Sensors for assessing target analytes in liquid beverage samples

Abstract

The present invention relates to a dry sensor for determining the concentration of an analyte in a liquid beverage sample. A novel dry sensor is described herein that is capable of receiving a liquid sample and adjusting the pH of the liquid to a level suitable for determining a target analyte without adding reagents to the sample and / or performing manual timing operations, and the sensor is capable of detecting a redox reaction in the presence of a liquid sample. When connected to the sensor disclosed herein, the meter disclosed herein is capable of adjusting the temperature of the liquid to a level suitable for determination, applying a series of potentials, measuring current at several time points, determining the diffusion coefficient of a limiting electrochemical species, calculating the concentration of one or more analytes, and quickly providing the user with the required liquid sample information.

Description

Technical Field

[0001] The present invention generally relates to a dry sensor for determining the concentration of an analyte in a liquid beverage sample, wherein the dry sensor is capable of receiving a liquid sample and adjusting the pH value of the liquid to a level suitable for determining the target analyte. Background Art

[0002] It is important to measure the concentration of analytes at various time points during the production and storage of beverages. The composition of beverages must be carefully monitored and controlled throughout the production and storage process so that the final product is tasty and free of microbial contamination. To achieve this, the concentration of preservatives, flavorings, etc. needs to be monitored and controlled at various stages of the process.

[0003] Sulfur dioxide (SO2) is an important preservative in the wine industry and its concentration must be strictly controlled. If the concentration of SO2 is too low, microbial growth will cause the wine to deteriorate. But if the concentration of SO2 is too high, the taste of the wine will be affected.

[0004] To date, the most reliable method for determining SO2 (aeration oxidation) requires acidification of the wine with phosphoric acid (to convert dissolved sulfite ions into SO2 gas), bubbling air through the acidified solution (thus leaving behind potential interfering substances), transferring the SO2 gas to a separate solution containing hydrogen peroxide (to oxidize the SO2 into sulfuric acid), and finally titrating the sulfuric acid with a known concentration of sodium hydroxide. These steps can be performed manually in 15 minutes by a trained operator or automatically using laboratory equipment (valued at $200,000).

[0005] Malic acid, glucose and fructose are important flavoring agents in wine. The standard method for measuring these substances involves mixing the wine with a pH buffer, adding one or more enzymes and a chromogenic compound, and then measuring the change in light absorbance with a spectrophotometer.

[0006] These standard procedures require the transportation of wine to a laboratory, the use of expensive specialized equipment that requires maintenance and calibration by trained laboratory personnel, and lengthy manual procedures. The overall results are accurate, but they are expensive and inconvenient, and the vintners may not know the results until hours later.

[0007] Test strips for sulfite are relatively inexpensive but are not accurate because the operator must manually read the intensity of the color (e.g., pink).

[0008] Laboratory electrochemical methods have been developed to determine sulfite (e.g., Caldwell et al. (1991) Analyst 116:253-256), but these methods require polishing of the glassy carbon electrode between measurements. This manual step is time-consuming and can add several minutes to each measurement. In addition, the polishing method requires specialized skills and is not suitable for thin-film electrodes.

[0009] The present invention can quickly and accurately measure several critical parameters of wine by using disposable test strip sensors. The present invention does not require the wine to be transported to a specialized laboratory with trained personnel, nor does it require equipment calibration and maintenance. In addition, no liquid waste is generated during the operation of the sensor. Summary of the invention

[0010] The present invention relates to methods and sensors for analyzing beverage samples (especially fermented beverages such as wine, spirits, kombucha, kefir or beer, or milk, buttermilk or juice, etc.) to determine the presence of target analytes. The target analytes include but are not limited to free sulfur dioxide, total sugar, glucose, fructose, malic acid, etc.

[0011] The present invention specifically refers to a sensor for determining the concentration of free sulfur dioxide, total sugar, glucose, fructose, and malic acid, but it should be understood that the sensor is not limited to this specific use and is suitable for other analytical determinations.

[0012] Embodiments of the present invention relate to a dry sensor for analyzing a liquid beverage sample (e.g., wine, spirits, kombucha, kefir, beer, milk, buttermilk, or juice, etc.). In certain embodiments, the apparatus and method disclosed herein include a disposable test strip sensor (hereinafter referred to as a "sensor") and a meter. In certain embodiments, the sensor is a dry sensor that can adjust the pH value of the liquid sample to a level suitable for measuring the target analyte.

[0013] In certain embodiments, the present invention further comprises a meter, wherein the meter is capable of adjusting the temperature of the liquid sample to a level suitable for measuring the analyte. In certain embodiments, the meter is capable of applying a series of potentials, measuring the current-time relationship, removing background signals, determining the diffusion coefficient of the limiting electrochemical species, calculating the concentration of one or more analytes, and providing the user with the desired liquid sample information without generating liquid waste.

[0014] In certain embodiments, the present invention is a system including a sensor and a meter, wherein the system operates over a range of ambient temperatures, solution pH values, and solution viscosities.

[0015] In certain embodiments, the present invention is a sensor for evaluating a target analyte in a liquid beverage sample, wherein the sensor comprises a first electrode, a second electrode and a counter surface. In certain embodiments, the counter surface may comprise the second electrode. Thus, certain embodiments relate to a counter electrode sensor, i.e., a first electrode and a second electrode that are opposite to each other. In certain embodiments, the counter surface is a smooth counter surface. For example, in certain embodiments, the electrodes are coplanar electrodes.

[0016] In an embodiment of the present invention, the first electrode is coated with a reagent containing electroactive particles, and the opposing surface is coated with a pH modifier, which is capable of adjusting the pH value of the liquid sample to a level suitable for determining the target analyte, wherein the sensor detects a redox reaction when a liquid beverage sample is present.

[0017] In some embodiments, the facing surface is coated with a reagent capable of changing the pH value of the liquid sample, which is referred to herein as a "pH modifier". In some embodiments, the desired pH value is between 1 and 3, such as SO2. In some embodiments, the desired pH value is between 4 and 5, 6 and 9, or other pH ranges, and the desired pH value is suitable for using a desired chemical reagent when the sensor is used for measurement.

[0018] In some embodiments, the pH modifier is an acidic reagent, such as oxalic acid or other acids, which are solid acids within the specified operating and storage temperature range of the sensor. Suitable examples of acids include oxalic acid, citraconic acid, waxy acid, camphorsulfonic acid, and other acids with suitable pH values ​​and stability within the operating and storage range of the sensor. In some embodiments, the pH modifier is a mixture of two or more acids. In some embodiments, the pH modifier is a mixture of oxalic acid and citraconic acid.

[0019] In some embodiments, the pH modifier is an alkaline agent, such as sodium hydroxide or other bases, and the other bases are solid bases within the specified operating and storage temperature range of the sensor. Suitable examples of bases include sodium hydroxide, potassium hydroxide, potassium phosphate, tris (hydroxymethyl) aminomethane, N-[tris (hydroxymethyl) methyl] -3-aminopropane sulfonic acid, N-cyclohexyl-3-aminopropane sulfonic acid, 4- (2-hydroxyethyl) -1-piperazineethane sulfonic acid, pyrophosphate and other bases, which have suitable pH values ​​and stability under the working and storage conditions of the sensor. In some embodiments, the base is a mixture of N-[tris (hydroxymethyl) methyl] -3-aminopropane sulfonic acid and sodium hydroxide. In some embodiments, a concentrated buffer is used. In some embodiments, the pH modifier includes a suitable buffer and a suitable base, or a combination or mixture thereof.

[0020] In some embodiments, the first electrode is coated with a dry reagent containing an electroactive species. In some embodiments, the electroactive species is nicotinamide adenine dinucleotide, potassium ferrocyanide, potassium ferrocyanide, ferric ethylenediaminetetraacetate, phenazine ethyl sulfate, 1,2-naphthoquinone-4-sulfonic acid, or a combination or mixture thereof. In some embodiments, the electroactive species is potassium ferrocyanide. In some embodiments, the electroactive species is potassium ferrocyanide.

[0021] In some embodiments, the target analyte is glucose, fructose and malic acid, and the electroactive species is ferrocyanide or ethylenediaminetetraacetic acid ferric. In some embodiments, the target analyte is SO2, and the electroactive species is ferrocyanide.

[0022] In certain embodiments, the sensor further comprises an enzyme, such as but not limited to malate dehydrogenase, glutamate oxaloacetate transaminase, aspartate transaminase, diaphorase, or a combination or mixture thereof. In certain embodiments, the sensor further comprises fructose dehydrogenase.

[0023] In some embodiments, the sensor further comprises a glucose-selective enzyme, such as glucose dehydrogenase or glucose oxidase. In some embodiments, the first electrode is coated with potassium ferrocyanide, and the sensor further comprises a glucose-selective enzyme, such as GDH-PQQ, FAD-GDH or GOD (GDH stands for glucose dehydrogenase; PQQ stands for pyrroloquinoline quinone; FAD stands for flavin adenine dinucleotide, and GOD stands for glucose oxidoreductase).

[0024] In an exemplary embodiment, the target analyte is SO2. In certain embodiments, the pH modifier is a mixture of N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid and sodium hydroxide, and the electroactive species is potassium ferrocyanide. In certain embodiments, the SO2 reacts directly with the electrode.

[0025] In an exemplary embodiment, the target analyte is glucose. In certain embodiments, the electroactive species is potassium ferrocyanide. In certain embodiments, the reagent further comprises a glucose selective enzyme. In certain embodiments, the reagent further comprises FAD-GAH. In certain embodiments, the reagent further comprises GOD. In certain embodiments, the pH modifier comprises N-[tris(hydroxymethyl)methyl]-3-aminopropane sulfonic acid.

[0026] In an exemplary embodiment, the target analyte is fructose. In certain embodiments, the electroactive species is potassium ferrocyanide, and the pH modifier includes N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid.

[0027] In an exemplary embodiment, the target analyte is malic acid. In certain embodiments, the electroactive species is ferric ethylenediaminetetraacetate, phenazine ethyl sulfate and / or nicotinamide adenine dinucleotide, and the pH modifier is a mixture of N-[tris(hydroxymethyl)methyl]-3-aminopropane sulfonic acid and sodium hydroxide.

[0028] In certain embodiments, the viscosity effect is corrected by the system and / or algorithm forming part of the present invention. Thus, in certain embodiments, it is not necessary to control the viscosity within a certain range.

[0029] In some embodiments, it is necessary to control the temperature of the test strip, for example, by heating the test strip. In some embodiments, the meter heats the test strip by passing an electric current through a resistive element.

[0030] In some embodiments, the desired temperature is about 5°C-45°C. In some embodiments, the desired temperature is about 10°C-45°C. In some embodiments, the desired temperature is about 15°C-45°C. In some embodiments, the desired temperature is about 20°C-45°C. In some embodiments, the desired temperature is about 25°C-45°C. In some embodiments, the desired temperature is about 30°C-45°C. In some embodiments, the desired temperature is about 35°C-45°C. In some embodiments, the desired temperature is about 40°C-45°C.

[0031] Other embodiments of the present invention relate to methods of using the sensors and test strips disclosed herein.

[0032] In the present invention, the sensor is electrically connected to the meter. In certain embodiments, the meter applies a predetermined voltage or current to the test strip and then measures the resulting current or voltage. Those skilled in the art will appreciate that these methods are referred to as amperometric titration or constant current electrochemical techniques, respectively.

[0033] In certain embodiments, the applied pulse sequence comprises a series of predetermined electrical potentials over a predetermined period of time.

[0034] In some embodiments, pulse voltammetry is used. In some embodiments, the instrument slowly increases the potential from about 0 V to about 1 V and applies a rectangular wave or sinusoidal pulse at the top of the ramp. In some embodiments, the amplitude of the applied pulse is about 1 mV-100 mV and the frequency is about 1 Hz-1000 Hz. In some embodiments, a DC chronopotentiometry is used, and the instrument applies a constant potential of about 50 mV-about 900 mV and lasts for about 0.1 seconds to about 1000 seconds.

[0035] In some embodiments, the meter is capable of adjusting the temperature of the liquid sample to a degree suitable for measurement. In some embodiments, the meter calculates, records, displays and / or transmits the results of the reaction. For example, in some embodiments, the current-time relationship is measured. In some embodiments, the meter measures the current-time relationship. In some embodiments, the measured value is corrected for the background signal, which improves the reading accuracy (reduces the error). In some embodiments, the meter removes the background signal. In some embodiments, the meter determines the diffusion coefficient of the limiting electrochemical species. In some embodiments, the meter calculates the concentration of one or more analytes. In some embodiments, the meter calculates the concentration of the target analyte. In some embodiments, the meter displays information to the user. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of an exemplary sensor with counter electrodes.

[0037] Figure 2 yes Figure 1 An exploded view of an exemplary test strip is shown.

[0038] Figure 3 is a graph showing free sulphur dioxide measured by an exemplary sensor compared to reference results obtained by the Australian Wine Research Institute using a Thermo Gallery reference analyser, n=471 wines.

[0039] Figure 4a shows the use of Figure 3 The sugar versus diffusion coefficient (D) data are plotted after fitting with a quadratic curve.

[0040] FIG. 4b is a plot showing the total concentration of sugars (sucrose + fructose + glucose).

[0041] Figure 5 is a plot showing the glucose concentration of two (2) wines spiked with various concentrations of glucose.

[0042] Figure 6 is a plot showing 12 red wines and 12 white wines spiked with various concentrations of glucose and tested on test strips.

[0043] Figure 7 is a plot showing white wines spiked with various concentrations of fructose and tested on a test strip.

[0044] Figure 8 is a plot showing 3 red wines spiked with various concentrations of malic acid and tested on test strips. DETAILED DESCRIPTION

[0045] The present invention adopts electrochemical sensor, and is used and produced by OneTouch ® Verio ® Electrochemical sensors can be mass-produced using methods similar to those used in glucose sensors (from LifeScan) and the Xprecia StrideTM blood coagulation sensor (from Siemens Healthineers).

[0046] As used herein, the term "pH modifying agent" or "pH modifier" refers to an agent that changes the concentration of protons and hydroxide ions present in a solution when mixed into an aqueous solution. For example, a pH modifier is a reagent that can release protons to a liquid sample (to lower the pH) or receive protons from a liquid sample (to increase the pH). Exemplary pH modifiers include, but are not limited to, acids (such as oxalic acid, citraconic acid, mesaconic acid, camphorsulfonic acid, and other acids with suitable pH values ​​and stability under the operating and storage conditions of the sensor) and bases, such as sodium hydroxide, potassium hydroxide, potassium phosphate, tris(hydroxymethyl)aminomethane, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, N-cyclohexyl-3-aminopropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pyrophosphates, and other bases with suitable pH values ​​and stability under the operating and storage conditions of the sensor.

[0047] As used herein, the term "electroactive species" refers to a species that can receive electrons or release electrons to an electrode. Electron transfer may occur between the electroactive species and an electrode, an analyte, an enzyme, an enzyme cofactor (coenzyme), or a secondary mediator. Exemplary electroactive species used herein include, but are not limited to, nicotinamide adenine dinucleotide, potassium ferrocyanide, potassium ferrocyanide, ferric ethylenediaminetetraacetate, phenazine ethyl sulfate, 1,2-naphthoquinone-4-sulfonic acid, or a combination or mixture thereof. Exemplary embodiments of the present invention are discussed in detail below. Although specific exemplary embodiments are discussed, it should be understood that their purpose is limited to illustration. Those skilled in the relevant art should recognize that other components and configurations may be used without departing from the spirit and scope of the present invention. References to "one embodiment", "embodiment", "exemplary embodiment", "various embodiments", etc. may indicate that the embodiments of the present invention described in this manner may include specific features, structures, or characteristics, but not every embodiment necessarily includes the specific features, structures, or characteristics. Further, repeated use of the phrase "in one embodiment" or "in an exemplary embodiment" does not necessarily refer to the same embodiment (although it may refer to the same embodiment).

[0048] The present invention relates to a sensor comprising at least two electrodes (e.g., electrodes that can be used as working electrodes, counter electrodes, or both during an assay); none, one or more enzymes; none, one or more electrochemical mediators; and none, one or more electroactive species that can complete an electrochemical circuit by giving up or receiving electrons at the sensor electrodes. In certain embodiments, one or more reagents are coated on one or more electrodes. As described below, these sensors can be used by adding a sample to the sensor. The electroactive species can be detected by a redox reaction at the electrodes.

[0049] The present invention has a wide range of uses, for example, a method for determining preservatives and flavoring agents (such as free sulfur dioxide, total sugar, glucose, fructose, malic acid, etc.) in fermented beverages (such as wine, spirits, kombucha, kefir or beer) or beverages (such as milk, buttermilk or juice, etc.).

[0050] In an embodiment of the present invention, the electrode is composed of a polyethylene terephthalate (PET) substrate sputtered with a thin layer of an inert conductive material, as is known in the art. Other substrate materials may also be used, but PET is particularly preferred because it is very stable under sensor operating and storage conditions and has a low thermal expansion coefficient. Examples of suitable inert conductive materials include platinum, palladium, iridium, indium oxide, tin oxide, a mixture of indium oxide and tin oxide, carbon and gold. In certain embodiments, gold is used as an inert conductive material. In certain embodiments, in order to extend the shelf life of the sensor, the inert conductive material is coated with a hydrophilic thiol. An example of a hydrophilic coating is provided in U.S. Patent No. 6,652,734, "Sensor with Improved Shelf Life", the disclosure of which is incorporated herein by reference in its entirety.

[0051] In certain embodiments, the sensor disclosed herein comprises counter electrodes, i.e., the electrodes face each other. In other embodiments, the sensor comprises coplanar electrodes. The advantage of the counter electrodes is that the electrode configuration facilitates the determination of the diffusion coefficient of the limiting electrochemical mediator, thereby correcting for the partial effects of viscosity and / or temperature changes on the sensor response in different determinations. This technology is described in International Publication No. WO99 / 09404 "Automatic Analysis Method and Apparatus", the disclosure of which is incorporated herein by reference in its entirety.

[0052] In embodiments using coplanar electrodes, it is advantageous that the sensor further includes a surface opposite to the surface on which the coplanar electrodes are placed. The surface on which the coplanar electrodes are placed is hereinafter referred to as the coplanar electrode surface, and the opposing surface is hereinafter referred to as the smooth opposing surface. It is advantageous that during the test, the positioning of the smooth opposing surface allows any reagent that can be applied to the smooth opposing surface and dissolved in the test solution to be mixed by diffusion through the depth of the solution layer above the coplanar electrode surface. It is only necessary to keep the smooth opposing surface at an appropriate distance from the coplanar electrode surface. An example of an appropriate distance range between the coplanar electrode surface and the smooth opposing surface is 5 microns to 500 microns, more preferably 20 microns to 200 microns, and most preferably 50 microns to 150 microns.

[0053] In some embodiments, the first electrode includes a dry reagent, the dry reagent including an electroactive species and one or more additional reagents, such as an enzyme that selectively reacts with the target analyte. For example, in some embodiments, glucose can be measured using a glucose selective enzyme and potassium ferrocyanide (e.g., see U.S. Pat. No. 9,632,054, "System and Method for High Precision Analyte Measurement", which is incorporated herein by reference in its entirety).

[0054] In certain embodiments, the second electrode or the smooth opposing surface may be optionally provided with an acidic or alkaline dry reagent. As is known in the art, the acidic or alkaline reagent may be a strong acid or a strong base (e.g., oxalic acid or sodium hydroxide, respectively), or may contain a compound having a buffering capacity within a desired pH range. These dry reagents need to be solid within the specified operating and storage temperature range of the sensor. This reagent is intended to change the pH value of the liquid sample so that the pH value of the liquid sample remains within a suitable range in which a reaction is expected to occur during the assay. For example, many enzymes are most active when approaching a neutral pH value, and are less active when in low pH conditions in many beverages (e.g., wine, spirits, kombucha, kefir, beer, milk, buttermilk, or juice, etc.).

[0055] In some embodiments, the two electrodes or the coplanar electrode surfaces and the smooth opposing surface are separated by a layer of double-sided tape with an adhesive side (referred to herein as double-sided adhesive tape), and a section is cut from the double-sided adhesive tape to define at least one area in the sensor to allow a liquid sample to pass or contain a liquid sample, or to exhaust gas from the sensor when a liquid sample is added. In some embodiments, the adhesive layer can be composed of a hot melt adhesive or a pressure sensitive adhesive. The former (i.e., hot melt adhesive) is advantageous because the hot melt adhesive is less viscous at ambient temperature and is less likely to transfer to the surface of the cutting tool during the sensor production process.

[0056] In some embodiments, the channels in the double-sided adhesive tape cover the dried reagents onto the electrodes and / or the smooth facing surface. In some embodiments, the sidewalls of the double-sided adhesive tape, the electrode surface, and / or the smooth facing surface define a chamber into which the liquid to be analyzed (e.g., wine, liquor, kombucha, kefir, beer, milk, buttermilk, or juice, etc.) will eventually be added.

[0057] In certain embodiments, the sensor disclosed herein comprises counter electrodes, i.e., the electrodes face each other. In other embodiments, the sensor comprises coplanar electrodes. The advantage of the counter electrodes is that the electrode configuration facilitates the determination of the diffusion coefficient of the limiting electrochemical mediator, thereby correcting for the partial effects of viscosity and / or temperature changes on the sensor response in different determinations. This technology is described in International Publication No. WO99 / 09404 "Automatic Analysis Method and Apparatus", the disclosure of which is incorporated herein by reference in its entirety.

[0058] In some embodiments, the first electrode includes a dry reagent that reacts with a specific target analyte. For example, in some embodiments, glucose can be measured using a glucose-selective enzyme and potassium ferrocyanide (see, for example, U.S. Pat. No. 9,632,054, "System and method for high-precision analyte measurement", which is incorporated herein by reference in its entirety).

[0059] In certain embodiments, the second electrode may be an acidic or alkaline dry reagent. As is known in the art, the acidic or alkaline reagent may be a strong acid or a strong base (e.g., oxalic acid or sodium hydroxide, respectively), or may contain a compound having a buffering capacity within a desired pH range. These dry reagents need to be solid within the specified operating and storage temperature range of the sensor. This reagent is intended to change the pH value of the liquid sample so that the pH value of the liquid sample remains within a suitable range for the desired reaction during the assay. For example, many enzymes are most active when approaching a neutral pH value, and are less active when in low pH conditions in many beverages (e.g., wine, spirits, kombucha, kefir, beer, milk, buttermilk, or juice, etc.).

[0060] The optimal location of the various reagents described herein can be determined by various factors, including optimizing the assay time and the instability of a particular component at a particular pH value. For example, in certain embodiments, a component of the reagents described herein is dried using a pH modifier, or is dried away from (or opposite to) the pH modifier, depending on the instability of each component at high or low pH values. In addition, in certain embodiments, a slowly diffusing reactant can be dried directly on the working electrode, and a rapidly diffusing reactant can be dried on the counter electrode / reference electrode (or smooth opposing surface) to optimize the assay time.

[0061] In some embodiments, the two electrodes are separated by a layer of double-sided tape with an adhesive surface (referred to herein as double-sided adhesive tape), and a section is cut from the double-sided adhesive tape to define at least one area in the sensor to allow a liquid sample to pass through or contain a liquid sample, or to exhaust gas from the sensor when a liquid sample is added. In some embodiments, the adhesive layer can be composed of a hot melt adhesive or a pressure sensitive adhesive. The former (i.e., hot melt adhesive) is advantageous because the hot melt adhesive is less viscous at ambient temperature and is less likely to transfer to the surface of the cutting tool during the sensor production process.

[0062] In some embodiments, the channels in the double-sided adhesive tape cover the dried reagents onto the electrodes. In some embodiments, the sidewalls of the double-sided adhesive tape and the electrode surface define a chamber into which the liquid to be analyzed (e.g., wine, liquor, kombucha, kefir, beer, milk, buttermilk, or juice, etc.) will eventually be added.

[0063] In some embodiments, it is advantageous to control the temperature of the test strip. Ambient temperature varies widely (e.g., 5-45°C), which affects the electrochemical response. Second, some reactions (e.g., enzyme-catalyzed reactions) are most efficient within a narrow temperature range: if the temperature is too low, the reaction is too slow; but if the temperature is too high, the enzyme will denature and fail. In some embodiments, it is best to heat the test strip to a specific temperature rather than trying to cool the test strip to a set temperature: this is because the heating component is less expensive and requires less energy to reach the desired temperature than the cooling component.

[0064] Figure 1 shows an example of a sensor made with opposing electrodes, Figure 2 It is described in more detail.

[0065] Figure 1A schematic diagram of the sensor is shown. A drop of liquid sample (e.g. wine, liquor, kombucha, kefir, beer, milk, buttermilk or juice) is placed on the indentation (1). Surface tension causes the liquid to flow up the capillary filling channel (2) into the reaction / detection chamber (3) where it is stopped by the "cut-off hole" (4). This allows a limited volume of liquid to react with the dried reagent in the reaction / detection chamber (3). In the rare case that the liquid passes through the cut-off hole, it flows into the "overflow zone" (5). This mechanism prevents liquid in the sensor from contaminating the meter. A cut-out in a portion of the sensor layer (6) allows the meter to contact the upper electrode. Two prongs (7) allow the meter to contact the lower electrode.

[0066] U.S. Patent No. 6,939,312, "Method and apparatus for sampling and analyzing tissue fluid and whole blood samples," describes the surface energy considerations required to ensure that the liquid moves along the filling channel and enters the reaction / detection chamber. The said U.S. Patent is incorporated by reference in its entirety by the present invention and becomes a part of the present invention.

[0067] Figure 2 An exploded view of the five layers in the sensor is shown. In some embodiments, the first (lowest) layer (8) is a "hydrophilic primer layer" made by coating PET with a water-soluble hot melt adhesive (Adhesives Research, JLF-19DEC19-1 or JLF-19DEC19-2 Eastman Kodak).

[0068] In some embodiments, the second layer is an electrode made of gold sputtered PET. The electrode has a perforated portion forming a "recess" (9), a "filling channel" (10), a "cutoff hole" (11) and a "gap" (12). A meter can be connected to the electrode using two prongs (13) on both sides of the gap. In some embodiments, the electrode is coated with a hydrophilic thiol layer. In some embodiments, the electrode area between the filling channel and the cutoff hole is further coated with one or more reagents that can react with the target analyte.

[0069] In some embodiments, the third layer is a double-sided adhesive tape with cutouts formed at several functional locations (including "recesses" (14), "filling channels" (15), "reaction / detection chambers" (16), "cutoff holes" (17), "overflow areas" (18), and "gap" (20)) to allow the electrodes to be connected to a potentiostat, such as a handheld meter (described further below). The two arms of the double-sided adhesive tape (19) on both sides of the gap separate the two electrodes so that they cannot touch each other, thereby preventing a short circuit.

[0070] In some embodiments, the fourth layer is an electrode made of gold sputtered PET. The electrode has a perforated portion forming a "notch" (21) and a "cutoff hole" (22). In some embodiments, the electrode is coated with a hydrophilic thiol layer and further coated with a pH-modifying species, such as oxalic acid, sodium hydroxide or potassium phosphate. In some embodiments, the pH-modifying species is coated in an area corresponding to a "reaction / detection chamber" or in an area corresponding to a "filling channel". The advantages of the latter approach will become apparent when antioxidant calibration is described below.

[0071] The fifth (top) layer is the "top layer" (23), which in some embodiments is made of PET coated with an adhesive. In some embodiments, the fifth layer may optionally include a barcode tape, which may contain information such as the identity of the analyte, production batch number, expiration date, calibration parameters, etc.

[0072] When all five layers are laminated together, they form Figure 1 The complete sensor is shown. In some embodiments, the two "tines" on the left side of the sensor are inserted into a meter (potentiostat). In some embodiments, the meter heats the sensor to the desired temperature. The liquid to be analyzed is placed on the "notch" on the right side of the sensor. The liquid moves along the filling channel (with the help of the hydrophilic bottom layer) and enters the detection chamber, but does not enter the interception hole. The liquid dissolves the reagent and adjusts it to the appropriate pH value, and then reacts with the reagent. The meter applies a sequence of voltage pulses between the upper and lower electrodes. The resulting current-time transient relationship is then analyzed using an algorithm. The analysis includes: determining the diffusion coefficient of the limiting electrochemical species, measuring the background current, removing the background current from the total current, correcting the current divided by the diffusion coefficient, and using calibration parameters to convert the signal to a final value (such as the concentration of a specific analyte).

[0073] The following examples illustrate specific embodiments of the sensor.

[0074] Example

[0075] Example 1. Sulfur dioxide sensor.

[0076] Using a rotary transfer process, kiss-cut the "reaction / detection chamber" and "overflow area" on a double-sided adhesive tape (with a release paper on each side of the tape).

[0077] Using an 8-gauge stainless steel blunt-tip pipette, pipette a solution of 80 mM potassium ferrocyanide + 1 mg / mL Pluronic F87 at 800 μL / min. The function of the ferrocyanide is to "complete the circuit," allowing the ferrocyanide to release electrons to the anode on the other side of the strip as the SO2 receives electrons from the cathode on one side of the strip.

[0078] A stream of ferrocyanide solution is deposited on the electrode, and the electrode is moved at a continuous speed of 10 m / min. The liquid is dried under an infrared bulb. This forms the lower electrode. Double-sided adhesive tape is laminated to the lower electrode so that the reaction / detection chamber overlaps the dried reagent. Using a male and female die set, the filling channels are punched out in the double-layer laminate. At the same time, the "gap" ( Figure 2 is marked as 12).

[0079] As described above, a solution of 4.5 M citraconic acid + 110 mM oxalic acid + 1 mg / mL Pluronic F87 was deposited on the top electrode and dried. In this case, the acid was dried on the area corresponding to the "reaction / detection chamber". However, in some embodiments, the acid can be dried on the area corresponding to the "filling channel". The top electrode was laminated to the double-sided adhesive tape so that the top electrode was facing the bottom electrode. Using a male and female die set, the notches and flow cutouts were punched simultaneously on the three-layer laminate.

[0080] The hydrophilic bottom layer is laminated below the lower electrode and the top layer is laminated above the upper electrode. The five-layer laminate is cut into individual test strips. The test strip is thus manufactured.

[0081] The final test strip size is 8 mm wide and 30 mm long. The area of ​​the bottom working electrode is 0.1128 square centimeters, and the distance between the electrodes is 95 microns. It is obvious to those skilled in the art that other sizes and geometries are also within the scope of the present invention.

[0082] The Australian Wine Research Institute tested a variety of wines using sulphur dioxide test strips. The tests were conducted using a meter that heated the sensor to 32°C. A drop of wine (≥ 6 μl) was loaded onto the notched area of ​​the sensor. After the sample was measured, the specified voltage sequence was applied. This voltage sequence consisted of a 5 second wait and a square wave pulse sequence (40 mV peak to valley, 38.5 Hz) on top of a linear voltage ramp (4 mV increase per cycle). In practice, at the start of each cycle the potential increased by 44 mV, was held constant for 13 milliseconds, then decreased by 40 mV and held constant for another 13 milliseconds. This pulse sequence was loosely based on that used by Caldwell et al. (1991) in Analyst 116:253-256. A square wave was used instead of a sine wave because it is easier to generate in an inexpensive potentiostat. The pulse sequence was followed by a 7 second wait and ended with a 600 mV potential.

[0083] Since the area of ​​the lower electrode is well defined by the various cutting and punching processes used to manufacture the sensor, the lower electrode is the working electrode.

[0084] The current was measured every 1 ms. Each time the potential increased by 44 mV, the current increased to a peak value and then decreased to a stable level. Similarly, each time the potential decreased by 40 mV, the current decreased to a trough value and then increased to a stable level. The target parameter is the difference between the higher stable current and the (previously) lower stable current. This difference is called the "triangular stable current" and is plotted versus potential in the figure (see Figure 5 ).

[0085] Examination of data from 253 red wines and 218 white wines tested on a Thermo Gallery reference analyser showed that the peak height at approximately 600 mV from the triangular steady current correlated with the concentration of free sulphur dioxide. The signal was refined by background subtraction which involved removing the fitted exponential using the triangular steady current at 420 mV and 100 mV beyond the trough of the sulphur dioxide peak.

[0086] This method removes the background signal in each wine from the total signal. The difference results in a signal from sulfites only. This method is beneficial because each wine may have different concentrations of background (interfering) electroactive species such as anthocyanins, polyphenols, and tannins.

[0087] The resulting sulfur dioxide signal is calibrated separately for red and white wines. Figure 3 Calibrated measurements are plotted against Thermo Gallery reference analyser results for all 471 wines trialled by the Australian Wine Research Institute.

[0088] The short sulfite test can be compared to the 10-12 minute test published by Caldwell et al. The rapid test described in the present invention can also be compared to the 15 minute aerated oxidation assay, the gold standard for sulfite assays.

[0089] The effect of interference was also evaluated. Since the sugar concentration of each wine was different (up to 200 g / L), the diffusion coefficient was affected, which further affected the current.

[0090] By using the measured diffusion coefficient in the algorithm, the error caused by the change of sugar concentration is reduced. At the end of the pulse voltammetric sequence, the instrument waits for 7 seconds (all species are randomized so that there is no concentration gradient), and then applies a continuous 600 mV pulse for 3 seconds. The current-time transient relationship is essentially analyzed according to the method disclosed in International Publication No. WO 99 / 09404, which is incorporated by reference in its entirety by this invention.

[0091] 1) Determine the maximum current obtained (shortly after application of the 600 mV constant pulse) as well as the minimum current (MINi; just before the end of the 600 mV pulse).

[0092] 2) Select the 600 mV transient portion between 1.05*MINi and 1.43*MINi. This portion corresponds to the decay range, which is well fitted by a single exponential function (plus an offset).

[0093] 3) Generate a plot of ln(i(t) / MINi-1) versus time.

[0094] 4) Calculate the slope of the relationship graph.

[0095] 5) D = -slope*0.0095 2 / (4π 2 ), where 0.0095 is the distance (cm) between the two electrodes. The unit of D is cm 2 s -1 .

[0096] 6) D multiplied by 10 6 Get more manageable values.

[0097] 7) Divide the previous raw signal (peak current in pulse voltammetry) by D.

[0098] 8) This forms the new raw signal required for the calibration process.

[0099] The improvement in the accuracy of SO2 measurement is as follows: Figure 3 When 220 g / L sugar is added to the wine, the SO2 measurement error is close to -30% (see Figure 3 Bold numbers in the fourth column). However, once the readings are corrected for the measured diffusion coefficient, the worst-case error is only 11%.

[0100] Example 2. Total Sugar Sensor

[0101] use Figure 3 Plot the relationship between sugar concentration and D using the data in the figure, and fit a quadratic curve to each point (see Figure 4A ).

[0102] like Figure 4B As shown, given the measured value of D and the fitted equation, the instrument is able to predict the total concentration of sugars (sucrose + fructose + glucose).

[0103] Total sugars are similar to the "Brix value" (based on density or refractive index) routinely measured by vintners.

[0104] Example 3. Glucose sensor containing GDH-PQQ

[0105] The glucose sensor was made by modifying the above sulfite sensor method. The glucose measurement chemistry and algorithm are derived from U.S. Patent No. 7,291,256 "Mediator-stabilized reagent composition and method for electrochemical analyte measurement", or U.S. Patent No. 8,404,102 "Method and apparatus for rapid electrochemical analysis", or U.S. Patent No. 8,916,040 "System and method for measuring analyte in sample", or U.S. Patent No. 9,632,054 "System and method for high precision analyte measurement", the disclosures of which are incorporated herein by reference in their entirety.

[0106] The lower electrode was striped with 900 mM potassium ferrocyanide and 20 mg / mL FAD-GDH (GDH stands for glucose dehydrogenase; FAD stands for flavin adenine dinucleotide). To adjust the pH of the wine to a range more suitable for GDH, the upper electrode was striped with 200 mM sodium hydroxide.

[0107] The following voltage pulse sequence was used. A voltage of +20 mV was applied for one (1) second (with the upper electrode being the anode), a voltage of +300 mV was applied for three (3) seconds, and finally a voltage of -300 mV was applied for one (1) second. The current was measured every ten (10) milliseconds and averaged over a fifty (50) millisecond period.

[0108] Various concentrations of glucose were added to two wines and tested on test strips. Figure 5 shown.

[0109] In this example, the sodium hydroxide is dried on the area corresponding to the "reaction / detection chamber". However, in some embodiments, the sodium hydroxide can be dried on the area corresponding to the "filling channel". The advantage of the latter method is that the invention described in U.S. Patent No. 8,163,162 "Method and apparatus for analyzing a sample in the presence of interferences" can be used for antioxidant correction, which is incorporated by reference in its entirety into this invention.

[0110] Example 4. Glucose sensor containing GOD

[0111] The glucose sensor was made by modifying the above sulfite sensor method. The glucose measurement chemistry and algorithm are derived from U.S. Patent No. 7,291,256 "Mediator-stabilized reagent composition and method for electrochemical analyte measurement", or U.S. Patent No. 8,404,102 "Method and apparatus for rapid electrochemical analysis", or U.S. Patent No. 8,916,040 "System and method for measuring analyte in sample", or U.S. Patent No. 9,632,054 "System and method for high precision analyte measurement", the disclosures of which are incorporated herein by reference in their entirety.

[0112] The lower electrode was striped using a reagent comprising 0.498 g / L Pluronic P103 (BASF), 0.258 g / L Pluronic F87 (BASF), 0.300 g / L citraconic acid, 10.00 g / L dipotassium citraconic acid, and 15.36 g / L sucrose dissolved in water to form a reagent buffer. 29.63 g / 100 g of potassium ferrocyanide and 112 mg / 100 g of Aspergillus niger Glucose Oxidoreductase (GOD) Reagent Buffer. To adjust the pH of the wine to a range more suitable for GOD-catalyzed glucose oxidation, the upper electrode was striped with 100 mM tris(hydroxymethyl)aminomethane base.

[0113] The following voltage pulse sequence was used. A voltage of +20 mV was applied for one (1) second (with the upper electrode being the anode), a voltage of +300 mV was applied for three (3) seconds, and finally a voltage of -300 mV was applied for ten (10) seconds. The current was measured every ten (10) milliseconds. The current was converted to glucose concentration using the following algorithm:

[0114] [Glucose] = ((-i(5) / 100)^r – a.ippmin / 100)-intercept) / slope

[0115] Where [glucose] is the measured glucose concentration, i(5) is the current 5 seconds after -300 mV is applied to the sensor, ippmin is the minimum current recorded during the application of +300 mV to the sensor, and r, a, intercept, and slope are adjustable parameters selected to optimize the agreement between [glucose] and the known glucose concentration measured using the reference instrument during the calibration experiment ([reference glucose]). The intercept and slope are the y-intercept and slope, respectively, determined by applying a linear least squares regression to the ([reference glucose], [glucose]) data pair. In this algorithm, the term containing i(5) gives a measure of the glucose in the sample, as well as the sample-specific, non-glucose related sensor signal. The term containing ippmin is only a measure of the sample-specific, non-glucose related sensor signal. Removing these two terms results in a correction for the non-glucose portion of the sensor signal.

[0116] Various concentrations of glucose were added to 12 red wines and 12 white wines and tested on test strips. Figure 6 shown.

[0117] Example 5. Fructose sensor

[0118] The fructose sensor can be prepared by referring to the chemical method proposed by Adachi et al. (Bioelectrochemistry (2019) 12:1-9), which is incorporated by reference in its entirety into the present invention. D-fructose dehydrogenase from Gluconobacter (Sigma) can receive electrons from fructose and transfer them to potassium ferrocyanide, thereby providing a simple electrochemical determination method for fructose.

[0119] White wine was spiked with various concentrations of fructose and tested on test strips stripped with 750 mM iron on the lower electrode. 10 uL of each [fructose] spiked wine + 2 uL 300 mM TRIS base + 2 uL 1000 U / mL FDH was tested on the test strips at 32°C.

[0120] The following sequence of voltage pulses was used. A voltage of +20 mV was applied for one (1) second (with the upper electrode being the anode), a voltage of +300 mV was applied for three (3) seconds, and finally a voltage of -300 mV was applied for one hundred (100) seconds. The current was measured every ten (10) milliseconds. The current was converted to fructose concentration using the following algorithm:

[0121] [Fructose] = ((-i(20)^a)-intercept) / slope

[0122] Where [fructose] is the measured fructose concentration, i(20) is the current 20 seconds after -300 mV was applied to the sensor, ippmin is the minimum current recorded during the application of +300 mV to the sensor, and a, intercept, and slope are the adjustable parameters selected to optimize the agreement between [fructose] and the spiked fructose concentration ([spike fructose]). The results are shown in Figure 7 shown.

[0123] Example 6. Malic acid sensor

[0124] Malate dehydrogenase catalyzes the following reaction:

[0125] Malic Acid + NAD + → Oxaloacetate + NADH

[0126] It is difficult to pick up NADH directly at the electrode, and a secondary mediator (such as ferrocyanide or diaphorase) is used to direct electrons from NADH to a secondary mediator (such as FeEDTA), or directly to the anode.

[0127] The addition of glutamate oxaloacetate transaminase (GOT, also known as aspartate transaminase, AST) and glutamate can promote the formation of NADH by catalyzing the transamination of oxaloacetate to aspartate.

[0128] The malic acid sensor was fabricated by making modifications to the above sensor method.

[0129] The lower electrode was striped with a reagent containing 200 mM FeEDTA, 200 mM nicotinamide adenine dinucleotide, and 1.00 g / L Pluronic F87 (BASF) dissolved in water. The upper electrode was striped with a reagent containing 1.00 g / L Pluronic F87 (BASF), 2000 U / mL malate dehydrogenase (Toyobo, MAD-211), 200 U / mL diaphorase (Sigma, D5540), 50 mM [tris(hydroxymethyl)methylamino]propane sulfonic acid, 37.5 mM sodium hydroxide, and 50 mM sucrose dissolved in water to form a reagent buffer.

[0130] The following voltage pulse sequence was used. 0 mV was applied for one (1) second, +300 mV was applied for three (3) seconds (with the upper electrode being the anode), -300 mV was applied for one hundred and eighty (180) seconds, 0 mV was applied for ten (10) seconds, and finally -300 mV was applied for seven (7) seconds. The current was measured every fifty (50) milliseconds. The current was converted to malic acid concentration using the following algorithm.

[0131] [Malic acid] = ((-i(60)^a)-intercept) / slope

[0132] Where [Malic Acid] is the measured malic acid concentration, i(60) is the current 60 seconds after an initial voltage of 0 mV is applied to the sensor, and a, intercept, and slope are adjustable parameters selected to optimize the agreement between [Malic Acid] and a known malic acid concentration determined using a reference instrument during the calibration experiment ([Reference Malate]). In this algorithm, the term containing i(60) gives a measure of the malic acid in the sample, as well as the sample-specific non-malic acid related sensor signal.

[0133] Three red wines with various concentrations of malic acid added were diluted 10-fold using 1 M TAPS buffer + 750 mM NaOH and tested on test strips. Figure 8 shown.

[0134] It will be apparent to those skilled in the art that the present invention can be used to determine many other electrolytes in beverages.

[0135] Although the present invention has been particularly shown and described with reference to certain embodiments thereof, it will be understood by those skilled in the art that these embodiments are exemplary rather than restrictive and that various changes may be made in form and detail without departing from the spirit and scope of the present invention. Therefore, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the claims and their equivalents. Unless otherwise expressly stated, any headings used herein are for organizational purposes only and are not intended to give any division or meaning to this document.

Claims

1. A sensor for evaluating a target analyte in a liquid beverage sample, characterized in that The sensor comprises a first electrode, a second electrode and a facing surface, wherein the facing surface may comprise the second electrode or may be a smooth facing surface, wherein the first electrode and the second electrode are coplanar electrodes or face each other, the two electrodes or the coplanar electrode surfaces and the smooth facing surface are separated by a layer of double-sided tape with an adhesive surface, a section is cut from the double-sided tape to define at least one area in the sensor to allow a liquid sample to pass through or to accommodate a liquid sample; The first electrode is coated with a reagent containing electroactive particles, and the opposing surface is coated with a pH modifier, which can adjust the pH value of the liquid sample to a level suitable for measuring the target analyte, wherein the sensor detects a redox reaction when a liquid beverage sample is present.

2. The sensor according to claim 1, characterized in that The target analyte is free sulfur dioxide, total sugar, glucose, fructose or malic acid.

3. The sensor according to claim 1, characterized in that The liquid sample is wine, liquor, kombucha, kefir, beer, milk, buttermilk or juice.

4. The sensor according to claim 1, characterized in that The pH modifier changes the pH value of the liquid sample to between 1 and 3, or between 4 and 5, or between 6 and 9.

5. The sensor according to claim 1, characterized in that The pH modifier comprises an acid that is a solid acid within the specified operating and storage temperature range of the sensor.

6. The sensor according to claim 5, characterized in that The acid is oxalic acid, citraconic acid, mesaconic acid, camphorsulfonic acid or a mixture of two or more acids.

7. The sensor according to claim 6, characterized in that The acid is a mixture of citraconic acid and oxalic acid.

8. The sensor according to claim 1, characterized in that The pH modifier comprises a base that is a solid base within the specified operating and storage temperature range of the sensor.

9. The sensor according to claim 8, characterized in that The base is selected from the group consisting of sodium hydroxide, potassium hydroxide, potassium phosphate, tris(hydroxymethyl)aminomethane, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, N-cyclohexyl-3-aminopropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and pyrophosphate.

10. The sensor according to claim 9, characterized in that The base is sodium hydroxide.

11. The sensor according to claim 9, characterized in that The base is a mixture of N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid and sodium hydroxide.

12. The sensor according to claim 1, characterized in that The electroactive species include nicotinamide adenine dinucleotide, potassium ferrocyanide, ethylenediaminetetraacetic acid ferric, phenazine ethyl sulfate, 1,2-naphthoquinone-4-sulfonic acid or a combination or mixture thereof.

13. The sensor according to claim 12, characterized in that The electroactive species includes potassium ferrocyanide.

14. The sensor according to claim 13, characterized in that The target analyte reacts directly with the electrode, and the electroactive species includes potassium ferrocyanide.

15. The sensor according to claim 1, characterized in that The first electrode or the counter surface is further coated with a glucose selective enzyme.

16. The sensor according to claim 15, characterized in that The glucose selective enzyme is glucose dehydrogenase or glucose oxidase.

17. The sensor according to claim 16, characterized in that The first electrode is coated with potassium ferrocyanide and a glucose-selective enzyme.

18. The sensor according to claim 16, characterized in that The first electrode or the opposite surface is coated with glucose dehydrogenase-pyrrole-quinoline-quinone, ie, GDH-PQQ, flavin adenine dinucleotide-glucose dehydrogenase, ie, FAD-GDH, or glucose oxidoreductase, ie, GOD.

19. The sensor according to claim 18, characterized in that The first electrode or the facing surface is coated with FAD-GDH.

20. The sensor according to claim 1, characterized in that The first electrode and the second electrode are coated on respective supports and separated by an insulating isolation layer.

21. The sensor according to claim 1, characterized in that Also included is a meter connected to the sensor.

22. The sensor according to claim 21, characterized in that The meter applies a predetermined voltage or current to the test strip and measures the resulting current or voltage.

23. The sensor according to claim 21, characterized in that The meter is capable of adjusting the temperature of a liquid sample to a level suitable for measurement.

24. The sensor according to claim 21, characterized in that The instrument is able to determine the current-time relationship.

25. The sensor according to claim 21, characterized in that The instrument is capable of removing background signals.

26. The sensor according to claim 21, characterized in that The meter is capable of calculating the concentration of the target analyte.

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