Disposable electrochemical biosensor based upon NAD(p)-dependent dehydrogenase and diaphorase

Abstract

To solve the problem that an alcohol sensor is not practical when used for a disposable sensor, and disadvantages thereof include sensor storage stability, response sensitivity, a concentration range, and the number of usable reduction-type redox mediators.SOLUTION: There is provided a disposable biosensor that has: a detection surface at least having a working electrode part and a reference electrode part; a first reagent which includes NAD(P)-dependent dehydrogenase, NAD(P)+, diaphorase, and an oxidized redox mediator, and is arranged on the working electrode part to form a working electrode; and a reference electrode material which is arranged on the reference electrode part to form a reference electrode.SELECTED DRAWING: Figure 7

Description

Detailed Description of the Invention

[0001] [Background of the invention] 1. Field of the Invention The present invention relates generally to electrochemical biosensors, and more particularly to electrochemical biosensors that use dehydrogenases as catalysts to quantify various analytes.

[0002] 2. Description of the Prior Art There are many electrochemical sensors that measure a variety of analytes, including but not limited to glucose, glutamate, lactate, cholesterol, d-hydroxybutyrate, glycerol, malate, leucine, alcohol, etc. Some electrochemical sensors are designed for liquid measurement and some are designed for gas measurement.

[0003] Those designed for liquid measurements typically include a working electrode, a reference electrode, and an optional counter electrode, and further involve the use of a respective oxidase as a catalyst to perform the analysis. For example, glucose oxidase is used when measuring glucose, glutamate oxidase is used when measuring glutamate, etc. Additionally, a redox mediator in its reduced state participates in a chemical reaction that changes the redox mediator to its oxidized state to produce a current proportional to the amount of analyte being measured.

[0004] Those designed for gas measurements typically include a working (or sensing) electrode, a counter electrode, and usually a reference electrode. These electrodes are sealed within the sensor housing in contact with the electrolyte. The working electrode is on the inner surface of a Teflon membrane that is permeable to gases but impermeable to the electrolyte. For any gas to be detectable by an electrochemical sensor, the gas must be electrically active. The gas diffuses into the sensor and through the membrane to the working electrode. When the gas reaches the working electrode, either an oxidation or reduction electrochemical reaction occurs, depending on the type of gas. For example, carbon monoxide may be oxidized to carbon dioxide, or oxygen may be reduced to water. The oxidation reaction results in the flow of electrons from the working electrode to the counter electrode through an external circuit.

[0005] Conversely, the reduction reaction results in the flow of electrons from the counter electrode to the working electrode. This electron flow constitutes a current, which is proportional to the concentration of the gas. Electronics within the associated device detect and amplify the current and scale the output according to a calibration. The device then displays the concentration of the gas, for example, in parts per million (ppm) for toxic gas sensors or volume percentages for oxygen sensors. Most alcohols are also electrically active, meaning that during the charge transfer step, either their oxidation state changes or chemical bonds are broken or formed. Considerable research has been conducted in the development of alcohol fuel cells, and some alcohol breath meters (i.e., breathalyzers) used by law enforcement agencies are based on electrochemical sensors.

[0006] Most breathalyzers use either fuel cell or semiconductor oxide sensor technology to measure the blood alcohol concentration (BAC) in a person's blood. Fuel cell sensors rely on an electrochemical process to oxidize the alcohol in a breath sample. The oxidation generates a current that the breathalyzer measures to determine the BAC. The strength of the current corresponds to the amount of alcohol in the sample. Semiconductor oxide sensors use tin oxide material to measure the BAC. Tin dioxide sensors use a heating coil to heat a steel mesh film, which changes resistance when exhaled alcohol comes into contact with the heated film. Sensor Resistance A change in resistance is measured as a change in the output voltage across a fixed or variable resistor. There is a direct relationship between sensor resistance and alcohol concentration. Breathalyzers that use semiconductor sensor technology are less accurate than those that use fuel cell sensors, and they are also less expensive.

[0007] [Summary of the Invention] Little research has been conducted to date to measure alcohol concentration in whole blood. As previously mentioned, the most common alcohol measurement technology has been in the breathalyzer industry. While the use of alcohol dehydrogenase to measure blood alcohol concentration has been mentioned in the prior art, various drawbacks of such sensors make them impractical for use in disposable sensors. These drawbacks include sensor storage stability, response sensitivity, concentration range, and the number of reduced redox mediators available. Sensor storage stability is not long enough for commercial use without more complex storage requirements. Response sensitivity is lower, resulting in reduced accuracy. The lower end of the concentration range is also compromised.

[0008] The present invention relates to a disposable electrochemical biosensor based on NAD(P)-dependent dehydrogenase and diaphorase. Diaphorase is a flavoprotein enzyme capable of oxidizing reduced forms of NAD and NADP, such as diphosphopyridine nucleotides and triphosphopyridine nucleotides. Because the diaphorase enzyme is included, an oxidized redox mediator can be used. Using a redox mediator in its oxidized form offers several advantages. The use of a redox mediator in its oxidized form is more stable in ambient environments than its reduced counterpart, thus benefiting the storage stability of the disposable biosensor. Another advantage is that the disposable biosensor will produce a more sensitive response. A further advantage is that the disposable biosensor can measure very low concentrations of the analyte being measured. Yet another advantage is that there are more options for oxidized redox mediators than reduced redox mediators. Another advantage is that oxidized redox mediators are more stable in ambient environments than reduced redox mediators in the same environment. This advantage can be translated to disposable biosensors that do not require the special storage requirements of disposable biosensors that use reduced redox mediators in order to exhibit similar storage stability.

[0009] An object of the present invention is to provide a disposable biosensor for whole blood that has good storage stability without requiring special storage conditions.

[0010] Another object of the present invention is to provide a disposable biosensor for whole blood that responds more sensitively than conventional disposable biosensors.

[0011] It is a further object of the present invention to provide a disposable biosensor for whole blood that is capable of measuring very low concentrations of a species / analyte of interest.

[0012] The present invention achieves these and other objectives by providing a disposable biosensor with longer shelf life stability, more sensitive response, and the ability to measure very low concentrations of a species / analyte of interest.

[0013] In one embodiment of the present invention, the disposable biosensor has a detection surface having at least a working electrode and a reference electrode, a first reagent containing NAD(P)-dependent dehydrogenase, NAD(P), diaphorase, and an oxidized redox mediator, and disposed on the working electrode portion to form the working electrode, and a reference electrode material disposed on the reference electrode portion to form the reference electrode.

[0014] In another embodiment of the present invention, the disposable biosensor includes a blank electrode and an NAD( and a second reagent containing (P), diaphorase, and an oxidized redox mediator, but not containing dehydrogenase, which is placed on the blank electrode portion to form a blank electrode.

[0015] In one embodiment of the present invention, the reference material for forming the reference electrode is either silver-silver chloride or a reference matrix containing at least one chemical oxidant selected from the group consisting of a reduced redox mediator, an oxidized redox mediator, and a mixture of a reduced redox mediator and an oxidized redox mediator.

[0016] In one embodiment, the NAD(P)-dependent dehydrogenase is one of alcohol dehydrogenase, glutamate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, D-3-hydroxybutyrate dehydrogenase, glycerol dehydrogenase, malate dehydrogenase, and leucine dehydrogenase.

[0017] In one embodiment, the oxidized redox mediator comprises a metal compound or an organic redox compound.

[0018] In one embodiment, the oxidized redox mediator comprises at least one of potassium ferricyanide, sodium ferricyanide, ferrocene and its derivatives, ruthenium compounds and its derivatives such as hexaammineruthenium(III) chloride, osmium complexes, 1,10-phenanthroline-5,6-dione, Meldola Blue, tetrathiafulvalene 7,7,8,8-tetracyanoquinodimethane, hydroquinone, dichlorophenol indophenol, p-benzoquinone, o-phenylenediamine, and 3,4-dihydroxybenzaldehyde.

[0019] In one embodiment, the first reagent further comprises a polymer, a surfactant, and a buffer. In a further embodiment, the first reagent further comprises an optional bulking reagent.

[0020] In one embodiment, the second reagent further comprises a polymer, a surfactant, and a buffer. In a further embodiment, the second reagent further comprises an optional bulking reagent.

[0021] In another embodiment of the present invention, a disposable biosensor further includes a base layer made of an insulating material and having at least two electrical circuits formed thereon; a channel-forming layer made of an insulating material and disposed on the base layer; and a cover layer made of an insulating material and disposed on the channel-forming layer. Each of the at least two electrical circuits extends longitudinally along the base layer, and each of the at least two circuits has a conductive contact pad formed at each of the base layer proximal end and the base layer distal end, along with a conductive trace electrically connecting the conductive contact pad at the base layer proximal end to a corresponding conductive contact pad at the base layer distal end. One conductive pad at the base layer distal end forms a working electrode, and the other conductive pad at the base layer distal end forms a reference electrode. The channel-forming layer has a slot extending from the channel layer distal end a predetermined distance sufficient to expose each conductive pad at the base layer distal end. The channel-forming layer has a length shorter than the length of the base layer so that each conductive pad at the base layer proximal end is exposed. A cover layer disposed over the channel forming layer forms a sample chamber defined by a slot in the channel forming layer, the cover layer having a vent opening spaced from a distal end of the cover layer, the vent opening at least partially communicating with the sample chamber.

[0022] In one embodiment, the disposable biosensor further includes a reagent holding layer between the base layer and the channel forming layer, the reagent holding layer having at least two through-openings at a distal end thereof, one of the at least two through-openings overlapping with the working electrode and another of the at least two through-openings overlapping with the reference electrode.

[0023] In one embodiment, the base layer includes a third electrical circuit extending longitudinally along the base layer, the third electrical circuit including conductive contact pads formed at each of the base layer proximal end and the base layer distal end, with conductive traces electrically connecting the conductive contact pads at the base layer proximal end to corresponding conductive pads at the base layer distal end, forming blank electrodes within slots in the channel-forming layer.

[0024] In one embodiment, the reagent holding layer has a third through opening, the third through opening overlapping the blank electrode.

[0025] In one embodiment, a method for preparing a disposable biosensor based on NADP-dependent dehydrogenase and diaphorase is disclosed. The method includes providing a sensing surface having at least a working electrode portion and a reference electrode portion; disposing a first reagent on the working electrode portion and drying the first reagent to form a first electrode matrix serving as the working electrode; and disposing a reference electrode material on the reference electrode portion to form the reference electrode. The first reagent includes NAD(P)-dependent dehydrogenase, NAD(P), diaphorase, and an oxidized redox mediator. The reference electrode material is one of silver-silver chloride and a reference electrode reagent including at least one chemical oxidant selected from the group consisting of a reduced redox mediator, an oxidized redox mediator, and a mixture of a reduced redox mediator and an oxidized redox mediator. The reference electrode reagent is then dried to form the reference electrode matrix.

[0026] In another embodiment, preparing the first reagent comprises adding together a predetermined amount of NAD(P)-dependent dehydrogenase, a predetermined amount of NAD(P), a predetermined amount of diaphorase, and a predetermined amount of oxidized redox mediator to a predetermined amount of water.

[0027] In another embodiment, the method includes weighing out 0.1 gram to 0.5 gram of NAD(P)-dependent dehydrogenase, weighing out 0.02 gram to 0.1 gram of NADP+, weighing out 0.02 gram to 0.5 gram of diaphorase, weighing out 0.1 gram to 0.5 gram of oxidized redox mediator, and adding the NAD(P)-dependent dehydrogenase, NAD(P)+, diaphorase, and redox mediator to 10 milliliters of water.

[0028] In one embodiment, the method includes weighing out 0.2 grams to 0.4 grams of NAD(P)-dependent dehydrogenase, weighing out 0.05 grams of NADP+, weighing out 0.05 grams to 0.2 grams of diaphorase, weighing out 0.3 grams of oxidized redox mediator, and adding the NAD(P)-dependent dehydrogenase, NAD(P)+, diaphorase, and redox mediator to 10 milliliters of water. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is an enlarged front perspective view of one embodiment of a disposable biosensor of the present invention. [Figure 2] FIG. 2 is an exploded perspective view of the disposable biosensor shown in FIG. 1. [Figure 3] FIG. 10 is an enlarged front perspective view of another embodiment of a disposable biosensor of the present invention. [Figure 4] FIG. 4 is an exploded perspective view of the disposable biosensor shown in FIG. 3. [Figure 5] FIG. 10 is an enlarged front perspective view of another embodiment of a disposable biosensor of the present invention. [Figure 6] FIG. 6 is an enlarged top view of the disposable biosensor shown in FIG. 5. [Figure 7]1 is a graphical representation of the data in Table 1 showing alcohol biosensor current results versus alcohol concentration using an alcohol biosensor incorporating hydrogenase, diaphorase, and the oxidized redox mediator potassium ferricyanide. [Figure 8] 1 is a graphical representation of the data in Table 2 showing the alcohol biosensor current results versus alcohol concentration using an alcohol biosensor incorporating hydrogenase, diaphorase, and the oxidized redox mediator hexaammineruthenium(III) chloride. [Figure 9] 1 is a graphical representation of the data in Table 3 showing the results of a precision test of an alcohol biosensor incorporating hydrogenase, diaphorase, and the oxidized redox mediator potassium ferricyanide. [Figure 10] 1 is a graphical representation of the data in Table 4 showing a comparison of the results of an alcohol biosensor containing hydrogenase and the reduced redox mediator 1,10-phenanthroline-5,6-dione, but no diaphorase, to the results of an alcohol biosensor containing hydrogenase, diaphorase, and the oxidized redox mediator potassium ferricyanide. [Figure 11] 1 is a graphical representation of the data in Table 5 showing the long-term storage stability of an alcohol biosensor containing hydrogenase and the reduced redox mediator 1,10-phenanthroline-5,6-dione, but no diaphorase, versus an alcohol biosensor containing hydrogenase, diaphorase, and the oxidized redox mediator potassium ferricyanide. DETAILED DESCRIPTION OF THE INVENTION

[0030] [Detailed Description of the Invention] Preferred embodiments of the present invention are shown in Figures 1-11. Figure 1 illustrates one embodiment of the present invention. Disposable biosensor 10 is a multi-layer integrated biosensor having laminate 12, electrode end 14, electrode end 14a, electrical contact end 16, electrical contact end 16a, and vent opening 52. Electrode end 14 has sample chamber 17 between sample inlet 18 of electrode end 14a and vent opening 52. Electrical contact end 16 has at least three electrical contact pads 16b, 16c, and 16d that electrically connect to respective electrodes located within sample chamber 17. In this embodiment, disposable biosensor 10 has a length of 30 mm (1.2 inches) and a width of 5.5 mm (0.22 inches), but is not limited to such dimensions.

[0031] 2, laminate 12 includes a base layer 20, a reagent holding layer 30, a channel-forming layer 40, and a cover 50. All layers of laminate 12 are made of a dielectric material, preferably a plastic. Examples of suitable dielectric materials include polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, nitrocellulose, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, polyimide, polypropylene, polyethylene, and polystyrene.

[0032] The base layer 20 has a conductive layer 21 on which three conductive paths 22, 24, and 26 are drawn. The conductive paths 22, 24, and 26 may be formed by scribing or scoring the conductive layer 21. Alternatively, the base layer 20 may have the conductive paths 22, 24, and 26 silk-screened thereon. A piece of gold-colored polyester film may be used and cut to the shape shown in FIG.

[0033] Scribing or scoring of the conductive layer 21 may be accomplished by mechanically scribing the conductive layer 21 sufficiently to form three independent conductive paths 22, 24, and 26. A preferred scribing or scoring method of the present invention is performed using a carbon dioxide laser, a YAG laser, or an excimer laser. The score lines are very thin, yet sufficient to create three separate electrical paths. The conductive layer 21 may be made of any conductive material, such as gold, tin oxide / gold, palladium, other precious metals or their oxides, or a carbon film composition. Preferred conductive materials are gold or tin oxide / gold. Additional score lines 28 (enlarged for illustrative purposes only and not to scale) may be formed along the outer edge of the base layer 20 where the conductive layer is removed to avoid potential static issues that could result in a noisy signal. However, it should be understood that the score lines 28 are not necessary for the function of the disposable biosensor 10. A suitable conductive material for base layer 20 is a polyester film coated with gold or tin oxide / gold.

[0034] The reagent holding layer 30 has a first electrode opening 32 exposing a portion of the first conductive pathway 22, a second electrode opening 34 exposing a portion of the second conductive pathway 24, and a third electrode opening 36 exposing a portion of the third conductive pathway 26. The reagent holding layer 30 is made of a plastic material, preferably a medical-grade single-sided adhesive tape available from Adhesive Research, Inc. of Glen Rock, Pennsylvania, or Global Instrument Corporation (GIC) of Taiwan. Acceptable tape thicknesses for use in the present invention range from about 0.001 inch (0.025 mm) to about 0.005 inch (0.13 mm). A preferred thickness is about 0.003 inch (0.075 mm). It should be understood that the use of tape is not required. Reagent holding layer 30 may be made of a plastic sheet, coated with a pressure-sensitive adhesive, a photosensitive resin, ultrasonically bonded to base layer 20, silkscreened onto base layer 20, or 3D printed onto base layer 20 to achieve the same results as using polyester tape described above.

[0035] Three electrode openings 32, 34, and 36 expose portions of the respective conductive paths 22, 24, and 26 that define the working electrode portion 20W, the reference electrode portion 20R, and the blank electrode portion 20B, respectively, which in turn define electrode wells W, R, and B, respectively, and hold chemical reagents that form the working electrode (W), the reference electrode (R), and the blank electrode (B). Preferably, electrode well W is filled with an NAD(P)-dependent dehydrogenase capable of catalyzing a reaction involving the enzyme's substrate or a substrate catalyzed by the enzyme, diaphorase, and an oxidized mediator that facilitates electron transfer between the enzyme-catalyzed reaction and the working electrode, to generate a current representative of the activity of the enzyme or the substrate (representing the alcohol being measured in the disclosed example), and is filled with at least a polymeric binder. Blank electrode well B is filled with chemistry similar to electrode well W, but without the dehydrogenase. One or more chemical components such as additional polymers, stabilizers and bulking agents may be included in the reagent matrix as needed. The reference reagent matrix is ​​loaded into electrode well R.

[0036] Preferably, the reference matrix contains at least a chemical oxidant such as a reduced redox mediator, an oxidized redox mediator, or a mixture of a reduced redox mediator and an oxidized redox mediator. For example, potassium ferricyanide, potassium ferrocyanide, or a mixture of potassium ferricyanide and potassium ferrocyanide may be filled to make the reference electrode function when a suitable conductive coating material is used. A mixture of potassium ferricyanide and potassium ferrocyanide is preferable because it is ferrocyanide. The potassium concentration may be prepared to range up to about 5%, while the potassium ferricyanide concentration may range up to about 10%. Alternatively, the reference electrode (electrode well R) may be filled with a silver / silver chloride layer (e.g., by applying a silver / silver chloride ink, or by (a) sputter-coating a silver layer followed by chlorinating the silver, or (b) sputter-coating a silver chloride layer on top of the silver layer), or with other reference electrode materials that do not require a redox mediator to function properly. It should be noted that the placement of the working, reference, and blank electrodes within the channel is not critical to obtaining usable results from the sensor.

[0037] The size of the reagent retention openings is preferably made as small as possible to minimize the amount of sample required for each test measurement and keep the sample chamber of the disposable biosensor as short as possible. The three reagent retention / electrode openings 32, 34, and 36 are aligned with one another and spaced apart by about 0.02 inches (0.508 mm) to about 0.05 inches (1.27 mm). The circular shape of the reagent retention openings is for illustrative purposes only. It should be understood that the shape of the reagent retention openings and the distance between the respective electrodes are not critical. In fact, the reagent retention openings may vary in surface area, so long as the surface area ratio remains substantially constant from sensor to sensor.

[0038] The placement of the working, substrate, and reference electrodes within the channel is not critical to obtaining usable results from the disposable biosensor. Possible electrode placements within the sample fluid channel may be WBR, WRB, RWB, BWR, BRW, or RBW, listing the placements as electrodes would be visible from the sample inlet 18 of the laminate 12 to the vent opening 52. The preferred location was found to be WRB, meaning that as the fluid sample enters the sampling end 14 of the laminate 12, it will spread first to the working electrode, then the reference electrode, and then the blank electrode.

[0039] Each of the three electrodes is electrically connected to a respective corresponding electrical contact pad, which is exposed for making electrical connection with a reader.

[0040] The channel-forming layer 40 has a channel notch 42 located at the electrode end 14. The length of the channel notch 42 is such that when the channel-forming layer 40 is laminated to the reagent holding layer 30, the electrode areas W, R, and B are within the space defined by the channel notch 42. The length, width, and thickness of the channel notch 42 define the volume of the capillary chamber. The channel-forming layer 40 is laminated to the reagent holding layer 30. Like the reagent holding layer 30, the channel-forming layer 40 may be made of a plastic sheet, coated with a pressure-sensitive adhesive, a photosensitive resin, ultrasonically bonded to the reagent holding layer 30, silkscreened onto the reagent holding layer 30, or 3D printed onto the reagent holding layer 30.

[0041] The channel-forming layer 40 is made of a plastic material, preferably a medical-grade double-sided pressure-sensitive adhesive tape available from Adhesive Research, Inc., Glen Rock, Pennsylvania, or Global Instruments Corporation (Taiwan). The thickness of the tape is preferably within the range of about 0.001 inch (0.025 mm) to about 0.010 inch (0.25 mm). The channel notches 42 can be created by laser or die-cutting (a more preferred method). The length of the channel notches 42 is about 0.22 inch (5.7 mm) to about 0.250 inch (6.4 mm), the width is about 0.05 inch (1.3 mm) to about 0.07 inch (1.8 mm), and the thickness is about 0.0039 inch (0.1 mm) to about 0.009 inch (0.225 mm). It should be understood that the thickness and size of the channel notch 42 is not critical.

[0042] A cover 50 laminated to the channel-forming layer 40 has a vent opening 52 spaced from the electrode end 14a of the disposable biosensor 10 to ensure that the sample in the sample chamber 17 completely covers the electrode areas W, R, and B. The vent opening 52 is positioned in the cover 50 so as to expose a portion of the channel notch 42 at or near the closed end of the channel notch 42 and overlap the channel notch 42. The vent opening 52 can be any shape, but is shown as a rectangle having dimensions of approximately 0.08 inches (2 mm) by approximately 0.035 inches (0.9 mm). A preferred material for the cover 50 is polyester film. To promote capillary action of the sample fluid in the sample chamber 17, the polyester film preferably has a highly hydrophilic surface on the portion of the polyester film that forms the capillary chamber. Transparent films from 3M or GIC may be used. The cover 50 may optionally have an inlet notch 54 to avoid unintentional blockage of the sample inlet 18 (which may prevent proper movement of the sample fluid to the electrode) when applying a blood sample to the sample chamber 17.

[0043] 3 shows a three-layer disposable biosensor 10'. Like the four-layer embodiment, the disposable biosensor 10' includes a laminate 12, an electrode end 14, an electrical contact end 16, and a vent opening 52. The electrode end 14 includes a sample chamber 17 between a sample inlet 18 and the vent opening 52. The electrical contact end 16 includes three individual electrical contact pads 16b, 16c, and 16d.

[0044] As seen in FIG. 4 , laminate 12 includes base layer 20, channel-forming layer 40, and cover 50. As previously mentioned, all layers of laminate 12 are made of a dielectric material, preferably plastic. Unlike the four-layer embodiment, the three-layer embodiment does not have a separate reagent-holding layer. Channel-forming layer 40 also defines areas where predetermined amounts of reagent mixtures are deposited onto the conductive pathways as three different reagent matrix coatings on the working electrode, reference electrode, and optional blank electrode, respectively.

[0045] 5, another embodiment of the present invention is shown showing a disposable biosensor 430. Disposable biosensor 430 includes a laminate 432, a sample receiving well 434, and an electrical contact end 436. Laminate 432 includes a base layer 450 and a cover 460. Cover 460 includes a sample opening 462 that, when combined with base layer 450, forms sample receiving well 434. Base layer 450 includes at least three electrical pathways 452, 454, and 456, each having a first portion exposed at electrical contact end 436 for connection to a meter device (not shown) and a second portion exposed by sample receiving well 434.

[0046] A second portion of electrical pathways 452, 454, and 456 exposed by sample receiving well 434 forms at least a working electrode W, a blank electrode B, and at least a reference / counter electrode R. A first reagent mixture 470 comprises the mixture previously described as a working electrode disposed on working electrode W. A second reagent mixture 472 comprises the mixture previously described as a blank electrode disposed on blank electrode B. Reference / counter electrode R may comprise any reference material 474 previously disclosed. In this embodiment of the invention, sample receiving well 434 functions as both a sample inlet and a sample chamber for receiving a fluid sample, such as blood, for quantitation of an analyte in the blood sample.

[0047] It should be understood that the conduit pathways of any of the embodiments disclosed herein may be made of any non-corrosive metal. Carbon deposits, such as carbon paste or carbon ink, may also be used as conduit pathways, all as known to those skilled in the art.

[0048] <Chemical reagents> [enzyme] The disposable biosensor of the present invention includes at least one chemical in the reagent matrix of the working electrode W that consumes the analyte to be measured in response to the dehydrogenase used in the reagent matrix. As a non-limiting example, alcohol dehydrogenase would be used when the analyte to be measured is ethanol. Another non-limiting example would be glutamate dehydrogenase when the analyte to be measured is glutamate. If the blood sample contains interferents, a blank electrode would be essential in the present invention to accurately quantify the alcohol concentration in the body fluid. As explained above, the use of such a blank electrode helps distinguish the oxidation current due to ethanol from that due to other oxidizable species in the sample fluid. Continuing with the alcohol example, commercially available alcohol dehydrogenase from Sigma Chemical Company of St. Louis, Missouri, or Worthington Biochemical Corporation of Lakewood, New Jersey, is used to prepare the alcohol working electrode. For example, the concentration of alcohol dehydrogenase in the reagent mixture is in the range of 5 mg / ml to 100 mg / ml, preferably in the range of 10 mg / ml to 50 mg / ml, and more preferably in the range of 20 mg / ml to 40 mg / ml.

[0049] [Diaphorase] Diaphorase is an essential part of the reagent matrix for the working electrode. Diaphorase is a flavoprotein enzyme capable of oxidizing the reduced coenzyme NAD. The concentration of diaphorase in the reagent matrix is ​​in the range of 2 mg / ml to 50 mg / ml, preferably in the range of 5 mg / ml to 20 mg / ml.

[0050] [Chemical oxidizing agents] A chemical oxidizing agent, such as a redox mediator, is included in the disposable biosensor. The redox mediator is preferably used in its oxidized form. It is also desirable that the reduced mediator be electrochemically oxidizable at the electrode surface at an applied potential. It is further desirable that the mediator be stable in the reagent matrix. It is also further desirable that when used in a reference electrode, the mediator enable the reference electrode to function properly. The redox mediator may be selected from, but is not limited to, various metal compounds and organic redox compounds. Examples of acceptable redox mediators include potassium (or sodium) ferricyanide, ferrocene and its derivatives, cupric compounds, nitrite compounds, ruthenium compounds and their derivatives, such as hexaammineruthenium(III) chloride, and osmium complexes, 1,10-phenanthroline-5,6-dione, Meldola Blue, tetrathiafulvalene 7,7.8.8-tetracyanoquinodimethane, tetrathiafulvalene, TCNQ, hydroquinone, dichlorophenol indophenol, p-benzoquinone, o-phenylenediamine, and 3,4-dihydroxybenzaldehyde. Suitable mediators are potassium ferricyanide or hexaammineruthenium(III) chloride. The concentration of potassium ferricyanide in the reagent mixture is preferably in the range of 0.5% to 10%, preferably 1% to 5%, and more preferably 3% (w / w) of the reagent mixture. The concentration of hexaammineruthenium(III) chloride in the reagent mixture is preferably in the range of 0.5% to 5%, more preferably in the range of 1% to 2%.

[0051] [Enzyme cofactor] The enzyme cofactor included in the reagent matrix of the disposable biosensor 10, 10', 430 is the organic cofactor nicotinamide adenine dinucleotide phosphate (NAD), which is used due to the use of dehydrogenase and diaphorase. The concentration of the cofactor in the solution is in the range of 0.1% to 2%, preferably in the range of 0.2 to 1%, more preferably equal to 0.5% (w / w).

[0052] [polymer] The polymer used as a binder in the reagent matrix should be sufficiently water-soluble and capable of stabilizing and binding all other chemicals in the reagent to the conductive surface layer in the electrode region. Suitable polymers include, but are not limited to, low and high molecular weight polyethylene oxide (PEO), polyethylene glycol, polyvinylpyrrolidone, starch, methylcellulose, hydroxypropyl cellulose, polyvinyl alcohol (PVA), carboxymethylcellulose (CMC), and polyamino acids. The reagent binder may be a single polymer or a combination of polymers, preferably in a concentration range of about 0.02% (w / w) to about 7.0% (w / w). A preferred binder in the reagent matrix of the present invention is a combination of polyethylene oxide (PEO) and methylcellulose. PEO has a molecular weight ranging from several thousand to several million and is available from Scientific Polymer Products, New York, USA. The concentration of PEO in the reagent matrix is ​​preferably about 0.04% (w / w) to about 2% (w / w). Methylcellulose, available under the brand name Methocel 60HG (catalog number 64655, Fluka Chemicals, Milwaukee, Wisconsin, USA), preferably has a concentration in the reagent matrix ranging from about 0.02% (w / w) to about 5% (w / w).

[0053] [Surfactants] A surfactant may be included in the reagent mixture to facilitate dispensing the mixture onto the electrode area, if necessary. The surfactant also helps rapidly dissolve the dry chemical reagents when the sample fluid enters the sample channel of the disposable biosensor. The amount and type of surfactant are selected to ensure the above-mentioned functions and avoid denaturing effects on the enzyme. The surfactant may be selected from various anionic, cationic, nonionic, and zwitterionic surfactants, but is not limited to these. Examples of acceptable surfactants include polyoxyethylene ether, Tween 20, sodium cholate hydrate, hexadecylpyridinium chloride monohydrate, and CHAPS. A preferred surfactant is polyoxyethylene ether. More preferably, it is t-octylphenoxypolyethoxyethanol, available under the brand name Triton X-100. The concentration of Triton X-100 in the reagent mixture is preferably about 0.01% (w / w) to about 2%.

[0054] [Buffer] If desired, a buffer solution may be present in the sensor strip of the present invention along with the dried redox mediator. The buffer solution is present in an amount sufficient to substantially maintain the pH of the reagent mixture. Examples of suitable buffer solutions include citrate, phosphate, Tris, and the like. In the present invention, the pH of the buffer solution is preferably within the range of about 5.0 to about 8.5.

[0055] [Increasing reagent] An optional bulking agent, which is a water-soluble and inert component, is preferably added to the reagent mixture / matrix. The use of a bulking agent is advantageous when an electrode-forming layer is used to contain the reagent matrix, so that the electrode openings of the electrode-forming layer will not trap air bubbles when the sample fluid fills the capillary channel. For example, trehalose, galactose, glucose, sucrose, lactose, mannitol, mannose, fructose, sucrose, lactose, lactitol, sorbitol, xylitol, nicotine, Various sugars, such as cellulose, maltose, and the like, can be added to the reagent mixture as long as they do not react with other components and are inert on the electrode surface. The bulking agent can be a single chemical or a combination of chemicals. The amount of bulking agent in the reagent mixture ranges from about 1% to about 15% (w / w).

[0056] <Creating a biosensor> As can be inferred from the above description, a disposable electrochemical biosensor based on NAD(P)-dependent dehydrogenase and diaphorase is constructed as follows.

[0057] A base layer 20 having a conductive layer 21 is formed and scribed as described above. A channel-forming layer 40 is formed as described above and laminated onto the base layer 20. A predetermined amount of a first reagent is disposed on the working electrode portion 20W and dried to form the working electrode W. A predetermined reference material is disposed on the reference electrode portion 20R to form the reference electrode R. If a reference electrode reagent is used, a predetermined amount of the reference reagent is disposed on the reference electrode portion 20R and dried. After the working electrode W and the reference electrode R are formed, a cover layer 50 is then laminated onto the channel-forming layer 40 such that the channel notch 42 is positioned so that the working electrode W and the reference electrode R are positioned within the channel notch 42, and the vent opening 52 is positioned so that the vent opening 52 at least partially overlaps a portion of the channel notch 42.

[0058] In embodiments including the reagent holding layer 30, the reagent holding layer 30 is disposed directly on the conductive layer 21 of the base layer 20 such that the through-openings 32, 34, and optionally 36 expose the working electrode portion 20W, the reference electrode portion 20R, and optionally the blank electrode portion 20B. A predetermined amount of a first reagent is disposed within the through-opening 32 and dried to form the working electrode W. A predetermined reference material is disposed within the through-opening 34 to form the reference electrode R. If the reference uses a reference electrode reagent, a predetermined amount of the reference reagent is disposed within the through-opening 34 and dried. The channel-forming layer 40 is then laminated onto the reagent holding layer 30 such that the through-openings 32, 34, and optionally 36 reside within the channel notch 42. The cover layer 50 is then laminated onto the channel-forming layer 40 as previously described.

[0059] <Quantitative determination of alcohol in blood samples> Although the following example provides data for quantifying alcohol in blood samples, it should be understood by those skilled in the art that, as disclosed above, other analytes in blood, such as glutamate, glucose, lactate, cholesterol, hydroxybutyrate, glycerol, malate, leucine, etc., can also be quantified using appropriate dehydrogenases and diaphorases, and as a result, these disposable biosensors would also have the advantages disclosed above.

[0060] With respect to an alcohol biosensor, the basic chemical reaction that occurs when a blood sample contains alcohol (in the practical case, the alcohol is ethanol) is as follows:

[0061] [ka]

[0062] Here, Med ox is an oxidative mediator, Med red is the reduced mediator and e represents the electrons that generate the current at the electrode surface.

[0063] The bias potential between the working and reference electrodes for an alcohol biosensor is in the range of 100 mV to 500 mV, preferably 300 mV to 400 mV. The voltage value within that range is not critical but should be constant. The bias potential between the working and reference electrodes causes the mediator to change from a reduced state to an oxidized state on the electrode surface (see Equation 3), thereby generating a current based on the alcohol concentration in the blood.

[0064] Blood sample concentration values ​​for alcohol were obtained using a blood analyzer known as a Dimension RxL Chemistry Analyzer sold by Siemens Healthcare Diagnostics Inc. of Tarrytown, NY. Analyte test strip test data were obtained using a CH Instruments Potentiostat, Model Number CHI812B or Model Number CHI660A.

[0065] <Test Data for Alcohol Determination Using an Alcohol Biosensor Containing Diaphorase and K3Fe(CN)6 as Mediators in the Reagent Matrix> To confirm the linearity of the response between blood alcohol concentration and the alcohol biosensor response, nine different concentration levels of ethanol were used in the test. The levels were 0%, 0.01%, 0.02%, 0.04%, 0.08%, 0.12%, 0.16%, 0.2%, and 0.25%. Samples were prepared to correspond to these ethanol levels, and amperometric measurements were performed for each ethanol concentration. A new alcohol biosensor strip was used for each measurement. Table 1 shows the test data obtained for the quantification of alcohol when the ethanol biosensor had a working electrode reagent matrix containing ethanol dehydrogenase, diaphorase, and an oxidized redox mediator. The oxidized redox mediator was potassium ferricyanide.

[0066] [Table 1]

[0067] Figure 7 shows the current response of the working electrode (i.e., the ethanol dehydrogenase-based electrode) measured for various alcohol concentrations. The current response is linear with respect to ethanol concentration throughout the range of alcohol concentrations tested.

[0068] <Test Data for Alcohol Determination Using an Alcohol Biosensor Containing Diaphorase and Hexaammineruthenium(III) Chloride as Mediators in a Reagent Matrix> In this example, the same levels and number of alcohol concentrations as in the previous example were used, except for one higher concentration level: 0%, 0.01%, 0.02%, 0.04%, 0.08%, 0.12%, 0.16%, 0.2%, 0.25%, and 0.35%. Samples were prepared corresponding to these ethanol levels, and amperometric measurements were performed for each ethanol concentration. A new alcohol biosensor strip was used for each measurement. Table 1 shows the test data obtained for the quantification of alcohol when the ethanol biosensor uses a hexaammineruthenium(III) chloride mediator.

[0069] [Table 2]

[0070] Figure 8 shows the current response of the working electrode (i.e., the ethanol dehydrogenase-based electrode) measured for various alcohol concentrations. The current response is linear with respect to ethanol concentration throughout the range of alcohol concentrations tested.

[0071] <Precision Test Results for an Ethanol Biosensor Using Diaphorase with K3Fe(CN)6 as a Mediator in the Reagent Matrix> To confirm the precise response of the ethanol biosensor, this test used two different concentration levels of ethanol: 0.05% and 0.18%. Samples were prepared to correspond to these ethanol levels, and current measurements were performed for each ethanol concentration. Twenty new alcohol biosensor strips were used for each concentration level measurement. Table 3 shows the test data obtained for the quantification of alcohol when the ethanol biosensor uses diaphorase and potassium ferricyanide mediator.

[0072] [Table 3]

[0073] Figure 9 shows the current response of the working electrode (i.e., the ethanol dehydrogenase-based electrode) measured for two alcohol concentrations. The averages of 20 ethanol biosensors for each alcohol concentration are 0.543 and 1.721, respectively. The coefficients of variation in percent are 1.55 and 1.81, respectively. The test data are for the alcohol This shows that the response of each individual biosensor is relatively accurate.

[0074] <Comparison of alcohol biosensor responses between oxidized and reduced mediators> To confirm the sensitivity response between blood alcohol concentration and the response based on (1) an alcohol biosensor using diaphorase and an oxidized redox mediator and (2) an alcohol biosensor using a reduced redox mediator but without diaphorase, nine different concentration levels of ethanol were used. The levels were 0%, 0.01%, 0.02%, 0.04%, 0.08%, 0.12%, 0.16%, 0.2%, and 0.25%. As before, the oxidized mediator used in one set of alcohol biosensors was potassium ferricyanide. The reduced mediator in the comparison set of alcohol biosensors was reduced 1,10-phenanthroline-5,6-dione.

[0075] For an alcohol biosensor containing the reduced redox mediator 1,10-phenanthroline-5,6-dione and no diaphorase, the basic chemical reaction that occurs when a blood sample contains alcohol (in the practical case, the alcohol is ethanol) is as follows:

[0076] [ka]

[0077] Here, Med ox is an oxidative mediator, Medred is the reduced mediator.

[0078] The bias potential between the working and reference electrodes for this diaphorase-free reduced redox mediator alcohol biosensor is in the range of -50 mV to -300 mV, preferably -100 mV to -200 mV. The voltage value within that range is not critical but should be constant. The bias potential between the working and reference electrodes causes the mediator in the comparison set to change from an oxidized to a reduced state on the electrode surface (see Equation 3a), thereby generating a current based on the alcohol concentration in the blood.

[0079] Samples were prepared to the above ethanol levels, and current measurements were performed for each ethanol concentration for each biosensor set. A new alcohol biosensor was used for each measurement. Table 4 shows the response comparison test data obtained for alcohol quantification using the different sets of ethanol biosensors described above.

[0080] [Table 4]

[0081] FIG. 10 shows the current response of the working electrode (i.e., the ethanol dehydrogenase-based electrode) measured for various alcohol concentrations for each alcohol biosensor set. The current response is linear with respect to ethanol concentration throughout the range of alcohol concentrations tested for each alcohol biosensor set. However, sensitivity shows significant differences when using alcohol dehydrogenase with an oxidized mediator and a reduced mediator. As shown in the graph, the alcohol biosensor with an oxidized mediator and diaphorase has a much higher sensitivity response of 0 to 2.6 microamps, compared to 0 to 0.8 microamps for the reduced mediator without diaphorase. Because the response is more sensitive, measurement accuracy is also improved. From the above equation, one skilled in the art will recognize that when using a reduced mediator, the presence of diaphorase will not affect the reaction because the reduced mediator loses electrons to NAD(P)H to form NAD(P) and the oxidized mediator.

[0082] <Storage stability of alcohol biosensor> To confirm the stability of an alcohol biosensor containing an NAD(P)-dependent dehydrogenase, diaphorase, and an oxidized redox mediator, a comparison was conducted over a 12-month period with an alcohol biosensor using a reduced redox mediator but without diaphorase. Each measurement was taken with a new alcohol sensor for a sample with an ethanol concentration of 0.15%. As before, the oxidized mediator used in one set of alcohol biosensors was potassium ferricyanide. The reduced mediator in the comparison set of alcohol biosensors was reduced 1,10-phenanthroline-5,6-dione.

[0083] Diaphorase and alcohols containing potassium ferricyanide as electron mediator The bias potential between the working electrode and the reference electrode for the biosensor is in the range of 100 mV to 500 mV, preferably in the range of 300 mV to 400 mV. The bias potential between the working electrode and the reference electrode for the alcohol biosensor containing the reduced redox mediator 1,10-phenanthroline-5,6-dione but not containing diaphorase is in the range of -50 mV to -300 mV, preferably in the range of -100 mV to -200 mV.

[0084] Samples were prepared at the above-mentioned ethanol levels, and current measurements were performed for a concentration of 0.15% for each biosensor set. A new alcohol biosensor was used for each measurement. Table 5 shows the response comparison test data obtained for the quantification of alcohol using the different sets of ethanol biosensors described above.

[0085] [Table 5]

[0086] Figure 11 shows the current response of the working electrode (i.e., the ethanol dehydrogenase-based electrode) measured under ambient storage conditions for each alcohol biosensor set. For the alcohol biosensor set containing an NAD(P)-dependent dehydrogenase, an oxidized redox mediator, and diaphorase, the current response is stable across the ethanol concentration throughout the 12-month period. As can be seen in the graph and Table 5 above, for the alcohol biosensor set containing a reduced redox mediator but not diaphorase, the current response decreases throughout the 12-month period. Indeed, after 4 months of storage for the alcohol biosensor containing a reduced redox mediator but not diaphorase, the current response begins to decrease, decreasing more rapidly as the biosensor ages. As shown in the graph, the alcohol biosensor containing a reduced redox mediator but not diaphorase decreases in response from 0.571 microamps (or an average of 0.574 microamps) at 4 months of storage to 0.398 microamps at 12 months of storage. In summary, the response of an alcohol biosensor decreases over time.

[0087] While preferred embodiments of the present invention have been described herein, the above description is by way of example only. Further modifications of the invention disclosed herein will occur to those skilled in the art, and all such modifications will be within the scope of the appended claims. Such modifications are considered to be within the scope of the present invention as defined by the appended claims.

Claims

1. 1. A disposable biosensor comprising: a detection surface having at least a working electrode portion and a reference electrode portion; a first reagent containing NAD(P)-dependent dehydrogenase, NAD(P)+, diaphorase, and an oxidized redox mediator, the first reagent being disposed on the working electrode portion to form a working electrode; a reference electrode material disposed on the reference electrode portion to form a reference electrode; A disposable biosensor comprising:

2. 10. The disposable biosensor of claim 1, The detection surface comprises: a blank electrode portion; a second reagent containing NAD(P)+, diaphorase, and an oxidized redox mediator, but not containing dehydrogenase, which is placed on the blank electrode portion to form a blank electrode; The disposable biosensor further comprises:

3. 10. The disposable biosensor of claim 1, A disposable biosensor, wherein the reference material is one of silver-silver chloride and a reference matrix containing at least one chemical oxidant selected from the group consisting of a reduced redox mediator, an oxidized redox mediator, and a mixture of a reduced redox mediator and an oxidized redox mediator.

4. 10. The disposable biosensor of claim 1, The disposable biosensor, wherein the NAD(P)-dependent dehydrogenase is one of alcohol dehydrogenase, glutamate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, D-3-hydroxybutyrate dehydrogenase, glycerol dehydrogenase, malate dehydrogenase, and leucine dehydrogenase.

5. 10. The disposable biosensor of claim 1, The disposable biosensor, wherein the oxidized redox mediator comprises a metal compound or an organic redox compound.

6. 10. The disposable biosensor of claim 1, the oxidized redox mediator includes at least one of potassium ferricyanide, sodium ferricyanide, ferrocene and its derivatives, ruthenium compounds and its derivatives, such as hexaammineruthenium(III) chloride, osmium complexes, 1,10-phenanthroline-5,6-dione, Meldola Blue, tetrathiafulvalene 7,7,8,8-tetracyanoquinodimethane, hydroquinone, dichlorophenol indophenol, p-benzoquinone, o-phenylenediamine, and 3,4-dihydroxybenzaldehyde.

7. 10. The disposable biosensor of claim 1, The disposable biosensor, wherein the first reagent further comprises a polymer, a surfactant, and a buffer.

8. 8. The disposable biosensor of claim 7, The disposable biosensor, wherein the first reagent further comprises a weight-increasing reagent.

9. 3. The disposable biosensor of claim 2, The disposable biosensor, wherein the second reagent further comprises a polymer, a surfactant, and a buffer.

10. 10. The disposable biosensor of claim 9, The disposable biosensor, wherein the second reagent further comprises a weight-increasing reagent.

11. 10. The disposable biosensor of claim 1, a base layer made of an insulating material and having at least two electrical circuits patterned thereon, each of the at least two electrical circuits extending longitudinally along the base layer, each of the at least two circuits having conductive contact pads formed at a respective base layer proximal end and a respective base layer distal end, with conductive traces electrically connecting the conductive contact pads at the base layer proximal end and the corresponding conductive contact pads at the base layer distal end, one conductive pad at the base layer distal end forming the working electrode and the other conductive pad at the base layer distal end forming the reference electrode; a channel forming layer made of an insulating material and disposed on the base layer, the channel forming layer having slots extending from a distal end of the channel layer a predetermined distance sufficient to expose each conductive pad at the distal end of the base layer, the channel forming layer having a length shorter than a length of the base layer so that each conductive pad at the proximal end of the base is exposed; a cover layer made of an insulating material and disposed over the channel forming layer to form a sample chamber together with the slot in the channel forming layer, the cover layer having a vent opening spaced from a distal end of the cover layer, the vent opening at least partially communicating with the sample chamber; The disposable biosensor further comprises:

12. 12. The disposable biosensor of claim 11, a reagent holding layer between the base layer and the channel forming layer; the reagent holding layer has at least two through openings at a reagent holding layer distal end; A disposable biosensor, wherein one of the at least two through-openings overlaps the working electrode and the other of the at least two through-openings overlaps the reference electrode.

13. 12. The disposable biosensor of claim 11, the base layer has a third electrical circuit extending longitudinally along the base layer; the third electrical circuit includes conductive contact pads formed on each of the base layer proximal end and the base layer distal end, with conductive traces electrically connecting the conductive contact pads at the base layer proximal end and the corresponding conductive pads at the base layer distal end, forming blank electrodes; The disposable biosensor, wherein the blank electrode is located within the slot of the channel-forming layer.

14. 14. The disposable biosensor of claim 13, the reagent holding layer has a third through opening; The disposable biosensor, wherein the third through opening overlaps a blank electrode.

15. 1. A method for making a disposable biosensor based on NADP-dependent dehydrogenase and diaphorase, comprising: providing a detection surface having at least a working electrode portion and a reference electrode portion; placing a first reagent containing NAD(P)-dependent dehydrogenase, NADP+, diaphorase, and an oxidized redox mediator on the working electrode portion, and drying the first reagent to form a first electrode matrix that serves as the working electrode; a reference electrode material, which is any one of silver-silver chloride and a reference electrode reagent containing at least one chemical oxidant selected from the group consisting of a reduced redox mediator, an oxidized redox mediator, and a mixture of a reduced redox mediator and an oxidized redox mediator, placed on the reference electrode portion forming a reference electrode, and drying the reference electrode reagent forming a reference electrode matrix; A method comprising:

16. 16. The method of claim 15, The method further includes preparing the first reagent, the method comprising combining a predetermined amount of the NAD(P)-dependent dehydrogenase, a predetermined amount of NADP+, a predetermined amount of diaphorase, and a predetermined amount of an oxidized redox mediator and adding them together to a predetermined amount of water.

17. 17. The method of claim 16, Weighing out 0.1 to 0.5 grams of the NAD(P)-dependent dehydrogenase; Weighing out 0.02 to 0.1 grams of the NAD(P)+; Weighing out 0.02 grams to 0.5 grams of the diaphorase; Weighing out 0.1 to 0.5 grams of the oxidized redox mediator; adding the NAD(P)-dependent dehydrogenase, the NAD(P)+, the diaphorase, and the redox mediator to 10 milliliters of water; The method further comprises:

18. 17. The method of claim 16, Weighing out 0.2 to 0.4 grams of the NAD(P)-dependent dehydrogenase; Weighing out 0.05 grams of the NAD(P)+; Weighing out 0.05 grams to 0.2 grams of the diaphorase; Weighing out 0.3 grams of the oxidized redox mediator; adding the NAD(P)-dependent dehydrogenase, the NAD(P)+, the diaphorase, and the redox mediator to 10 milliliters of water; The method further comprises:

Images