes not only ascorbic acid but also the additional interfering chemical component simultaneously. Here, neither the first nor the second working electrode includes a membrane to block the additional interfering chemical component. Thus, the current from the first working electrode 4103A represents the oxidation of glucose, ascorbic acid, and acetaminophen, and the current from the second working electrode 4103B represents the oxidation of ascorbic acid and acetaminophen. The difference between the currents represents the oxidation of glucose, and the interference from acetaminophen and ascorbic acid is canceled.

[0234] bias voltage In embodiments, any bias voltage value in the range of 0.2-0.45 V can be used to address interferences. In some embodiments, bias voltage values in the range of 0.2-0.32 V can be used to address ascorbic acid-only interferences, assuming that acetaminophen cannot be oxidized within the nanoporous metal layer in that bias voltage range, as described in more detail below.

[0235] Different bias voltages In embodiments, the two-electrode system 4101 can employ different bias voltages for the first and second working electrodes. For example, a first bias voltage is applied to the first working electrode 4103A and a second bias voltage is applied to the second working electrode 4103B. With different bias voltages, the current resulting from the oxidation of ascorbic acid at the second working electrode 4103B may not be the same as or equivalent to the current component due to the oxidation of ascorbic acid at the first working electrode 4103A. Thus, the current resulting from glucose oxidation may not be a simple difference between the currents from the two electrodes. However, in embodiments, the two-electrode system 4101 has or is connected to hardware and software for calculating an accurate glucose concentration using the different bias voltages, the current values from the first and second working electrodes 4103A and 4103B, data indicating the oxidation potential of ascorbic acid at the different bias voltages, etc.

[0236] Simultaneous detection In some embodiments, the detection of current from the first working electrode 4103A and the detection of current from the second working electrode 4103B occur at the same time, simultaneously, in parallel, or concomitantly. In other embodiments, using either one current sensor or two current sensors, the detection can occur at different times with a time gap, as long as the concentration fluctuations of the relevant chemical components are negligible over the time gap. One skilled in the art will recognize how long a time gap is possible without significant risk of inaccuracy. For example, the time gap can be less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, or the time gap can be less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0237] Recording concentrations of interfering chemicals In embodiments, two-electrode system 4101 includes or is connected to hardware and software (not shown) configured to store current values from first and second working electrodes 4103A and 4103B and / or store individual concentrations of glucose and ascorbic acid derived from the current values. In some embodiments where oxidation of both ascorbic acid and acetaminophen occurs at second working electrode 4103B, the hardware and software is configured to store the concentration of glucose and the combined concentration of ascorbic acid and acetaminophen.

[0238] Applicable to CGM The two-electrode system 4101 can be incorporated into a CGM electrode unit for in vivo glucose sensing. Figure 41 shows a CGM electrode unit 4201 including first and second working electrodes 4103A and 4103B, which are connected to first and second working electrode terminals 4103AT and 4103BT, respectively.

[0239] Applicable to background music The two-electrode system 4101 may be incorporated into a BGM disposable cartridge or strip for in vitro glucose sensing. The ridge 901 may include two working electrodes. In such an embodiment, the cartridge working electrode 905 serves as the first working electrode 4103A. A second working electrode 4103B may be attached to the base 907 for contacting the test fluid. Additionally, the corresponding sensing module 911 may include circuitry for receiving signals from the first and second working electrodes from the BGM disposable cartridge.

[0240] The first and second working electrodes must work together In a two-electrode system 4101, to obtain the glucose level in the test fluid, two current values must be present: one from the first working electrode 4103A and one from the second working electrode 4103B. In a CGM, each of the first and second working electrodes 4103A and 4103B must operate sequentially or repeatedly to provide the glucose level. The system is therefore distinguished from any electrochemical sensing system that has a spare sensing electrode that is occasionally used for various reasons.

[0241] Acetaminophen Interference Acetaminophen is one of the most commonly used over-the-counter medications. In addition, acetaminophen is widely used as the active ingredient in combination medications.

[0242] Commonly recognized problems Given the popularity of acetaminophen, the drug could potentially be ingested by patients who also need to detect their blood glucose levels. Given that many glucose sensing devices are used by patients themselves, rather than by medical professionals, inaccurate readings caused by acetaminophen could have serious consequences. The electrochemical glucose sensing industry is aware of this problem and has expressed interest in solving it.

[0243] No good solution Many attempts have been made to solve this problem. However, to date, no solution has been able to convince the industry to adopt it. No membrane has been employed to selectively screen acetaminophen from reaching the electrode. Thus, a long-standing, unmet need exists.

[0244] Explanation for no good solution Commercially available electrochemical glucose sensing technologies cannot easily address this problem at all. This is at least in part because electrochemical glucose sensing systems are highly technically complex. The working electrode has stacked components, each of which has its own function and does not interfere with the other components. It would be difficult to find a solution to address this problem involving acetaminophen without affecting the function of the other components and the overall performance of the working electrode. In addition to the complexity of the technology, the rigorous regulatory approval process in this industry makes developing such a product for market release very expensive. Therefore, once a working product is approved and released on the market, it would be difficult to make significant changes to any of the working components of the approved product.

[0245] A non-enzymatic glucose sensing system for dealing with acetaminophen. In embodiments, the non-enzymatic electrochemical glucose sensing system selectively oxidizes glucose and simultaneously does not oxidize acetaminophen without introducing an additional membrane to this end. Referring back to Figures 3 and 31, the working electrode 103NE, 501 includes a conductive layer 110 and a nanoporous layer 117. The working electrode may include one or more additional functional layers on the nanoporous layer 117.

[0246] Acetaminophen Cleaning Filmless In embodiments, the working electrode 103NE does not include a membrane, film, or layer over the nanoporous layer 117 that is designed to selectively screen, repel, or block acetaminophen while allowing glucose to pass therethrough. Thus, when the working electrode 103NE contacts a test fluid containing acetaminophen, both glucose and acetaminophen contact the nanoporous layer 117 and can enter the nano-sized pores therein for oxidation.

[0247] Bias voltage for the oxidation of glucose and acetaminophen In glucose sensing systems according to embodiments, glucose is oxidized at the nanoporous layer 117 at bias voltages between about 0.2 V and about 0.45 V. On the other hand, acetaminophen is oxidized at bias voltages greater than 0.33, 0.34, 0.35, or 0.36 V. The bias voltage can be adjusted to cause oxidation of glucose while simultaneously avoiding oxidation of acetaminophen.

[0248] Bias voltage for selective oxidation of glucose and no oxidation of acetaminophen In embodiments, the bias voltage applied to the conductive layer 110 relative to the reference electrode 106 is set to cause oxidation of glucose but not oxidation of acetaminophen when both are in contact with the nanoporous layer 117. For selective oxidation of glucose and selective non-oxidation of acetaminophen, in embodiments, the bias voltage is set at or about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, or 0.32 V. In embodiments, the bias voltage may be within a range formed by selecting any two numbers (two voltage values) listed in the immediately preceding sentence, e.g., 0.28 V to 0.30 V, about 0.27 V to about 0.31 V, 0.26 V to 0.30 V, about 0.28 V to about 0.32 V, etc. In an embodiment, the bias voltage is less than 0.30, 0.31 or 0.32V.

[0249] Bias voltage in enzyme-sensing electrodes For comparison, an enzymatic glucose sensor applies a bias voltage in the range of 0.5-0.6 V. In an enzymatic sensing sensor, this bias voltage does not cause oxidation of glucose at the sensing electrode or elsewhere. Rather, the glucose-specific enzyme oxidizes the glucose molecule, thereby releasing electrons for the electron carrier, which is oxidized by the bias voltage at the conductive layer. Thus, the bias voltage causes oxidation of the electron carrier at the enzymatic electrode.

[0250] Example Various aspects and features of the invention will now be further described in connection with examples and experiments.

[0251] Preparation of reverse micellar phase Example 1.1 An aqueous platinum solution was prepared by dissolving 0.500 g (0.965 mmol) of chloroplatinic acid hexahydrate HPtCl6·6H2O (Sigma-Aldrich) in 24.5 g of purified water with stirring. 25 g of the surfactant Triton X-100™ (Sigma-Aldrich) was added to the aqueous platinum solution to provide an aqueous composition containing the surfactant and platinum ions. The concentration of platinum ions in the aqueous composition was approximately 0.02 M. A reverse micellar phase was prepared in the aqueous composition by adjusting the temperature to 70°C with stirring.

[0252] Example 1.2 A reverse micellar phase is prepared by repeating Example 1.1, except that PtCl4·6H2O is used instead of H2PtCl6·6H2O in an amount to provide a platinum ion concentration of about 0.02 M in the aqueous composition.

[0253] Example 1.3 A reverse micellar phase is prepared by repeating Example 1.1, except that HPtCl(OH) is used instead of HPtCl·6H0 in an amount to provide a platinum ion concentration of about 0.02 M in the aqueous composition.

[0254] Example 1.4 A reverse micellar phase is prepared by repeating Example 1.1, except that HPt(SO)(OH)·6H0 is used instead of HPtCl·6H0 in an amount to provide a platinum ion concentration of about 0.02 M in the aqueous composition.

[0255] Example 1.5 A reverse micellar phase is prepared by repeating Example 1.1, except that TiCl4·6H2O is used instead of H2PtCl6·6H2O in an amount to provide a titanium ion concentration of about 0.02 M in the aqueous composition.

[0256] Example 1.6 A reverse micelle phase is prepared by repeating Example 1.1, except that NP-40™ is used as the surfactant instead of Triton X-100 to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and further, the amount of surfactant and temperature are adjusted to achieve a reverse micelle phase of the surfactant.

[0257] Example 1.7 A reverse micelle phase is prepared by repeating Example 1.1, except that polysorbate 80 is used as the surfactant instead of Triton X-100 to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and further, the amount of surfactant and temperature are adjusted to achieve the reverse micelle phase of the particular surfactant.

[0258] Example 1.8 A reverse micelle phase is prepared by repeating Example 1.1, except that Isoceteth-20 is used as the surfactant instead of Triton X-100 to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and further, the amount of surfactant and temperature are adjusted to achieve the reverse micelle phase of the particular surfactant.

[0259] Example 1.9 A reverse micelle phase is prepared by repeating Example 1.1, except that poloxamer 407 is used as the surfactant in place of Triton X-100 to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and further, the amount of surfactant and temperature are adjusted to achieve the reverse micelle phase of the particular surfactant.

[0260] Example 1.10 A reverse micelle phase is prepared by repeating Example 1.1, except that monolaurin is used as the surfactant instead of Triton X-100 to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and further, the amount of surfactant and temperature are adjusted to achieve the reverse micelle phase of the particular surfactant.

[0261] Preparation of reducing agent Example 2.1 An aqueous reducing agent solution was prepared by adding 30 g (0.170 mol) of ascorbic acid to 250 ml of purified water with stirring. The reducing agent solution was heated to 70°C. The concentration of ascorbic acid in the aqueous reducing agent solution was set to 0.6 M, which is 60 times the concentration of the metal ions in Examples 1.1 to 1.10.

[0262] Example 2.2 An aqueous reducing agent solution was prepared by repeating Example 2.1, except that formaldehyde was used as the reducing agent instead of ascorbic acid. The amount of formaldehyde was adjusted to provide a concentration of about 0.6 M in the aqueous reducing agent solution.

[0263] Example 2.3 An aqueous reducing agent solution was prepared by repeating Example 2.1, except that acetic acid was used as the reducing agent instead of ascorbic acid. The amount of acetic acid was adjusted to provide a concentration of about 0.6 M in the aqueous reducing agent solution.

[0264] Example 2.4 An aqueous reducing agent solution was prepared by repeating Example 2.1, except that hypophosphite was used as the reducing agent instead of ascorbic acid. The amount of hypophosphite was adjusted to provide a concentration of about 0.6 M in the aqueous reducing agent solution.

[0265] Formation of Nanoparticle Colloids Example 3.1 The aqueous solution of reducing agent prepared in Example 2.1 was added to the aqueous composition of Example 1.1 at 70°C immediately after preparing the reverse micellar phase. In the resulting liquid composition, the concentration of platinum ions was about 0.0028M and the concentration of ascorbic acid was about 0.50M. The resulting liquid composition was continuously stirred at 70°C for about 4 hours. A black platinum colloid was obtained.

[0266] Examples 3.2-3.10 Example 3.1 is repeated using the reverse micellar phases prepared in Examples 1.2-1.10 in place of the reverse micellar phase prepared in Example 1.1. This provides the metal colloids of Examples 3.2-3.10, respectively.

[0267] Particle Size Analysis of Nanoparticle Colloids Example 4.1 The Korea Polymer Testing and Research Institute (KOPTRI) performed dynamic light scattering particle size analysis on the platinum colloid obtained from Example 3.1 using a Zeta Potential & Particle Size Analyzer ELS-Z2 from Otsuka Electronics. The analysis showed that a sample of the Example 3.1 platinum colloid was dispersed in purified water and had a refractive index of 1.3328, a viscosity of 0.8878 cp, and a dielectric constant of 78.3 at 25°C.

[0268] Figure 14 shows the particle size distribution for the colloids obtained from Example 3.1. The particle diameters are primarily between about 9 nm and about 14 nm. This size distribution is interpreted as representing reverse micelles. The size distribution does not show a diameter size of 1-5 nm, which would be interpreted as most platinum nanoparticles being contained or encapsulated inside the reverse micelles. Similar results were obtained from multiple runs of experiments according to Examples 1.1, 2.1, and 3.1.

[0269] Examples 4.2-4.10 The analysis of Example 4.1 is repeated using each of the colloids prepared in Examples 3.2-3.10 in place of the colloid prepared in Example 3.1. The particle size distribution for each of the colloids prepared in Examples 3.2-3.10 is obtained.

[0270] Detergent Removal Example 5.1 50 ml of 0.3 M aqueous HCl solution was added to 60 ml of the platinum colloid prepared in Example 3.1. The acid-added platinum colloid was centrifuged at 3800 rpm for 10 minutes. The clear supernatant was then discarded and the black bottom was collected. The sequence of adding aqueous HCl solution, centrifugation, and collecting the black bottom was repeated four more times to remove the surfactant and obtain the platinum colloid.

[0271] The resulting platinum colloid was then washed with purified water to remove HCl. 50 ml of purified water was added to the collected platinum colloid. The water-added platinum colloid was centrifuged at 3800 rpm for 10 minutes. The clear supernatant was then discarded and the black bottom was collected. The sequence of adding purified water, centrifugation, and collecting the black bottom was repeated four more times to remove HCl and obtain HCl-washed platinum colloid.

[0272] Examples 5.2-5.10 Example 5.1 is repeated using the nanoparticle colloids obtained from Examples 3.2-3.10 in place of the nanoparticle colloids prepared in Example 3.1, and colloids from Examples 5.2-5.10 are collected, respectively.

[0273] Example 5.11 Example 5.1 is repeated using 0.3 M aqueous HNO3 instead of aqueous HCl.

[0274] Example 5.12 Example 5.1 is repeated using 0.3 M aqueous NaOH instead of aqueous HCl.

[0275] Particle Size Analysis of Cluster Colloids Example 6.1 Similar to Example 4.1, the Korea Polymer Testing and Research Institute (KOPTRI) performed dynamic light scattering particle size analysis on the platinum colloid obtained from Example 5.1 using a Zeta Potential & Particle Size Analyzer ELS-Z2 from Otsuka Electronics. For the analysis, a sample of the Example 5.1 colloid was dispersed in water and found to have a refractive index of 1.3328, a viscosity of 0.8878 cp, and a dielectric constant of 78.3 at 25°C.

[0276] Figure 15 shows the particle size distribution for the colloids obtained from Example 5.1. The particle diameters are primarily between about 60 nm and about 200 nm. This size distribution is interpreted as representing irregularly shaped clusters formed from nanoparticles. Considering that the particle sizes in Example 4.1 are primarily between about 9 nm and about 14 nm (the size of the reverse micelles, not the size of the clusters), it is understood that clusters were formed in the process of Example 5.1, where surfactant molecules were desorbed from the platinum nanoparticles by the addition of an acidic solution, and the surfactant was removed by centrifugation and bottom collection. Similar results were obtained from multiple runs of experiments according to Examples 1.1, 2.1, 3.1, and 5.1.

[0277] Examples 6.2-6.10 Example 6.1 is repeated using each of the colloids prepared in Examples 3.2-3.10 in place of the colloid prepared in Example 3.1. The particle size distribution for each of the colloids prepared in Examples 3.2-3.10 is obtained.

[0278] Platinum Recovery - Yield Example 7 The cluster colloid obtained in Example 5.1 was subjected to drying. The dry weight of the colloid was 0.143 g. The resulting colloid in Example 5.1 was prepared from 60 ml of the nanoparticle colloid prepared in Example 3.1 (which contained 0.188 g). In the overall process, the platinum yield was 76.1%.

[0279] Fabrication of an Electrode with a Clustered Nanoporous Layer Example 8.1 - Electrode Base As shown in Figure 16A, a silver layer 1603 and a conductive carbon layer 1605 were formed on a polyimide substrate 1601. The silver layer 1603 was formed to a thickness of about 20 μm by printing a silver ink containing silver particles. The conductive carbon layer 1605 was formed to a thickness of about 20 μm by printing a carbon ink containing carbon particles. A polyimide insulating film 1602 was laminated on the substrate 1601 around the silver layer 1603 and the conductive carbon layer 1605 to provide an electrode base 1606.

[0280] fruit Example 8.2 - Formation of nanoporous layer The cluster colloid obtained in Example 5.1 was diluted to a concentration of 60 mg / ml. Using a microsyringe, 0.2 μL of the diluted cluster colloid was dropped onto the conductive carbon layer of electrode base 1606. The electrode base with the colloid dropped onto it was placed in an oven at 60° C. for 30 minutes to form electrode 1607 including platinum nanoporous layer 1609, as shown in FIG. 16B.

[0281] Example 8.3 - Roughness Factor The electrochemical cell of FIG. 1 was prepared using a CH Instruments Inc. electrochemical analyzer CHI660 as the potentiostat 104, the electrode 1607 prepared in Example 8.2 as the working electrode 103, a platinum wire as the counter electrode 105, and Ag / AgCl (3 M KCl) as the reference electrode 106. The silver layer 1603 of the electrode 1607 was connected to the potentiostat 104. Instead of the test fluid 102, a 1 M H2SO4 aqueous solution was added to the electrochemical cell of FIG. 1.

[0282] Cyclic voltammetry was performed using a potential sweep from -0.2 V to +1.2 V. The actual surface area of the platinum nanoporous layer was obtained by measuring the amount of protons adsorbed on the surface of the platinum nanoporous layer using cyclic voltammetry. The top surface area (geometric area) of the platinum nanoporous layer was measured. The roughness factor was calculated by dividing the actual surface area by the geometric area. The roughness factor of the nanoporous layer obtained from Example 8.2 was 1147.

[0283] Example 8.4 - Repeat of Examples 8.1-8.2 Example 8.1 was repeated multiple times to prepare additional electrode bases. Example 8.2 was repeated multiple times using additional electrode bases to prepare additional electrodes 1607 including platinum nanoporous layers 1609.

[0284] Example 8.5 - Repeat of Example 8.3 Example 8.3 was repeated for the five electrodes 1607 prepared in Example 8.4. The roughness coefficient values of the nanoporous layers were 1187, 1171, 1143, 1190, and 1119.

[0285] Example 8.6 - SEM Photo Figure 17A is an SEM photograph taken from the top of electrode 1607 obtained from Example 8.4. The darker center represents the area of the conductive carbon layer. Figure 17B is an SEM photograph of a cross section of electrode 1607 showing, from top to bottom, platinum nanoporous layer 1609, carbon conductive layer 1605, and silver layer 1603. Figure 17C includes three SEM photographs of another electrode 1607 prepared in Example 8.4. These three photographs were taken from the top at different magnifications.

[0286] Sensing Glucose in PBS Example 9.1 - Preparation of Solutions of Glucose and Other Test Materials D-(+)-glucose powder purchased from Sigma-Aldrich was dissolved in purified water to prepare a 1 M glucose stock solution. Ascorbic acid purchased from Sigma-Aldrich was dissolved in purified water to prepare a 0.05 M ascorbic acid solution. Acetaminophen purchased from Sigma-Aldrich was dissolved in purified water to prepare a 0.05 M acetaminophen solution. Maltose purchased from Sigma-Aldrich was dissolved in purified water to prepare a 0.5 M maltose solution.

[0287] Example 9.2 - Preparation of PBS A 500 ml solution of 0.1 M NaH2PO4 and 0.15 M NaCl in purified water was prepared. A 500 ml solution of 0.1 M Na2HPO4 and 0.15 M NaCl in purified water was prepared. The two solutions were mixed to prepare 1 L of stock phosphate buffered saline (PBS), pH 7.4.

[0288] Example 9.3 - Preparation of glucose sensing system in PBS 20 ml of the PBS prepared in Example 9.2 was placed in a beaker, in which the temperature of the PBS was maintained at 37° C. The electrochemical cell of FIG. 1 was prepared using a CH Instruments Inc. electrochemical analyzer CHI660 as the potentiostat 104, the electrode 1607 prepared in Example 8.4 as the working electrode 103, a platinum wire as the counter electrode 105, and Ag / AgCl (3 M KCl) as the reference electrode 106. The silver layer 1603 of the electrode 1607 was connected to the potentiostat 104. The electrode was immersed in the PBS and electrically connected to the electrochemical analyzer.

[0289] Example 9.4 - Current Measurement For the system prepared in Example 9.3, a bias voltage of 0.4 V was applied between the working electrode 103 (electrode 1607) and the reference electrode 106. When the bias voltage was applied, the current from the working electrode 103 was continuously measured. The electrochemical cell was maintained for 12 minutes in PBS without adding any substances thereto to condition the glucose sensing system. A current value of 0.087 μA was then obtained for no glucose in the PBS. Figure 18 shows the current profiles obtained from the electrochemical cell for Examples 9.5-9.11 below. In Figure 18, "AA" stands for ascorbic acid, and "AP" stands for acetaminophen.

[0290] Example 9.5 - Sensing 1 mM glucose in PBS After conditioning the glucose sensing system, 20 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS of Example 9.3 to produce PBS containing 1 mM glucose. Immediately after addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 0.54 μA was obtained for 1 mM glucose in PBS.

[0291] Example 9.6 - Sensing 3 mM glucose in PBS After the current stabilized in Example 9.5, 40 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained from Example 9.4 to produce PBS containing 3 mM total glucose. Immediately after addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 1.19 μA was obtained for 3 mM glucose in PBS.

[0292] Example 9.7 - Sensing 6 mM glucose in PBS After the current stabilized in Example 9.6, 60 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained from Example 9.5 to produce PBS containing 6 mM total glucose. Immediately after addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 2.09 μA was obtained for 6 mM glucose in PBS.

[0293] Example 9.8 - Sensing 10 mM glucose in PBS After the current stabilized in Example 9.7, 80 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained from Example 9.6 to produce PBS containing 10 mM total glucose. Immediately after addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 2.89 μA was obtained for 10 mM glucose in PBS.

[0294] Example 9.9 - Sensing of 0.11 mM Ascorbic Acid in PBS After the current stabilized in Example 9.8, 44 μl of the aqueous ascorbic acid solution prepared in Example 9.1 was added to the PBS obtained from Example 9.7 to produce PBS containing 0.11 mM ascorbic acid (AA). Immediately after addition, the PBS containing the added ascorbic acid was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 2.93 μA was obtained for the sum of 10 mM glucose and 0.11 mM ascorbic acid in PBS.

[0295] Example 9.10 - Sensing of 0.17 mM Acetaminophen in PBS After the current stabilized in Example 9.9, 68 μl of the acetaminophen aqueous solution prepared in Example 9.1 was added to the PBS obtained from Example 9.8 to produce PBS containing 0.17 mM acetaminophen (AP). Immediately after addition, the PBS containing acetaminophen was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 3.21 μA was obtained for the sum of 10 mM glucose, 0.11 mM ascorbic acid, and 0.17 mM acetaminophen in PBS.

[0296] Example 9.11 - Sensing 13.9 mM Maltose in PBS After the current stabilized in Example 9.10, 556 μl of the maltose solution prepared in Example 9.1 was added to the PBS obtained from Example 9.9 to produce PBS containing 13.9 mM maltose. Immediately after the addition, the maltose-added PBS was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 4.74 μA was obtained for the sum of 10 mM glucose, 0.11 mM ascorbic acid, 0.17 mM acetaminophen, and 13.9 mM maltose in PBS.

[0297] Example 9.12 - Glucose Level Formula In Examples 9.5-9.11, the current values represent and correspond to the concentration of glucose in PBS. Similar experiments are performed many more times with identically prepared glucose sensing systems using the same and other glucose concentrations to obtain data on current values and corresponding glucose concentrations. A correlation between the glucose concentration in PBS and the current values is obtained by processing the data. The glucose concentration is calculated using the correlation and the current values obtained from Examples 9.5-9.11.

[0298] Glucose Sensing in Serum Example 10.1 - Preparation of a Glucose Sensing System in Serum Human serum was purchased from Sigma-Aldrich. The glucose content in the serum was measured using a YSI. The serum was determined to contain 5.8 mM glucose, corresponding to a blood glucose level of 104 mg / dL. 10 ml of serum was placed in a beaker, in which the temperature of the serum was maintained at 37°C. An electrochemical cell was prepared similarly to Example 9.3, except that one electrode 1607 prepared in Example 8.4 was used as the working electrode 103, and further, the working electrode, reference electrode, and counter electrode were immersed in the serum.

[0299] Example 10.2 - Preconditioning of the glucose sensing system in serum A 0.4 V bias voltage was applied between the working electrode 103 and the reference electrode 106 of the electrochemical cell prepared in Example 10.1. The bias voltage was maintained in the electrochemical system for more than 3 hours for system conditioning, i.e., until the background current was low enough to sense glucose oxidation. The bias voltage was then disconnected from the system.

[0300] Example 10.3 - Current Measurement Immediately after removing the bias voltage in Example 10.2, the same bias voltage was reapplied to the system and measurement of the current from the working electrode began. The electrochemical cell was maintained for 1.2 hours for further conditioning of the glucose sensing system in serum without adding any substances to it. When the current stabilized, a current value of 96 nA was obtained for 5.8 mM glucose originally contained in serum. Figure 19 shows the current profiles measured from the electrochemical cells of Examples 10.4-10.9 below. In Figure 19, "AA" stands for ascorbic acid and "AP" stands for acetaminophen.

[0301] Example 10.4 - Sensing 10 mM glucose in serum After conditioning the glucose sensing system, 42 μl of the glucose stock solution prepared in Example 9.1 was added to the serum from Example 10.2 to produce serum containing 10 mM total glucose. Immediately after addition, the glucose-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 110 nA was obtained for 10 mM glucose in serum.

[0302] Example 10.5 - Sensing 15 mM glucose in serum After the current stabilized in Example 10.4, 50 μl of the glucose stock solution prepared in Example 9.1 was added to the serum in Example 10.3 to produce serum containing 15 mM total glucose. Immediately after addition, the glucose-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 132 nA was obtained for 15 mM glucose in the serum.

[0303] Example 10.6 - Sensing 20 mM glucose in serum After the current stabilized in Example 10.5, 50 μl of the glucose stock solution prepared in Example 9.1 was added to the serum in Example 10.4 to produce serum containing 20 mM total glucose. Immediately after addition, the glucose-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 159 nA was obtained for 20 mM glucose in the serum.

[0304] Example 10.7 - Sensing 0.11 mM Ascorbic Acid in Serum After the current stabilized in Example 10.6, 22 μl of the aqueous ascorbic acid solution prepared in Example 9.1 was added to the serum obtained from Example 10.5 to produce serum containing 0.11 mM ascorbic acid (AA). Immediately after addition, the ascorbic acid-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 163 nA was obtained for the sum of 20 mM glucose and 0.11 mM ascorbic acid in the serum.

[0305] Example 10.8 - Sensing 0.17 mM Acetaminophen in Serum After the current stabilized in Example 10.7, 34 μl of the aqueous acetaminophen solution prepared in Example 9.1 was added to the serum obtained from Example 10.6 to produce serum containing 0.17 mM acetaminophen (AP). Immediately after addition, the acetaminophen-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 223 nA was obtained for the sum of 20 mM glucose, 0.11 mM ascorbic acid, and 0.17 mM acetaminophen in the serum.

[0306] Example 10.9 - Sensing 13.9 mM Maltose in Serum After the current stabilized in Example 10.8, 278 μl of the aqueous maltose solution prepared in Example 9.1 was added to the serum obtained from Example 10.7 to produce serum containing 13.9 mM maltose. Immediately after addition, the maltose-spiked serum was stirred for 3-4 seconds, which caused a transient peak in the current. The current from the working electrode was continuously measured. When the current stabilized, a current value of 231 nA was obtained for the sum of 20 mM glucose, 0.11 mM ascorbic acid, 0.17 mM acetaminophen, and 13.9 mM maltose in the serum.

[0307] Example 10.10 - Glucose Level Formula In Examples 10.4-10.9, the current values represent and correspond to the concentration of glucose in serum. Similar experiments are performed many more times using the same and other glucose concentrations on similarly prepared glucose sensing systems to obtain data on current values and corresponding glucose concentrations. A correlation between serum glucose concentration and current values is obtained by processing the data. The glucose concentration is calculated using the correlation and the current values obtained from Examples 10.4-10.9.

[0308] Non-clustered nanoporous layers Example 11.1 - Electroplating from a reverse micellar phase This disclosure hereby incorporates in its entirety the examples and descriptions of U.S. Patent No. 8,343,690 (the '690 patent). The experiments appearing in columns 6-9 of the '690 patent are specifically incorporated herein as examples for fabricating nanoporous layers by electroplating and using the layers for glucose sensing.

[0309] Example 11.2 - Electroplating from the Hexagonal Phase This disclosure hereby incorporates the disclosure of U.S. Patent No. 7,892,415 (the '415 patent) in its entirety. The experiments appearing in columns 5-6 of the '415 patent are specifically incorporated herein as examples for fabricating hexagonally structured nanoporous layers by electroplating and using the layers for glucose sensing.

[0310] Example 11.3 - Electroplating from the Hexagonal Phase This disclosure hereby incorporates in its entirety the disclosure of "Electrochemistry Communications, Vol. 4, No. 8, August 2002, pp. 610-612."

[0311] Example 11.4 - Chemical precipitation from the hexagonal phase This disclosure hereby incorporates in its entirety the disclosure of Science, Vol. 278, October 31, 1997, pp. 838-840.

[0312] Preparation of Maltose-Blocking Layer Example 12.1 - Preparation of mPD Aqueous Solution M-phenylenediamine (mPD) purchased from Sigma-Aldrich was dissolved in purified water to provide aqueous mPD solutions containing mPD at 0.1, 0.3, 0.5, 1.0, 2.0, and 5.0 mM.

[0313] Example 12.2 - Preparation for Cyclic Voltammetry An electrochemical cell was prepared using a CH Instruments Inc. electrochemical analyzer, CHI Multi 1030C, as the potentiostat 104, and using the electrode 1607 prepared in Example 8.4 as the working electrode 103, a platinum wire as the counter electrode 105, and Ag / AgCl (3M KCl) as the reference electrode 106. The counter electrode 105 and the reference electrode 106 were electrically connected to form a two-electrode system.

[0314] Example 12.3 - Electrochemical Polymerization at 0.1 mM, 10 mV / sec In the electrochemical cell prepared in Example 12.2, the 0.1 mM mPD aqueous solution prepared in Example 12.1 was added instead of the test fluid 102. Cyclic voltammetry was performed using a potential sweep from +0.5 V to +1.0 V at a scan rate of 10 mV / s for two sweep segments, as shown in Figure 22. A poly-mPD maltose-blocking layer 301 was obtained on the nanoporous layer 117.

[0315] Example 12.4 - Electrochemical Polymerization at 0.1 mM, 100 mV / sec Example 12.3 was repeated except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0316] Example 12.5 - Electrochemical polymerization at 0.1 mM, 200 mV / sec Example 12.3 was repeated except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0317] Example 12. Electrochemical Polymerization at 6-0.3 mM, 10 mV / sec Example 12.3 was repeated, except that the 0.3 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0318] Example 12.7 - Electrochemical polymerization at 0.3 mM, 100 mV / sec Example 12.6 was repeated except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0319] Example 12. Electrochemical polymerization at 8-0.3 mM, 200 mV / sec Example 12.6 was repeated except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0320] Example 12. Electrochemical Polymerization at 9-0.5 mM, 10 mV / sec Example 12.3 was repeated, except that the 0.5 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0321] Example 12. Electrochemical Polymerization at 10-0.5 mM, 100 mV / sec Example 12.6 was repeated except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0322] Example 12.11 - Electrochemical Polymerization at 0.5 mM, 200 mV / sec Example 12.6 was repeated except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0323] Example 12.12 - Electrochemical Polymerization at 1.0 mM, 10 mV / sec Example 12.3 was repeated, except that the 1.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0324] Example 12.13 - Electric Shock The electrochemical cell of Figure 23 was prepared for chronoamperometry using the poly-mPD layer prepared in Example 12.12 as the porous polymer layer 302 and 1 M aqueous H2SO4 solution as the electrolyte solution. An electric shock was applied to the porous polymer layer 302 by applying a single pulse from +0.0 V to +1.0 V with a pulse width of 1.0 second.

[0325] Example 12.14 - Electrochemical Polymerization and Electroshock at 1.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0326] Example 12. Electrochemical Polymerization and Electroshock at 15-1.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0327] Example 12.16 - Electrochemical Polymerization and Electroshock at 2.0 mM, 10 mV / sec Example 12.3 was repeated, except that the 2.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated using the poly-mPD layer formed on the nanoporous layer.

[0328] Example 12.17 - Electrochemical Polymerization and Electroshock at 2.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0329] Example 12.18 - Electrochemical Polymerization and Electroshock at 2.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0330] Example 12.19 - Electrochemical Polymerization and Electroshock at 5.0 mM, 10 mV / sec Example 12.3 was repeated, except that the 5.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated using the poly-mPD layer formed on the nanoporous layer.

[0331] Example 12. Electrochemical Polymerization and Electroshock at 20-5.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scan rate was 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0332] Example 12.21 - Electrochemical Polymerization and Electroshock at 5.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scan rate was 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Example 12.13 was then repeated with the poly-mPD layer formed on the nanoporous layer.

[0333] Glucose Sensing Without Maltose Interference Example 13.1 - Serum Preparation Human serum was purchased from Sigma-Aldrich. The glucose content in the serum was measured using a YSI. The serum was determined to contain 5.8 mM glucose, which corresponds to a blood glucose level of 104 mg / dl.

[0334] Example 13.2 - Preparation of a glucose sensing system in serum Ten ml of serum prepared in Example 13.1 was placed in a beaker in which the temperature of the serum was maintained at 37° C. An electrochemical cell was prepared similarly to Example 10.2, except that the working electrode 103 included a poly-mPD maltose-blocking layer 301 on a nanoporous layer prepared in Example 12.3 using a 0.1 mM mPD solution and a scan rate of 10 mV / sec.

[0335] Example 13.1 - Preparation of a glucose sensing system in serum An electrochemical cell was prepared by repeating Example 10.2, except that the working electrode 103 included a poly-mPD maltose-blocking layer 301 on the nanoporous layer prepared in Example 12.3 (using a 0.1 mM mPD solution and a scan rate of 10 mV / sec), and further, the working, reference, and counter electrodes were immersed in serum.

[0336] Example 13.2 - Conditioning of the glucose sensing system in serum In the electrochemical cell system prepared in Example 13.1, a bias voltage of 0.4 V was applied between the working electrode 103 and the reference electrode 106. The bias voltage was maintained in the electrochemical system for more than 3 hours to precondition the system. The bias voltage was then disconnected from the system and reconnected. When the bias voltage was reapplied, current measurement from the working electrode began. The electrochemical cell was maintained in serum to further condition the glucose sensing system. When the current stabilized, a current value of 96 nA was measured for 5.8 mM glucose originally contained in serum.

[0337] Example 13.3 - Electrode with Maltose-Blocking Layer (0.1 mM, 10 mV / sec) In the system prepared in Example 13.2, a glucose stock solution prepared as in Example 9.1 was added to serum to produce serum containing a total glucose concentration of 10 mM. Further additions of glucose stock solution were then made to produce serum containing total glucose concentrations of 15 mM and 20 mM (with a time interval between additions). The ascorbic acid solution prepared in Example 9.1 was then added to the serum to produce serum containing 0.11 mM ascorbic acid. The acetaminophen solution prepared in Example 9.1 was then added to the resulting serum to produce serum containing 0.17 mM acetaminophen. The maltose solution prepared in Example 9.1 was then added to the resulting serum to produce serum containing 13.9 mM maltose. Immediately after each addition, the serum was stirred for 3-4 seconds, which caused a transient peak in the current. Figure 25 shows the current monitored in this example in red. Changes in current were observed in response to the addition of glucose, ascorbic acid (AA), and acetaminophen (AP). However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond this was observed. Thus, the maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0338] Example 13.4 - Electrode with Maltose-Blocking Layer (0.1 mM, 100 mV / sec) Examples 13.1-13.3 were repeated (using a 0.1 mM mPD solution at a scan rate of 100 mV / sec) except that the working electrode 103 contained a maltose-blocking layer prepared as in Example 12.4. Figure 25 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0339] Example 13.5 - Electrode with Maltose-Blocking Layer (0.1 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode 103 included a maltose-blocking layer prepared as in Example 12.5 (using a 0.1 mM mPD solution at a scan rate of 200 mV / sec). Figure 25 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0340] Example 13.6 - Electrode with Maltose-Blocking Layer (0.3 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.6 (using a 0.3 mM mPD solution at a scan rate of 10 mV / sec). Figure 26 shows in red the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0341] Example 13.7 - Electrode with Maltose-Blocking Layer (0.3 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.7 (using a 0.3 mM mPD solution at a scan rate of 100 mV / sec). Figure 26 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0342] Example 13.8 - Electrode with Maltose-Blocking Layer (0.3 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.8 (using a 0.3 mM mPD solution at a scan rate of 200 mV / sec). Figure 26 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0343] Example 13.9 - Electrode with Maltose-Blocking Layer (0.5 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.9 (using a 0.5 mM mPD solution at a scan rate of 10 mV / sec). Figure 27 shows in red the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0344] Example 13.10 - Electrode with Maltose-Blocking Layer (0.5 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.9 (using a 0.5 mM mPD solution at a scan rate of 100 mV / sec). Figure 27 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0345] Example 13.11 - Electrode with Maltose-Blocking Layer (0.5 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode contained a maltose-blocking layer prepared in Example 12.11 (using a 0.5 mM mPD solution at a scan rate of 200 mV / sec). Figure 27 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0346] Example 13.12 - Electrode with Maltose-Blocking Layer (1.0 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.12 (using a 1.0 mM mPD solution at a scan rate of 10 mV / sec) and further included a maltose-blocking layer that was subjected to an electric shock as in Example 12.13. Figure 28 shows in red the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0347] Example 13.13 - Electrode with Maltose-Blocking Layer (1.0 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.14 (using a 1.0 mM mPD solution at a scan rate of 100 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.13. Figure 28 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0348] Example 13.14 - Electrode with Maltose-Blocking Layer (1.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.15 (using a 1.0 mM mPD solution at a scan rate of 200 mV / sec) and further included a maltose-blocking layer with an electric shock. Figure 28 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0349] Example 13.15 - Electrode with Maltose-Blocking Layer (2.0 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.16 (using a 2.0 mM mPD solution at a scan rate of 10 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 29 shows in red the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0350] Example 13.16 - Electrode with Maltose-Blocking Layer (2.0 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.17 (using a 2.0 mM mPD solution at a scan rate of 100 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 29 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0351] Example 13.17 - Electrode with Maltose-Blocking Layer (2.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.18 (using a 2.0 mM mPD solution at a scan rate of 200 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 29 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0352] Example 13.18 - Electrode with Maltose-Blocking Layer (5.0 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.19 (using a 5.0 mM mPD solution at a scan rate of 10 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 30 shows in red the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0353] Example 13.19 - Electrode with Maltose-Blocking Layer (5.0 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.20 (using a 5.0 mM mPD solution at a scan rate of 100 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 30 shows in green the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0354] Example 13.20 - Electrode with Maltose-Blocking Layer (5.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared as in Example 12.21 (using a 5.0 mM mPD solution at a scan rate of 200 mV / sec) and further included a maltose-blocking layer with an electric shock as in Example 12.15. Figure 30 shows in purple the current monitored in this example. Changes in current were observed in response to the addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, the current was below 5 nA / mMcm, except for the peak caused by stirring. 2 No current change beyond 0.05 was observed. The maltose-blocking layer in this example effectively blocked maltose but did not interfere with glucose sensing.

[0355] Example 13.21 - Electrode with Maltose-Blocking Layer (1.0 mM, 10 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.12 (using a 1.0 mM mPD solution at a scan rate of 10 mV / sec) is not subjected to electric shock.

[0356] Example 13.22 - Electrode with Maltose-Blocking Layer (1.0 mM, 100 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.14 (using a 1.0 mM mPD solution at a scan rate of 100 mV / sec) is not subjected to electrical shock.

[0357] Example 13.23 - Electrode with Maltose-Blocking Layer (1.0 mM, 200 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.15 (using a 1.0 mM mPD solution at a scan rate of 200 mV / sec) is not subjected to electrical shock.

[0358] Example 13.24 - Electrode with Maltose-Blocking Layer (2.0 mM, 10 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.16 (using a 2.0 mM mPD solution at a scan rate of 10 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0359] Example 13.25 - Electrode with Maltose-Blocking Layer (2.0 mM, 100 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.17 (using a 2.0 mM mPD solution at a scan rate of 100 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0360] Example 13.26 - Electrode with Maltose-Blocking Layer (2.0 mM, 200 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.18 (using a 2.0 mM mPD solution at a scan rate of 200 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0361] Example 13.27 - Electrode with Maltose-Blocking Layer (5.0 mM, 10 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.19 (using a 5.0 mM mPD solution at a scan rate of 10 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0362] Example 13.28 - Electrode with Maltose-Blocking Layer (5.0 mM, 100 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.20 (using a 5.0 mM mPD solution at a scan rate of 100 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0363] Example 13.29 - Electrode with Maltose-Blocking Layer (5.0 mM, 200 mV / sec) Example 13.12 is repeated, except that the poly-mPD layer prepared in Example 12.21 (using a 5.0 mM mPD solution at a scan rate of 200 mV / sec) is not subjected to electrical shock. No change in current is observed with each addition of glucose, indicating that the poly-mPD layer effectively blocks glucose.

[0364] Another shock example 14.1 - Shock with two pulses Example 12.13 is repeated, except with a pulse width of 0.5 seconds and two pulses spaced 0.5 seconds apart.

[0365] Example 14.2 - Electric shock with two pulses Repeat Example 14.1 except each pulse is from +0.0V to +2.0V.

[0366] Example 14.3 - Multiple Pulse Electric Shock Example 12.13 is repeated except for a series of 10 pulses with a pulse width of 0.1 seconds and an interval of 0.1 seconds between two pulses.

[0367] Example 14.4 - Multiple Pulse Electric Shock Repeat Example 14.1 except each pulse is from +0.0V to +2.0V.

[0368] Example 14.5 - Electrical shock at a single ramp Example 12.13 is repeated except that the potential is gradually increased from +0.0 V to +1.0 V over a period of 1 second.

[0369] Example 14.6 - Electrical shock at multiple gradients Repeat Example 14.5 except that the ramp potential is repeated 5 times with an interval of 0.1 between two ramps.

[0370] Example 14.7 - Electrical shock at a single ramp Example 12.13 is repeated except that the potential is gradually increased from +0.0 V to +2.0 V over a period of 2 seconds.

[0371] Example 14.8 - Electrical shock at multiple gradients Repeat Example 14.7 except that the ramp potential is repeated 5 times with an interval of 0.1 between two ramps.

[0372] Conditioning the Working Electrode Example 15.1 - Preparation of a Glucose Sensing System in Serum Example 10.2 was repeated to prepare an electrochemical cell for glucose sensing in serum. The working electrode 103 was one of the electrodes 1607 prepared in Example 8.4 (including a platinum nanoporous layer 1609) and did not include an electrolyte ion-blocking layer.

[0373] Example 15.2 - Conditioning of the Working Electrode (No Electrolyte Ion-Blocking Layer) In the electrochemical cell prepared in Example 15.1, a bias voltage of 0.4 V was applied between the working electrode 103 and the reference electrode 106. Unlike in Example 10.3, the current from the working electrode was continuously measured immediately upon application of the bias voltage. Figure 42A shows the current profile measured from the electrochemical cell, where the working electrode 103 does not include an electrolyte ion-blocking layer. Referring to Figure 42A, at 10,000 seconds (approximately 3 hours), 20,000 seconds, and 30,000 seconds, the current still decreases at a significant rate. Figure 42B is an expanded view of the profile in Figure 42A, showing that a glucose stock solution prepared as in Example 9.1 was successfully added after conditioning of the working electrode was complete.

[0374] Example 15.3 - Preparation of Working Electrode with PMMA Electrolyte Ion-Blocking Layer PMMA (product number 445746) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to obtain a 2 wt% PMMA solution. Using a microsyringe, 0.2 μL of the PMMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. Once the solvent had completely dried, a PMMA electrolyte ion-blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0375] Example 15.4 - Preparation of a glucose sensing system in serum Example 10.2 was repeated to prepare an electrochemical cell for glucose sensing in serum, except that the working electrode with the PMMA electrolyte ion-blocking layer prepared in Example 15.1 was used as the working electrode 103.

[0376] Example 15.5 - Conditioning the Working Electrode In the electrochemical cell prepared in Example 15.4, a bias voltage of 0.4 V was applied between the working electrode 103 and the reference electrode 106. Immediately after the bias voltage was applied, the current from the working electrode was continuously measured. Figure 43 shows the current profile measured from the electrochemical cell, where the working electrode 103 includes an electrolyte ion-blocking layer. A glucose stock solution prepared as in Example 9.1 was successfully added after conditioning of the working electrode was completed. The peaks in Figure 43 represent the stirring after each addition.

[0377] Example 15.6 - Conditioning Time Comparison Figure 44 is an overlay of the current profiles from Figure 42 (Example 15.2) and Figure 43 (Example 15.5). The current for Example 15.5 (with an electrolyte ion-blocking layer) settles and stabilizes by about 600 seconds, while the current for Example 15.2 (without an electrolyte ion-blocking layer) decreases at a significantly faster rate over the same time frame.

[0378] Example 15.7 - Preparation of working electrode with PHEMA layer PHEMA (product number 529265) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to obtain a 2 wt% PHEMA solution. Using a microsyringe, 0.2 μL of the PHEMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. Once the solvent had completely dried, a PHEMA electrolyte ion-blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0379] Example 15.8 - Preparation of a working electrode with a PMMA-EG-PMMA layer PMMA-EG-PMMA (product number 463183) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to obtain a 2 wt% PMMA-EG-PMMA solution. Using a microsyringe, 0.2 μL of the PMMA-EG-PMMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. Once the solvent had completely dried, a PMMA-EG-PMMA electrolyte ion-blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0380] Example 15.8 - Preparation of a glucose sensing system and conditioning in serum An electrochemical cell for glucose sensing in serum was prepared by repeating Example 15.4, except that the working electrode prepared in Examples 15.7 and 15.8 was used as working electrode 103. Additionally, Example 15.5 was repeated with the prepared electrochemical cell.

[0381] Fabrication of CGM Subcutaneous Electrode Unit Example 16.1 - Formation of Conductive Layer on Base A polyimide film having a thickness of 150 μm was used as the base substrate 503. A silver layer 1603 was printed on the polyimide film to obtain silver conductive elements 110C, 110W, and 110R having a thickness of about 20 μm in the shape shown in Figure 35. Then, a conductive carbon layer 1605 was printed on the silver conductive elements 110C and 110W to a thickness of about 20 μm. No carbon layer was formed on the silver layer conductive element 110R.

[0382] Example 16.2 - Placement and cutting of insulating layers A polyimide film with a thickness of 50 μm was used as the insulating layer 707. The polyimide film was cut to a size that covered the intermediate product of FIG. 35 while exposing the terminal portion 705. Holes were drilled in the polyimide film to provide three openings to expose areas for the working electrode, reference electrode, and counter electrode. Then, pre-cut polyimide was placed on the intermediate product of FIG. 35 so that the adhesive layer was in contact with the polyimide base 503 to provide the intermediate product of FIG. 36. Then, the polyimide base 503 and the polyimide insulating layer 707 outside the conductive elements were cut to provide the intermediate product of FIG. 37.

[0383] Example 16.3 - Formation of a clustered nanoporous layer The cluster colloid obtained in Example 5.1 was diluted to 60 mg / ml with purified water. Using a microsyringe, 0.2 μL of the diluted cluster colloid was dropped onto the exposed carbon layer 1605 through one opening for the working electrode 501 of the intermediate product prepared in Example 16.2. The cluster colloid dropped onto the carbon layer 1605 was dried to provide a clustered nanoporous layer 117, resulting in the intermediate product shown in FIG. 38A.

[0384] Example 16.4 - Formation of an Electrolyte Ion-Blocking Layer PMMA (product number 445746) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to obtain a 2 wt% PMMA solution. Using a microsyringe, 0.2 μL of the PMMA solution was dropped onto the nanoporous layer 117 of the intermediate product prepared in Example 16.3. Once the solvent had completely dried, a PMMA electrolyte ion-blocking layer 505 was formed on the nanoporous layer 117.

[0385] Example 16.5 - Formation of a Biocompatible Layer A biocompatible layer (pHEMA) is formed on the electrolyte ion-blocking layer 505 as shown in FIG. 38B to obtain the non-enzymatic CGM electrode unit of FIG.

[0386] Example 16.6 - Formation of a Biocompatible Layer pHEMA (product number 192066) purchased from Sigma-Aldrich was dissolved in dimethyl sulfoxide (DMSO) to obtain a 0.5 wt% pHEMA solution. Using a microsyringe, 1.0 μL of the pHEMA solution was dropped onto the electrolyte ion-blocking layer 505 of the intermediate product prepared in Example 16.4. Once the solvent had completely dried, a pHEMA biocompatible layer 507 was formed as shown in FIG. 38B, yielding the non-enzymatic CGM electrode unit 701 of FIG. 33.

[0387] CGM Animal Testing Example 17.1 - Preparation for CGM Animal Testing The non-enzymatic CGM electrode unit prepared in Example 16.6 was subcutaneously inserted into the rat's body so that electrodes 103, 105, and 106 were in contact with the rat's interstitial fluid. The device was connected to a UXN potentiostat developed by Co., Ltd. (the applicant of the present application) and Seoul National University Hospital. Figure 45A is a photograph of the UXN potentiostat. Figure 45B is a photograph showing the CGM electrode unit 701 connected to the UXN potentiostat of Figure 45A. Figure 45C is a photograph showing the UXN potentiostat together with its case. The UXN potentiostat includes a wireless module for wireless communication with a computer and can be wirelessly controlled by the computer. A glucose solution was prepared for injection into the rat's vein to induce changes in glucose levels in the rat's blood and interstitial fluid.

[0388] Example 17.2 - Continuous monitoring of glucose levels in rats The CGM electrode unit 701 remained subcutaneously inserted for five consecutive days. On the first day, the rats were injected with glucose solution twice. On subsequent days, the glucose solution was injected once daily. The UXN potentiostat measured the current from the CGM electrode unit 701 for a period of approximately 1.5 hours after the (first) injection each day. Also, every 2-5 minutes during the approximately 1.5-hour period, a small amount of blood was collected from the rats' tails and applied to a Roche Accu-Chek® blood glucose meter test strip, which provided the glucose concentration in the blood.

[0389] Example 17.3 - Rat CGM Readings and Blood Glucose Plotting Figure 46 shows in blue the current from the CGM electrode module measured by the UXN potentiostat in Example 17.2. The red dots in Figure 46 represent blood glucose concentrations obtained from a Roche Accu Chek® blood glucose meter. Assuming a time lag of approximately 10 minutes exists between the glucose levels in the interstitial fluid and the glucose levels in the blood, the data was calibrated by time-shifting the blue signal, which was shifted relative to the red dots. It is understood that the sharp peaks in the blue signal are primarily due to the rat's body movements during the measurement. Based on the graph in Figure 45, it appears that there is a strong correlation between blood glucose concentrations using the Roche Accu Chek® blood glucose meter and CGM monitoring using the non-enzymatic CGM electrode unit 701 prepared in Example 16.6.

[0390] Example 17.4 - Clark Error Grid Analysis FIG. 47 is a Clarke Error Grid for the non-enzymatic CGM electrode unit 701 prepared in Example 16.6, based on the measurements presented in the graph of FIG. 46. The reference sensor for this Clarke Error Grid analysis is a Roche Accu-Chek® blood glucose meter. The grid has five regions. Region A contains values within 20% of the reference sensor; Region B contains values that are outside 20% of Region A but would not lead to improper treatment; Region C contains values that potentially lead to unnecessary treatment; Region D contains values that indicate a potentially dangerous failure to detect hypoglycemia or hyperglycemia; and Region E contains values that would disrupt hypoglycemia treatment for hyperglycemia, and vice versa. As summarized in the table below the grid, the analysis shows that over 91% of the points were within Regions A and B.

[0391] Combination of features This disclosure provides extensive discussion and information about many features related to nanoporous structures and / or glucose sensing technology. It is an object of this disclosure to provide many devices, systems, and methods related to those features. Two or more features disclosed above can be combined together to form a device, system, or method, to the extent that the specific combination is not presented in this disclosure but can be combined. It is also an object of this disclosure to pursue claims directed to many of the features disclosed herein. Some of those features are presented in claim form in the following section. Many claims are presented in a dependent form by referencing one or more other claims. Applicant notes that some claims that reference multiple claims may encompass combinations of features that are inconsistent with one another (hereinafter "incorrect combinations"). However, Applicant recognizes that such claims may still encompass one or more combinations of features that are not inconsistent with one another (hereinafter "correct combinations"). By presenting a claim that may encompass both correct and incorrect combinations, Applicant intends to confirm its or the inventor's possession of a correct combination and to provide specific support for the correct combination for any subsequent assertion of a correct combination.

Claims

1. providing a liquid composition comprising a surfactant and a metal ion, wherein the surfactant is in a reverse micellar phase comprising a plurality of hydrophilic spaces; adding a reducing agent to the liquid composition to cause reduction of at least a portion of the metal ions and form nanoparticles, thereby providing a first colloid, wherein at least a portion of the nanoparticles are within at least some of the plurality of hydrophilic spaces and no electric potential is applied for the reduction of the at least a portion of the metal ions; and removing the surfactant from the first colloid to form a second colloid, such that the second colloid comprises the surfactant in an amount of 0 to 2 parts by weight per 100 parts by weight of the nanoparticles, and such that the second colloid comprises a number of irregularly shaped bodies dispersed in a liquid; 1. A method for producing a colloid, comprising: each of the irregularly shaped bodies comprises a nanoparticle cluster including many nanoparticles having a generally oval or spherical shape with a length in the range of about 2 nm to about 5 nm; In each cluster, adjacent ones of the nanoparticles are spaced apart from one another and form interparticle gaps, the interparticle gaps being distributed substantially throughout each cluster; the irregularly shaped bodies comprise first and second clusters discretely dispersed in the liquid, each of the first and second irregularly shaped bodies having a length in the range of about 50 nm to about 300 nm; the first cluster comprises a first nanoparticle and a second nanoparticle, each of which has a generally oval or spherical shape with a length in the range of about 2 nm to about 5 nm, and within the first cluster, the first and second nanoparticles are adjacent to each other with no intervening nanoparticles between them, and are separated from each other by a first interparticle gap having a size in the range of about 0.5 nm to about 3 nm.

2. 2. The method of claim 1, wherein some molecules of the surfactant are bound to nanoparticles in the first colloid, and removing the surfactant further comprises adding an acid or a base to the first colloid to cause at least some of the molecules to detach from the nanoparticles.

3. 3. The method of claim 1, wherein after removing the surfactant, the method further comprises adjusting the concentration of the nanoparticles in the second colloid to provide a colloid composition, the nanoparticles contained in the colloid composition being in an amount of about 0.01 wt % to about 2 wt %, based on the total weight of the colloid composition.

4. Implementing the method of any one of claims 1 to 3 to provide said second colloid; thereafter, adjusting the concentration of the nanoparticles in the second colloid to provide a colloid composition, such that the nanoparticles are contained in the colloid composition in an amount of about 0.01 wt % to about 2 wt %, based on the total weight of the colloid composition; dispensing the colloidal composition onto a substrate; and subjecting the dispensed colloidal composition to drying to form a nanoporous layer.

1. A method for producing a nanoporous layer, comprising: No electric potential is applied to the liquid composition to form the nanoporous layer; when the dispensed colloidal composition is subjected to drying, the irregularly shaped bodies contained in the dispensed colloidal composition are deposited on the substrate such that adjacent ones of the irregularly shaped bodies contact one another over some surfaces or portions thereof while forming unoccupied spaces between the non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer; the three-dimensional interconnected network of irregularly shaped bodies originating from the irregularly shaped bodies of the colloidal composition comprises many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.5 nm to about 3 nm; The method, wherein at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps having a size ranging from about 100 nm to about 500 nm.

5. A colloid comprising many nanoparticle clusters dispersed in a liquid, Each cluster comprises an irregularly shaped body formed from many nanoparticles; In each cluster, adjacent ones of the nanoparticles are spaced apart from one another and form interparticle gaps distributed generally throughout each cluster; the colloid comprises first clusters and second clusters discretely dispersed in the liquid; each of the first and second clusters has a length in the range of about 50 nm to about 300 nm; the first clusters include first nanoparticles and second nanoparticles, each of which has a generally oval or spherical shape with a length in the range of about 2 nm to about 5 nm; within the first cluster, the first and second nanoparticles are adjacent to each other with no intervening nanoparticles therebetween and are separated from each other by a first interparticle gap having a size ranging from about 0.5 nm to about 3 nm; The colloid further comprises nanoparticles that are individually dispersed in the liquid without forming clusters; the colloid contains a surfactant in an amount greater than 0 and less than 2 parts by weight per 100 parts by weight of the total amount of nanoparticles contained in the colloid; A colloid, wherein the nanoparticles contained in the colloid are in an amount of about 0.01 wt % to about 2 wt % based on the total weight of the colloid.

6. Dispensing onto the colloidal substrate of claim 5; and subjecting the distributed colloid to drying to form a nanoporous layer.

1. A method for producing a nanoporous layer, comprising: No electrical potential is applied to form the nanoporous layer; subjecting the dispensed colloid to drying, such that the irregularly shaped bodies contained in the dispensed colloid are deposited on the substrate, with adjacent ones of the irregularly shaped bodies abutting one another over some surfaces or portions thereof, while forming unoccupied spaces between the non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; The interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer.

7. providing a liquid composition comprising a surfactant and a metal ion, wherein the surfactant is in a reverse micellar phase comprising a plurality of hydrophilic spaces; adding a reducing agent to the liquid composition to cause reduction of at least a portion of the metal ions and form nanoparticles, thereby providing a first colloid, wherein at least a portion of the nanoparticles are within at least some of the plurality of hydrophilic spaces, and no electric potential is applied for the reduction of the at least a portion of the metal ions; and removing the surfactant from the first colloid to form a second colloid, such that the second colloid comprises the surfactant in an amount of 0 to 2 parts by weight per 100 parts by weight of the nanoparticles, and such that the second colloid comprises a number of irregularly shaped bodies dispersed in a liquid; 1. A method for producing a colloid, comprising: each of the irregularly shaped bodies comprises a nanoparticle cluster including many nanoparticles having a generally oval or spherical shape with a length in the range of about 2 nm to about 5 nm; a first one of the irregularly shaped bodies comprising a first nanoparticle and a second nanoparticle adjacent to each other with no intervening nanoparticle therebetween and separated from each other by a first interparticle gap having a size ranging from about 0.5 nm to about 3 nm.

8. removing the surfactant centrifuging the first colloid; collecting the bottoms from the centrifuged composition; adding a solvent to the collected bottoms; and repeating a series of centrifuging the solvent-added colloid, collecting the bottoms from the centrifuged composition, and adding the solvent thereto. The method of claim 7, comprising:

9. Some molecules of the surfactant are bound to nanoparticles in the first colloid, 9. The method of claim 7 or 8, wherein removing surfactant further comprises adding an acid or a base to the first colloid prior to centrifugation to separate at least some of the molecules from the nanoparticles.

10. 10. The method of claim 7, further comprising, after removing the surfactant, adjusting the concentration of the nanoparticles in the second colloid to provide a colloid composition, such that the nanoparticles contained in the colloid composition are in an amount of about 0.01 wt % to about 2 wt %, based on the total weight of the colloid composition.

11. Implementing the method of any one of claims 7 to 10 to provide said second colloid; thereafter, adjusting the concentration of the nanoparticles in the second colloid to provide a colloid composition, such that the nanoparticles are contained in the colloid composition in an amount of about 0.01 wt % to about 2 wt %, based on the total weight of the colloid composition; dispensing the colloidal composition onto a substrate; and subjecting the dispensed colloidal composition to drying to form a nanoporous layer; 1. A method for producing a nanoporous layer, comprising: The method, wherein no electric potential is applied to the liquid composition to form the nanoporous layer.

12. 12. The method of claim 11, further comprising storing the colloid composition in a container for a period of more than one week after adjusting the concentration and before dispensing.

13. 13. The method of claim 11 or 12, wherein the colloidal composition is dispensed in a predetermined amount to form the nanoporous layer having a roughness factor of about 100 to about 2500.

14. subjecting the dispensed colloid composition to drying, and depositing the irregularly shaped bodies contained in the dispensed colloid composition onto the substrate, such that adjacent ones of the irregularly shaped bodies abut one another over some surfaces or portions thereof, while forming unoccupied spaces between the non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; 14. The method of any one of claims 11 to 13, wherein the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer.

15. the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.5 nm to about 3 nm; at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps with sizes ranging from about 100 nm to about 500 nm; 15. The method of any one of claims 11 to 14, wherein the three-dimensional interconnected network of irregularly shaped bodies originates from the irregularly shaped bodies of the colloidal composition and comprises many nanoparticles having a generally ovoid or spherical shape with lengths in the range of about 2 nm to about 5 nm.

16. A nanoporous layer comprising a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths in the range of about 2 nm to about 5 nm, adjacent ones of the irregularly shaped bodies abut one another over some surfaces or portions thereof, while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between said adjacent ones of said irregularly shaped bodies connect said adjacent ones to one another, which continues to other ones of said irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.5 nm to about 3 nm; A nanoporous layer, wherein at least some of said irregularly shaped spaces of said three-dimensional interconnected network of irregularly shaped spaces include gaps having a size ranging from about 100 nm to about 500 nm.

17. 17. The nanoporous layer of claim 16, wherein the interparticle nanopores are distributed substantially throughout the interior of the three-dimensional interconnected network of irregularly shaped bodies, the unoccupied spaces of the three-dimensional interconnected network of irregularly shaped spaces are distributed substantially throughout the nanoporous layer, and the interparticle nanopores are substantially interconnected within the three-dimensional interconnected network of irregularly shaped bodies and further connected to the three-dimensional interconnected network of irregularly shaped spaces.

18. 18. The nanoporous layer of claim 16 or 17, wherein the nanoporous layer does not contain organic molecules therein, or contains organic molecules, if any, in an amount of less than 0.5 parts by weight per 100 parts by weight of the deposit.

19. The nanoporous layer according to any one of claims 16 to 18, wherein the nanoporous layer has a roughness factor of from about 100 to about 2500.

20. a substrate comprising an electrically conductive surface; and 20. A glucose sensing electrode comprising the nanoporous layer of any one of claims 16 to 19 formed on the conductive surface, the glucose-sensing electrode does not contain a glucose-specific enzyme; A glucose sensing electrode, wherein the nanoporous layer does not contain organic molecules therein or contains organic molecules in an amount of less than 0.5 parts by weight per 100 parts by weight of the deposit.

21. 21. The glucose sensing electrode of claim 20, wherein the substrate comprises a conductive metal layer and a conductive carbon layer formed on the conductive metal layer, the substrate comprising a conductive or semiconductive material that provides the conductive surface.

22. when a bias voltage of 0.2-0.45 V is applied between the glucose-sensing electrode and a reference electrode in contact with a glucose-containing solution, the glucose-sensing electrode is configured to cause oxidation of glucose in the nanoporous layer and to generate a current that is the sum of a glucose-oxidation current caused by the oxidation of glucose alone and a background current caused by other electrochemical interactions between the glucose-containing solution and the glucose-sensing electrode; When the glucose-containing solution contains glucose at a concentration of 4-20 mM (72-360 mg / dL), at steady state, the glucose-oxidation current is 0.1 μA / mMcm ２ (10nA / mMcm ２ 22. The glucose sensing electrode of claim 20 or 21, wherein the glucose sensing electrode is at a level higher than the glucose sensing electrode.

23. an electrolyte ion-blocking layer formed on the nanoporous layer; and a biocompatible layer formed on the electrolyte ion-blocking layer; further comprising Glucose, Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ When the electrolyte ion-blocking layer comes into contact with a liquid containing Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ from diffusing toward the nanoporous layer, thereby preventing Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ 23. The glucose sensing electrode of claim 20, wherein there is a substantial discontinuity in the total concentration of

24. 24. The glucose sensing electrode of claim 20, wherein the total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 10% of the total concentration above the electrolyte ion-blocking layer.

25. The electrolyte ion-blocking layer is formed to prevent Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ 25. The glucose sensing electrode of claim 20, comprising a porous and hydrophobic polymer layer configured to restrict the mobility of a glucose molecule therethrough but not restrict the mobility of a glucose molecule therethrough.

26. 26. The glucose sensing electrode of claim 20, wherein the electrolyte ion-blocking layer comprises at least one selected from the group consisting of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

27. Single body; a first electrode formed on the single body, the first electrode comprising the glucose sensing electrode of any one of claims 20 to 26; and a second electrode formed on the single body and configured to contact the liquid when the first electrode contacts the liquid; 1. A glucose sensing device comprising: The glucose sensing device does not contain a glucose-specific enzyme.

28. 28. The device of claim 27, wherein the nanoparticles of the nanoporous layer are made from at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and oxides of one or more of the foregoing metals, and wherein the first electrode does not include a biocompatible layer configured to prevent immune rejection.

29. 29. The device of claim 27 or 28, wherein the nanoparticles comprise at least one of platinum (Pt) and gold (Au), and the first electrode comprises a biocompatible layer configured to prevent immune rejection.

30. Providing a device according to any one of claims 27 to 29; applying a bias voltage between the first electrode and the second electrode while a test fluid is in contact with both the first electrode and the second electrode, thereby causing oxidation of glucose contained in the test fluid at the glucose sensing electrode; and measuring the current flowing from the first electrode; and and processing the measured value of the current, with or without additional data, to provide a glucose level corresponding to the glucose contained in the test fluid. A method for non-enzymatic glucose sensing comprising:

31. a conductive layer; a nanoporous layer formed on the conductive layer; an electrolyte ion-blocking layer formed on the nanoporous layer; and a biocompatible layer formed on the electrolyte ion-blocking layer; 1. A glucose sensing electrode comprising: the glucose-sensing electrode does not contain a glucose-specific enzyme; Glucose, Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ When the electrolyte ion-blocking layer comes into contact with a liquid containing Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ from diffusing toward the nanoporous layer, thereby preventing Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２ A glucose sensing electrode in which there is a substantial discontinuity in the total concentration of

32. the nanoporous layer comprises a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another over some surfaces or portions thereof, while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.25 nm to about 4.5 nm; 32. The glucose sensing electrode of claim 31, wherein at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps ranging in size from about 25 nm to about 750 nm.

33. the nanoporous layer comprises a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to each other, thereby forming a three-dimensional interconnected network of irregularly shaped bodies within the nanoporous layer; 33. The glucose sensing electrode of claim 31 or 32, wherein the unoccupied spaces between the adjacent but non-contacting surfaces of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces, thereby forming a three-dimensional interconnected network of irregularly shaped spaces inside the nanoporous layer.

34. the nanoparticles comprise at least one of platinum (Pt) and gold (Au); within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; 34. The glucose sensing electrode of claim 31, wherein the interparticle nanopores are distributed substantially throughout the interior of the three-dimensional interconnected network of irregularly shaped bodies, and the unoccupied space of the three-dimensional interconnected network of irregularly shaped spaces is distributed substantially throughout the nanoporous layer.

35. When a bias voltage of 0.2-0.45 V is applied between the glucose sensing electrode and a reference electrode in contact with the liquid, the glucose sensing electrode is configured to cause oxidation of glucose in the nanoporous layer and generate a current that is the sum of a glucose-oxidation current caused by the oxidation of glucose alone and a background current caused by other electrochemical interactions between the liquid and the glucose sensing electrode, and when the liquid contains glucose at a concentration of 4-20 mM (72-360 mg / dL), the glucose-oxidation current is less than 0.1 μA / mMcm at steady state. ２ (10nA / mMcm ２ 35. The glucose sensing electrode of claim 31, wherein the glucose sensing electrode is at a level higher than the reference glucose level.

36. 36. The glucose sensing electrode of claim 31, wherein the total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 10% of the total concentration above the electrolyte ion-blocking layer.

37. 37. The glucose sensing electrode of claim 31, wherein the total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 5% of the total concentration above the electrolyte ion-blocking layer.

38. The electrolyte ion-blocking layer is formed to prevent Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ 38. The glucose sensing electrode of claim 31, comprising a porous and hydrophobic polymer layer configured to restrict the mobility of a glucose molecule therethrough but not restrict the mobility of a glucose molecule therethrough.

39. 39. The glucose sensing electrode of any one of claims 31 to 38, wherein the electrolyte ion-blocking layer comprises at least one selected from the group consisting of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

40. The electrolyte ion-blocking layer comprises at least one selected from the group consisting of copolymers of methyl methacrylate and butyl methacrylate, and polymers obtained from the polymerization of one or more monomers including branched or unbranched C1-C8 alkyl methacrylate, branched or unbranched C1-C8 cycloalkyl methacrylate, branched or unbranched C1-C8 alkyl acrylate, branched or unbranched C1-C8 cycloalkyl acrylate, and branched or unbranched C1-C8 cycloalkyl methacrylate.

40. The glucose sensing electrode of any one of claims 31 to 39, comprising one or more monomers selected from the group consisting of methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, and 2-ethylhexyl acrylate.

41. The glucose sensing electrode of any one of claims 31 to 40, wherein the glucose sensing electrode is a continuous glucose monitoring (CGM) electrode and the liquid is a body fluid of a subject.

42. 42. The glucose sensing electrode of claim 31, wherein the glucose sensing electrode is configured to cause oxidation of glucose in the nanoporous layer and generate a current that is the sum of a glucose-oxidation current caused solely by glucose oxidation and a background current caused by other electrochemical interactions between the fluid and the glucose sensing electrode when a bias voltage of 0.2-0.45 V is applied to the glucose sensing electrode relative to a reference electrode, and the electrolyte ion-blocking layer is configured to facilitate conditioning of the glucose sensing electrode such that conditioning is completed within 30 minutes of contact with the subject's body fluid using application of a bias voltage of 0.2-0.45 V.

43. 43. The glucose sensing electrode of any one of claims 31 to 42, wherein conditioning of the glucose sensing electrode is considered complete when the rate of decrease of the current is less than a first predetermined value.

44. 44. The glucose sensing electrode of claim 31, wherein conditioning of the glucose sensing electrode is considered complete when the rate of decrease of the current becomes less than a first predetermined value and the current remains less than a second predetermined value.

45. 45. The glucose sensing electrode of claim 31, wherein the reference electrode is configured to provide a reference level of potential for a bias voltage applied to the glucose sensing electrode, regardless of whether reduction of a chemical component occurs at the reference electrode. 。

46. Single, integrated body containing the subcutaneous tissue and terminal area 1. A continuous glucose monitoring device comprising: The subcutaneous tissue comprises a glucose sensing electrode and a reference electrode according to any one of claims 31 to 45, each of which is exposed to contact with interstitial fluid of the subject when the subcutaneous tissue is inserted subcutaneously into the body of the subject; and The terminal portion is configured to couple with a compatible device and include a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode.

47. The glucose sensing electrode of any one of claims 31 to 45; and a reference electrode configured to contact the test solution when the glucose sensing electrode contacts the test solution; 1. A glucose sensing device comprising: The nanoporous layer is configured to cause oxidation of glucose molecules therein in the absence of glucose-specific enzymes when a bias voltage in the range of about 0.2 V to about 0.45 V is applied between the glucose-sensing electrode and the reference electrode.

48. When the bias voltage is applied between the glucose-sensing electrode and the reference electrode, the glucose-sensing electrode is configured to cause oxidation of glucose in the nanoporous layer and to generate a current that is the sum of a glucose-oxidation current caused by the oxidation of glucose and a background current caused by at least one other electrochemical interaction between the liquid and the glucose-sensing electrode and the reference electrode, and when the liquid contains glucose at a concentration of 4-20 mM (72-360 mg / dL), the glucose-oxidation current is greater than or equal to 0.1 μA / mMcm at steady state. ２ (10nA / mMcm ２ 48. The apparatus of claim 47, wherein the first electrode is at a level higher than the second electrode.

49. 49. An apparatus according to claim 47 or 48; a first corresponding terminal and a second corresponding terminal, a circuit network, and a corresponding device including a power source connected to the circuit network; A system comprising: the corresponding device further includes a corresponding terminal configured to connect or engage with the device; the first and second mating terminals are arranged within the mating terminal portions such that, when the mating terminal portions of the device and the mating device are coupled or engaged, the first terminals are electrically connected to the first mating terminals and the second terminals are electrically connected to the second mating terminals; the circuitry of the corresponding device is configured to provide a bias voltage between the first corresponding terminal and the second corresponding terminal, whereby the bias voltage is applied between the glucose sensing electrode and the reference electrode.

50. 50. The system of any one of claims 47-49, wherein the capable device includes a wireless communication module configured to communicate wirelessly with a wirelessly paired computing device including at least one processor and at least one memory, and the capable device is configured to receive a current indicative of glucose oxidation at the nanoporous layer of the glucose sensing electrode.

51. Providing an apparatus according to any one of claims 47 to 49; applying a bias voltage between the glucose-sensing electrode and the reference electrode while a test fluid contacts both the glucose-sensing electrode and the reference electrode to cause oxidation of glucose contained in the test fluid at the nanoporous layer; and measuring the current flowing from the glucose sensing electrode; and and processing the measured value of the current, with or without additional data, to provide a glucose level corresponding to the glucose contained in the test fluid. A method for non-enzymatic glucose sensing comprising:

52. Providing a device according to any one of claims 47 to 49; contacting the glucose sensing electrode and the reference electrode with a fluid containing glucose; applying a bias voltage of 0.2-0.45 V to the glucose sensing electrode relative to the reference electrode; measuring the current generated from the glucose sensing electrode; calculating a glucose level using a current value obtained by measuring the current within less than one hour after contacting the glucose sensing electrode and the reference electrode with the fluid; and presenting the calculated glucose level on a display within a range of about 4 mM to about 20 mM (about 72 mg / dL to about 360 mg / dL).

1. A method for continuous glucose monitoring, comprising:

53. Base material; a nanoporous layer formed on the substrate and capable of oxidizing both glucose and maltose without glucose- or maltose-specific enzymes at the glucose-sensing electrode; and A maltose-blocking layer formed on the nanoporous layer 1. A glucose sensing electrode comprising: The maltose-blocking layer has a porosity that allows glucose to pass therethrough and blocks maltose from passing therethrough toward the nanoporous layer, such that the current caused by the oxidation of glucose alone in the nanoporous layer is 10 nA / mMcm ２ Furthermore, the current caused by the oxidation of maltose alone in the nanoporous layer was 5 nA / mMcm ２ The glucose sensing electrode is made lower.

54. the nanoporous layer comprises a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another over some surfaces or portions thereof, while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.25 nm to about 4.5 nm; 54. The glucose sensing electrode of claim 53, wherein at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps ranging in size from about 25 nm to about 750 nm.

55. the nanoporous layer comprises a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to each other, thereby forming a three-dimensional interconnected network of irregularly shaped bodies within the nanoporous layer; 55. The glucose sensing electrode of claim 53 or 54, wherein the unoccupied spaces between the adjacent but non-contacting surfaces of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces, thereby forming a three-dimensional interconnected network of irregularly shaped spaces inside the nanoporous layer.

56. 56. The glucose sensing electrode of any one of claims 53-55, wherein within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween and are spaced apart from one another, defining interparticle nanopores therebetween, and at least some of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.5 nm to about 3 nm.

57. 57. The glucose sensing electrode of any one of claims 53 to 56, wherein at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps ranging in size from about 100 nm to about 500 nm.

58. The nanoporous layer can oxidize glucose, such that when a bias voltage of 0.2-0.45 V is applied between the glucose-sensing electrode and the reference electrode, and the glucose-sensing electrode and the reference electrode are in contact with a liquid containing glucose at a concentration of 4-20 mM, the current caused by the oxidation of glucose alone is 10 nA / mMcm in the absence of the maltose-blocking layer. ２ It becomes higher, The nanoporous layer can also oxidize maltose, such that when a bias voltage of 0.2-0.45 V is applied between the glucose-sensing electrode and the reference electrode, and the glucose-sensing electrode and the reference electrode are in contact with a liquid containing maltose at a concentration of 4-20 mM, the current caused by the oxidation of maltose alone is 10 nA / mMcm in the absence of the maltose-blocking layer. ２ 58. The glucose sensing electrode of any one of claims 53 to 57, wherein the glucose sensing electrode has a higher

59. 59. The glucose sensing electrode of any one of claims 53 to 58, wherein the maltose-blocking layer comprises poly-phenylenediamine (poly-PD) and has a thickness of 10 nm to 40 nm.

60. 60. Any one of claims 53 to 59, wherein the maltose-blocking layer consists essentially of poly-phenylenediamine (poly-PD) and has a thickness of 10 nm to 35 nm.

2. The glucose sensing electrode according to claim 1 .

61. 61. The glucose sensing electrode of any one of claims 53 to 60, wherein the maltose-blocking layer is composed of poly-phenylenediamine (poly-PD) and has a thickness of 10 nm to 40 nm.

62. further comprising an electrolyte ion-blocking layer formed on the maltose-blocking layer and a biocompatible layer formed on the electrolyte ion-blocking layer; Glucose, Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ When the electrolyte ion-blocking layer comes into contact with a liquid containing Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ is configured to prevent diffusion of Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ 62. The glucose sensing electrode of claim 53, wherein a substantial discontinuity in the total concentration of exists between above the electrolyte ion-blocking layer and below the electrolyte ion-blocking layer.

63. the glucose sensing electrode is configured to cause oxidation of glucose in the nanoporous layer when a bias voltage of 0.2-0.45 V relative to a reference electrode is applied thereto, and configured to generate a current that is the sum of a glucose-oxidation current caused by the oxidation of glucose alone and a background current caused by other electrochemical interactions between the liquid and the glucose sensing electrode; When the fluid contains glucose at a concentration of 4-20 mM (72-360 mg / dL), at steady state, the glucose-oxidation current is 0.1 μA / mMcm ２ (10nA / mMcm ２ 63. The glucose sensing electrode of any one of claims 53 to 62, wherein the glucose sensing electrode is at a level higher than the reference glucose level.

64. 64. The glucose sensing electrode of any one of claims 53 to 63, wherein the total concentration below the electrolyte ion-blocking layer is greater than 0% and less than about 10% of the total concentration above the electrolyte ion-blocking layer.

65. The electrolyte ion-blocking layer is formed to prevent Na ＋ , K. ＋ , Ca ２＋ , Cl － , P.O. ４ ３－ and CO ３ ２－ 65. The glucose sensing electrode of any one of claims 53 to 64, comprising a porous and hydrophobic polymer layer configured to restrict the mobility of a glucose molecule therethrough but not restrict the mobility of a glucose molecule therethrough.

66. 66. The glucose sensing electrode of any one of claims 53 to 65, wherein the electrolyte ion-blocking layer comprises at least one selected from the group consisting of poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

67. 67. The electrode of any one of claims 53 to 66, wherein the electrolyte ion-blocking layer is configured to facilitate conditioning of the glucose sensing electrode such that conditioning of the glucose sensing electrode is completed within 30 minutes of contact with the subject's body fluid using application of a bias voltage of 0.2-0.45 V to the glucose sensing electrode relative to a reference electrode.

68. A continuous glucose monitoring device comprising a single, integral body including a subcutaneous portion and a terminal portion; The subcutaneous layer comprises a glucose sensing electrode and a reference electrode according to any one of claims 53 to 67; The terminal portion is configured to couple with a corresponding device and includes a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode.

69. 68. A single-use glucose sensing device comprising a single, integral body including the glucose sensing electrode and the reference electrode of any one of claims 53 to 67, wherein the single, integral body further comprises a reservoir configured to at least temporarily hold a test fluid therein; A single-use glucose sensing device, wherein the glucose sensing electrode and the reference electrode are arranged within the single integral body, such that when the test fluid is held within the reservoir, each of the glucose sensing electrode and the reference electrode is configured to contact the test fluid.

70. 69. The apparatus of claim 68; a first corresponding terminal and a second corresponding terminal, a circuit network, and a corresponding device including a power source connected to the circuit network; 1. A system comprising: the corresponding device further includes a corresponding terminal configured to couple with or engage the terminal of the device; the first and second mating terminals are arranged within the mating terminal portion such that when the terminal portion of the device and the mating terminal portion of the mating device are coupled or engaged, the first terminal electrically connects to the first mating terminal and the second terminal electrically connects to the second mating terminal; the circuitry of the corresponding device is configured to provide a bias voltage between the first corresponding terminal and the second corresponding terminal, whereby the bias voltage is applied between the glucose sensing electrode and the reference electrode.

71. 71. The system of claim 70, wherein the capable device includes a wireless communication module configured to communicate wirelessly with a wirelessly paired computing device including at least one processor and at least one memory, and the capable device is configured to receive a current indicative of glucose oxidation in the nanoporous layer of the glucose sensing electrode.

72. providing said nanoporous layer capable of oxidizing both glucose and maltose; and forming the maltose-blocking layer on the nanoporous layer, the maltose-blocking layer comprising a poly-phenylenediamine (poly-PD) film, the poly-PD film having a porosity that allows glucose to pass therethrough and blocks maltose from passing therethrough toward the nanoporous layer, such that when a bias voltage of 0.2-0.45 V is applied to the nanoporous layer relative to a reference electrode, and the poly-PD film is in contact with a liquid containing glucose at a concentration of 4-20 mM and maltose at a concentration of 4-20 mM, the current caused by the oxidation of glucose alone in the nanoporous layer is 10 nA / mMcm ２ Furthermore, the current caused by the oxidation of maltose alone in the nanoporous layer was 5 nA / mMcm ２ lower 68. A method of manufacturing the glucose sensing electrode of any one of claims 53 to 67, comprising:

73. 73. The method of claim 72, wherein forming the poly-PD film comprises performing electrochemical polymerization using the nanoporous layer as an electrode for electrochemical polymerization.

74. Forming the poly-PD film includes providing a polymer layer containing poly-PD, and determining whether the current caused by the oxidation of glucose alone in the nanoporous layer is 10 nA / mMcm. ２ 74. The method of claim 72 or 73, comprising adjusting the porosity of the polymer layer if it is expected to be lower.

75. 75. The method of any one of claims 72 to 74, wherein adjusting the porosity comprises subjecting the polymer layer to at least one electric shock while the polymer layer is in contact with an acidic solution.

76. 76. The method of any one of claims 72 to 75, wherein forming the poly-PD film comprises polymerizing poly-PD from a liquid composition comprising a phenylenediamine at a concentration, and when the concentration is higher than a predetermined value, forming the poly-PD film further comprises adjusting the porosity of the polymer layer.

77. 77. The method of any one of claims 72 to 76, wherein adjusting the porosity comprises subjecting the polymer layer to at least one electric shock while the polymer layer is in contact with an acidic solution.

78. The formation of the poly-PD film resulted in a current caused by the oxidation of glucose alone in the nanoporous layer of 10 nA / mMcm. ２ 78. The method of any one of claims 72 to 77, further comprising providing a polymer layer comprising poly-PD without adjusting the porosity of said polymer layer if it is expected that the porosity of said polymer layer will be higher.

79. 79. The method of any one of claims 72-78, wherein forming the poly-PD film comprises polymerizing poly-PD from a liquid composition comprising a concentration of phenylenediamine, and when the concentration is below a predetermined value, the method does not include adjusting the porosity of the polymer layer to form the poly-PD film.

80. Base material; a first electrode comprising a first conductive layer formed on a first electrode location of the substrate and a glucose-oxidation layer formed on the first conductive layer; a first terminal formed on a first terminal location of the substrate and electrically connected to the first electrode; a second electrode including a second conductive layer formed on the substrate at a second electrode location; a second terminal formed on the substrate at a second terminal location and electrically connected to the second electrode; a reference electrode including a third conductive layer formed on the substrate at a reference electrode location; and a third terminal formed on the substrate at a third terminal location and electrically connected to the reference electrode, the glucose-oxidation layer of the first electrode is configured to cause oxidation of glucose and at least one of ascorbic acid and acetaminophen therein when the first electrode and the reference electrode are in contact with a liquid containing glucose, ascorbic acid, and acetaminophen, and when a first bias voltage sufficient to oxidize glucose in the glucose-oxidation layer is applied between the first electrode and the reference electrode; and configured to generate a first current in the glucose-oxidation layer, the first current including a glucose component caused by the oxidation of glucose and a first interference component caused by the oxidation of at least one of ascorbic acid and acetaminophen; the second electrode is arranged within the device such that when the first electrode contacts the liquid, the second electrode also contacts the same liquid, the second electrode does not include a layer configured to cause oxidation of glucose therein, such that application of a second bias voltage between the second electrode and the reference electrode causes oxidation of at least one of ascorbic acid and acetaminophen therein, but not oxidation of glucose therein, and is further configured to generate a second current at the second electrode that includes a second interferent caused by oxidation of at least one of ascorbic acid and acetaminophen, but not caused by oxidation of glucose; A sensor device, wherein the device is configured to provide the first current at the first terminal and the second current at the second terminal.

81. the glucose-oxidation layer comprises a nanoporous layer comprising a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another over some surfaces or portions thereof, while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; the junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to one another, which continues to other ones of the irregularly shaped bodies, forming a three-dimensional interconnected network of irregularly shaped bodies; the unoccupied spaces between non-contacting surfaces or portions of the adjacent ones of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces formed by others of the irregularly shaped bodies; the interconnections between the unoccupied spaces form a three-dimensional interconnected network of irregularly shaped spaces outside of and geometrically complementary to the three-dimensional interconnected network of irregularly shaped bodies inside the nanoporous layer; within the three-dimensional interconnected network of irregularly shaped bodies, at least some of the nanoparticles are adjacent to one another with no intervening nanoparticles therebetween, and are spaced apart with interparticle nanopores therebetween; the nanoporous layer comprises, inside the three-dimensional interconnected network of irregularly shaped bodies, the interparticle nanopores, and, outside the three-dimensional interconnected network of irregularly shaped bodies, the three-dimensional interconnected network of irregularly shaped spaces; at least a portion of the interparticle nanopores within the three-dimensional interconnected network of irregularly shaped bodies comprise gaps in a size range from about 0.25 nm to about 4.5 nm; 81. The apparatus of claim 80, wherein at least some of the irregularly shaped spaces of the three-dimensional interconnected network of irregularly shaped spaces include gaps with sizes ranging from about 25 nm to about 750 nm.

82. the glucose-oxidation layer comprises a nanoporous layer comprising a deposit of irregularly shaped bodies formed from many nanoparticles having a generally ovoid or spherical shape with lengths ranging from about 2 nm to about 5 nm; adjacent ones of the irregularly shaped bodies abut one another while forming unoccupied spaces between non-abutting surfaces or portions of the adjacent ones of the irregularly shaped bodies; junctions between adjacent ones of the irregularly shaped bodies connect the adjacent ones to each other, thereby forming a three-dimensional interconnected network of irregularly shaped bodies within the nanoporous layer; 82. The device of claim 80 or 81, wherein the unoccupied spaces between the adjacent but non-contacting surfaces of the irregularly shaped bodies are irregularly shaped and connect with other unoccupied spaces, thereby forming a three-dimensional interconnected network of irregularly shaped spaces inside the nanoporous layer.

83. 83. Apparatus according to any one of claims 80 to 82, wherein the apparatus is configured to generate the first current and the second current simultaneously.

84. 84. The apparatus of any one of claims 80 to 83, wherein the apparatus is configured to provide the first current and the second current together with information indicative of a time at which the first current and the second current were generated.

85. 85. Apparatus according to any one of claims 80 to 84, wherein the apparatus is configured to provide the second current together with the first current whenever the apparatus provides the first current.

86. 86. The device of any one of claims 80 to 85, wherein the first current further includes a first background current caused by other electrochemical interactions between the liquid and the glucose-oxidation layer, and the second current further includes a second background current caused by other electrochemical interactions between the liquid and the second electrode.

87. when a voltage of 0.2 V to 0.32 V is applied as a first bias voltage, the glucose-oxidation layer is configured to oxidize glucose and ascorbic acid but not acetaminophen, and the first interfering component is caused mainly by the oxidation of ascorbic acid between ascorbic acid and acetaminophen; 87. The device of any one of claims 80 to 86, wherein when a voltage of 0.2 V to 0.32 V is applied as a second bias voltage, the second electrode is configured to oxidize ascorbic acid but not acetaminophen, and the second interfering component between ascorbic acid and acetaminophen is mainly caused by the oxidation of ascorbic acid.

88. when a voltage of 0.34 V to 0.45 V is applied as a first bias voltage, the glucose-oxidation layer is configured to oxidize glucose, ascorbic acid, and acetaminophen, and the first interfering component is caused by the oxidation of both ascorbic acid and acetaminophen; 88. The device of any one of claims 80 to 87, wherein when a voltage of 0.34 V to 0.45 V is applied as a second bias voltage, the second electrode is configured to oxidize ascorbic acid and acetaminophen, and the second interfering component is caused by the oxidation of both ascorbic acid and acetaminophen.

89. the first electrode further comprises a maltose-blocking layer formed on the glucose-oxidation layer, the maltose-blocking layer comprising poly-phenylenediamine (poly-PD); The maltose-blocking layer is configured to allow glucose to pass therethrough and to substantially block maltose from passing therethrough, so that, at steady state, when the first bias voltage is 0.2-0.45 V and the first electrode and the reference electrode are in contact with a liquid containing glucose at a concentration of 4-20 mM and maltose at a concentration of 4-20 mM, the glucose-oxidation current is 0.1 μA / mMcm ２ (10nA / mMc ２ ), whereas the maltose oxidation current caused by maltose oxidation alone was 0.05 μA / mMcm ２ (5 nA / mMcm ２ 89. The device according to any one of claims 80 to 88, wherein the temperature is lower than

90. 90. The device of any one of claims 80 to 89, wherein the device is a continuous glucose monitoring (CGM) electrode module including a subcutaneous portion configured to subcutaneously contact a body fluid of a subject, and wherein the first electrode, second electrode and reference electrode are formed within the subcutaneous portion.

91. 91. The device of any one of claims 80-90, wherein the first bias voltage is between 0.2V and 0.45V, the second bias voltage is the same as or different from the first bias voltage, and the glucose-oxidation layer comprises a nanoporous metallic material or a glucose-specific enzyme configured to oxidize glucose.

92. The glucose-oxidation layer is an irregularly shaped body including many nanoparticles clustered together locally and interparticle gaps formed between adjacent ones of the nanoparticles in said irregularly shaped body; Including, the nanoparticles are generally ovoid or spherical in shape with a diameter of about 2 nm to about 5 nm, and the interparticle gap has an interparticle gap distance of about 0.25 nm to about 4.5 nm; the irregularly shaped bodies are interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies; the irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nano-sized or micro-sized; 92. Apparatus according to any one of claims 80 to 91, wherein the irregularly shaped spaces are interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces.

93. The sensor device according to any one of claims 80 to 92, further comprising a terminal portion in which the first, second, and third terminals are arranged; a corresponding device including a first corresponding terminal, a second corresponding terminal, a third corresponding terminal, a circuit network, and a power source connected to the circuit network; 1. A system comprising: the corresponding device further includes a corresponding terminal portion configured to couple with or engage with the terminal portion; the first, second, and third corresponding terminals are arranged within the corresponding terminal portion such that, when the terminal portion of the sensor device and the corresponding terminal portion of the corresponding device are coupled or engaged, the first terminal is electrically connected to the first corresponding terminal, the second terminal is electrically connected to the second corresponding terminal, and the third terminal is electrically connected to the third corresponding terminal; the circuitry of the corresponding device is configured to provide a first bias voltage between the first corresponding terminal and the third corresponding terminal; The circuitry of the corresponding device is further configured to provide a second bias voltage between the second corresponding terminal and the third corresponding terminal.

94. 94. The system of claim 93, wherein the capable device includes a wireless communication module configured to communicate wirelessly with a wirelessly paired computing device, the wireless communication module including at least one processor and at least one memory, the capable device configured to receive the first current at the first corresponding terminal and the second current at the second corresponding terminal, the capable device further configured to transmit the second current together with the first current when transmitting the first current.

95. 95. The system of claim 94, wherein the first current is communicated with a first timestamp and the second current is communicated with a second timestamp, the first and second timestamps indicating the same time.

96. Software installed and executable by the at least one processor of the wirelessly paired computing device. further comprising When executed, the software: causing at least one memory of the computing device to store the first current and the second current received together or in association with each other from the corresponding device; processing the first current and the second current to provide a value indicative of glucose oxidation in the glucose-oxidation layer of the first electrode of the sensor device; and causing the value or corresponding information to be presented on a display of said computing device.

96. A system according to claim 94 or 95, configured to carry out a method comprising:

97. 97. The system of any one of claims 94 to 96, wherein the first current and the second current are stored in association with each other in the at least one memory, and processing includes subtracting the second current from the first current.

98. software installed and executable by the at least one processor of the wirelessly paired computing device; further comprising A system as described in any one of claims 94 to 97, wherein when executed, the software is configured to perform data processing using the first current and the second current received from the compatible device to obtain the level of glucose contained in the liquid, and the software requires the second current when processing to obtain the level of glucose.

99. the enabled device further includes at least one processor, at least one memory, and software stored in the at least one memory and executable by the at least one processor; When executed, the software: causing the at least one memory to store the first current and the second current received together or in association with one another from the sensor device; and processing the first current and the second current to provide a value indicative of glucose oxidation in the glucose-oxidation layer of the first electrode of the sensor device; 99. A system according to any one of claims 94 to 98, configured to perform a method comprising:

100. one or both of the first current and the second current are in the form of a continuous signal; 100. The system of any one of claims 94 to 99, wherein processing the first current and the second current comprises processing values of the first current and the second current obtained simultaneously.

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