Colloids with nanoporous structures and devices and systems 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] This disclosure relates to glucose sensing. [Background technology]

[0002] In the healthcare society and industry, there is a high level of interest in improving technologies for sensing and monitoring blood glucose levels. Today, most glucose sensors use electrochemical methods. Most, though not all, electrochemical sensors use enzyme-based electrochemical sensors. [Overview of the project]

[0003] One aspect of the invention provides a colloidal composition comprising clusters of many nanoparticles dispersed in a liquid, each cluster comprising many nanoparticles that cluster together to form an irregularly shaped body having a nanoscale or microscale length, the individual nanoparticles having separate bodies that are generally oval or spherical with a diameter of about 2 nm to about 5 nm, and interparticular gaps are formed between adjacent nanoparticles inside each cluster, with an interparticular gap distance of about 0.5 nm to about 2 nm.

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

[0005] In the aforementioned colloidal composition, the nanoparticles may be mainly produced 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 aforementioned elements. The nanoparticles may be mainly produced from platinum (Pt), the interparticle gap may be distributed almost throughout each cluster, the colloidal composition may contain a surfactant in an amount less than 1 part by weight per 100 parts by weight of nanoparticles contained therein, and the amount of nanoparticles contained in the colloidal composition may be about 0.1 wt% to about 1 wt% of the total weight of the colloidal composition.

[0006] Another aspect of the invention provides a method for producing a nanoporous layer. The method comprises distributing the aforementioned colloidal composition onto a substrate; subjecting the distributed colloidal composition to drying so that the clusters contained in the distributed 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 disordered shapes formed from the clusters stacked on top of each other, the disordered shapes comprising many nanoparticles locally clustered together and interparticle gaps formed between adjacent nanoparticles in the disordered shapes, the disordered shapes being interconnected, providing a three-dimensional interconnected network of disordered shapes, where disordered spaces are formed between adjacent portions of the disordered shapes, and they are nano-sized or micro-sized.

[0007] In the method described above, the nanoparticles may be generally oval or spherical in shape with a diameter of about 2 nm to about 5 nm. The interparticle gap may have an interparticle gap distance of about 0.5 nm to about 2 nm. The disordered spatial arrangements may be interconnected, providing a three-dimensional interconnected network of disordered spatial arrangements. The colloidal composition may be distributed in predetermined amounts to form a nanoporous layer having a roughness coefficient of about 100 to about 2500. The nanoporous layer may contain a surfactant in an amount 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. The method comprises: providing a liquid composition comprising a metal ion, a surfactant, and a solvent, wherein the surfactant is an inverse micelle phase defining hydrophilic spaces; adding a reducing agent to the liquid composition to cause reduction of the metal ion, thereby forming a first colloid comprising metal nanoparticles and the surfactant, wherein in the first colloid, the metal nanoparticles are dispersed together with the inverse 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, wherein each cluster is clustered together to form an irregular shape having a nanoscale or microscale length.

[0009] In the manufacturing method described above, potential may not be applied to the liquid composition for the reduction of metal ions therein. The surfactant may be a nonionic surfactant capable of forming an isotropic inverse micelle phase. Individual nanoparticles may have separate bodies that are generally oval or spherical with a diameter 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 considerable amount of surfactant from the first colloid so that the second colloid is substantially free of surfactant. Removing the surfactant removes a considerable amount of surfactant from the first colloid so that the second colloid contains less than 1 part by weight of surfactant per 100 parts by weight of nanoparticles contained therein.

[0010] In the aforementioned manufacturing method, the removal of the surfactant may further include: centrifuging the first colloid; and collecting the bottom from the centrifuged composition. The removal of the surfactant may further include repeating a series of centrifugation and collections multiple times. The removal of the surfactant may further include adding an acid or base to the first colloid before centrifugation. The removal of the surfactant may further include repeating a series of additions, centrifugation, and collections multiple times. The nanoparticles contained in the second colloid may be in an amount of about 10 wt% to about 40 wt% of the total weight of the composition. The nanoparticles can be mainly manufactured 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 aforementioned metals. Nanoparticles can be mainly produced from platinum (Pt), interparticle gaps can be distributed throughout each cluster, and the composition can contain a surfactant in an amount less than 2 parts by weight per 100 parts by weight of nanoparticles contained therein, and the amount of nanoparticles contained in the composition may be about 0.1 wt% to about 2 wt% of the total weight of the composition.

[0011] Another aspect of the invention provides a method for producing a nanoporous layer. This method comprises the aforementioned method for producing a colloidal composition to provide a second colloid; distributing the second colloid onto a substrate; drying the distributed second colloid so that the clusters contained in the distributed composition are deposited and stacked on the substrate, providing a nanoporous layer on the substrate, the nanoporous layer comprising disordered shapes formed from the stacked clusters, the disordered shapes comprising many nanoparticles locally clustered together and interparticle gaps formed between adjacent nanoparticles in the disordered shapes. The disordered shapes are interconnected, providing a three-dimensional interconnected network of disordered shapes, the disordered shapes are formed between adjacent portions of the disordered shapes and are nano-sized or micro-sized, and the disordered shapes are interconnected, providing a three-dimensional interconnected network of disordered shapes.

[0012] In the aforementioned method for producing the nanoporous layer, the nanoparticles may be generally oval 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 0.5 nm to about 2 nm. The colloidal composition may be distributed in predetermined amounts to form a nanoporous layer having a roughness coefficient 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: many nanoparticles locally clustered together and an irregular shape comprising interparticle gaps formed between adjacent nanoparticles in the irregular shape, where the nanoparticles may be generally oval or spherical with a diameter 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, the irregular shapes may be interconnected, providing a three-dimensional interconnected network of irregular shapes, the irregular shape spaces are formed between adjacent parts of the irregular shapes and are nanoscale or microscale, and the irregular shape spaces are interconnected to provide a three-dimensional interconnected network of irregular shape spaces.

[0014] The aforementioned nanoporous structure may substantially contain no surfactant molecules. In the aforementioned nanoporous structure, the interparticle gaps may substantially contain no nanosized organic molecules. The three-dimensional network of disordered shapes and the three-dimensional network of gaps between disordered clusters may be complementary to form a nanoporous structure. The interparticle gaps may be substantially interconnected within themselves and may be further interconnected in a three-dimensional interconnected network of gaps between disordered clusters. The nanoporous structure may be formed by distributing a solid-liquid colloid containing separate clusters of disordered shapes dispersed in a liquid and drying the distributed solid-liquid colloid, where the separate clusters of disordered shapes can be stacked, providing a three-dimensional interconnected network of disordered shapes and a three-dimensional interconnected network of gaps between disordered clusters. The gaps between disordered clusters have an average inter-cluster gap distance. Nanoparticles can be manufactured 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 aforementioned metals. The nanoporous structure has a roughness coefficient of about 100 to about 2500.

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

[0016] In the aforementioned device or electrode, 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] A further 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 come into contact with the test solution while the test solution is held in the reservoir. In the single-use glucose sensing device, the electrodes do not contain any biocompatible polymer material formed on the nanoporous layer.

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

[0019] A further 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 contain glucose-specific enzymes, the nanoporous layer may comprise an irregular shape comprising many nanoparticles locally clustered together, interparticle gaps may be formed between adjacent nanoparticles in the irregular shape, the nanoparticles may be generally oval or spherical with a diameter 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, and the irregular shape comprises mutual A three-dimensional interconnected network of disordered shapes is provided, which can be linked together and extend over substantially the entire nanoporous layer, and the disordered shape spaces can be formed between adjacent parts of the disordered shapes and can be nano-sized or micro-sized, and the disordered shape spaces can be interconnected, providing a three-dimensional interconnected network of disordered shape spaces extending over substantially the entire nanoporous layer, and the nanoporous layer can be configured to induce oxidation of glucose molecules therein without glucose-specific enzymes, with a bias voltage applied to it of about 0.2V to about 0.45V.

[0020] In the aforementioned non-enzymatic glucose sensing device, the nanoporous layer may be substantially free of surfactant molecules, and the substrate may include at least one conductive layer containing a conductive or semiconducting material. The interparticle gaps may be substantially free of nano-sized organic molecules. The three-dimensional network of disordered shapes and the three-dimensional network of inter-cluster gaps of disordered shapes may be complementary, forming the nanoporous layer. The interparticle gaps may be substantially interconnected within themselves and may be further interconnected in the three-dimensional interconnected network of inter-cluster gaps of disordered shapes.

[0021] In the aforementioned non-enzymatic glucose sensing device, the nanoporous layer may further be formed by distributing a solid-liquid colloid containing separate clusters of irregular shapes dispersed in a liquid, and then drying the distributed solid-liquid colloid, where the separate clusters of irregular shapes can be stacked, providing a three-dimensional interconnected network of irregular shapes and a three-dimensional interconnected network of gaps between irregular shapes. The nanoparticles may be manufactured 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 aforementioned metals. The nanoporous layer has a roughness coefficient 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 maltose contained in the test fluid from passing through, while allowing glucose to pass through. The maltose-blocking layer may include a form of polyphenylenediamine (poly-PD) that allows glucose molecules to pass through and effectively blocks maltose molecules from passing through. The bias voltage may be set to be in the range of 0.2V to 0.45V.

[0022] A further aspect of the invention provides a non-enzymatic glucose sensing system comprising: the aforementioned non-enzymatic glucose sensing device; a counter electrode; and a bias voltage source electrically connected between the working electrode and the counter electrode to supply a bias voltage between the working electrode and the counter electrode.

[0023] A further aspect of the invention provides a method for non-enzymatic glucose sensing. The method includes: providing the aforementioned non-enzymatic glucose sensing device; applying a bias voltage between a working electrode and a counter electrode while bringing a test fluid into contact with both electrodes, thereby inducing oxidation of glucose contained in the test fluid in a 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 may be set to be in the range of 0.2V to 0.45V.

[0024] Another aspect of the invention provides a glucose-sensing electrode, which includes: a substrate; a nanoporous metal layer formed on the substrate and capable of oxidizing both glucose and maltose within the glucose-sensing electrode without enzymes specific to glucose or maltose; and a maltose-blocking layer formed on the nanoporous metal layer. In the glucose-sensing electrode, the maltose-blocking layer has a degree of porosity that allows glucose to pass through it and prevents maltose from passing through it toward the nanoporous metal layer, so that when a bias voltage of 0.2-0.45V is applied to the nanoporous metal layer relative to a reference electrode, and 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 Furthermore, the current induced by the oxidation of maltose alone in the nanoporous metal layer was 5 nA / mMcm. 2 It will become lower.

[0025] In the aforementioned glucose-sensing electrode, the nanoporous metal layer can oxidize glucose, and therefore, the current induced by the oxidation of glucose alone is 10 nA / mMcm when a bias voltage of 0.2-0.45 V is applied and the liquid containing glucose at a concentration of 4-20 mM is in contact with it without the maltose-blocking layer on top. 2Higher. The nanoporous metal layer can further oxidize maltose. Therefore, the current caused by the oxidation of only maltose is 10 nA / mM cm when a bias voltage of 0.2 - 0.45 V is applied and it contacts a liquid containing maltose at a concentration of 4 - 20 mM without a maltose-blocking layer thereon. 2 Higher. The maltose-blocking layer may contain poly-phenylenediamine (poly-PD) and have a thickness of 10 - 40 nm. The maltose-blocking layer may be essentially composed of poly-phenylenediamine (poly-PD) and have a thickness of 10 nm - 35 nm. The maltose-blocking layer is composed of poly-phenylenediamine (poly-PD) and may have a thickness of 10 - 40 nm.

[0026] In the aforementioned glucose-sensing electrode, the nanoporous metal layer may include irregularly shaped bodies containing many nanoparticles locally clustered together and interparticle gaps formed between adjacent ones of the nanoparticles in the irregularly shaped bodies. Here, the nanoparticles are generally oval or spherical in shape and have 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 bodies may be interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies. Irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and be nano-sized or micro-sized. The irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces.

[0027] The aforementioned 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 blocks Na + , K + , Ca 2+ , Cl - , PO4 3- and CO3 2-However, it is configured to prevent diffusion toward the nanoporous metal layer, and therefore Na between the area 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. The electrolyte ion-blocking layer can facilitate the conditioning of the glucose-sensing electrode so that the conditioning of the glucose-sensing electrode is completed within 30 minutes of contact with the subject's body fluids by applying a bias voltage of 0.2-0.45V.

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

[0029] A further aspect of the invention provides an apparatus comprising a single, integrated body including the aforementioned glucose-sensing electrode and reference electrode, the single, integrated body further comprising a reservoir configured to hold a test fluid therein, at least temporarily, the glucose-sensing electrode and reference electrode arranged within the single, integrated body, so that when the test fluid is held in the reservoir, each of the glucose-sensing electrode and reference electrode is configured to come into contact with the test fluid.

[0030] A further aspect of the invention provides a method for manufacturing a glucose-sensing electrode. The method comprises: providing a nanoporous metal layer within the glucose-sensing electrode that can oxidize both glucose and maltose without enzymes specific to glucose or maltose; forming a polyphenylenediamine (poly-PD) film on the nanoporous platinum layer such that the poly-PD film allows glucose to pass through it and blocks maltose from passing through it. Here, the poly-PD film has a porosity that allows glucose to pass through it and prevents maltose from passing through it and toward the nanoporous metal layer, so that when a bias voltage of 0.2-0.45 V is applied to the nanoporous metal 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 induced by the oxidation of glucose alone in 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 for manufacturing glucose-sensing electrodes, forming a poly-PD film may include carrying out electrochemical polymerization using a nanoporous metal layer as an electrode for electrochemical polymerization. Forming a poly-PD film provides a polymer layer containing poly-PD, and the polymer layer may not have sufficient porosity to allow glucose to pass through it, and therefore the current induced by the oxidation of glucose alone in the nanoporous metal layer is 10 nA / mMcm. 2If the porosity is lower, this 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 it is in contact with an acidic solution. Forming a poly-PD film may involve polymerizing poly-PD from a liquid composition containing phenylenediamine at a certain concentration, and if the concentration is higher than a predetermined value, forming a poly-PD film may further include adjusting the porosity of the polymer layer. Adjusting the porosity may include subjecting the polymer layer to at least one electric shock while it is in contact with an acidic solution.

[0032] In the aforementioned method for manufacturing glucose-sensing electrodes, forming a poly-PD film allows the polymer layer to have sufficient porosity to allow glucose to pass through, thus enabling a current of 10 nA / mMcm induced solely by the oxidation of glucose in the nanoporous metal layer. 2 If higher is expected, the method may include providing a polymer layer containing poly-PD without further adjustment of the porosity of the polymer layer. Forming a poly-PD film may include polymerizing poly-PD from a liquid composition containing phenylenediamine at a certain concentration, and if the concentration is lower than a predetermined value, the method does not involve adjusting the porosity of the polymer layer for forming the poly-PD film.

[0033] One aspect 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 glucose-specific enzymes. Glucose, Na + , K + Ca 2+ Cl - , PO4 3- and CO3 2- When in contact with a liquid containing Na, the electrolyte ion-blocking layer will react with the Na contained in the liquid. + , K + Ca 2+Cl - , PO4 3- and CO3 2- It is configured to prevent Na from diffusing toward the nanoporous metal layer, and therefore Na is prevented from diffusing between the area above the electrolyte ion-blocking layer and the area below the electrolyte ion-blocking layer. + , K + Ca 2+ Cl - , PO4 3- and CO3 2- There is a substantial discontinuity in the total concentration.

[0034] In the aforementioned glucose-sensing electrode, when a bias voltage of 0.2-0.45V is applied to the reference electrode, the glucose-sensing electrode is configured to induce glucose oxidation in the nanoporous metal layer and to generate a current that is the sum of the glucose-oxidation current caused solely by glucose oxidation and the 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 in the steady state is 、1 0 nA / mMcm 2 Yo It is at a very high 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 approximately 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 approximately 5% of the total concentration above the electrolyte ion-blocking layer. The electrolyte ion-blocking layer is where Na passes through. + , K + Ca 2+ Cl - , PO4 3- and CO3 2- It may include a porous and hydrophobic polymer layer configured to restrict the mobility of one component but not the mobility of glucose molecules passing through it.

[0036] In the glucose-sensing electrode described above, the electrolyte ion-blocking layer may 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 may contain at least one polymer selected from the group consisting of a copolymer 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, 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 the subject's body fluid. The electrolyte ion-blocking layer is configured to facilitate the conditioning of the glucose-sensing electrode so that the conditioning of the glucose-sensing electrode is completed within 30 minutes of contact with the subject's body fluid using the application of a bias voltage of 0.2-0.45 V. Conditioning of the glucose-sensing electrode can be considered complete when the rate of decrease of the 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 may further include a maltose-blocking layer inserted between a nanoporous metal layer and an electrolyte ion-blocking layer, the maltose-blocking layer may contain polyphenylenediamine (poly-PD). The maltose-blocking layer may be configured to allow glucose to pass through it and substantially block maltose from passing through it, so that in a steady state the glucose-oxidation current is 、1 0 nA / mMcm 2 Yo While it is at a high level, on the other hand, the maltose oxidation current caused solely by the oxidation of maltose. is 5 nA / mMcm 2 Yo It's low.

[0039] The reference electrode may be configured to provide a reference potential level for the bias voltage applied to the glucose-sensing electrode, regardless of whether or not reduction of chemical components occurs at the reference electrode. In a three-electrode electrochemical cell, in addition to the reference electrode, a counter electrode is provided for the reduction of chemical components within it, whereas in a two-electrode electrochemical cell, the reduction of chemical components occurs at the reference electrode.

[0040] In the glucose-sensing electrode described above, the nanoporous metal layer may include: many nanoparticles locally clustered together and disordered shapes containing interparticle gaps formed between adjacent nanoparticles in the disordered shapes, where the nanoparticles are generally oval or spherical with a diameter 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. Here, the disordered shapes may be interconnected, providing a three-dimensional interconnected network of disordered shapes. Disordered shape spaces may be formed between adjacent parts of disordered shapes, are nano-sized or micro-sized, and the disordered shape spaces are interconnected, providing a three-dimensional interconnected network of disordered shape spaces.

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

[0042] Another aspect of the invention provides a method for continuous glucose monitoring. The method includes: providing a sensor device; subcutaneously inserting the subcutaneous portion of a glucose-sensing electrode into the body of a first subject so that the glucose-sensing electrode and a reference electrode are in contact with the interstitial fluid of the body of the first subject; 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 the 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 displaying on a display the glucose level calculated for the first subject, in the range of about 4 mM to about 20 mM (approximately about 72 mg / dL to about 360 mg / dL). The glucose-sensing electrode may include one or more features of the glucose-sensing electrode described above.

[0043] Further aspects of the invention provide a sensor device comprising: a substrate; a first electrode (or glucose sensing electrode) comprising a first conductive layer formed on the substrate and a glucose-oxide layer formed on the first conductive layer; a first terminal formed on the substrate and electrically connected to the first electrode; a second electrode comprising 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 comprising 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 comes into 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, the glucose-oxidation layer of the first electrode is configured to cause oxidation of glucose and at least one of ascorbic acid and acetaminophen within it, and is further configured to generate a first current in the glucose-oxidation layer 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. The second electrode is arranged in the device such that when the first electrode comes into contact with the liquid, the second electrode also comes into contact with the same liquid. The second electrode does not contain a layer configured to induce the oxidation of glucose, and therefore, when a second bias voltage is applied between the second electrode and the reference electrode, the second electrode is configured to induce the oxidation of at least one of ascorbic acid and acetaminophen, but not of glucose, and is further configured to generate a second current containing a second interfering component induced by the oxidation of at least one of ascorbic acid and acetaminophen, but not by the oxidation of glucose. The apparatus is configured to provide a first current at the first terminal and a second current at the second terminal.

[0045] The aforementioned sensor device may be configured to provide a second current in association with a first current when providing a first current. The sensor device may be configured to generate a first current and a second current simultaneously. The sensor device may be configured to provide a first current and a second current along with information indicating the time at which the first current and the second current were generated. The sensor device may be configured to provide a second current together with the first current whenever the first current is provided. In the aforementioned sensor device, 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 aforementioned sensor device, when the first bias voltage is 0.2V to 0.32V, 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 0.2V to 0.32V, 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 aforementioned sensor device, when the first bias voltage is 0.34V to 0.45V, 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 0.34V to 0.45V, 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 aforementioned sensor device, the first electrode may further include a maltose-blocking layer containing polyphenylenediamine (poly-PD) formed on the glucose-oxidation layer. When brought into contact with a liquid containing glucose at 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 through it and substantially block maltose from passing through it, so that in a steady state the glucose-oxidation current is 、1 0 nA / mMcm 2 Yo While it is at a high level, on the other hand, the maltose oxidation current caused solely by the oxidation of maltose. is 5 nA / mMcm 2 Yo It's low.

[0048] The aforementioned sensor device may also be a continuous glucose monitoring (CGM) electrode module including a subcutaneous layer configured to contact the subject's bodily fluids subcutaneously, with the first, second, and reference electrodes formed subcutaneously. In the aforementioned 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 contains Na in the liquid. + , K + Ca 2+ Cl - , PO4 3- and CO3 2- It may be configured to prevent Na from diffusing toward the nanoporous metal layer, thus preventing Na from diffusing between the area 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.

[0049] In the aforementioned sensor device, the electrolyte ion-blocking layer contains Na passing through it. + , K + Ca 2+ Cl - , PO4 3- and CO3 2- The electrolyte ion-blocking layer may include a porous and hydrophobic polymer layer configured to restrict the mobility of the electrolyte but not the mobility of glucose molecules passing through it, and the electrolyte ion-blocking layer may 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).

[0050] In the aforementioned sensor device, the electrolyte ion-blocking layer may be configured to facilitate the conditioning of the glucose-sensing electrode so that the conditioning of the glucose-sensing electrode is completed within 30 minutes of contact with the subject's body fluids by applying a bias voltage of 0.2-0.45V, and the conditioning of the glucose-sensing electrode is considered to be completed when the rate of decrease of the current becomes less than a first predetermined value, and when the current remains less than a second predetermined value, or both.

[0051] The aforementioned sensor device is a blood glucose monitoring (BGM) electrode module including a reservoir configured to receive blood, in which case the first electrode, second electrode, and reference electrode are configured to come into contact with the blood once blood is received in the reservoir. The first bias voltage is 0.2V to 0.45V, and the second bias voltage is the same as or different from the first bias voltage. The glucose-oxidation layer may include a nanoporous metallic material or a glucose-specific enzyme configured to oxidize glucose. The glucose-oxidation layer may include a disordered shape containing many nanoparticles locally clustered together and interparticle gaps formed between adjacent nanoparticles in the disordered shape, the nanoparticles being generally oval or spherical with a diameter 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. Here, the disordered shapes may be interconnected, providing a three-dimensional interconnected network of disordered shapes. Irregular shape spaces can be formed between adjacent parts of an irregular shape body, are nano-sized or micro-sized, and are interconnected, providing a three-dimensional interconnected network of irregular shape spaces.

[0052] A further aspect of the invention provides the aforementioned sensor device further comprising a terminal section having first, second, and third terminals arranged therein; a system comprising a corresponding device including a first corresponding terminal, a second corresponding terminal, a third corresponding terminal, a network, and power connected to the network, the corresponding device further comprising a corresponding terminal section configured to connect to or engage with the terminal section, wherein 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 connected 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 network of the corresponding device is configured to provide a first bias voltage between the first corresponding terminal and the third corresponding terminal, and the network of the corresponding device is further configured to provide a second bias voltage between the second corresponding terminal and the third corresponding terminal.

[0053] In the aforementioned system, the corresponding device may include a wireless communication module configured to communicate wirelessly with a wirelessly paired computing device, which includes 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 the second current together with or in association 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, and the first and second timestamps indicate the same time.

[0054] The aforementioned system is installed on the processor of at least one wirelessly paired computing device and may further include executable software. When executed, the software is configured to perform the following: store in the memory of at least one of the computing devices a first current and a second current received together or in association from the corresponding device; process the first current and the second current to provide a value indicating the oxidation of glucose in the glucose-oxide layer of the first electrode of the sensor device; and display that value or the corresponding information on the display of the computing device.

[0055] In the aforementioned system, either or both of the first and second currents may be in the form of continuous signals, and processing the first and second currents may include processing the values ​​of the first and second currents obtained simultaneously, where processing the values ​​may include subtracting the second current from the first current. The first and second currents may be stored in association with each other in at least one memory. The aforementioned system may further include executable software installed on wirelessly paired computing devices. When executed, the software is configured to use the first and second currents received from the corresponding device to perform data processing to obtain the level of glucose contained in the liquid in which the first electrode of the sensor device is in contact, where the software requires the second current when processing to obtain the glucose level.

[0056] In the aforementioned system, the corresponding device may further include at least one processor, at least one memory, and software stored in at least one memory and executable by at least one processor. When executed, the software is configured to perform a method including: storing in at least one memory a first current and a second current received from the sensor device together or in association with each other; and processing the first current and the second current to provide a value indicating the oxidation of glucose in the glucose-oxide layer of the first electrode of the sensor device, where 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 the values ​​of the first current and the second current obtained simultaneously. The corresponding device may further include a display, and the method may further include displaying the value or corresponding information on the display. The compatible device may further include a wireless communication module configured to wirelessly pair with a device including a display, and the method may further include transmitting data to the wirelessly paired device in order to present its value or corresponding information on the display of the wirelessly paired device.

[0057] A further aspect of the invention provides a method for electrochemical sensing. The method includes: providing a sensor device comprising a first electrode having a glucose-oxidation layer capable of oxidizing glucose, a second electrode not having a glucose-oxidation layer, and a reference electrode; bringing the first electrode, the second electrode, and the reference electrode into contact with a liquid containing glucose, ascorbic acid, and acetaminophen; applying a first bias voltage between the first electrode and the reference electrode sufficient to oxidize glucose in the glucose-oxidation layer so that glucose and at least one of ascorbic acid and acetaminophen are oxidized in the glucose-oxidation layer; and further, generating a first current from the first electrode, wherein the first current is the glucose component induced by glucose oxidation. , further comprising a first interfering component caused by the oxidation of at least one of ascorbic acid and acetaminophen; a second bias voltage is applied between a second electrode and a reference electrode so that at least one of ascorbic acid and acetaminophen is oxidized at the second electrode, but glucose is not oxidized therein; further comprising causing a second current to be generated from the second electrode, the second current comprising a second interfering component caused by the 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, the first current being provided for processing and the second current also being provided in association with the first current.

[0058] In the method described above, the first and second currents may be generated simultaneously or successively within a reasonable period in which the glucose level does not change substantially or beyond a predetermined tolerance level. The first current may be provided with information indicating the generation time of the first current, and the second current may be provided with information indicating the generation time of the second current. The second current may be provided together with the first current whenever the first current is provided. In the method described above, the first bias voltage is applied at 0.2V to 0.32V, thereby 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 and not by the oxidation of acetaminophen; the second bias voltage is applied at 0.2V to 0.32V, thereby 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 and not by the oxidation of acetaminophen. In another case, a first bias voltage of 0.34V to 0.45V is applied, thereby oxidizing glucose, ascorbic acid, and acetaminophen in the glucose-oxidation layer, in which case the first interfering component is caused by the oxidation of ascorbic acid and acetaminophen; a second bias voltage of 0.34V to 0.45V is applied, thereby oxidizing ascorbic acid and acetaminophen in the second electrode, in which case the second interfering component is caused by the oxidation of both ascorbic acid and acetaminophen.

[0059] In the method described above, the sensor device may further include a maltose-blocking layer formed on a glucose-oxidation layer and containing polyphenylenediamine (poly-PD). The sensor device may also be a continuous glucose monitoring (CGM) electrode module including a subcutaneous layer configured to contact the subject's bodily fluids subcutaneously, wherein the first, second, and reference electrodes are formed subcutaneously, and contacting the first, second, and reference electrodes with the liquid may include subcutaneous insertion of the subcutaneous layer into the subject's body. 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 contains Na in the liquid. + , K + Ca 2+ Cl - , PO4 3- and CO3 2- This prevents Na from diffusing toward the nanoporous metal layer, thus preventing Na from diffusing between the area 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.

[0060] In the method described above, the sensor device is a blood glucose monitoring (BGM) electrode module including a reservoir, and contacting a first electrode, a second electrode, and a reference electrode with a liquid may include providing a blood sample into the reservoir. The glucose-oxidation layer may include many nanoparticles locally clustered together and disordered shapes including interparticle gaps formed between adjacent nanoparticles in the disordered shapes, the nanoparticles being generally oval or spherical with a diameter 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 disordered shapes may be interconnected, providing a three-dimensional interconnected network of disordered shapes. Disordered shape spaces may be formed between adjacent parts of disordered shapes, being nano-sized or micro-sized, and the disordered shape spaces may be interconnected, providing a three-dimensional interconnected network of disordered shape spaces.

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

[0062] A further aspect of the invention provides a method for providing or determining glucose levels. The method includes: providing software stored in at least one memory and executable by at least one processor provided in a sensor device or another device; using at least one processor, executing the software to process a first current and a second current and provide a value indicating the oxidation of glucose in the glucose-oxidation layer of the first electrode of the sensor device; and displaying the value or corresponding information on a display provided in the sensor device, another device or yet another device.

[0063] In the method described above, at least one memory and at least one processor are provided in the other device. The method may further include: transmitting a first current and a second current to the other device; and, before execution, storing the first current and the second current, which are received together or in association, in at least one memory. In the method described above, the first current is transmitted with a first timestamp, and the second current is transmitted with a second timestamp, and the first and second timestamps indicate the same time. In the method described above, 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 the values ​​of the first current and the second current obtained simultaneously. In the method described above, processing may include subtracting the second current from the first current.

[0064] 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 a glucose-specific enzyme is not present at the working electrode.

[0065] In the sensor device, the nanoporous metal layer includes irregularly shaped bodies containing many nanoparticles locally clustered together and interparticle gaps formed between adjacent ones of the nanoparticles in the irregularly shaped bodies. The nanoparticles are generally oval or spherical in shape, having a diameter 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 with each other to provide a three-dimensional interconnected network of the irregularly shaped bodies. Irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies, which are nanosized or microsized, and the irregularly shaped spaces are interconnected with each other to provide a three-dimensional interconnected network of the irregularly shaped spaces. In the sensor device, the bias voltage is set to be sufficient to cause oxidation of glucose in the nanoporous metal layer but not sufficient to cause oxidation of acetaminophen in the nanoporous metal layer, and the bias voltage is set within the range of about 0.20 V to about 0.32 V.

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

[0067] The aforementioned sensor device may further include a maltose-blocking layer, which comprises polyphenylenediamine (poly-PD) and is inserted between a nanoporous metal layer and an electrolyte ion-blocking layer. When brought into contact with a liquid containing maltose and glucose at concentrations of 4-20 mM (approximately 72-360 mg / dL) and a bias voltage is applied, the maltose-blocking layer is configured to allow glucose to pass through and substantially block maltose from passing through, so that in a steady state the glucose-oxidation current is 、1 0 nA / mMcm 2 Yo While it is at a high level, on the other hand, the maltose oxidation current caused solely by the oxidation of maltose. is 5 nA / mMcm 2 Yo It's low.

[0068] Another aspect of the invention provides a method for sensing glucose. The method includes: providing one of the aforementioned sensor devices; and applying a bias voltage between a working electrode (or glucose sensing electrode) and a reference electrode in the range of about 0.20 V to about 0.32 V. Here, the application of the bias voltage causes oxidation of glucose in the nanoporous metal layer, and thus a glucose-oxidation current is induced solely by glucose oxidation. is 1 0 nA / mMcm 2 Yo The level is high, while the application of a bias voltage does not cause sufficient oxidation of acetaminophen in the nanoporous metal layer, and therefore the acetaminophen oxidation current caused by acetaminophen oxidation in the nanoporous metal layer is 5 nA / mMcm 2 Yo It's low.

[0069] The patent or application file includes color drawings of the completed work. A copy of this patent or patent application publication containing the color drawings will be provided by the office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0070] [Figure 1] This shows a conceptual electrochemical glucose sensing system according to an embodiment of the invention. [Figure 2] This shows a working electrode for an enzyme glucose sensing system according to one embodiment. [Figure 3] One embodiment of a working electrode for a non-enzyme sensing system includes a nanoporous layer. [Figure 4] The top surface and depth of the nanoporous layer are shown. [Figure 5A] This shows the clustering morphology of a nanoporous layer according to one embodiment. [Figure 5B] This is a TEM image of a cluster according to one embodiment. [Figure 5C] This is a zoomed-in image of the TEM photograph shown in Figure 5B. [Figure 5D] This is an SEM image of a nanoporous layer taken from its upper surface according to one embodiment. [Figure 6A] This is a flowchart for manufacturing a clustered nanoporous layer according to one embodiment. [Figure 6B] This is a flowchart for manufacturing a clustered nanoporous layer according to another embodiment. [Figure 7] This is an example of a phase diagram for surfactants exhibiting different phases. [Figure 8] An inverse micelle phase and nanoparticle-surfactant colloid according to one embodiment are shown. [Figure 9] Includes a TEM image of a nanoparticle cluster according to one embodiment. [Figure 10A] This shows a non-clustered form of a nanoporous layer according to one embodiment. [Figure 10B]This is a TEM image of a non-clustered form of a nanoporous layer formed on a metal surface according to one embodiment. [Figure 11] This is a flowchart for manufacturing a non-clustered nanoporous layer according to one embodiment. [Figure 12] This is a flowchart for manufacturing a hexagonal nanostructure according to one embodiment. [Figure 13A] This shows the formation of a hexagonal arrangement according to one embodiment. [Figure 13B] This shows the deposition of metal using a hexagonal arrangement of liquid crystal phases. [Figure 14] The particle size distribution for a nanoparticle-surfactant colloid prepared according to one embodiment is shown. [Figure 15] The particle size distribution for a cluster colloid prepared according to one embodiment is shown. [Figure 16A] The images show cross-sections of the electrode base and the non-enzymatic glucose-sensing electrode according to the respective embodiments. [Figure 16B] The images show cross-sections of the electrode base and the non-enzymatic glucose-sensing electrode according to the respective embodiments. [Figure 17A] This is an SEM image of a glucose-sensing electrode according to an embodiment. [Figure 17B] This is an SEM image of a glucose-sensing electrode according to an embodiment. [Figure 17C] This is an SEM image of a glucose-sensing electrode according to an embodiment. [Figure 18] This is a profile of the current generated by the oxidation of glucose and other materials in PBS according to the embodiment. [Figure 19] This is a profile of the current generated by the oxidation of glucose and other materials in human serum according to the embodiment. [Figure 20] This is the structural formula of a maltose molecule. [Figure 21] A non-enzymatic electrode containing a maltose-blocking layer according to one embodiment is shown. [Figure 22]This shows the scanning of the oxidation voltage during cyclic voltammetry electrochemical polymerization of phenylenediamine according to the embodiment. [Figure 23] This describes a chronoamperometry setup for an electric shock treatment to adjust the porosity of a porous polymer layer according to one embodiment. [Figure 24] This is a flowchart for manufacturing a maltose-blocking layer according to one embodiment. [Figure 25] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 26] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 27] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 28] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 29] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 30] The current monitored using a glucose-sensing electrode having a maltose-blocking layer according to the embodiment is shown, and in this case, the current signal is not easily discernible in black and white, so it is presented in color. [Figure 31] This shows a CGM working electrode according to one embodiment. [Figure 32]This demonstrates the decrease in electrolyte concentration across the thickness of the electrolyte ion-blocking layer according to one embodiment. [Figure 33] A CGM electrode unit according to one embodiment is shown. [Figure 34] This is a flowchart for fabricating a CGM electrode unit according to one embodiment. [Figure 35] Figure 33 shows top and cross-sectional views of intermediate products at various stages in the fabrication of the CGM electrode, where each cross-section is taken along line 3501 and viewed in the direction of the arrow. [Figure 36] Figure 33 shows top and cross-sectional views of intermediate products at various stages in the fabrication of the CGM electrode, where each cross-section is taken along line 3501 and viewed in the direction of the arrow. [Figure 37] Figure 33 shows top and cross-sectional views of intermediate products at various stages in the fabrication of the CGM electrode, where each cross-section is taken along line 3501 and viewed in the direction of the arrow. [Figure 38A] The images show cross-sections of the intermediate product and the functional layer of the CGM working electrode after the formation of the nanoporous layer according to the embodiment. [Figure 38B] The images show cross-sections of the intermediate product and the functional layer of the CGM working electrode after the formation of the nanoporous layer according to the embodiment. [Figure 39] A disposable glucose-sensing cartridge according to an embodiment is shown. [Figure 40] A two-electrode glucose sensing system according to one embodiment is shown. [Figure 41] A CGM electrode unit for a two-electrode glucose sensing system according to one embodiment is shown. [Figure 42A] This is a profile of the current generated by the oxidation of glucose according to one embodiment, in which case the working electrode does not include an electrolyte ion-blocking layer. [Figure 42B] Figure 42B is a magnified view of a portion of the profile shown in Figure 42A. [Figure 43]This is a profile of the current generated by the oxidation of glucose according to one embodiment, in which case the working electrode includes an electrolyte ion-blocking layer. [Figure 44] This is a comparison of the time required to condition working electrodes with and without an electrolyte ion-blocking layer. [Figure 45A] This is a photograph of a potentiostat according to one embodiment. [Figure 45B] This is a photograph of a potentiostat according to one embodiment. [Figure 45C] This is a photograph of a potentiostat according to one embodiment. [Figure 46] This graph shows CGM monitoring of glucose levels in rats using a non-enzymatic CGM electrode module according to one embodiment. [Figure 47] This is a Clark error grid for a non-enzymatic CGM electrode module according to one embodiment. [Modes for carrying out the invention]

[0071] The subject matter disclosed herein will be described and explained in more detail below with reference to the accompanying drawings, in terms of several specific embodiments and examples. Some, but not all, embodiments of the invention are shown here. Throughout, similar figures indicate similar elements or parts. The subject matter disclosed herein can be embodied in many different forms, but should not be construed as being limited to the specific embodiments expressed herein. Rather, these embodiments are provided to satisfy the applicable legal requirements of this disclosure. Indeed, many modifications and other embodiments of the subject matter disclosed herein will be conceivable to a person skilled in the art to which the subject matter relates. Therefore, it should be understood that the subject matter disclosed herein is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the accompanying claims.

[0072] Electrochemical glucose sensing system Electrochemical glucose detection Electrochemical glucose sensing measures the glucose concentration in an electrolyte solution. Figure 1 conceptually shows an electrochemical glucose sensing system 101 for detecting the glucose concentration 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 in contact with the test fluid 102. In embodiments, the potentiostat includes an electrical network for functioning as a voltage source 109 and a current sensor 108. The voltage source 109 provides a bias voltage to drive redox reactions at the working electrode 103 and the counter electrode 105. The potentiostat further includes an electrical network 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 redox reactions involving glucose contained in the test fluid 102.

[0073] Enzyme glucose sensing electrode Most, though not all, electrochemical glucose sensing systems utilize glucose-specific enzymes for the detection of glucose molecules. Figure 2 shows a working electrode 103E for an enzyme glucose sensing system, i.e., an enzyme glucose sensing electrode. The terms “glucose sensing electrode” and “working electrode” are used interchangeably in this disclosure. The enzyme working electrode 103E comprises a conductive layer 110 and an enzyme layer 111. Optionally, the enzyme working electrode 103E may include at least one functional layer 112 on the enzyme layer 111, as shown in Figure 2. Alternatively, although not shown, at least one functional layer may be positioned between the enzyme layer 111 and the conductive layer 110. The enzyme layer 111 contains a glucose-specific enzyme molecule 115, which is maintained therein by a fixture 113. When a glucose molecule comes into contact with the glucose-specific enzyme, the enzyme catalyzes the oxidation of glucose to gluconolactone. Electrons derived from glucose oxidation are eventually transferred to the conductive layer 110, generating an electric current in the electrical circuit of the electrochemical sensing system 101.

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

[0075] Glucose dehydrogenase In other enzyme glucose sensing systems, the enzyme-acting electrode 103E contains 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, this enzyme oxidizes maltose and glucose, which hinders accurate sensing of glucose concentration.

[0076] Non-enzymatic glucose sensing electrode Non-enzymatic electrochemical glucose sensing systems do not use glucose-specific enzymes or any enzymes at all to detect glucose. Instead, non-enzymatic glucose sensing systems have a non-enzymatic working electrode that detects glucose without glucose-specific enzymes. In embodiments, the non-enzymatic working electrode includes at least one glucose oxidation layer that allows oxidation of glucose molecules at a medium bias voltage. Generally, the higher the bias voltage, the more likely glucose oxidation is to occur in at least one glucose oxidation layer. However, there are limitations to the bias voltage, as high bias voltages also oxidize other chemical components. Therefore, non-enzymatic electrochemical glucose sensing relies on a material that oxidizes glucose at a bias voltage that does not cause oxidation of other chemical components in the test fluid.

[0077] Nanoporous layer for non-enzymatic glucose sensing electrodes Figure 3 shows a non-enzymatic working electrode (simply put, “working electrode”) 103NE comprising a conductive layer 110 and a nanoporous glucose oxidation layer (or nanoporous layer) 117. In embodiments, the nanoporous layer 117 includes a nanoporous internal structure to induce, enable, or promote glucose oxidation at a medium bias voltage. When glucose oxidation occurs, the conductive layer 110 extracts electrons from the glucose oxidation, generating a current in the electrical circuit. The current can be detected by a current sensor 108 and interpreted by the system’s 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 Using a bias voltage, the conductive layer 110 in Figures 2 and 3 extracts electrons from the glucose oxide layer and transfers them to the current sensor 108. In embodiments, the conductive layer 110 comprises or is fabricated from at least one conductive material and is connected to the electrical circuit of system 101. In some embodiments, assuming a small conductive layer 110, a semiconducting material may be used instead of a conductive material. Non-limiting examples of materials for the conductive layer include platinum (Pt), gold (Au), silver (Ag), ruthenium (Ru), stainless steel, silicon (amorphous, polycrystalline, and single crystal), conductive carbon materials such as graphite, graphene, fluorene, and carbon nanotubes. In embodiments, the conductive layer 110 does not contain the nanoporous internal structure of the glucose oxide layer 117.

[0079] Conductive layer-structure In some embodiments, the conductive layer 110 may be formed from a single layer of homogeneous material. In other embodiments, 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 beneath the top sublayer. In some embodiments, the top sublayer does not include silver, copper, aluminum, or other conductive materials that are more prone to oxidation than silver, copper, or aluminum. The top sublayer may have lower conductivity than the other sublayers. In some embodiments, the conductive layer 110 includes a conductive carbon layer as the top sublayer and a silver layer as another sublayer beneath the carbon layer. The conductive layer 110 has a thickness that can vary considerably depending on the particular example. In some embodiments, the conductive layer 110 may be omitted, and the nanoporous layer is connected directly to the current sensor via a conductive wire or connection.

[0080] Counter electrode Using a bias voltage, reduction of chemical components occurs at the counter electrode 105. In embodiments, the counter electrode 105 comprises at least one conductive or semiconducting material and is connected to the electrical circuit of system 101. In embodiments, the counter electrode 105 may be formed from a single layer of homogeneous material or multiple layers manufactured from different materials. Conductive or semiconducting material for the conductive layer 110 may also be used in the counter electrode 105, although in certain systems, different materials may be 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 the reduction at the counter electrode 105 is not at the same rate as the oxidation at the sensing electrode 103. In some embodiments, the counter electrode 105 can be omitted, and the reference electrode 106 can perform the dual function of both the counter electrode and the reference electrode. In embodiments, the reference electrode 106 may be formed from a single layer of homogeneous material or multiple layers manufactured from different materials. Conductive or semiconducting material for the conductive layer 110 may also be used in the reference electrode 105, although in certain systems, different materials may be used in the conductive layer 110 and the reference electrode 106. In some embodiments, the reference electrode 106 may include a salt layer on top of the conductive or semiconducting material layer. For example, the salt layer may be manufactured from or contain silver chloride (AgCl).

[0082] Current sensor The current sensor 108 measures the current flowing from the working electrode 103. The current sensor 108 can detect the current flowing at a specific time point by amperometry. Alternatively, the current sensor 108 may be an electrometer.

[0083] Test fluid In some embodiments, the test fluid is a human or animal biofluid, but is not limited thereto. In some embodiments, the test fluid is a liquid mixture comprising the biofluid and at least one additional substance added to the biofluid. The biofluid includes, but is not limited to, blood, interstitial fluid, cerebrospinal fluid, lymph, or urine. In some embodiments, the test fluid includes 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 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 0.46V, or approximately those values. In the embodiment, the applied bias voltage may be within a range formed by selecting any two numbers (two voltage values) listed in the preceding sentence, for example, approximately 0.20V to approximately 0.30V, approximately 0.30V to approximately 0.40V, approximately 0.28V to approximately 0.40V, approximately 0.30V to approximately 0.38V, approximately 0.28V to approximately 0.36V, etc.

[0085] Nanoporous layer The nanoporous layer 117 for the working electrode 103NE includes nanoscale internal structures, such as cavities, spaces, and openings (collectively, "nanopores" or "nano-pores"). In embodiments, the nanopores of the nanoporous layer 117 enable or promote glucose oxidation, and the glucose concentration can be measured based on the current induced by glucose oxidation. No aspect of the invention is bound by any theory or belief, but it is thought that glucose oxidation occurs when glucose molecules enter the nanopores and come into contact with the inner surface more often and for longer periods within the nanoporous layer 117 than on the non-porous surface of the electrode.

[0086] No enzymes, and no electron mediators. By incorporating the nanoporous layer 117, the working electrode 103NE can avoid requiring a more complex fabrication process and providing a less stable glucose-specific enzyme than a solid material of the nanoporous layer 117. Furthermore, the enzyme-sensing electrode 103NE can operate without an electron mediator that facilitates electron transfer between different materials. In this embodiment, the working electrode 103NE contains neither an enzyme nor an electron mediator.

[0087] Materials for nanoporous layers In some embodiments, the nanoporous layer 117 is manufactured from, or includes, 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 aforementioned elements. In other embodiments, the nanoporous layer 117 is manufactured from, or includes, alloy materials of two or more metallic elements listed in the preceding sentence, including Pt-Ir, Pt-Ru, and Pt-Pd.

[0088] The specified roughness coefficient The roughness coefficient, or surface roughness, is the ratio of the actual surface area to the geometric surface area of ​​an object. Here, the geometric surface area represents the projected area of ​​the object projected onto a plane without considering the internal surface within the object. The actual surface area represents the total surface area considering the internal surface. Referring to Figure 4, for example, if the nanoporous layer 117 is a rectangular block having a height or depth 118 and a top rectangle 119, the projected area or geometric surface area of ​​the nanoporous layer is the area of ​​the top rectangle exposed to the outside. The actual surface area of ​​the nanoporous layer can be measured electrochemically, for example, using the well-known cyclic voltammetry technique, which detects current from proton adsorption on the actual surface.

[0089] Roughness coefficient of nanoporous layer The roughness coefficient value indicates the total amount of internal pores within the nanoporous layer 117. The roughness coefficient of the nanoporous layer 117 may be related to the sensitivity of the nanoporous layer 117 to glucose oxidation. Generally, the higher the roughness coefficient, the more glucose oxidation is likely to occur. The roughness coefficients of the nanoporous layer 117 are 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 or approximately those values. In the embodiment, the roughness coefficient may be within a range formed by selecting any two numbers (two roughness coefficient values) listed in the preceding sentence, for example, about 100 to about 2500, about 750 to about 1250, or about 850 to about 1150.

[0090] Thickness of the nanoporous layer The roughness coefficient value does not indicate the level of porosity or density of the nanoporous material per unit volume, but its value may indicate the total amount of internal pores. Therefore, depending on the level of porosity of the nanoporous material, in embodiments, the thickness of the nanoporous layer can be adjusted to achieve a target value for the roughness coefficient. 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 preceding sentence, for example, 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 different internal morphologies in each particular manufacturing. In some embodiments, the nanoporous layer 117 may contain, or be manufactured from, nanoparticles that are deposited together and form nanopores (interparticle nanopores) between themselves. In other embodiments, the nanoporous layer 117 may contain, or be manufactured from, clusters of deposited nanoparticles that form interparticle nanopores within the cluster, and also spaces between clusters (intercluster gaps or spaces). In other embodiments, the nanoporous layer 117 may contain, or be manufactured from, repeats of nanostructures of a particular shape, such as a hexagonal structure containing nanopores. Furthermore, in each particular manufacturing, the nanoporous layer 117 may have different levels of porosity and different roughness coefficient values ​​per unit volume.

[0092] Manufacturing of nanoporous layers The nanoporous layer 117 can be prepared using a liquid composition containing metal ions and a surfactant. In embodiments, different forms of the nanoporous layer can be formed using different phases of the surfactant. A micelle phase, inverse micelle phase, liquid crystal phase, or another phase of the surfactant can be used to generate a particular form of the nanoporous layer. In these different phases, the metal ions are aligned adjacent to or locally concentrated in the hydrophilic portion of the surfactant. The localized metal ions in the liquid composition are subjected to additional processes for reduction and deposition on the surface to provide the nanoporous layer 117 having different forms.

[0093] Clustered nanoporous layer clustering morphology Figure 5A shows a vertical cross-section of a nanoporous layer having 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 clustered morphology 120, many nanoparticles 121 come together to form irregularly shaped clusters 125. Different shading or shading is used in different clusters 125 for illustrative purposes. These irregularly shaped clusters 125 are irregularly stacked to form a nanoporous layer. Figure 5B is a transmission electron microscope (TEM) image of several clusters 125 before they are deposited to form a nanoporous layer. Figure 5C is a zoomed-in image of the circled area in Figure 5B. Figure 5D is a scanning electron microscope (SEM) image of a nanoporous layer having clustered morphology taken from the top surface of the nanoporous layer.

[0094] Clustered morphology of pores and spaces Using an irregular stack of irregularly shaped clusters 125, adjacent clusters form intercluster gaps or spaces 127 between them. These intercluster gaps 127 may be nano-sized or micro-sized. In this disclosure, nano-sized means greater than 1 nm and less than 100 nm, and micro-sized means greater than 100 nm and less than 100 μm. Each cluster 125 contains or is fabricated from approximately spherical or oval nanoparticles 121. In each cluster, the individual nanoparticles are generally spaced apart from each other, forming small gaps 123 between them. These small gaps are nano-sized and are called 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] Formation of inter-cluster gaps / spaces In this embodiment, irregularly shaped clusters 125 are first prepared as a liquid suspension to generate a clustered morphology. The suspension is then distributed onto a substrate 129, which is subjected to drying. As the liquid dries, the clusters naturally deposit on the substrate and on top of other clusters. No external forces can be applied to the clusters during drying. Therefore, the clusters do not clog as they deposit. As the clusters deposit and stack on top of other clusters, each cluster may come into contact with the substrate surface or adjacent clusters. After drying is complete, the clusters touch or come into contact with adjacent or neighboring clusters. The deposited clusters are linked or merged with each other through junctions or contacts. Due to the irregular shape of the individual clusters, irregularly shaped gaps and spaces are formed between adjacent clusters, in which case the gaps and spaces define the irregular shape of the deposited clusters, as if the surface and outline of the deposited clusters were surrounded by 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 inter-cluster gaps In this embodiment, irregularly shaped clusters 125 are distributed throughout the clustered form 120 of the nanoporous layer 117. The irregularly shaped clusters 125 are interconnected via junctions, meaning that these clusters are in contact with themselves and form a three-dimensional network of clusters over substantially the entire nanoporous layer 117. Intercluster gaps 127 define and surround the surface of the irregularly shaped clusters, interconnecting them with themselves and forming a three-dimensional interconnected or networked channel throughout the nanoporous layer 117. The intercluster gaps and spaces 127 are well distributed throughout the nanoporous layer 117 from the top surface to the bottom surface (on or directly above the substrate 129) (not shown). The three-dimensional network of irregularly shaped clusters 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 clusters and channels may be analogous to the three-dimensional internal geometry of a sponge, except that the interparticle gaps and spaces are networked together throughout the entire nanoporous layer 117.

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

[0098] Intercluster gaps / spaces for glucose diffusion In the embodiment, the interconnection of the inter-cluster 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 primarily occurs within nano-sized inter-particle nanopores rather than within micro-sized spaces. As the inter-cluster gaps 127 are networked or interconnected throughout the entire nanoporous layer 117, they become large in scale considering the size of glucose molecules. cluster Through the inter-cluster spaces, glucose molecules can reach almost anywhere within the nanoporous layer 117. Furthermore, since the inter-cluster gaps 127 are well interconnected with the inter-particle nanopores 123, some inter-particle nanopores 123 within the nanoporous layer 117 may be exposed and open for glucose oxidation. Therefore, the three-dimensional interconnected or networked channels of the inter-cluster gaps can provide more glucose oxidation, i.e., a stronger signal (higher current) of glucose oxidation, than a nanoporous layer without such interconnected channels formed in the inter-cluster gaps.

[0099] Two types of particles and two types of pores As described, the clustered form 120 comprises two different types of particles that define two different types of pores. From the perspective of particles, one is nanoparticles 121, and the other is clusters 125 made from nanoparticles 121. From the perspective of pores, one is interparticle nanopores 123 between nanoparticles 121 within clusters 125, and the other is intercluster gaps 127 between clusters 125.

[0100] Nanoparticle clusters The TEM image in Figure 5B shows irregularly shaped clusters. The number of nanoparticles 121 within each cluster can vary considerably, and the size of the clusters 125 can vary accordingly. In the clustered morphology, some clusters 125 are nano-sized (smaller than 100 nm), while others are micro-sized (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 cluster 125 may be within a range formed by selecting any two numbers (two length or diameter values) listed in the preceding sentence, for example, about 20 nm to about 300 nm, or about 60 nm to about 240 nm. Cluster 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 cluster 125 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, 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 separated and generally spherical (ball-like) or oval (egg-like) in shape, but are not limited to these. The nanoparticles 121 have diameters 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 preceding sentence, for example, 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. In the embodiment, the average diameter of the nanoparticles 121 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, 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 the embodiment, 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 a cluster. The interparticle nanopores are networked and interconnected within the cluster. The interparticle gap or nanopore 123 has an interparticle gap distance between two directly adjacent nanoparticles within the same cluster. The interparticle gap distances are 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 preceding sentence, for example, approximately 0.5 nm to approximately 4.5 nm, or approximately 1.5 nm to approximately 4.0 nm. The interparticle nanopore 123 may have an average interparticle gap distance of approximately 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 nanopore 123 is within a range formed by selecting any two numbers listed in the preceding sentence, for example, approximately 0.75 nm to approximately 1.5 nm, or approximately 1.0 nm to approximately 2.5 nm. m It may be present. In the embodiment, interparticle nanopores 123 having an average interparticle gap distance of 1-2.5 nm are found throughout the nanoporous layer 117.

[0103] Inter-cluster gap / space The SEM image in Figure 5D shows the opening of networked inter-cluster gaps visible from the top surface of the nanoporous layer. Although the three-dimensional shape is not well shown in the two-dimensional image in Figure 5D, the top surface of the nanoporous layer contains valleys and hills formed by stacked clusters. Inside the nanoporous layer, the valleys and hills form inter-cluster gaps. The inter-cluster gaps or spaces have an irregular shape. The inter-cluster gaps 127 are nano- to micro-sized. The inter-cluster gaps 127 have inter-cluster gap distances 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 an embodiment, the inter-cluster gap distance may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, about 100 nm to about 1000 nm. The inter-cluster gap 127 has an average inter-cluster gap distance of about 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm. In an embodiment, the average inter-cluster gap distance may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, about 150 nm to about 400 nm.

[0104] The entire process for manufacturing clustered nanoporous layers In embodiments, nanoporous layers having a clustered morphology can be prepared using an isotropic inverse micelle phase (or “inverse micelle phase”) of a surfactant. Referring to Figure 6A, in step 601, an aqueous liquid composition is prepared using a metal ion source and a surfactant in the inverse micelle phase. The metal ions are locally concentrated within the hydrophilic spaces of individual inverse micelles. Then, in step 603, a reducing agent is added to the inverse micelle phase to form metal nanoparticles dispersed in the surfactant-containing liquid composition (“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 (“cluster colloid” or “cluster-liquid colloid”) are collected. Optionally, in step 607, the collected cluster colloid is mixed with a non-surfactant liquid. In step 609, the cluster colloid is distributed onto a surface, for example, by printing technology, without the use of electroplating. Subsequently, in step 611, the liquid is dried, and a nanoporous layer 117 is formed on the surface 129.

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

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

[0107] Examples of surfactants The surfactant is selected from those capable of forming an isotropic inverse micelle phase under reasonable conditions for processing. In some embodiments, nonionic surfactants are used, but are not limited thereto. Non-limiting examples of surfactants include alkylbenzene sulfonates, alkyl polyglycosides, alkyl sulfates, carboxylates, carboxylic acid esters, cetomacrogol 1000™, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, disodium cocoamphodiacetate, ethoxylated aliphatic alcohols, glycerol monostearate, glycol esters of fatty acids, and IGEPAL. CA-630™, Isoceteth-20, Lauryl Glucoside, Maltoside, Monolaurin, Mycosbutyrin, 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, Polodocanol Examples include Xamer, Poloxamer 407, polyethoxylated tallow amines, polyethylene glycol esters, polyglycerol polyricinolates, polyoxyethylene fatty acid amides, polyoxyethylene surfactants, polysorbates, polysorbate 20, polysorbate 80, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, sulfated alkanolamides, sulfonates, Triton X-100 (trademark), and Tween 80 (trademark). Those skilled in the art will recognize what constitutes reasonable conditions.

[0108] Conditions for the reverse micelle phase After selecting a surfactant, its concentration and temperature are adjusted to form an isotropic inverse micelle phase. The concentration and temperature of the surfactant may be determined in relation to the surfactant's phase diagram. If a phase diagram is unavailable, several experiments 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, concentrations of 10–60 wt% and temperatures of 40–80°C may provide an inverse 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 compounds containing ionic metals, such as acids, bases, or salts. Non-limiting examples of metal source compounds include H2PtCl6, H2Pt(OH)6, H2PtCl2(OH)4, H2Pt(SO4)(OH)4, PtCl4, K2PtCl6, PdCl2, and TiCl4.

[0110] metal ion concentration The concentration of metal ions is also adjusted for optimal 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 concentrations of metal ions are approximately 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 preceding sentence, for example, about 0.01 to about 0.03 M, about 0.02 to 0.03 M, etc. Within a suitable concentration range, it has been observed that the concentration level affects the rate of nanoparticle formation.

[0111] Different from a plating bath The inverse micelle phase prepared in step 601 is different from the plating bath composition for electroplating. Unlike the plating bath, a metal chelating agent may not be required.

[0112] Formation of nanoparticles In step 603, a reducing agent is mixed into the aqueous liquid composition of the inverse micelle phase. When the reducing agent enters the hydrophilic core 143 of the inverse micelle 141, it reduces the metal ions to metal atoms within the hydrophilic core 143. Since 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 solidify together and grow to form metal nanoparticles. One metal nanoparticle can grow from one inverse micelle, but is not limited to that. 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 reduction of metal ions and the growth of nanoparticles, some inverse micelles rupture or disintegrate, and thus nanoparticles from these disintegrated inverse micelles can be dispersed in the hydrophobic space. Some of these nanoparticles 151 can float freely outside the hydrophilic core of the inverse micelles in the resulting colloidal composition. Some other nanoparticles 153 may be surrounded or bound by the hydrophilic heads of surfactant molecules outside the hydrophilic core of the inverse micelles. Some nanoparticles 155 remain inside the inverse micelles 141. Overall, in the resulting nanoparticle colloid, the solid nanoparticles 151, 153, and 155 are dispersed in a liquid composition containing inverse micelles 141, water, and surfactant molecules. Since the nanoparticles 151, 153, and 155 are significantly separated from each other in the nanoparticle colloidal composition, it is unlikely that the nanoparticles will aggregate and grow into larger particles.

[0114] Reducing agent A reducing agent is a chemical component that can donate one or more electrons to metal ions contained within a nanoparticle colloid. The reducing agent is a hydrophilic compound in order to enter the hydrophilic core of the inverse micelle. Non-exclusive examples of hydrophilic reducing agents include ascorbic acid, acetic acid, formaldehyde, citric acid, hydroxylamine, and hypophosphate.

[0115] Amount of reducing agent A 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 excess amount greater than the stoichiometric amount required to reduce the total metal ions contained in the nanoparticle colloid. Here, "substantially greater" means only 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, or 400% greater.

[0116] stirring During and / or after the addition of the reducing agent, the mixture may be stirred to promote the distribution of the reducing agent. Stirring can facilitate the entry of the reducing agent into the hydrophilic space of the reverse micelles. Thus, the time required for complete reduction of metal ions within the hydrophilic space may be reduced. Stirring may be carried out continuously or intermittently. In embodiments, stirring is carried out for a period of 1 to 10 hours.

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

[0118] Chemical bond between surfactant and nanoparticles Due to surfactants, some nanoparticles have strong chemical bonds with the hydrophilic heads of some surfactant molecules. Surfactant molecules with negatively charged hydrophilic heads can form coordination bonds with the nanoparticle surface. Also, if surfactant molecules have electron-rich hydrophilic heads (even if they are uncharged), they can form coordination 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 of chemical bonds In some embodiments, an acidic or basic solution is used as shown in Figure 6B. After nanoparticle formation in step 603, In process 604 of Before centrifugation, the nanoparticles are added to the surfactant colloid. The acid or base of the added solution triggers a chemical reaction that breaks the coordination bonds between the surfactant and the nanoparticles, freeing the nanoparticles. For example, protons from the acid can bind to the negatively charged or electron-rich surfactant heads, freeing the nanoparticles. Subsequent centrifugation and bottom collection separate the nanoparticles that have been freed from the surfactant molecules. In embodiments, the addition of an acidic or basic solution may be carried out at least once before centrifugation. In some embodiments, the addition of an acidic or basic solution may be carried out before each centrifugation. In embodiments, the acid and base may be washed with water or other solvent after centrifugation.

[0120] Acidic or basic solution In the embodiments, the acid or base is selected with respect to the surfactant, so that the surfactant molecules are effectively detached from the nanoparticles. In the 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 the acidic solution include HCl, HNO3, H2SO4, HClO4, etc. In the 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 the basic solution include NaOH, KOH, Ca(OH)2, etc.

[0121] Cluster colloid After or during the process of removing surfactants and collecting nanoparticles, the nanoparticles tend to cluster together or aggregate to form clusters of nanoparticles. In liquid, the clusters are dispersed, forming cluster colloids. Each cluster contains metal nanoparticles that interact with each other, and from these, a larger body is formed. Individual nanoparticles within a cluster are most likely to be electrically neutral. While the invention is not bound by any theory or belief, it is thought that protons, hydroxides, and other charged electrolytes can bind to the surface of nanoparticles, and that the ionic interactions of these electrolytes with adjacent nanoparticles can keep adjacent nanoparticles together and form clusters. In fact, although the surfactant molecules are substantially removed, the liquid of the cluster colloid contains a sufficient amount of electrolytes derived from the metal ion source and acidic or basic solution used in the previous preparation step.

[0122] Clusters and nanoparticles Figure 9 provides TEM images of nanoparticle clusters from a diluted sample of the cluster colloid. Two of the images in Figure 9 are also seen in Figures 5B and 5C. In these images, the clusters do not have an orderly shape and are approximately 30–500 nm in length. The nanoparticles 121 within the clusters are separated, generally spherical or oval, and have a diameter of approximately 2–3 nm. Between adjacent or neighboring nanoparticles 121, there are interparticle gaps 125 with a gap distance of approximately 1–2 nm. These interparticle nanopores 125 are primarily involved in glucose oxidation in glucose-sensing electrodes having a clustered nanoporous layer.

[0123] Centrifugal separation Centrifugation may be performed at a rotational speed of 3000–5000 rpm. Centrifugation may continue for a period of 3–15 minutes. After centrifugation, the supernatant is removed and the bottom containing 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 surfactants By using multiple centrifugation treatments, the surfactant is substantially removed. The resulting cluster colloid will have a significantly lower concentration of surfactant, although it cannot be completely removed. Initially, the reverse micelle phase contains approximately 10 to 60 wt of surfactant. The resulting cluster colloid can be surfactant-free. In fact, the resulting cluster colloid is substantially surfactant-free. The resulting cluster colloid or the remaining surfactant in the final bottom collection may be greater than 0.0001 parts by weight per 100 parts by weight of nanoparticles, and less than approximately 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 the embodiment, the remaining surfactant may be in an amount 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 centrifugation treatments, the total amount of nanoparticles in the final collection at the bottom (as part of the clusters and as free nanoparticles) may 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 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, about 20 to about 30 wt%, about 15 to 25%, etc.

[0126] Storage of cluster colloids The clusters are dispersed in the cluster colloid without treatment for a long period, for example, one week or longer than one month. The cluster colloid may be stored in a container for a while after preparation and before subsequent processing. Immediately after preparation, the cluster colloid may be made available for sale and transport for processing by others or at other locations. To maintain colloidal properties over a long period, the concentration of nanoparticles may be adjusted after the final collection at the bottom. In embodiments, the cluster colloid of the final collection at the bottom may be stored or transported in a container with or without adjustment of concentration.

[0127] Adjustment of concentration for distribution In step 607, the collected cluster colloid may be stored for a period of time, either diluted with a solvent or without dilution. Dilution may be to adjust the concentration of clusters in the cluster colloid for subsequent processing, such as distribution. The solvent may be water or an organic compound. One or more additive compounds may be added. Through dilution, the concentration of nanoparticles or clusters can be 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 a range formed by selecting any two numbers listed in the preceding sentence, for example, 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] Distribution of cluster colloids In step 609, the cluster colloid is distributed onto the substrate 129 to form a nanoporous layer, while maintaining its colloidal properties. Various distribution techniques can be used to distribute the cluster colloid. Distribution can be controlled to form a certain thickness of the distributed cluster colloid or to provide an appropriate thickness for the resulting nanoporous layer after subsequent drying. Alternatively, distribution can be controlled to provide an appropriate roughness coefficient value for the resulting nanoporous layer.

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

[0130] Drying of liquid to form clustered nanoporous layers In step 611, the distributed cluster colloids are subjected to conditions for drying the liquid. Once distributed, the nanoparticle clusters float in the liquid and move freely horizontally and vertically. As the liquid dries, the height of the cluster colloids decreases. As the liquid continues to dry, the clusters come into contact with adjacent clusters vertically and horizontally between the lower substrate 129 and the upper surface of the cluster colloids. The mobility of the clusters becomes significantly restricted. After some time, the liquid level becomes lower than the clusters located on or near the upper surface. As soon as drying is complete, the nanoparticle clusters deposit on the substrate 129, forming a nanoporous layer having clustered morphology 120, as shown in Figure 5A.

[0131] Thickness of the nanoporous layer The resulting nanoporous layer has a thickness of approximately 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 preceding sentence, for example, approximately 1 μm to approximately 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 in the case of subsequent processing to add layers on 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 within it are reduced, forming metal atoms, which then coagulate and form nanoparticles. Subsequent treatment to remove the surfactant also collects most of the nanoparticles within the clusters. Thus, most of the metal ions added to the aforementioned process are ultimately collected and deposited in the form of nanoparticle clusters, resulting in the nanoporous layer 117. In this embodiment, the input metal ions 89, 90, 91, 92, 93, 94, 95, 96, 9 7 More than 98% of the material is collected before distribution in the form of nanoparticle clusters.

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

[0135] No electroplating or no application of electricity Throughout the process, electroplating is not used to form clustered morphologies for the nanoporous layer. 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. Similar to 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 image of the non-clustered morphology of the nanoporous layer formed on a metal surface, where the dark areas are parts of the metal surface. The nanoparticles and interparticle pores in the TEM image are similar to those illustrated in Figure 10A.

[0137] No clusters and no gaps between clusters Unlike the clustered form 120, the non-clustered form 161 does not contain clusters 123 or inter-cluster gaps 127. To generate the non-clustered form, nanoparticles are deposited on the substrate 129 by electroplating without preparing clusters before electroplating. As a result, the resulting structure, i.e., the non-clustered form 161, does not form clusters or inter-cluster gaps. Therefore, the non-clustered form 161 does not possess the characteristics of the clustered form derived from clusters 123 or inter-cluster gaps 127.

[0138] Non-clustered cavities Although there are no intercluster gaps, the non-clustered form 161 may contain internal cavities 133 that are significantly larger than the interparticle nanopores 123. These internal cavities 133 may be formed during the electroplating process because the nanoparticles are not necessarily stacked sequentially on the surface immediately below them. The internal cavities 133 are irregular in shape and size. They may be found throughout the entire nanoporous layer 117.

[0139] Cavities distinct from inter-cluster gaps or spaces. The non-clustered voids 133 are distinguished from the inter-cluster gaps 127 in the clustered void 120. The voids 133 are formed because the electroplating and nanoparticle deposition do not occur at the same rate on the surface of the substrate 129. The voids 133 do not surround or define one or more clusters 125 of nanoparticles 121. Rather, each void 133 is surrounded or defined by aggregates or aggregates of nanoparticles 121. The voids 133 may be interconnected by interparticle nanopores 123, but the voids 133 themselves are not interconnected across the entire nanoporous layer 117 or any substantial portion thereof. Furthermore, the voids 133 do not occupy the same volume of nanoporous layer 117 (lower roughness coefficient in the non-clustered void) as the inter-cluster gaps 127 (higher roughness coefficient in the clustered void).

[0140] Substrate substantially coated with nanoparticles Referring to Figures 10A and 10B, the upper surface of the substrate 129 is substantially coated with nanoparticles 121. In some embodiments, but not limited to, substantial internal space is not formed on or directly above the substrate 129.

[0141] Comparison of clustered and non-clustered configurations Overall, the clustered form 120 has a much lower density than the unclustered form 161. At the same thickness, the clustered form 120 has a higher roughness coefficient than the unclustered form 161, and therefore, to produce the same roughness coefficient, the clustered form 120 can be made thinner than the unclustered form. Also, assuming an irregular shape of clusters, the intercluster gaps 127 in the clustered form 120 are interconnected throughout almost the entire nanoporous layer 117, whereas the internal cavities 133 in the unclustered form 161 are not interconnected in the same way as the intercluster gaps 127. Therefore, the interparticle nanopores 125 within the clusters 123 are connected to the network of intercluster gaps 127 in the clustered form 120, but there are no intercluster gaps in the unclustered form 161, and the interparticle nanopores 125 cannot be connected as in the clustered form 120.

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

[0143] Preparation of the plating bath In step 1101, the plating bath is similar to the inverse micelle phase of step 601 in Figure 6A for producing a clustered nanoporous layer without electroplating. The plating bath, like the production of the clustered nanoporous layer, contains surfactants and metal ion source materials for the inverse micelle phase. All descriptions of the surfactants and metal ion source materials for step 601 in Figure 6A are applicable to step 1101 in Figure 11. However, the plating bath in step 1101 is not identical to the inverse micelle phase of step 601. One important difference is 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 spontaneously reduced, the plating bath may require chelating agents to prevent the metal ions from spontaneously reducing during and before electroplating. In contrast, such chelating agents may not be required in the inverse micelle phase of step 601.

[0144] Electroplating In step 1103, electroplating is performed in an aqueous liquid composition of an inverse micelle phase containing metal ions. The cathode and anode electrodes are submerged in the 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 into the hydrophilic space immediately adjacent to the inverse micelle, where positively charged metal ions can be reduced to metal atoms. The metal atoms come together to form metal particles, which can be deposited on the surface of the cathode electrode. In the process, the inverse micelle may burst. The electrons supplied to the cathode electrode move 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, which are deposited on top of the already deposited nanoparticles.

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

[0146] Formation of successive layers and cavities In electroplating reduction, nanoparticles adjacent to the cathode electrode are initially deposited on the cathode surface. Subsequently, additional nanoparticles are deposited on top of the previously deposited nanoparticles 121. Thus, nanoparticles generally deposit in successive layers on the cathode electrode. However, since the deposition of nanoparticles cannot occur at the same rate on the cathode surface and through the layers deposited before the nanoparticles, internal cavities 133 may be formed within the resulting nanoporous layer. The deposition of nanoparticles can grow horizontally or laterally in space, where nanoparticles are not deposited, and some cavities 133 may be enclosed by nanoparticles formed on top of them. The cavities 133 may eventually be interconnected via interparticle nanopores 125, but micro-sized channels are not formed throughout the entire nanoporous layer 117 or in substantial parts thereof to interconnect the cavities 133.

[0147] Surfactants deposited together During the electroplating process, the inverse micelles encapsulating these nanoparticles may rupture, and the nanoparticles are deposited on the cathode electrode. A considerable amount of surfactant molecules from the ruptured inverse micelles are deposited on the cathode electrode along with the nanoparticles. During the electroplating process, the surfactant molecules may bind to the surface of the nanoparticles, and nanoparticle-surfactant molecule complexes may be deposited together. The surfactant molecules may be inserted or trapped between the nanoparticles in the resulting nanostructure.

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

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

[0150] No nanoparticle colloids In electroplating, no reducing agent is added to reduce metal ions and form nanoparticles. During the electroplating process, nanoparticles may form in the hydrophilic space of inverse 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 space of inverse micelles throughout the entire liquid composition. Therefore, no nanoparticle colloids are formed, as illustrated in Figure 8.

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

[0152] Yield - Metal Recovery Upon completion of electroplating, the plating bath contains a considerable amount of metal ions. Therefore, metal recovery in electroplating may not be as effective as reduction achieved by adding an excess amount of reducing agent, as in the process for clustered nanoporous layers.

[0153] Manufacturing of nanoporous layers using liquid crystal phase Nanoporous metal layers can be fabricated from the liquid crystal phase of a surfactant. Referring to Figure 12, in step 1201, an aqueous liquid composition is prepared to contain metal ions and a liquid crystal phase surfactant, for example, in a hexagonal arrangement. Then, in step 1203, the aqueous liquid composition is subjected to electroplating to deposit a nanoporous layer, in which case the metal atoms are deposited using the liquid crystal phase as a template. In step 1205, the surfactant is removed from the deposited hexagonal nanostructure. Figure 13A shows the formation of the hexagonal arrangement. Figure 13B shows the deposition of metal using a hexagonal arrangement of the liquid crystal 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 the test fluid can interfere with the accurate sensing of glucose levels in both enzymatic and non-enzymatic glucose sensing systems.

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

[0156] Maltose interference in non-enzymatic glucose sensing The nanoporous layer 117 of the working electrode 103NE can oxidize maltose at the same bias voltage as glucose. As shown in Figure 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 together with glucose. Example 9.11 and Figure 18 confirm that maltose can be detected in PBS along with glucose and other interfering chemical components. Furthermore, Example 10.9 and Figure 19 confirm that maltose can be detected in serum along with glucose and other interfering chemical components.

[0157] Non-enzymatic electrode with maltose-blocking layer Referring to Figure 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 this embodiment, the nanoporous layer 117 can oxidize both maltose and glucose, whether or not they include clustered or non-clustered forms. The maltose-blocking layer 301 can be in contact with the underlying nanoporous layer 117 or separated by an intervening layer. The working electrode 103NE may also include an additional functional layer 112 on the maltose-blocking layer 301. In another case, the additional functional layer 112 may be inserted 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 it, while allowing glucose molecules to pass through. With the maltose-blocking layer 301, maltose molecules present in the test fluid cannot reach the underlying nanoporous layer 117, either entirely or at concentrations significant enough to interfere with glucose sensing. Given the selective maltose-blocking effect of the maltose-blocking layer 301, even if the nanoporous layer 117 could oxidize maltose at the same bias voltage as it does for glucose oxidation, the presence of maltose in the test fluid is unlikely to affect glucose sensing. In addition, the maltose-blocking layer 301 effectively blocks or prevents other molecules and components of the test fluid that are 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 the embodiment, the maltose-blocking layer 301 is made from or includes a porous polymer material through which glucose can pass but maltose cannot. The porous polymer material contains at least one polyphenylenediamine (poly-PD), including poly(m-phenylenediamine) (poly-mPD), poly(o-phenylenediamine) (poly-oPD), and poly(p-phenylenediamine) (poly-pPD).

[0161] Nano-sized 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 approximately 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 this description, the thickness of the maltose-blocking layer represents the average thickness of the polymer layer, excluding the upper and lower 10% of the thickness variation. In embodiments, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the preceding sentence, for example, 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] Level of porosity In embodiments, the maltose-blocking layer 301 has porosity that allows glucose molecules to pass through its thickness but effectively blocks maltose molecules from passing through it. To achieve the goal of allowing glucose to pass through and blocking maltose from passing through, the overall porosity of the maltose-blocking layer needs to 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 the material 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 specified or described using the concentration of the material 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 unit volume. Thus, the level of porosity needs to be specified in different ways.

[0163] Sensitivity (current density) for glucose and maltose without maltose-blocking layer For glucose monitoring, in a test fluid having a glucose concentration of 4 - 20 mM (typical glucose levels in human body fluids) in a steady state, when applying a bias voltage of 0.2 - 0.45 V, the nanoporous layer 117 in contact with the test fluid (i.e., without the maltose-blocking layer) , Minimum current density (sensitivity) for glucose This is 10 nA / mMcm 2 It is necessary to generate a higher level of glucose-oxidation current (current caused by oxidation of only glucose). According to an embodiment, without the maltose-blocking layer, the same nanoporous layer 117, in a test fluid containing maltose at a concentration of 4 - 20 mM (the same as the above glucose concentration), in a steady state using the application of a bias voltage of 0.2 - 0.45 V, will generate a current of a similar level (i.e., 10 nA / mMcm 2 Higher).

[0164] Porosity of the maltose-blocking layer based on the current density of glucose and maltose According to an embodiment, the maltose-blocking layer 301 has a porosity that allows glucose to move through it, so that the glucose oxidation current is still higher than the minimum current density for glucose. Thus, when applying a bias voltage of 0.2 - 0.45 V in a test fluid having a glucose concentration of 4 - 20 mM, in a steady state, the working electrode 103NE having the maltose-blocking layer 301 has a glucose-oxidation current of, Minimum current density (sensitivity) for glucose This is 10 nA / mMcm 2To generate at a higher level. On the other hand, the maltose blocking layer 301 has a porosity that effectively blocks maltose from passing through it, and therefore, when a bias voltage of 0.2-0.45V is applied in a test fluid with a maltose concentration of 4-20 mM, in the steady state, the current caused solely by maltose (maltose-oxidation current) teeth, Maximum current density for maltose when using a maltose-blocking layer This is 5 nA / mMcm 2 It is at a lower level.

[0165] Electrochemical polymerization The porous polymer material for the maltose-blocking layer 301 can be formed on the nanoporous layer 117 using cyclic voltammetry techniques by electrochemical polymerization (electrolytic polymerization). In an embodiment, the working electrode including the nanoporous layer is immersed in a reaction mixture solution containing a monomer for cyclic voltammetry electrochemical polymerization. Polymerization occurs by applying a bias voltage within the range of the oxidation voltage of the monomer between the working electrode and the reference electrode, 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: Kinetic, 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, 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, but is not limited thereto. For phenylenediamine, the bias voltage is applied at 0.5V to 1.0V. FIG. 22 shows an example of the scan of the bias voltage during the cyclic voltammetric electrochemical polymerization of phenylenediamine.

[0167] Bias voltage scan rate Together with the concentration of the monomers described below, the scan rate of the bias voltage between the lower and upper limits of the oxidation voltage range 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 / second. In embodiments, the scan rate can be within a range formed by selecting any two numbers listed in the previous sentence, for example, about 5 mV / second to about 200 mV / second.

[0168] Concentration of monomers The concentration of the monomers 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 monomers can be within a range formed by selecting any two numbers listed in the previous sentence, for example, about 0.05 mM to about 0.8 mM, about 1.0 mM to about 5.0 mM, etc. The aforementioned concentrations are applicable to three types of phenylenediamine.

[0169] Porosity considering monomer concentration The monomer concentration in the reaction mixture solution is related to the porosity of the resulting maltose-blocking layer. In the flowchart for producing the maltose-blocking layer in Figure 24, the monomer concentration is first determined in step 2401, and polymerization is carried out in step 2403. In embodiments, monomer concentrations of about 0.7 mM, about 0.6 mM, or less than about 0.5 mM may provide a desirable level of overall porosity for the maltose-blocking layer. In embodiments, if 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 will not have sufficient porosity to allow glucose to pass through, i.e. , Minimum current density (sensitivity) for Lucos This is 10 nA / mMcm 2 A glucose-oxidation current is generated at a lower level. In step 2405, the resulting polymer layer is subjected to a treatment to adjust its porosity.

[0170] Electric shock to adjust porosity If the overall porosity of the polymer layer 302 is not at a desired level, the polymer layer may be further treated to adjust its porosity. For example, the polymer layer may be subjected to an electric shock. In an embodiment, the electric shock may be applied to the polymer layer 302 using the chronoamperometry setting shown in Figure 23, in which case the electric shock electrode 309 and the polymer layer 302 formed on the nanoporous layer 117 are immersed in an electrolyte solution 311. A voltage source 305 and a switch 307 are connected between the substrate 303 and the electric shock electrode 309. The operation of the switch 307 causes an electric current to flow through the porous polymer layer 302, causing a morphological change, thereby increasing 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 and effectively blocks maltose from passing through it.

[0171] Acidic solution The electrolyte solution for electric shock may be, but is not limited to, an acidic solution having a pH of about 2, 3, or less than 4. In some embodiments, the acidic solution may contain 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, and the like.

[0172] Waveform for electric shock The potential can be applied in various waveforms. In some embodiments, the potential is applied as AC or DC. In some embodiments, the potential is applied as multiple pulses or a single pulse. In some embodiments, the potential can be applied in other forms of voltage signals.

[0173] Electric potential for electric shock The potential applied to the polymer layer 302 is approximately 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 4.0V. In embodiments, the maximum voltage may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, approximately 0.5 to approximately 2.5V, approximately 1.0 to approximately 2.0V, etc.

[0174] Period for electric shock The duration for applying the potential is between approximately 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 preceding sentence, for example, approximately 0.5 to approximately 2.5 seconds, approximately 1.0 and approximately 2.0 seconds, etc.

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

[0176] CGM system with CGM (Continuous Gamma Temperature) A continuous glucose monitoring (CGM) system includes a glucose-sensing electrode that comes into in vivo contact with the subject's biofluid for measuring glucose levels in the biofluid. In practice, the CGM electrode is inserted or implanted in the subject's body for measurements over an extended period, such as several days, weeks, weeks, or months.

[0177] Non-enzymatic CGM working electrode Figure 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 a support for the laminated structure of the CGM working electrode 501. In embodiments, the base 503 is an electrically insulating layer and may be made from, or include, materials such as, polyimide, polypropylene, polyethylene glycol, polyhydroxyethyl methacrylate (pHEMA), and other biocompatible polymers, but is not limited to these. 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 about 30 μm to about 200 μm, but is not limited thereto. The base 503 is an optional layer for the CMG sensing electrode 501 and may be omitted in some embodiments.

[0179] conductive layer The conductive layers 110 may be arranged on the base 503 with or without intervening layers between them. In embodiments, the conductive layers 110 are formed by printing or distributing conductive or semiconducting material on the base 503, but are not limited thereto. In the CGM working electrode 501, the conductive layers 110 may have a thickness in the range of approximately 100 nm to 100 μm, but are not limited thereto. In some embodiments, the conductive layers 119 may include two or more sublayers of conductive or semiconducting material. In embodiments where the base 503 is omitted, the conductive layers 119 may function as a support for the laminated structure thereon.

[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 in the range of approximately 500 nm to approximately 10 μm, but is not limited thereto. The nanoporous layer 117 may have at least one of clustered morphology, non-clustered morphology, hexagonal nanostructures, or other nanoporous morphology.

[0181] Maltose-blocking layer The maltose-blocking layer 301 can be formed on the nanoporous layer 117, which blocks maltose molecules from reaching the underlying nanoporous layer 117 while allowing glucose molecules to pass through. In an embodiment, the maltose-blocking layer 301 includes a polymeric material such as poly-PD having nanosize pores to allow glucose molecules to pass through and block maltose molecules from passing through. The maltose-blocking layer can have a thickness in the range of 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 effectively restricts or blocks small electrolyte ions, such as Na + , K + , Ca 2+ , Cl - , PO4 3- and CO3 2- from passing through it or diffusing towards the underlying nanoporous layer 117. As will be described later, 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 glucose molecules can freely pass through it. When implemented, the electrolyte ion-blocking layer 505 is hydrophobic so as not to swell immediately by absorbing water contained in the test fluid. The electrolyte ion-blocking layer 505 can have a thickness in the range of about 0.1 μm to about 10 μm, but is not limited thereto.

[0183] Materials for the electrolyte ion-blocking layer The electrolyte ion-blocking layer 505 may contain, 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), or may be manufactured from them. Alternatively, the electrolyte ion-blocking layer 505 may be formed from a copolymer of methyl methacrylate and butyl methacrylate, and a polymer 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, and 2-ethylhexyl acrylate, or may additionally contain them.

[0184] Biocompatible layer Once the CGM sensor is implanted or inserted into the subject's body, the biocompatible or biological protective layer 507 comes into contact with the subject's tissues and bodily fluids. The biocompatible layer 507 comprises 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. Furthermore, at least one material of the biocompatible layer 507 must allow bodily fluids to pass through it to the underlying nanoporous layer 117 so that glucose concentration sensing is not significantly impaired by its presence. The biocompatible layer 507 may, but is not limited to, have a thickness ranging from approximately 5 μm to approximately 30 μm.

[0185] Materials for biocompatible layers The biocompatible layer 507 may contain, or be manufactured from, at least one of the following: poly(vinyl alcohol), poly(ethylene oxide-copropylene 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 a single layer, or their positions may be changed.

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

[0188] No oxygen uptake layer The CGM working electrode 501 does not contain any oxygen uptake material or layer that would be required to collect and supply molecular oxygen when glucose oxidase is used for glucose oxidation.

[0189] No electron carrier The CGM working electrode 501 does not contain electron transfer material, which would be necessary to transfer electrons if glucose dehydrogenase were used for the oxidation of 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 fabricated by applying a 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 derived 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. Subsequently, the rate of decrease slows down. Eventually, the current settles at a certain level, i.e., a steady state, although 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 the CGM working electrode are in a steady state. In other words, the current from the CGM working electrode should not change much over time if the glucose concentration does not change (i.e., it should settle at a certain level after the initial decrease). Furthermore, for accurate glucose sensing, the background current (noise) should not be too high compared to the current caused solely by glucose oxidation. In other words, the total current should not be too high compared to the current derived solely from glucose oxidation.

[0192] Conditioning of CGM working electrodes or electrochemical cells The CGM working electrode requires conditioning before glucose sensing. Here, conditioning refers to the process of stabilizing the CGM working electrode for accurate glucose sensing. Once the CGM working electrode is conditioned, the current emanating 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 the CGM working electrode can take a long time. Commercially available enzyme CGM working electrodes require several hours to several days for conditioning.

[0193] Desired rate of current change Assuming the current derived from glucose oxidation in vivo is approximately several tens of nanoamperes, for accurate glucose sensing, the rate of decrease of the 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 at points of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 nA or less per minute. In embodiments, the rate of current change may be determined over shorter or longer periods.

[0194] Desired level of current The current derived from glucose oxidation in vivo is typically in the tens of nanoamperes. The desirable level of total current can vary depending on various factors, including measurement accuracy, signal processing capability, and data processing capability. These factors can be further developed, so the desirable level may increase. Nevertheless, assuming that the current derived from glucose oxidation in vivo is approximately in the tens of nanoamperes, the current from the CGM working electrode must be less than, for example, 500 nA, for accurate glucose sensing. To provide reference points, the desired currents should be at 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 or less.

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

[0196] Notification of completion of conditioning A CGM system can notify its user of the completion of conditioning. Upon forming an electrochemical cell for glucose oxidation, or some time after its formation, the CGM system can begin monitoring the current from its CGM working electrode. When the current meets one or more requirements for the completion of conditioning, the CGM system can provide a notification to its user to indicate the completion of conditioning. The notification may be in any form, including sound, vibration, light, or information display. In addition, or otherwise, the CGM system may not provide information indicating glucose levels before the completion of conditioning.

[0197] Reducing the time required for conditioning the CGM working electrode, small electrolyte ion concentration discontinuities. Human body fluids contain a considerable amount of Na + , K + Ca 2+ Cl - , PO4 3- and CO3 2- It contains electrolyte ions. In the embodiment, the electrolyte ion-blocking layer 505 is Na + , K + Ca 2+ Cl - , PO4 3- and CO3 2- This restricts or blocks the passage of electrolyte ions through it. As a result, the concentrations of these electrolyte ions differ significantly between the area above and below the electrolyte ion-blocking layer 505. Figure 32 conceptually illustrates the concentration discontinuity on both sides of the electrolyte ion-blocking layer 505. With the electrolyte ion-blocking layer 505, the total concentration of small electrolyte ions is significantly lower 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 ion - Low concentration of electrolyte ions under the blocking layer In the embodiment, the total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 is greater than 0%, but lower than approximately 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 preceding sentence. As shown in Figure 32, for example, the total concentration of electrolyte ions in human interstitial fluid (i.e., above the electrolyte ion-blocking layer 505) is approximately 0.1 M or greater; in contrast, the total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 is approximately 0.01 M or less. The total concentration of electrolyte ions below the electrolyte ion-blocking layer 505 can be obtained by measuring the bilayer volume of the nanoporous layer 117 and applying the measured value to the Gouy-Chapman formula, 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 ion equilibrium 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 the electrolyte ion-blocking layer 505 and the layer below the same layer. The lower concentration of small electrolyte ions is significantly better for conditioning the CGM working electrode 501, particularly the nanoporous layer 117. Although none of the embodiments of the invention is bound by any theory or belief, the lower concentration of small electrolyte ions can accelerate ionic equilibrium in the nanoscale structure and surface of the nanoporous layer 117, which would not occur at larger scales such as microscale structures and surfaces. As ionic equilibrium is accelerated in the nanoporous layer 117, the time to reach ionic equilibrium or a steady state inside the nanostructure of the nanoporous layer 117 will be shorter at lower concentrations of electrolyte ions in the presence of the electrolyte ion-blocking layer 505 than at higher concentrations without the electrolyte ion-blocking layer 505.

[0200] Significantly shorter time for conditioning By utilizing the acceleration of ion equilibrium in the nanoporous layer 117, the electrolyte ion-blocking layer 505 significantly enhances and accelerates the conditioning of the non-enzymatic CGM working electrode 501 in Figure 31, i.e., shortening the time to reach the desired current and / or desired rate of current change, i.e., the steady state. According to the embodiment, when using the non-enzymatic CGM working electrode 505 having the electrolyte ion-blocking layer 505, a little less time is required to complete the conditioning compared to when using the same non-enzymatic CGM working electrode without the electrolyte ion-blocking layer 505.

[0201] Conditioning time When the desired current change rate is 5 nA / min or less, a non-enzymatic CGM working electrode without the electrolyte ion-blocking layer 505 takes approximately 3 hours in serum containing electrolyte ions at 0.1 M or higher; in contrast, a non-enzymatic CGM working electrode with the electrolyte ion-blocking layer 505 takes approximately 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 current change rate 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 electrolyte ions at 0.1 M or higher; in contrast, a non-enzymatic CGM working electrode with an electrolyte ion-blocking layer 505 takes approximately 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 current change rate is 2 nA / min or less, a non-enzymatic CGM working electrode without an electrolyte ion-blocking layer 505 takes more than 5 hours or 10 hours in serum containing electrolyte ions at 0.1 M or higher; in contrast, a non-enzymatic CGM working electrode with an electrolyte ion-blocking layer 505 takes approximately 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 current for accurate glucose levels. Reducing the time required for conditioning is a crucial practical consideration when developing and manufacturing CGM working electrodes. This is because proper conditioning of a CGM working electrode can take several hours, if not several tens of minutes, and people tend to want to know their glucose levels immediately after inserting the electrode into their body. As will be shown in the examples described later, the time required for conditioning a CGM working electrode can be reduced from approximately 3, 5, or 10 hours to less than 30 minutes simply by including the electrolyte ion-blocking layer 505, while all other conditions remain the same. This is a very significant improvement and an unexpectedly high success rate.

[0203] Details of the electrolyte ion-blocking layer The electrolyte ion-blocking layer 505 of the non-enzymatic CGM working electrode contains, or is produced 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 approximately 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, and 2. These are 00,000, 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, or 400,000. In embodiments, the molecular weight may be within a range formed by selecting any two numbers listed in the preceding sentence. The electrolyte ion-blocking layer may have a thickness of approximately 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 the embodiment, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the preceding sentence, for example, a range of about 2 to about 5 μm, about 1 to about 3 μm, etc.

[0204] Ion concentration reduction has no effect on enzyme glucose-sensing electrodes. In an enzyme-mediated CGM system, the CGM working electrode contains a glucose-specific enzyme for the oxidation of glucose molecules. The enzyme-mediated CGM working electrode may include a functional layer containing a porous hydrophobic material that can effectively lower the concentration of electrolyte ions beneath the functional layer. However, in an enzyme-mediated CGM system, the concentration reduction by the functional layer cannot provide a reduction in the time required for conditioning the CGM electrode with respect to ion equilibrium at nanoscale surfaces or structures. This is because the enzyme-mediated CGM system uses an enzyme to oxidize glucose molecules and does not require a nanoporous layer for glucose oxidation. Therefore, even if a porous hydrophobic layer were included in the enzyme-mediated CGM working electrode, and even if such a layer caused discontinuities in electrolyte ion concentration across its thickness, and even if there was some reduction in the time required for conditioning the enzyme-mediated CGM working electrode, such a reduction would not be equivalent to the reduction in the time required for conditioning in a non-enzymatic CGM working electrode 501 having 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 comes into subcutaneous contact with the subject's bodily fluids. The electrode unit may include a single body housing one or more electrodes that come into contact with bodily fluids when inserted into the subject's body. The single body may be flexible.

[0206] Configuration of the CGM electrode unit Figure 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 the subject's body and includes a working electrode 501, a counter electrode 105, and a reference electrode 106, which are exposed through openings formed through an insulating layer 707 to make subcutaneous contact with bodily fluids. The contact terminal portion 705 remains outside the subject's body and is for engaging with or connecting to a corresponding device. The contact terminal portion 703 includes working electrode terminals 501T, counter electrode terminal 105T, and reference electrode terminal 106T, respectively, which are electrically connected to the working electrode 501, counter electrode 105, and reference electrode 106 below the insulating layer 707. Here, each of the working electrode 501, counter electrode 105, and reference electrode 106 may have, but are not limited to, the features and properties described herein.

[0207] Fabrication of CGM electrode units Figure 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 Figure 31). Then, in step 3403, a conductive layer is formed on the base 503 in predetermined shapes 110R, 110W, and 110C, as shown in Figure 35. Then, as step 3405, an insulating film 707 is applied over the conductive layer, and a portion or area of ​​the conductive layer is selectively exposed as shown in Figure 36. Then, in step 3407, the intermediate product is cut, providing the shape shown in Figure 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, providing a laminated configuration of the non-enzymatic CGM working electrode 501, as shown in Figure 31. Furthermore, a salt layer may be formed on the exposed area for the reference electrode 106. In the embodiment, the cutting of the intermediate product in step 3407 may be carried out after step 3409 or 3411.

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

[0209] Manufacturing of conductive layers The conductive layer may be a single layer of conductive material, or it may be formed from multiple sublayers of different conductive materials. In embodiments, 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 embodiments, 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 conductive ink onto or above the base 503 and then drying it. Sublayers formed on other sublayers may also be formed by printing conductive material for those sublayers. The conductive layer elements 110W, 110C, and 110R in Figure 35 are all single layers; however, for the purpose of illustrating alternatives, in Figures 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 Figure 16A).

[0210] insulating film Figure 36 shows an intermediate product after the insulating film has been placed according to one embodiment. The insulating film 707 may be pre-cut with an opening in the subcutaneous 703 of Figure 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 Figure 33 and therefore exposes the respective terminal portions of the conductive layer elements 110C, 110W, and 110R (which will be 105T, 501T, and 106T, respectively). The conductive connections of the conductive layer elements 110C, 110W, and 110R are covered by 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] Cutting In step 3407, the intermediate product shown in Figure 36 is subjected to cutting, and any unnecessary portions of the insulating film 707 and base 503 are removed, for example, by die cutting. Figure 37 shows the resulting product, in which case the contact terminal portion 705 (proximal end portion of the CGM electrode unit 701) is wider than the subcutaneous portion 703 (distal end portion of the 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 preceding sentence, for example, approximately 1.0 mm to approximately 1.5 mm. In the embodiment, the CGM electrode unit 701 has a length 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 the direction between its distal and proximal ends. In the embodiment, the length may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, about 10 mm to about 20 mm.

[0212] Formation of nanoporous layers In step 3409, the nanoporous layer 117 is formed on the conductive layer element 110W exposed for the working electrode. Figure 38A shows a cross-section of the intermediate product in the direction of the arrow with respect to line 3501 after the formation of the nanoporous layer 117. In some embodiments, the nanoporous layer 117 is formed by distributing a cluster colloid containing nanoparticle clusters dispersed in a liquid onto the conductive layer 110 and then drying the liquid therefrom. In other cases, other 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 the formation of 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 shown in Figure 31. A maltose blocking layer 301 may, but is not limited to, be formed on the nanoporous layer 117. An electrolyte ion-blocking layer 505 is obtained as a result. CGM The working electrode 501 may, but is not limited to, be formed on the nanoporous layer 117 to improve its conditioning. Furthermore, the biocompatible layer 507 may, but is not limited to, be formed on the nanoporous layer 117, or more specifically, on the electrolyte ion-blocking layer 505. Figure 38B shows a cross-section of the CGM working electrode 501 including the electrolyte ion-blocking layer 505 and the biocompatible layer 507.

[0214] Reference electrode and counter electrode In the embodiment, a salt layer, such as AgCl, may be formed on the conductive layer element 110R exposed for the reference electrode 106. The formation of the salt layer can be carried out at any time after the conductive layer element 110R has been formed. In the embodiment, the counter electrode 105 may not require any additional processing on the conductive layer element 110C.

[0215] Subcutaneous insertion of CGM electrode unit In this embodiment, the subcutaneous portion 703 (distal portion) of the CGM electrode unit 701 is subcutaneously inserted into the subject's body with or without using insertion tools known in the art or that may 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 are in contact with the subject's interstitial fluid, while the terminal portion 705 of the CGM electrode unit 701 remains outside the subject's body.

[0216] Compatible devices Subsequently, in some embodiments, the terminal portion 705 is engaged with or connected to a corresponding device (not shown) which includes corresponding ports or terminals corresponding to the working electrode terminal 501T, the counter electrode terminal 105T, and the reference electrode terminal 106T. In some embodiments, the corresponding device further includes electrical circuits that complete the electrochemical cell of Figure 1 together with the CGM electrode unit 701 for a continuous monitoring glucose module. In some embodiments, in addition to the electrical circuits for completing the electrochemical cell, the corresponding device may include at least one processor for processing data, including currents obtained from the electrochemical cell to be converted into standardized numbers representing glucose levels. In some embodiments, the corresponding 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. A single-time point glucose sensing system measures glucose levels in a test fluid, most commonly in the blood. Therefore, the system is called a blood glucose monitoring (BGM) system. BGM systems include single-use, disposable cartridges or strips.

[0218] Disposable cartridges Figure 39 shows a BGM disposable cartridge 901 and a 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 supports for electrodes 105, 106 and 905. An electrical connection (not shown) is formed between the electrode and a connector 909 via the base 907.

[0219] Sensing module In this embodiment, the disposable cartridge 901 is designed to be electrically and / or mechanically coupled to the sensing module 911 via a connector 909. The sensing module 911 may include an electrical network (not shown) for a voltage source 109 and a current sensor 108. Once the disposable cartridge 901 is properly connected to the sensing module 911, electrodes 105, 106 and 905 are connected to the circuitry of the sensing module 911, as shown in Figure 1.

[0220] working electrode In one embodiment, the working electrode 905 comprises a conductive layer 110 and a nanoporous layer 117. The working electrode 905 further comprises a filter layer 913 for filtering and screening cells, lipids, and large molecules contained in the test fluid. In the embodiment, the filter layer 913 may be made from or include woven fabric, cotton, or other materials that allow glucose to pass through, although the filter layer 913 may screen for cells, lipids, and other large components of blood.

[0221] Working electrode is not included. In this embodiment, the working electrode 905 does not contain glucose-specific enzymes. Furthermore, the working electrode 905 does not contain surfactants and electron carriers that may be required in enzyme glucose sensing. Moreover, assuming 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 the embodiment, a non-enzymatic electrode having a nanoporous glucose-oxidation layer generates an electric current caused by the oxidation of glucose contained in the test solution. In practice, the current from the non-enzymatic electrode includes 1) a current caused solely 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 the interaction 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). In diabetic patients, glucose levels may range from 4.0–20 mM (72–360 mg / dL).

[0224] Glucose-oxidation current In the embodiment, in a steady state (after conditioning) in a test fluid containing 4.0-20 mM glucose, applying a bias voltage of approximately 0.2V to approximately 0.45V results in a current that originates solely from glucose oxidation (glucose-oxidation current). is 1 0 nA / mMcm 2 YoIt is at a very high level. In the glucose concentration range of 4.0–20 mM, the nanoporous glucose-oxidation layer (and thus the non-enzymatic electrode) generates a glucose-oxidation current of approximately 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 contained in the test fluid. In embodiments, the glucose-oxidation current derived from 1 mM glucose contained in the test fluid may be within the range formed by any two numbers in the preceding sentence, for example, approximately 1.5 nA to 2.5 nA. Therefore, in the glucose concentration range of 4.0–20 mM, the glucose-oxidation current from the non-enzymatic electrode can be approximately 2.0 nA (4.0 × 0.5) to approximately 120 nA (20 × 6.0). In this embodiment, the glucose-oxidation currents can be approximately 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, and 118, which can be 120Na. In the embodiment, the glucose-oxidation current derived from 4.0-20 mM glucose contained in the test fluid may be within the range formed by any two numbers in the preceding sentence, for example, about 1.5 nA to 2.5 nA.

[0225] Calibration of current and glucose concentration In the embodiment, for the same glucose concentration in the test fluid, the glucose-oxidation current may differ between one nanoporous glucose-oxidation layer and another, depending on their specific manufacturing conditions. Furthermore, in a particular nanoporous glucose-oxidation layer, the glucose-oxidation current may generally correlate linearly with glucose concentration, but may not be so linear across the entire concentration or current range. In the embodiment, 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 a particular batch. Later in the glucose sensing or monitoring process using the nanoporous glucose-oxidation layers from the same batch, the correlation profile is used to calculate or determine 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. It may interfere with glucose sensing from body fluids.

[0227] Currently, there is no layer available that can block ascorbic acid. Assuming that ascorbic acid has a negative charge, it has been proposed that a negatively charged layer would repel ascorbic acid while allowing glucose to pass through. However, to date, no glucose-sensing electrodes that block ascorbic acid are commercially available.

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

[0229] 2. Operation of the 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, at the second working electrode 4103B, oxidation of ascorbic acid occurs, but not 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 shown in Figure 3. The nanoporous layer 117 may include, but is not limited to, a clustered nanoporous structure. In other embodiments, the first working electrode 4103A may include an enzyme layer containing a glucose-specific enzyme for oxidizing glucose, instead of the nanoporous layer 117 in Figure 3, as shown in Figure 2. 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 through it.

[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 layer or feature for effectively inducing glucose oxidation. In the embodiment, the second working electrode 4103B does not include a nanoporous layer 117 or a glucose-specific enzyme for glucose oxidation. However, oxidation of ascorbic acid occurs in the conductive layer 110. In the embodiment, the conductive layer 110 includes, but is not limited to, a conductive carbon layer formed on a silver layer.

[0232] Same bias voltage for both electrodes In this 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 roughly 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 interference from additional chemical components The 2-electrode system 4101 can be used to address interference from more than one chemical component. In the embodiment, by adjusting the bias voltage, the first working electrode 4103A can oxidize not only glucose and ascorbic acid but also additional interfering chemical components, such as acetaminophen. Similarly, the second working electrode 4103B oxidizes not only ascorbic acid but also the additional interfering chemical component at the same time. Here, neither the first nor the second working electrode contains a film 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, canceling out the interference of acetaminophen and ascorbic acid.

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

[0235] Different bias voltages In the embodiment, the two-electrode system 4101 can employ different bias voltages for the first and second working electrodes. For example, the first bias voltage is applied to the first working electrode 4103A, and the second bias voltage is applied to the second working electrode 4103B. Using different bias voltages, the current derived 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 derived from glucose oxidation may not be a simple difference between the currents from the two electrodes. However, in the embodiment, the two-electrode system 4101 has, or is connected to, hardware and software for calculating an accurate glucose concentration using different bias voltages, current values ​​from the first and second working electrodes 4103A and 4103B, data showing the oxidation potential of ascorbic acid at 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 concurrently. In other embodiments, using either one or two current sensors, detection may occur at different times with time gaps, as long as the concentration fluctuations of the chemical component in question are negligible over the time gap. Those skilled in the art will recognize what length of time gap is possible with little risk of inaccuracy. For example, the time gap may be less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

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

[0238] Applicable to CGM The 2-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 the first and second working electrode terminals 4103AT and 4103BT, respectively.

[0239] Applicable to background music The 2-electrode system 4101 can be incorporated into a BGM disposable cartridge or strip for in vitro glucose sensing. In an embodiment, the disposable cart shown in Figure 39 The ridge 901 may include two working electrodes. In such an embodiment, the cartridge working electrode 905 acts as the first working electrode 4103A. A second working electrode 4103B may be added to the base 907 to come into contact with the test fluid. Furthermore, the corresponding sensing module 911 may include a network for receiving signals from the first and second working electrodes from a BGM disposable cartridge.

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

[0241] Interference by acetaminophen Acetaminophen is one of the most commonly used over-the-counter medications. Furthermore, acetaminophen is widely used as an active ingredient in concomitant medications.

[0242] Commonly recognized problems Given the popularity of acetaminophen, the drug may be ingested by patients who also need to have their blood glucose levels monitored. Considering that many glucose sensing devices are used by patients themselves, rather than 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 finding a solution.

[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 adopted to selectively screen for acetaminophen to prevent it from reaching the electrode. Therefore, there is a long-standing but unmet need.

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

[0245] Non-enzymatic glucose sensing systems for dealing with acetaminophen In this embodiment, the non-enzymatic electrochemical glucose sensing system selectively oxidizes glucose and, at the same time, does not oxidize acetaminophen without introducing an additional membrane for this result. Referring back to Figures 3 and 31, the working electrodes 103NE, 501 include 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 membrane not included In this embodiment, the working electrode 103NE does not include a membrane, film, or layer on the nanoporous layer 117 that is designed to selectively screen, repel, or block acetaminophen, but allow glucose to pass through. Therefore, when the working electrode 103NE comes into contact with a test fluid containing acetaminophen, both glucose and acetaminophen come into contact with 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 the glucose sensing system according to the embodiment, glucose is oxidized in the nanoporous layer 117 at a bias voltage of approximately 0.2V to approximately 0.45V. On the other hand, acetaminophen is oxidized at a bias voltage greater than 0.33, 0.34, 0.35, or 0.36V. 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 the embodiment, the bias voltage applied to the conductive layer 110 relative to the reference electrode 106 is set such that, when both are in contact with the nanoporous layer 117, they cause oxidation of glucose but not oxidation of acetaminophen. For selective oxidation of glucose and selective non-oxidation of acetaminophen, in the embodiment, the bias voltage is set to 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, or about them. In the embodiment, the bias voltage may be within a range formed by selecting any two numbers (two voltage values) listed in the preceding sentence, for example, 0.28V to 0.30V, about 0.27V to about 0.31V, 0.26V to 0.30V, about 0.28V to about 0.32V, etc. In the embodiment, the bias voltage is lower than 0.30, 0.31, or 0.32V.

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

[0250] Examples Here, various aspects and features of the invention will be further described in relation to examples and experiments.

[0251] Preparation of the reverse micelle phase Example 1.1 A platinum aqueous solution was prepared by dissolving 0.500 g (0.965 mmol) of hexahydrate chloroplatinic acid H2PtCl6·6H2O (manufactured by Sigma-Aldrich) in 24.5 g of purified water with stirring. 25 g of the surfactant Triton X-100 (trademark) (manufactured by Sigma-Aldrich) was added to the platinum aqueous 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. An inverse micelle phase was prepared in the aqueous composition by adjusting the temperature to 70°C with stirring.

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

[0253] Example 1.3 The reverse micelle phase is prepared by repeating Example 1.1, except that H2PtCl2(OH)4 is used instead of H2PtCl6·6H2O in an amount that provides a platinum ion concentration of about 0.02 M in the aqueous composition.

[0254] Example 1.4 The reverse micelle phase is prepared by repeating Example 1.1, except that H2Pt(SO4)(OH)4·6H2O is used instead of H2PtCl6·6H2O in an amount that provides a platinum ion concentration of about 0.02 M in the aqueous composition.

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

[0256] Example 1.6 The 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 the reverse micelle phase of the surfactant.

[0257] Example 1.7 The 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 a specific surfactant.

[0258] Example 1.8 The 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 a specific surfactant.

[0259] Example 1.9 The reverse micelle phase is prepared by repeating Example 1.1, except that Poloxamer 407 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 a specific surfactant.

[0260] Example 1.10 The 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 a specific surfactant.

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

[0262] Example 2.2 A reducing agent aqueous 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 approximately 0.6 M in the reducing agent aqueous solution.

[0263] Example 2.3 A reducing agent aqueous 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 approximately 0.6 M in the reducing agent aqueous solution.

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

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

[0266] Examples 3.2-3.10 Example 3.1 is repeated using the reverse micelle phase prepared in Examples 1.2-1.10 instead of the reverse micelle 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 Photal Otsuka Electronics zeta potential and particle size analyzer ELS-Z2. In the analysis, the platinum colloid sample from Example 3.1 was dispersed in purified water and found to have a refractive index of 1.3328, a viscosity of 0.8878 cp, and a relative permittivity of 78.3 at 25°C.

[0268] Figure 14 shows the particle size distribution for the colloid obtained from Example 3.1. The particle diameter is mainly approximately 9 nm to 14 nm. This size distribution is interpreted to represent inverse micelles. The size distribution does not show a diameter size of 1-5 nm, which is interpreted to mean that most platinum nanoparticles are contained within or enclosed inside the inverse micelles. Similar results were obtained from multiple runs of the 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 colloid prepared in Examples 3.2-3.10 instead 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] Surfactant 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 platinum colloid with the added acid was centrifuged at 3800 rpm for 10 minutes. The clear supernatant was then discarded, and the black bottom layer was collected. The sequence of adding aqueous HCl solution, centrifugation, and collecting the black bottom layer was repeated four more times to remove the surfactant and obtain platinum colloid.

[0271] Subsequently, the resulting platinum colloid was 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 layer was collected. The sequence of adding purified water, centrifugation, and collecting the black bottom layer was repeated four more times to remove HCl and obtain HCl-washed platinum colloid.

[0272] Examples 5.2-5.10 Example 5.1 was repeated, using the nanoparticle colloids obtained from Examples 3.2-3.10 instead of the nanoparticle colloids prepared in Example 3.1, and the colloids of Examples 5.2-5.10 were collected in each case.

[0273] Example 5.11 Repeat Example 5.1 using a 0.3 M HNO3 aqueous solution instead of an HCl aqueous solution.

[0274] Example 5.12 Repeat Example 5.1 using a 0.3 M NaOH aqueous solution instead of an HCl aqueous solution.

[0275] Particle size analysis of cluster colloids Example 6.1 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 Photal Otsuka Electronics ELS-Z2 zeta potential and particle size analyzer, similar to Example 4.1. In the analysis, the colloid sample from Example 5.1 was dispersed in water and found to have a refractive index of 1.3328, a viscosity of 0.8878 cp, and a relative permittivity of 78.3 at 25°C.

[0276] Figure 15 shows the particle size distribution for the colloid obtained from Example 5.1. The particle diameter is mainly about 60 nm to 200 nm. This size distribution is interpreted as representing irregularly shaped clusters formed from nanoparticles. Considering that the particle size in Example 4.1 is mainly about 9 nm to 14 nm (reverse micelle size, not cluster size), it is understood that the clusters were formed by the process in Example 5.1, in which case the surfactant molecules were detached 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 the 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 colloid prepared in Examples 3.2-3.10 instead of the colloid prepared in Example 3.1. The particle size distribution for each colloid prepared in Examples 3.2-3.10 is obtained.

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

[0279] Electrode fabrication example 8.1 - Electrode base using clustered nanoporous layer 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 approximately 20 μm by printing silver ink containing silver particles. The conductive carbon layer 1605 was formed to a thickness of approximately 20 μm by printing 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 a 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 a 60°C oven for 30 minutes to form electrode 1607 containing a platinum nanoporous layer 1609, as shown in Figure 16B.

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

[0282] Cyclic voltammetry was performed using a potential sweep from -0.2V to +1.2V. 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 coefficient was calculated by dividing the actual surface area by the geometric area. The roughness coefficient 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 the additional electrode bases to prepare additional electrodes 1607 containing a platinum nanoporous layer 1609.

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

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

[0286] Glucose sensing in PBS Example 9.1 - Preparation of solutions of glucose and other test materials A 1M glucose stock solution was prepared by dissolving D-(+)-glucose powder purchased from Sigma-Aldrich in purified water. A 0.05M ascorbic acid aqueous solution was prepared by dissolving ascorbic acid purchased from Sigma-Aldrich in purified water. A 0.05M acetaminophen aqueous solution was prepared by dissolving acetaminophen purchased from Sigma-Aldrich in purified water. A 0.5M maltose aqueous solution was prepared by dissolving maltose purchased from Sigma-Aldrich in purified water.

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

[0288] Example 9.3 - Preparation of a glucose sensing system in PBS 20 ml of PBS prepared in Example 9.2 was placed in a beaker, and the temperature of the PBS was maintained at 37°C. An electrochemical cell as shown in Figure 1 was prepared using a CH660 electrochemical analyzer from CH Instruments Inc. as potentiostat 104, 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 silver layer 1603 of electrode 1607 was connected to potentiostat 104. The electrode was immersed in PBS and electrically connected to the electrochemical analyzer.

[0289] Example 9.4 - Measurement of Current In the system prepared in Example 9.3, a bias voltage of 0.4V was applied between the working electrode 103 (electrode 1607) and the reference electrode 106. Upon application of the bias voltage, the current from the working electrode 103 was continuously measured. The electrochemical cell was maintained for 12 minutes to condition the glucose-sensing system in PBS without adding any substances. A current value of 0.087 μA was then obtained for the absence of 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" represents ascorbic acid and "AP" represents 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 in Example 9.3 to produce PBS containing 1 mM glucose. Immediately after addition, the glucose-added PBS was stirred for 3-4 seconds, which induced a temporary 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 the PBS.

[0291] Example 9.6 - Sensing 3 mM glucose in PBS In Example 9.5, after the current stabilized, 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 the addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a temporary 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 the PBS.

[0292] Example 9.7 - Sensing 6 mM glucose in PBS In Example 9.6, after the current stabilized, 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 a total of 6 mM glucose. Immediately after the addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a temporary 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 the PBS.

[0293] Example 9.8 - Sensing 10 mM glucose in PBS In Example 9.7, after the current stabilized, 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 the addition, the glucose-added PBS was stirred for 3-4 seconds, which caused a temporary 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 the PBS.

[0294] Example 9.9 - Sensation of 0.11 mM ascorbic acid in PBS In Example 9.8, after the current stabilized, 44 μl of the ascorbic acid aqueous 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 with added ascorbic acid was stirred for 3-4 seconds, which caused a temporary 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 the PBS.

[0295] Example 9.10 - Sensation of 0.17 mM acetaminophen in PBS In Example 9.9, after the current stabilized, 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 with added 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 the PBS.

[0296] Example 9.11 - Detection of 13.9 mM maltose in PBS In Example 9.10, after the current stabilized, 556 μl of the maltose aqueous 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 addition, the PBS with added maltose was stirred for 3-4 seconds, which caused a temporary 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 the PBS.

[0297] Example 9.12 - Glucose Level Formula In Examples 9.5-9.11, the current value represents and corresponds to the glucose concentration in PBS. Similar experiments are performed many more times using the same and other glucose concentrations with similarly prepared glucose sensing systems to obtain data on current values ​​and corresponding glucose concentrations. The correlation between glucose concentration in PBS and current value 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] Sensing of glucose 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 YSI. The serum was determined to contain 5.8 mM glucose, which corresponds to a blood glucose level of 104 mg / dl. 10 ml of serum was placed in a beaker, where the serum temperature was maintained at 37°C. An electrochemical cell was prepared in the same manner as in 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 serum.

[0299] Example 10.2 - Preconditioning of a glucose-sensing system in serum A bias voltage of 0.4V 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 to condition the system; that is, until the background current became low enough to sense glucose oxidation. The bias voltage was then disconnected from the system.

[0300] Example 10.3 - Measurement of Current In Example 10.2, immediately after removing the bias voltage, the same bias voltage was reapplied to the system, and measurement of the current from the working electrode was started. The electrochemical cell was maintained for 1.2 hours in serum with the glucose sensing system, without adding any substances, to further condition it. When the current stabilized, a current value of 96 nA was obtained relative to the 5.8 mM glucose originally present in the serum. Figure 19 shows the current profiles measured from the electrochemical cells in Examples 10.4-10.9 below. In Figure 19, "AA" represents ascorbic acid and "AP" represents 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-added serum was stirred for 3-4 seconds, which induced a temporary 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 relative to the 10 mM glucose in the 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 the addition, the serum with added glucose was stirred for 3-4 seconds, which caused a temporary 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 relative to the 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 the addition, the serum with added glucose was stirred for 3-4 seconds, which caused a temporary 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 relative to the 20 mM glucose in the serum.

[0304] Example 10.7 - Sensation of 0.11 mM ascorbic acid in serum After the current stabilized in Example 10.6, 22 μl of the ascorbic acid aqueous 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 serum with 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 163 nA was obtained for the sum of 20 mM glucose and 0.11 mM ascorbic acid in the serum.

[0305] Example 10.8 - Sensation of 0.17 mM acetaminophen in serum After the current stabilized in Example 10.7, 34 μl of the acetaminophen aqueous 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 serum with added 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 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 - Detection of 13.9 mM maltose in serum After the current stabilized in Example 10.8, 278 μl of the maltose aqueous 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 serum with added maltose 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 value represents and corresponds to the concentration of glucose in the serum. Similar experiments are performed many more times using the same and other glucose concentrations with similarly prepared glucose sensing systems to obtain data on current values ​​and corresponding glucose concentrations. The correlation between serum glucose concentration and current value 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 layer Example 11.1 - Electroplating from reverse micelle phase This disclosure thereby incorporates the examples and descriptions of U.S. Patent No. 8,343,690 ('690 Patent) in their entirety herein. The experiments appearing in paragraphs 6-9 of '690 Patent are specifically incorporated herein as examples for fabricating a nanoporous layer by electroplating and using the layer for glucose sensing.

[0309] Example 11.2 - Electroplating from Hexagonal Phase This disclosure thereby incorporates the disclosure of U.S. Patent No. 7,892,415 ('415 Patent) in its entirety herein. The experiments described in paragraphs 5-6 of '415 Patent are specifically incorporated herein as an example of fabricating a hexagonal nanoporous layer by electroplating and using the layer for glucose sensing.

[0310] Example 11.3 - Electroplating from a hexagonal phase This disclosure thereby incorporates the disclosure in its entirety as “Electrochemistry Communications, Vol. 4, No. 8, August 2002, pp. 610–612.”

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

[0312] Maltose-blocking layer preparation Example 12. Preparation of 1-mPD aqueous solution M-phenylenediamine (mPD), purchased from Sigma-Aldrich, was dissolved in purified water to provide aqueous solutions of mPD containing mPD at concentrations of 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 CHI Multi 1030C electrochemical analyzer manufactured by CH Instruments Inc. as the potentiostat 104, with 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 2-electrode system.

[0314] Example 12.3 - Electrochemical polymerization at 0.1 mM and 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 of +0.5V to +1.0V with a scanning speed of 10mV / sec 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 and 100 mV / sec Example 12.3 was repeated, except that the scanning speed was set to 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0316] Example 12. Electrochemical polymerization at 0.1 mM and 200 mV / sec. Example 12.3 was repeated, except that the scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0317] Example 12.6 - Electrochemical polymerization at 0.3 mM and 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 and 100 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0319] Example 12.8-0.3 mM, electrochemical polymerization at 200 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0320] Example 12.9 - Electrochemical polymerization at 0.5 mM and 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 12.10 - Electrochemical polymerization at 0.5 mM and 100 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 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 scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117.

[0323] Example 12.12 - Electrochemical polymerization at 1.0 mM and 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 shown in Figure 23 was prepared for chronoamperometry using the poly-mPD layer prepared in Example 12.12 as the porous polymer layer 302, and a 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 of +0.0 V to +1.0 V with a pulse width of 1.0 second.

[0325] Example 12.14 - Electrochemical polymerization and electric shock at 1.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0326] Example 12.15 - Electrochemical polymerization and electric shock at 1.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0327] Example 12.16 - Electrochemical polymerization and electric shock 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. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0328] Example 12.17 - Electrochemical polymerization and electric shock at 2.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0329] Example 12.18 - Electrochemical polymerization and electric shock at 2.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0330] Example 12.19 - Electrochemical polymerization and electric shock 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. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0331] Example 12.20 - Electrochemical polymerization and electric shock at 5.0 mM, 100 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 100 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0332] Example 12.21 - Electrochemical polymerization and electric shock at 5.0 mM, 200 mV / sec Example 12.6 was repeated, except that the scanning speed was set to 200 mV / sec, thereby forming a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0333] Sensation of glucose without interference by maltose Example 13.1 - Serum preparation Human serum was purchased from Sigma-Aldrich. The glucose content in the serum was measured using 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, and the serum temperature was maintained at 37°C. The electrochemical cell was prepared in the same manner as in Example 10.2, except that the working electrode 103 contained 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 scanning speed 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 a nanoporous layer prepared in Example 12.3 (using a 0.1 mM mPD solution and a scanning speed of 10 mV / sec), and further, the working electrode, reference electrode, and counter electrode were immersed in serum.

[0336] Example 13.2 - Conditioning of a glucose-sensing system in serum In the electrochemical cell system prepared in Example 13.1, a bias voltage of 0.4V 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. After that, the bias voltage was disconnected from the system and reconnected. Upon reapplying the bias voltage, measurement of the current from the working electrode was started. The electrochemical cell was maintained to further condition the glucose sensing system in serum. When the current stabilized, a current value of 96nA was measured for 5.8mM glucose originally present in the serum.

[0337] Example 13.3 Electrode with a maltose-blocking layer (0.1 mM, 10 mV / sec) In the system prepared in Example 13.2, a glucose stock solution prepared in the same manner as in Example 9.1 was added to serum to produce serum containing a total glucose concentration of 10 mM. Subsequently, the glucose stock solution was further added to produce serum containing total glucose concentrations of 15 mM and 20 mM (with a certain time interval between additions). Then, an aqueous ascorbic acid solution prepared in Example 9.1 was added to the serum to produce serum containing 0.11 mM ascorbic acid. Next, an aqueous acetaminophen solution prepared in Example 9.1 was added to the resulting serum to produce serum containing 0.17 mM acetaminophen. Finally, an aqueous maltose solution prepared in Example 9.1 was 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 induced a temporary peak in the current. Figure 25 shows the current monitored in this example in red. The change in current was observed in response to the addition of glucose, ascorbic acid (AA), and acetaminophen (AP). However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding this value were observed. Therefore, the maltose-blocking layer in this embodiment effectively blocked maltose but did not interfere with glucose sensing.

[0338] Example 13.4 Electrode with a maltose-blocking layer (0.1 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode 103 contained a maltose-blocking layer prepared in the same manner as in Example 12.4 (using a 0.1 mM mPD solution at a scanning speed of 100 mV / sec). Figure 25 shows the current monitored in this example in green. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0339] Example 13.5 Electrode with a maltose-blocking layer (0.1 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode 103 contained a maltose-blocking layer prepared in the same manner as in Example 12.5 (using a 0.1 mM mPD solution at a scanning speed of 200 mV / sec). Figure 25 shows the current monitored in this example in purple. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0340] Example 13.6 Electrode with a 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 scanning speed of 10 mV / sec). Figure 26 shows the current monitored in this example in red. The change in current was observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0341] Example 13.7 Electrode with a 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 scanning speed of 100 mV / sec). Figure 26 shows the current monitored in this example in green. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0342] Example 13.8 Electrode with a 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 scanning speed of 200 mV / sec). Figure 26 shows the current monitored in this example in purple. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0343] Example 13.9 Electrode with a 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 scanning speed of 10 mV / sec). Figure 27 shows the current monitored in this example in red. The change in current was observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0344] Example 13.10 Electrode with a 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 scanning speed of 100 mV / sec). Figure 27 shows the current monitored in this example in green. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer 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 scanning speed of 200 mV / sec). Figure 27 shows the current monitored in this example in purple. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0346] Example 13.12 Electrode with a maltose-blocking layer (1.0 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.12 (using a 1.0 mM mPD solution at a scanning speed of 10 mV / sec), and further included a maltose-blocking layer subjected to electric shock as in Example 12.13. Figure 28 shows the monitored current in red in this example. Changes in current were observed upon the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0347] Example 13. Electrode with 13-maltose blocking layer (1.0 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.14 (using a 1.0 mM mPD solution at a scanning speed of 100 mV / sec), and further included a maltose-blocking layer with electric shock, as in Example 12.13. Figure 28 shows the monitored current in green in this example. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0348] Example 13.14 Electrode with a maltose-blocking layer (1.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.15 (using a 1.0 mM mPD solution at a scanning speed of 200 mV / sec) and further included a maltose-blocking layer with electric shock. Figure 28 shows the current monitored in this example in purple. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, except for a peak caused by stirring, the current was 5 nA / mMcm. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0349] Example 13.15 Electrode with a maltose-blocking layer (2.0 mM, 10 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.16 (using a 2.0 mM mPD solution at a scanning speed of 10 mV / sec), and further, a maltose-blocking layer with electric shocks was included, as in Example 12.15. Figure 29 shows the monitored current in red in this example. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0350] Example 13.16 Electrode with a maltose-blocking layer (2.0 mM, 100 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.17 (using a 2.0 mM mPD solution at a scanning speed of 100 mV / sec), and further, a maltose-blocking layer with electric shocks was included, as in Example 12.15. Figure 29 shows the monitored current in green in this example. Changes in current were observed upon the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0351] Example 13.17 Electrode with a maltose-blocking layer (2.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.18 (using a 2.0 mM mPD solution at a scanning speed of 200 mV / sec), and further included a maltose-blocking layer with electric shock, as in Example 12.15. Figure 29 shows the current monitored in this example in purple. Changes in current were observed upon the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer 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 in Example 12.19 (using a 5.0 mM mPD solution at a scanning speed of 10 mV / sec), and further, a maltose-blocking layer with electric shocks was included, as in Example 12.15. Figure 30 shows the monitored current in red in this example. Changes in current were observed upon the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer 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 in Example 12.20 (using a 5.0 mM mPD solution at a scanning speed of 100 mV / sec), and further included a maltose-blocking layer with electric shock, as in Example 12.15. Figure 30 shows the monitored current in green in this example. Changes in current were observed with the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0354] Example 13. Electrode with 20-maltose blocking layer (5.0 mM, 200 mV / sec) Examples 13.1-13.3 were repeated, except that the working electrode was prepared in Example 12.21 (using a 5.0 mM mPD solution at a scanning speed of 200 mV / sec), and further included a maltose-blocking layer with electric shock, as in Example 12.15. Figure 30 shows the current monitored in this example in purple. Changes in current were observed upon the addition of glucose, ascorbic acid, and acetaminophen, respectively. However, after the addition of maltose, the current was 5 nA / mMcm, except for a peak caused by stirring. 2 No current changes exceeding a certain threshold were observed. In this embodiment, the maltose-blocking layer effectively blocked maltose but did not interfere with glucose sensing.

[0355] Example 13.21 Electrode with a maltose-blocking layer (1.0 mM, 10 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.12 (using a 1.0 mM mPD solution at a scanning speed of 10 mV / sec) is not subjected to electric shock.

[0356] Example 13.22 Electrode with a maltose-blocking layer (1.0 mM, 100 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.14 (using a 1.0 mM mPD solution at a scanning speed of 100 mV / sec) is not subjected to electric shock.

[0357] Example 13.23 Electrode with a maltose-blocking layer (1.0 mM, 200 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.15 (using a 1.0 mM mPD solution at a scanning speed of 200 mV / sec) is not subjected to electric shock.

[0358] Example 13.24 Electrode with a maltose-blocking layer (2.0 mM, 10 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.16 (using a 2.0 mM mPD solution at a scanning speed of 10 mV / sec) is not subjected to electric shock. No change in current is observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0359] Example 13.25 Electrode with a maltose-blocking layer (2.0 mM, 100 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.17 (using a 2.0 mM mPD solution at a scanning speed of 100 mV / sec) is not subjected to electric shock. No change in current is observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0360] Example 13.26 Electrode with a maltose-blocking layer (2.0 mM, 200 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.18 (using a 2.0 mM mPD solution at a scanning speed of 200 mV / sec) is not subjected to electric shock. No change in current is observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0361] Example 13.27 Electrode with a maltose-blocking layer (5.0 mM, 10 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.19 (using a 5.0 mM mPD solution at a scanning speed of 10 mV / sec) is not subjected to electric shock. No change in current is observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0362] Example 13.28 Electrode with a maltose-blocking layer (5.0 mM, 100 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.20 (using a 5.0 mM mPD solution at a scanning speed of 100 mV / sec) is not subjected to electric shock. No change in current is observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0363] Example 13.29 Electrode with a maltose-blocking layer (5.0 mM, 200 mV / sec) Repeat Example 13.12, except that the poly-mPD layer prepared in Example 12.21 (using a 5.0 mM mPD solution at a scanning speed of 200 mV / sec) is not subjected to electric shock. No change in current was observed with each addition of glucose, which means that the poly-mPD layer effectively blocks glucose.

[0364] Another electric shock example 14.1-2 pulses of electric shock Repeat Example 12.13, except for two pulses with a pulse width of 0.5 seconds and an interval of 0.5 seconds.

[0365] Example 14.2-2 Electric shock with pulses Repeat Example 14.1, except that each pulse is between +0.0V and +2.0V.

[0366] Example 14.3 - Electric shock with multiple pulses Repeat Example 12.13, except for a series of 10 pulses having a pulse width of 0.1 seconds and an interval of 0.1 seconds between any two pulses.

[0367] Example 14.4 - Electric shock with multiple pulses Repeat Example 14.1, except that each pulse is between +0.0V and +2.0V.

[0368] Example 14.5 - Electric shock at a single incline Repeat Example 12.13, except that the potential gradually increases from +0.0V to +1.0V over a period of 1 second.

[0369] Example 14.6 - Electric shock with multiple inclines Repeat Example 14.5, except that the gradient potential is repeated five times, with an interval of 0.1 between the two gradients.

[0370] Example 14.7 - Electric shock on a single incline Repeat Example 12.13, except that the potential gradually increases from +0.0V to +2.0V over a period of 2 seconds.

[0371] Example 14.8 - Electric shock with multiple inclines Repeat Example 14.7, except that the gradient potential is repeated five times, with an interval of 0.1 between the two gradients.

[0372] Conditioning of 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 (including the platinum nanoporous layer 1609) prepared in Example 8.4, and did not include the electrolyte ion-blocking layer.

[0373] Example 15.2 - Conditioning of the working electrode (without 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 measured continuously immediately after the bias voltage was applied. Figure 42A shows the current profile measured from the electrochemical cell, in which case the working electrode 103 does not include an electrolyte ion-blocking layer. In 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 a magnified view of the profile in Figure 42A, showing that the glucose stock solution prepared in the same manner as in Example 9.1 was successfully added after the working electrode conditioning was complete.

[0374] Example 15.3 - Preparation of a working electrode having a 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 one platinum nanoporous layer 1609 of electrode 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 having the PMMA electrolyte ion-blocking layer prepared in Example 15.1 was used as the working electrode 103.

[0376] Example 15.5 - Conditioning of 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, in which case the working electrode 103 includes an electrolyte ion-blocking layer. The glucose stock solution, prepared in the same manner as in Example 9.1, was successfully added after the working electrode conditioning was complete. The peaks in Figure 43 represent stirring after each addition.

[0377] Example 15.6 - Comparison of Conditioning Times Figure 44 shows the superimposed current profiles of Figure 42 (Example 15.2) and Figure 43 (Example 15.5). The current in Example 15.5 (including the electrolyte ion-blocking layer) settles and stabilizes in approximately 600 seconds, while the current in Example 15.2 (without the electrolyte ion-blocking layer) decreases at a significantly faster rate within the same time frame.

[0378] Example 15.7 - Preparation of a working electrode having a 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 one platinum nanoporous layer 1609 of electrode 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. Preparation of a working electrode having an 8-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 one platinum nanoporous layer 1609 of electrode 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. Furthermore, Example 15.5 was repeated with the prepared electrochemical cell.

[0381] Manufacturing Example 16.1 of a CGM subcutaneous electrode unit - Formation of a conductive layer on the base A polyimide film with a thickness of 150 μm was used as the base substrate 503. A silver layer 1603 was printed onto the polyimide film to obtain silver conductive elements 110C, 110W, and 110R with a thickness of approximately 20 μm in the shape shown in Figure 35. Subsequently, a conductive carbon layer 1605 was printed onto the silver conductive elements 110C and 110W with a thickness of approximately 20 μm. No carbon layer was formed on the silver conductive element 110R.

[0382] Example 16.2 - Arrangement and cutting of the insulating layer 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 would cover the intermediate product shown in Figure 35, while exposing the terminal portion 705. Holes were made in the polyimide film to provide three openings for exposing areas for the working electrode, reference electrode, and counter electrode. The pre-cut polyimide was then placed on the intermediate product of Figure 35 so that the adhesive layer was in contact with the polyimide base 503 to provide the intermediate product of Figure 36. Subsequently, the polyimide insulating layer 707 outside the polyimide base 503 and the conductive element was cut to provide the intermediate product of Figure 37.

[0383] Example 16.3 - Formation of clustered nanoporous layers 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 carbon layer 1605, which was exposed 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, and the intermediate product shown in Figure 38A was obtained.

[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 When a biocompatible layer (pHEMA) is formed on the electrolyte ion-blocking layer 505 as shown in Figure 38B, the non-enzymatic CGM electrode unit shown in Figure 33 is obtained.

[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, the pHEMA biocompatible layer 507 was formed as shown in Figure 38B, and the non-enzymatic CGM electrode unit 701 shown in Figure 33 was obtained.

[0387] CGM Animal Test Example 17.1 - Preparation for CGM Animal Test 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 CGM electrode unit 701 was then connected to UXN It was connected to a UXN potentiostat developed by Co., Ltd. (the applicant of this 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 in Figure 45A. Figure 45C is a photograph showing the UXN potentiostat with its case. The UXN potentiostat includes a wireless module for wireless communication with a computer and can be controlled wirelessly by the computer. A glucose solution was prepared for injection into the veins of rats to induce changes in glucose levels in the rats' blood and interstitial fluid.

[0388] Example 17.2 - Continuous monitoring of glucose levels in rats Subcutaneous implantation of the CGM electrode unit 701 was maintained for 5 consecutive days. On day 1, 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 approximately 1.5 hours after the (first) injection each day. Also, small amounts of rat blood were collected from the tail every 2-5 minutes during the approximately 1.5-hour period and applied to a test strip for the Roche Accu-Chek® blood glucose meter. This provided the glucose concentration in the blood.

[0389] Example 17.3 - Rat CGM measurement and blood glucose plotting Figure 46 shows the current from the CGM electrode module measured by the UXN potentiostat in Example 17.2, in blue. The red dots in Figure 46 represent blood glucose concentrations obtained from the Roche Accu Chek® blood glucose meter. Assuming a time lag of approximately 10 minutes between interstitial fluid glucose levels and blood glucose levels, the data was calibrated by shifting the blue signal, which was shifted relative to the red dots, over time. The sharp peak in the blue signal is understood to be mainly due to the movement of the rat's body during measurement. Based on the graph in Figure 45, a strong correlation is considered to exist between blood glucose concentrations measured 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 Figure 47 is the Clark error grid for the non-enzymatic CGM electrode unit 701 prepared in Example 16.6, based on the measurements presented in the graph of Figure 46. The reference sensor for this Clark error grid analysis is the 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 ​​outside 20% of Region A but which would not lead to inappropriate treatment; Region C contains values ​​which would lead to potentially unnecessary treatment; Region D contains values ​​which indicate a potentially dangerous failure to detect hypoglycemia or hyperglycemia; and Region E contains values ​​which would confuse the treatment of hypoglycemia for hyperglycemia, and vice versa. As summarized in the table below the grid, the analysis shows that more than 91% of the points were within Regions A and B.

[0391] combination of features This disclosure provides much discussion and information on many features relating to nanoporous structures and / or glucose sensing technologies. The purpose of this disclosure is to provide many devices, systems, and methods relating to those features. Two or more features disclosed above can be combined to form devices, systems, or methods to the extent that a particular combination can be formed even if that combination is not presented in this disclosure. It is also the purpose of this disclosure to pursue claims directed to many of those features disclosed herein. Some of those features are presented in the form of claims in the following sections. Many claims are presented in reference form by referring to one or more other claims. The applicant notes that some claims referring to multiple claims may contain combinations of features that are inconsistent (hereinafter, “inappropriate combinations”). However, the applicant recognizes that such claims may still contain one or more combinations of features that are not inconsistent (hereinafter, “appropriate combinations”). By presenting claims that may contain both appropriate and inappropriate combinations, the applicant intends to confirm its or the inventor’s possession of appropriate combinations and to provide specific support to appropriate combinations for subsequent claims of appropriate combinations.

Claims

1. A nanoporous layer containing numerous nanoparticles that form an irregularly shaped body, At least a portion of the irregular shape is connected to each other to form a three-dimensional network of interconnected irregular shapes. Inside the three-dimensional network of interconnected irregular shapes, adjacent nanoparticles among the nanoparticles, where no interposing nanoparticles exist between them, define an interparticle nanopore between them. At least a portion of the aforementioned interparticle nanopores have a size in the range of about 0.5 nm to about 3 nm. A nanoporous layer in which the three-dimensional network of interconnected irregular shapes defines spaces between multiple parts of the irregular shapes that are not occupied by the irregular shapes.

2. The interparticle nanopores are distributed throughout almost the entire interior of the irregularly shaped body. The nanoporous layer according to claim 1, wherein the unoccupied space is distributed over substantially the entire nanoporous layer.

3. The nanoporous layer either does not contain organic molecules, or, if it does contain organic molecules, it contains them in an amount less than 0.5 parts by weight per 100 parts by weight of the nanoparticles. The nanoporous layer according to claim 1 or claim 2, wherein the nanoporous layer has a roughness coefficient of about 100 to about 2500.

4. The nanoporous layer according to any one of claims 1 to 3, wherein at least a portion of the nanoparticles have a generally egg-shaped or spherical shape with a length in the range of about 2 nm to about 5 nm.

5. A substrate including a conductive surface, A nanoporous layer according to any one of claims 1 to 4 formed on the conductive surface, A glucose-sensing electrode comprising, A glucose-sensing electrode that does not contain glucose-specific enzymes.

6. The substrate includes a conductive metal layer and a conductive carbon layer formed on the conductive metal layer. The glucose-sensing electrode according to claim 5, wherein the substrate comprises at least one of a conductive material and / or a semiconducting material that provides the conductive surface.

7. When a bias voltage of 0.2 to 0.45 V is applied between the glucose-sensing electrode in contact with the glucose-containing solution and the reference electrode, the glucose-sensing electrode is configured to induce 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 to 20 mM (72 to 360 mg / dL), in a steady state, the glucose-oxidation current is 10 nA / mMcm. ２ A glucose-sensing electrode according to claim 5 or claim 6, which is at a higher level.

8. An electrolyte ion-blocking layer formed on the nanoporous layer, A biocompatible layer formed on the electrolyte ion-blocking layer, Furthermore, it is equipped with, Glucose, Na ＋ , K ＋ , Ca ２＋ , Cl － , PO ４ ３- and CO3 ２－ When contacted with a liquid containing, the electrolyte ion-blocking layer prevents Na ＋ , K ＋ , Ca ２＋ , Cl － , PO ４ ３- and CO ３ ２－ from diffusing towards the nanoporous layer, whereby there is a substantial discontinuity in the total concentration of Na ＋ , K ＋ , Ca ２＋ , Cl － , PO ４ ３- and CO ３ ２－ across both sides of the electrolyte ion-blocking layer. The glucose-sensing electrode according to any one of claims 5 to 7.

9. The glucose-sensing electrode according to claim 8, wherein the total concentration on one side of the electrolyte ion-blocking layer is greater than 0% and less than about 10% of the total concentration on the other side of the electrolyte ion-blocking layer.

10. The electrolyte ion-blocking layer comprises a porous and hydrophobic polymer layer. The polymer layer does not restrict the mobility of glucose molecules passing through the polymer layer, while the Na passing through the polymer layer... ＋ _K ＋ Ca ２＋ , Cl － , PO ４ ３- and CO ３ ２－ A glucose-sensing electrode according to claim 8, configured to limit the mobility of the glucose-sensing electrode.

11. The glucose-sensing electrode according to any one of claims 8 to 10, 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).

12. A single body, A first electrode formed on the single body, including the glucose-sensing electrode described in claim 5, A second electrode is formed on the single body and configured to contact the liquid when the first electrode comes into contact with the liquid, A glucose sensing device equipped with the following features.

13. The device according to claim 12, wherein the nanoparticles of the nanoporous layer include 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 these metals.

14. To provide the device according to claim 12 or claim 13, The method involves applying a bias voltage between the first electrode and the second electrode while the test fluid is in contact with both the first electrode and the second electrode, thereby causing oxidation of the glucose contained in the test fluid at the glucose sensing electrode. Measuring the current flowing from the first electrode, The measured value of the current is processed with or without additional data to provide the glucose level corresponding to the glucose contained in the test fluid. A non-enzymatic glucose sensing method, including...

15. A method for producing colloids, To provide a liquid composition containing a surfactant and a metal ion, wherein the surfactant is in an inverse micelle phase that defines a plurality of hydrophilic spaces containing at least a portion of the metal ion, The present invention provides a first colloid by adding a reducing agent to the liquid composition to induce the reduction of at least some of the metal ions, thereby forming nanoparticles, wherein no potential is applied for the reduction of at least some of the metal ions. To remove at least a portion of the surfactant from the first colloid and provide a second colloid containing many irregularly shaped clusters of nanoparticles dispersed in a liquid, Includes, A method wherein the irregularly shaped cluster comprises nanoparticles having a generally egg-shaped or spherical shape with a length in the range of about 2 nm to about 5 nm.

16. Some molecules of the surfactant bind to the nanoparticles in the first colloid. The method according to claim 15, further comprising removing the surfactant by adding an acid or base to the first colloid to separate at least a portion of the molecules from the nanoparticles.

17. The method according to claim 15, further comprising, after removing the surfactant, adjusting the concentration of the nanoparticles in the second colloid to provide a colloidal composition.

18. A method for manufacturing a nanoporous layer, To provide a colloidal composition containing many irregularly shaped clusters dispersed in a liquid, wherein the irregularly shaped clusters include nanoparticles having a generally egg-shaped or spherical shape with a length in the range of about 2 nm to about 5 nm, Distributing the colloidal composition onto a substrate, The distributed colloidal composition is dried to form a nanoporous layer, Includes, No potential is applied to the liquid composition in order to form the aforementioned nanoporous layer. When the distributed colloidal composition is dried, the irregularly shaped clusters contained in the distributed colloidal composition are deposited on the substrate, and adjacent clusters of the irregularly shaped clusters are in contact with each other and further in contact with other clusters of the irregularly shaped clusters, thereby forming an irregularly shaped body comprising the nanoparticles, wherein the irregularly shaped unoccupied spaces are defined between at least a portion of the irregularly shaped body. A method wherein, inside the irregularly shaped body, interparticle nanopores are defined between adjacent particles of the nanoparticles, where no interposing nanoparticles exist between the adjacent particles.

19. The method according to claim 18, wherein the nanoporous layer does not contain organic molecules, or if it does contain organic molecules, it does so in an amount less than 0.5 parts by weight per 100 parts by weight of the nanoparticles.

20. The method according to claim 18 or claim 19, wherein at least a portion of the interparticle nanopores have a size in the range of about 0.5 nm to about 3 nm.

21. The nanoporous layer according to claim 1, wherein at least a portion of the unoccupied space is interconnected to form a three-dimensional network of interconnected, irregularly shaped spaces.

22. The nanoporous layer according to claim 21, wherein the three-dimensional network of interconnected irregularly shaped spaces includes a size in the range of 100 nm to 500 nm.

Images