HK1147525B - Reagents and methods for detecting analytes - Google Patents

Description

Reagent and method for detecting analyte

Technical Field

The present invention relates generally to reagents, methods and devices for measuring analytes. More particularly, the present invention relates to reagents, methods and devices for measuring glucose in a blood sample.

Background

The quantitative determination of analytes in body fluids is of great importance for the diagnosis and maintenance of certain physiological conditions. For example, lactate, cholesterol and bilirubin should be monitored in certain individuals. In particular, it is important that diabetics frequently check the glucose level in their body fluids to regulate the glucose intake in their diets. The results of such a test can be used to determine the insulin or other drug, if any, that should be administered. In one type of blood glucose testing system, a test sensor is used to test a blood sample.

The test sensor contains a biosensing or reagent material that reacts with, for example, blood glucose. The testing end of the sensor is adapted to be placed within the fluid being tested (e.g., blood that accumulates on a person's finger after the finger has been pricked). Fluid may be drawn into a capillary channel extending from the testing end to the reagent material in the sensor by capillary action, such that a sufficient amount of fluid under test is drawn into the sensor. Testing is typically performed using optical or electrochemical testing methods.

Electrochemical test sensors are based on an enzyme-catalyzed chemical reaction in which the analyte of interest participates. In the case of glucose monitoring, the relevant chemical reaction is the oxidation of glucose to gluconolactone or its corresponding acid. This oxidation is catalyzed by various enzymes, some of which may use coenzymes such as nicotinamide adenine dinucleotide (phosphate) (nad (p)), and others may use coenzymes such as Flavin Adenine Dinucleotide (FAD) or pyrroloquinoline quinone (PQQ).

In test sensor applications, redox equivalents generated during oxidation of glucose are transported to the electrode surface, thereby generating an electrical signal. The amplitude of the electrical signal is related to the glucose concentration. The transfer of the redox equivalent from the chemical reaction site in the enzyme to the electrode surface is accomplished using an electron transfer mediator.

Previously used electron transfer mediators FAD-glucose dehydrogenases (FAD-GDH) include potassium ferricyanide, phenazine-methyl sulfate (PMS), methoxyphenazine-methyl sulfate, phenazine methyl sulfate, and Dichloroindophenol (DCIP). However, these compounds have proven to be highly susceptible to environmental conditions including temperature and moisture, which can result in poor stability of the test sensor reagents. For example, reduced mediator may be generated from the interaction between the oxidized mediator and the enzyme system during storage. The greater the amount of mediator or enzyme, the greater the amount of reduced mediator produced. Due to the high concentration of reduced mediator, the background current, which increases over time, generally increases towards the end of the shelf life of the sensor strip. Increased background current may reduce the measurement accuracy and precision of the test sensor, and therefore, the test sensor may have a limited shelf life.

Another disadvantage associated with existing test sensors is the relatively slow fill rate. It is desirable to obtain a fast sensor fill rate so that rehydration of the reagent can be faster and more uniform. Thus, a faster fill rate generally results in a more accurate, more stable test sensor, and less variability.

It is therefore desirable to have agents that can address one or more of these disadvantages.

Disclosure of Invention

According to one aspect of the present invention, a reagent for detecting an analyte, the reagent comprising a flavoprotein and a mediator selected from the group consisting of the formula or a combination thereof,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸and R⁹May be the same or different and is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, halo, haloalkyl, carboxy, alkoxy,carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic ketonyl, aliphatic ketonyl, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfonic acid group, dihydroxyboron, and combinations thereof. The reagent further comprises at least one surfactant, a polymer, and a buffer. At least one of the surfactant and the buffer includes an inorganic salt, wherein the ratio of total inorganic salt to mediator is less than about 3: 1.

According to another embodiment of the present invention, a reagent for detecting an analyte in a fluid sample comprises FAD-glucose dehydrogenase having an activity of about 0.1-10U/. mu.L. The reagent further comprises a 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator at a concentration of about 5-120 mM. The reagent further comprises a surfactant at a concentration of about 0.05 to 0.5 wt.%. The reagent also includes a hydroxyethyl cellulose polymer at a concentration of about 0.1 to 4 wt.% and a buffer. At least one of the surfactant and the buffer includes an inorganic salt, wherein the ratio of total inorganic salt to mediator is less than about 3: 1.

In accordance with another embodiment of the present invention, an electrochemical test sensor includes a working electrode having a surface. The test sensor also includes a counter electrode having a surface. The test sensor further includes a reagent coating at least a portion of a surface of the working electrode and at least a portion of a surface of the counter electrode. The reagent comprises a flavoprotein, a phenothiazine mediator or a phenoxazine mediator, a buffer, at least one surfactant, and a polymer. At least one of the surfactant and the buffer includes an inorganic salt, wherein the ratio of total inorganic salt to mediator is less than about 3: 1.

According to one method of the present invention, a method for detecting an analyte in a fluid sample, the analyte undergoing a chemical reaction, includes the step of providing an electrode surface. The method further comprises the step of using a surfactant to cause the fluid sample to flow to the electrode surface. The method further comprises the step of catalyzing the chemical reaction with a flavoprotein enzyme. The method further comprises the step of producing a redox equivalent by the chemical reaction. The method further comprises the step of transferring the redox equivalent to the electrode surface using a phenothiazine or phenoxazine mediator. The maximum kinetic performance is less than about 3 seconds.

According to another method, an analyte in a fluid sample is detected, the method comprising providing an electrode surface. Providing a reagent comprising a flavoprotease, a mediator selected from the group consisting of the formula or a combination thereof,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹May be the same or different and is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, halogen, haloalkyl, carboxyl, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic ketonyl, aliphatic ketonyl, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfonic acid, dihydroxy boron, and combinations thereof; the reagent is in contact with the electrode surface. Contacting the fluid sample with the reagent. The concentration of the analyte is determined. Maximum kinetic performance of less than about 3 seconds

The above summary of the present invention is not intended to represent each embodiment, or every aspect, of the present invention. Other features and advantages of the present invention will be apparent from the following detailed description and the accompanying drawings.

Drawings

FIG. 1a is a test sensor according to one embodiment.

FIG. 1b is a side view of the test sensor of FIG. 1 a.

FIG. 2 is a line graph plotting current measurements versus glucose concentration.

Figure 3 is a bar graph comparing the fill times of sensors containing heptanoyl-N-methylglucamide (MEGA 8) surfactant and sensors without MEGA 8 surfactant.

Fig. 4 is a bar graph comparing background current for sensors containing MEGA 8 surfactant and sensors without MEGA 8 surfactant.

Fig. 5 is a bar graph of fill time of a test sensor versus different formulations with and without surfactant.

Fig. 6 is a plot of current values versus time measured in a formulation containing a surfactant.

FIG. 7 is a bar graph of the peak time of the mediator using 50mg/dL glucose, different sulfate concentrations, and different phosphate buffer concentrations.

FIG. 8 is a bar graph of the peak time of the mediator using 100mg/dL glucose, different sulfate concentrations, and different phosphate buffer concentrations.

FIG. 9 is a bar graph of the peak time of the mediator using 400mg/dL glucose, different sulfate concentrations, and different phosphate buffer concentrations.

Fig. 10a and 10b are graphs of current values versus time measured in formulations with different inorganic salt concentrations.

Figure 11 is a graph of CV% for low salt reagent solutions and high salt reagent solutions.

While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Detailed Description

The present invention relates to reagents, methods and devices for analyte measurement. More specifically, the present invention relates to a test sensor reagent for detecting an analyte, the reagent comprising: (1) a flavoprotease, (2) a phenothiazine or phenoxazine mediator, (3) a buffer, (4) a surfactant or surfactant mixture, and/or (5) a cellulosic polymer.

The reagents described herein may be used to assist in determining the concentration of an analyte in a fluid sample. The nature of the analyte monitored according to the present invention is not limited as long as the analyte can undergo a chemical reaction catalyzed by flavoprotein enzymes. Some examples of the types of analytes that can be collected and analyzed include glucose, the lipid repertoire (e.g., cholesterol, triglycerides, LDL and HDL), microalbumin, hemoglobin, A_1CFructose, lactate or bilirubin. It is contemplated that other analyte concentrations may be determined. The analyte may be in, for example, a whole blood sample, a serum sample, a plasma sample, other bodily fluids such as ISF (interstitial fluid), urine, and non-bodily fluids.

The test sensors described herein are electrochemical test sensors. A meter for an electrochemical test sensor may have an optical aspect for detecting calibration information and an electrochemical aspect for determining information (e.g., analyte concentration) related to an analyte of a fluid sample. FIG. 1a shows one non-limiting example of an electrochemical test sensor. FIG. 1a shows a test sensor 10 comprising a base 11, a capillary channel and a plurality of electrodes 16 and 18. Region 12 is shown as the region defining the capillary channel (e.g., after placing the lid on the base 11). The plurality of electrodes includes a counter electrode 16 and a working electrode 18. The electrochemical test sensor may also include at least 3 electrodes, such as a working electrode, a counter electrode, a trigger electrode, or other electrodes used to detect interfering substances (e.g., hematocrit, ascorbate, uric acid) in the fluid sample. The working electrode used in the electrochemical sensor according to the embodiment of the present invention may be changed to a suitable electrode including, but not limited to, carbon, platinum, palladium, gold, ruthenium, rhodium, a combination thereof, and the like.

The electrodes 16 and 18 are connected to a plurality of leads 15a, b which, in the embodiment shown, terminate in a larger area referred to as the test-sensor contacts 14a, b. The capillary channel is generally located within the fluid receiving zone 19. Examples of electrochemical test sensors, including their operation, can be found, for example, in U.S. Pat. No.6,531,040 to Bayer Corporation. It is contemplated that other electrochemical test sensors may be used with embodiments of the present invention.

The fluid-receiving zone 19 contains at least one reagent for converting an analyte of interest (e.g., glucose) in a fluid sample (e.g., blood) into a chemical species that is electrochemically measurable according to the current generated by the components utilizing the electrode pattern. The reagent typically comprises an analyte-specific enzyme that reacts with the analyte and the electron acceptor to produce an electrochemically measurable species that can be detected by the electrode. The reagent contains a mediator or other substance that facilitates the transfer of electrons between the analyte and the electrode, a binder that holds the enzyme and the mediator together, other inert components, or combinations thereof.

A fluid sample (e.g., blood) may be applied to the fluid-receiving zone 19. The fluid sample is reacted with at least one reagent. Upon reaction with the reagent and binding to the plurality of electrodes, the fluid sample generates an electrical signal that facilitates determination of the analyte concentration. The leads 15a, b carry the electrical signal back to the opposite second end 42 of the test sensor 10 where the test sensor contacts 14a, b transfer the electrical signal to the meter.

Referring to FIG. 1b, a side view of the test sensor 10 of FIG. 1a is shown. As shown in FIG. 1b, the test sensor 10 of FIG. 1b also includes a cover 20 and a spacer 22. The base 11, lid 20 and spacer 22 may be made of various materials, such as a polymeric material. Non-limiting examples of polymeric materials that may be used to form the base 11, lid 20, and spacer 22 include polycarbonate, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyimide, and combinations thereof. It is contemplated that other materials may be used to form the base 11, lid 20, and/or spacer 22.

To form the test sensor 10 of fig. 1a and 1b, the base 11, spacer 22, and lid 20 can be joined using, for example, an adhesive or a heat seal. When the base 11, lid 20 and spacer 22 are adhered, a fluid receiving area 19 is formed. The fluid receiving area 19 provides a fluid channel for introducing a fluid sample into the test sensor 10. The fluid receiving area 19 is formed at a first or testing end 40 of the test sensor 10. The test sensor in embodiments of the present invention may be formed from a base and a lid without a spacer, wherein the fluid-receiving zone is formed directly within the base and/or lid.

The flavoproteins according to the invention include any enzyme with a flavofactor. Some non-limiting examples of flavoproteins include FAD-glucose oxidase (EC (enzyme Classification) No.1.1.3.4), flavin-hexose oxidase (EC No.1.1.3.5), and FAD-glucose dehydrogenase (EC No. 1.1.99.10). Other oxidases useful according to the present invention include, but are not limited to, lactate oxidase, cholesterol oxidase, alcohol oxidase (e.g., methanol oxidase), d-amino acid oxidase, choline oxidase, and FAD derivatives thereof. A preferred flavoprotein for use according to the present invention is FAD-glucose dehydrogenase (FAD-GDH).

Mediators according to the invention comprise phenothiazines of the formula,

and a phenoxazine of the formula,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹May be the same or different and is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, halogen, haloalkyl, carboxyl, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic ketonyl, aliphatic ketonyl, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfonic acid, dihydroxy boron, and combinations thereof. It is contemplated that their isomers may also be formed.

A preferred example of a phenothiazine which has been prepared and found to have suitable properties as a mediator for NADH is the water soluble sodium or ammonium salt of 3- (2 ', 5' -disulfonylphenylimino) -3H-phenothiazine of the formula,

3- (2 ', 5' -disulfonylphenylimino) -3H-phenothiazine is associated with a particularly low background current, which leads to an increase in the signal-to-noise ratio. Another preferred example that has been prepared and found to have suitable properties as NADH mediators is the 3- (3 ', 5' -dicarboxy-phenylimino) -3H-phenothiazine mediator. The background current of these phenothiazines was found to be much lower than previously used mediators.

Other phenothiazines and phenoxazines that have been found to have suitable properties as mediators of NADH are: 3- (4' -chloro-phenylimino) -3H-phenothiazine; 3- (4' -diethylamino-phenylimino) -3H-phenothiazine; 3- (4' -ethyl-phenylimino) -3H-phenothiazine; 3- (4' -trifluoromethyl-phenylimino) -3H-phenothiazine; 3- (4' -methoxycarbonyl-phenylimino) -3H-phenothiazine; 3- (4' -nitro-phenylimino) -3H-phenothiazine; 3- (4' -methoxy-phenylimino) -3H-phenothiazine; 7-acetyl-3- (4' -methoxycarbonylphenylimino) -3H-phenothiazine; 7-trifluoromethyl-3- (4' -methoxycarbonyl-phenylimino) -3H-phenothiazine; 3- (4' - ω -carboxy-n-butyl-phenylimino) -3H-phenothiazine; 3- (4' -aminomethyl-phenylimino) -3H-phenothiazine; 3- (4 ' - (2 "- (5" - (p-aminophenyl-1, 3, 4-oxadiazolyl) phenylimino) -3H-phenothiazine, 3- (4 ' -beta-aminoethyl-phenylimino) -3H-phenothiazine, 6- (4 ' -ethylphenyl) amino-3- (4 ' -ethylphenylimino) -3H-phenothiazine, 6- (4 ' - [2- (2-ethanoxy) ethoxy ] -ethoxyphenyl) amino-3- (4 ' - [2- (2-ethanoxy) ethoxy ] ethoxyphenylimino) -3H-phenothiazine, 3- (4 ' - [2- (2-ethanoxy) ethoxy ] ethoxy-phenylimino) -3H-phenothiazine -3H-phenothiazine; 3- (4' -phenylimino) -3H-phenothiazineboronic acid; 3- (3 ', 5' -dicarboxy-phenylimino) -3H-phenothiazine; 3- (4' -carboxyphenylimino) -3H-phenothiazine; 3- (3', 5-dicarboxy-phenylimino) -3H-phenoxazine; 3- (2 ', 5' -phenylimino) -3H-phenothiazinedisulfonic acid; and 3- (3' -phenylimino) -3H-phenothiazinesulfonic acid.

In one embodiment, a 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator is prepared by dissolving phenothiazine (1.53 moles, 1.1 equivalents, 306g) in 6.0L Tetrahydrofuran (THF) with stirring, then cooling to 0 ℃. Aniline 2, 5-disulfonic acid (1.38 moles, 350g) was dissolved in 7.0L of water and 1M sodium hydroxide (NaOH) (128ml) was added with stirring. The aniline 2, 5-disulfonic acid solution was slowly added to the phenothiazine solution over about 2 hours to give a white turbid suspension. The phenothiazine/aniline suspension is at a temperature of about 0-4 ℃. Sodium persulfate (5.52 moles, 4 equivalents, 1314g) was dissolved in 4.0L of water to form a sodium persulfate solution.

Sodium persulfate solution was added dropwise over 3 hours to a phenothiazine/aniline suspension at a temperature of about 0-3 ℃ to give a dark black solution. The dark black solution was kept cool using an ice bath and stirred overnight. The contents were then transferred to a Buchi rotary evaporator and the tetrahydrofuran removed in about 2 hours at a temperature below 35 ℃. After evaporation, the residual solution was transferred to a 25L separator and washed back with ethyl acetate. The remaining solution was washed 3 times in reverse, using 2L of ethyl acetate each time. Reaction fluid in acetone/CO₂The bath was cooled to-3 ℃ with stirring. In the same day, pass through twoThe precipitated solid was filtered through two cloths on a 24cm Buchner funnel. The precipitated solid was left to dry overnight in the funnel and then transferred to a flask containing 2L of acetonitrile and stirred at room temperature for about 1 hour. To remove residual water, the sample was filtered and washed with more acetonitrile. The mediator was dried in a vacuum oven at 35 ℃ to constant weight.

Since low background currents are achieved using reagents having 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediators, the same reagent formulations can be applied to the working and counter electrodes of electrochemical test sensors. Applying the same reagents to the working and counter electrodes simplifies the manufacturing process and thus reduces the associated costs. In addition, low background currents help to obtain accurate glucose readings, especially with samples having low glucose concentrations, which is particularly important for analyzing blood glucose assays in newborns.

The reagents of embodiments of the invention also comprise a surfactant or mixture of surfactants and/or a cellulosic polymer. The surfactant or surfactant mixture facilitates the blood fill rate of the sensor and rehydration of the dried reagent. To achieve rapid assays (e.g., assays in less than 5 seconds) in the hematocrit range of about 20% to 70%, faster blood fill rates and reagent rehydration rates are desirable.

The surfactant is preferably selected from biocompatible surfactants, including sugar surfactants or phosphorylcholine based surfactants. One non-limiting example of a saccharide surfactant is heptanoyl-N-methylglucamide (MEGA 8 from Sigma-Aldrich of St.Louis, Mo.). Surfactants such as MEGA 8 help to improve the thermal stability of the test sensors. Furthermore, surfactants such as MEGA 8 contribute to a fast fill rate even for blood samples with high hematocrit levels. The use of a surfactant such as MEGA 8 and other inert ingredients (e.g., hydroxyethyl cellulose polymer and/or neutral pH buffer) in the reagent formulation provides a sensor with high stability, even at high temperatures. Non-limiting examples of phosphorylcholine based surfactants include the Lipidure series (NOF Corporation, Japan).

The surfactant may also be selected from conventional neutral surfactants such as ethoxylated oleyl alcohol (Rhodasurf ON870 from Rhodia inc. of Cranbury, NJ). The surfactant may also be selected from anionic surfactants such as sodium methyl cocoyl taurate (Geropon TC-42 from Rhodia Inc.) and alkylphenol ethoxylated phosphate esters (Phospholan CS131 from Akzo-Nobel surface Chemistry LLC of Chicago, IL). It is contemplated that other surfactants may be used to form the agent.

Alternatively or additionally, the reagents of embodiments of the invention comprise a polymer. The reagent may comprise a cellulosic polymer, such as a hydroxyethylcellulose polymer. In some embodiments, the cellulosic polymer is a low to medium molecular weight cellulosic polymer. Polymers such as cellulose-based polymers help provide increased stability and sufficient viscosity to the reagent so that the reagent remains in an initial position on the sensor substrate when dried. It is contemplated that other polymers may be used, such as polyvinylpyrrolidone (PVP).

The reagents may also include buffers (e.g., phosphate buffers) and/or other inert ingredients. Non-limiting examples of suitable buffer solutions include, but are not limited to, Good's buffers (e.g., HEPES (i.e., N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid), MOPS (i.e., 3- (N-morpholinyl) propanesulfonic acid), TES (i.e., N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)), McIlvaine's buffers, or combinations thereof, and the like.

To provide the desired assay accuracy, thermal stability and maximum kinetic performance, the ratio of inorganic salt to mediator should be less than about 3: 1. The source of the inorganic salt may be from the buffer and/or mediator. Even more preferably, the ratio of inorganic salt to mediator is less than about 2: 1 or even less than about 1.5: 1.

According to one embodiment of the invention, the reagent comprises FAD-GDH, a low background phenothiazine mediator, a surfactant or surfactant mixture, a cellulosic polymer, and a buffer for improving sensor performance and stability. The reagent can be used to determine the glucose concentration in a biological sample such as blood, plasma, serum or urine. In one embodiment, the phenothiazine mediator is 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine. In another embodiment, the surfactant is MEGA 8 and the polymer is hydroxyethyl cellulose. In one embodiment, the reagent comprises FAD-GDH having an activity of about 0.1 to 10U/. mu.L, about 5 to 120mM of 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator, about 0.05 to 0.5 wt.% of MEGA 8 surfactant, about 0.1 to 4 wt.% of hydroxyethyl cellulose, and about 25 to 200mM of buffer having a pH of about 4 to 8. In another embodiment, the reagent comprises FAD-GDH having an activity of about 0.5 to 2.5U/. mu.L, about 30 to 60mM of 3- (2 ', 5' -disulfonylphenylimino) -3H-phenothiazine mediator, about 0.1 to 0.4 wt.% of MEGA 8 surfactant, 0.01 to 0.1% of Geropon TC-42, about 0.2 to 0.5 wt.% of hydroxyethylcellulose, and about 50 to 150mM of buffer having a pH of about 6 to 7.

Example 1

As shown in fig. 2, the chemical reactivity of 4 sensor batches was analyzed by generating a glucose dose-response curve for a sensor comprising FAD-GDH enzyme with an activity of about 1.75U/μ L, about 40mM of 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator, about 0.2 wt.% of MEGA 8 surfactant, about 0.25 wt.% of hydroxyethyl cellulose, and about 100mM of phosphate buffer at a pH of about 6.5. The sensor was tested using a whole blood sample at a hematocrit level of about 40%. Blood glucose concentrations of the blood samples were approximately 0mg/dL, 38mg/dL, 67mg/dL, 112mg/dL, 222mg/dL, 339mg/dL, and 622 mg/dL. For each blood sample, 10 replicates were collected for each sensor batch. As shown in fig. 2, the average current for each sample was plotted versus the Glucose concentration (mg/dL) of the sample measured using Yellow Springs Glucose Analyzer (YSI, inc., Yellow Springs, Ohio) for each sensor batch. The slope of the dose response line was about 20nA/mg/dL, indicating that the sensitivity of the test sensors was relatively high. The y-intercept was relatively close to 0nA, indicating a low background noise level. These results indicate that accurate readings can be obtained using test sensors that include the reagents described herein.

The coefficient of variation was determined for each of the 10 replicates used to produce the sensor lot of the graph of fig. 2. Table 1 below shows the percentage of the mean coefficient of variation (CV%) for 4 sensor batches.

TABLE 1

Glucose concentration

38mg/dL 67mg/dL 112mg/dL 222mg/dL 339mg/dL 622mg/dL

CV％ 3.1 2.1 3.4 2.5 2.1 1.2

Due to the low background noise of sensors comprising reagents according to embodiments of the invention, the average assay CV% is less than 3.5%, even for samples with low glucose concentrations. Therefore, the CV% value is less than 5%, which is generally considered as a standard allowable limit. This low CV% indicates high accuracy of the test sensor. Furthermore, a low CV% correlates with low variation between test sensors, which is preferable for obtaining consistent test results.

Example 2

Figure 3 shows the effect of MEGA 8 surfactant on sensor fill rate using 60% hematocrit whole blood. The test sensor used in FIG. 3 included FAD-GDH with an activity of about 1U/μ L (about 192U/mg), about 4 wt.% (about 120mM) of potassium ferricyanide mediator, about 1.6-4 wt.% hydroxyethyl cellulose, and about 35mM citrate buffer at a pH of about 5.0. Potassium ferricyanide mediator was used to test whether MEGA 8 surfactant without 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator had the desired effect on the test sensors. The fill rate of a group of 30 test sensors containing about 0.2 wt.% MEGA 8 surfactant was compared to a control group of 30 test sensors without MEGA 8 surfactant. The initial fill rate of 10 sensors of each group was measured. Then, 10 sensors of each group were placed at a temperature of about-20 ℃ for about two weeks. Finally, 10 sensors of each group were placed at a temperature of about 50 ℃ for about two weeks. The average fill time for each group and subgroup of test sensors was calculated and is shown in fig. 3. Using a reagent with a surfactant and a 60% hematocrit whole blood sample, the blood fills the reaction chamber of the test sensor in less than 0.3 seconds. As shown in fig. 3, the fill rate of the sensor containing MEGA 8 surfactant was at least 2 times faster, up to about 4 times faster than the fill rate of the sensor without MEGA 8 surfactant.

Example 3

The background current of the heat stress sensor containing MEGA 8 surfactant was compared with the background current of the heat stress sensor without MEGA 8 surfactant. The test sensor used in FIG. 4 included FAD-GDH with an activity of about 1U/. mu.L, about 50mM of 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator, about 0.75 wt.% hydroxyethyl cellulose, and about 50mM buffer at a pH of about 7. The first set of 40 test sensors contained no MEGA 8 surfactant. The second set of 40 test sensors contained about 0.2 wt.% MEGA 8 surfactant. The first and second groups each comprise 2 packets: the first group included 20 test sensors with FAD-GDH from AmanoEnzyme inc. (Nagoya, Japan), and the second group included 20 test sensors with FAD-GDH from Toyobo co. (Osaka, Japan). Each group of 10 test sensors was stored at about 50 ℃ for about two weeks. The remaining test sensors were stored at about-20 ℃ for about two weeks. The sensor was then tested for background current using a 40% hematocrit whole blood sample with a glucose concentration of about 0 mg/dL. 10 replicates of each sample were collected. Fig. 4 shows the average sensor background current for 10 replicates. As shown in fig. 4, the test sensor containing the MEGA 8 surfactant had a very low change in sensor background current compared to the test sensor without the MEGA 8 surfactant, indicating that the reagent was more stable when the MEGA 8 surfactant was added to the reagent.

Example 4

The thermal stability of the test sensors according to embodiments of the present invention was also tested. The test sensor used in this example included FAD-GDH having an activity of about 2U/μ L, about 40mM of 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator, about 0.2 wt.% of MEGA 8 surfactant, about 0.25 wt.% of hydroxyethyl cellulose, and about 100mM of buffer having a pH of about 6.5. The first set of test sensors was stored at about 50 ℃ for about two weeks. The second set of test sensors was stored at a temperature of about-20 c for about two weeks. The performance of the sensors in each group was then evaluated using whole blood samples of 40% hematocrit having glucose concentrations of about 50mg/dL, about 100mg/dL, and about 400 mg/dL. 10 replicates of each sample were collected. The average difference in glucose concentration between the test sensor stored at 50 ℃ and the test sensor stored at-20 ℃ was calculated and compared to several different types of self-test blood glucose monitoring systems. The test sensors according to embodiments of the present invention have negligible glucose assay bias. Thus, the results of the glucose assay did not change significantly even after storage of the sensor for two weeks at relatively extreme temperatures. In contrast, commercial test sensors for comparison typically have a dextrose assay bias of about 5% to 12%. Thus, the thermal stability of the test sensors of embodiments of the present invention is significantly superior to existing test sensors.

Example 5

A test is performed using the test sensor to determine the fill rate of a high hematocrit blood sample. Specifically, as shown in fig. 5, formulations without surfactant (formulation 1) and formulations with surfactant (formulations 2-7) were tested. The formulations are listed in table 2 below.

TABLE 2

	Formulation of	Formulation of	Formulation of	Formulation of	Formulation of	Formulation of	Formulation of
									1	2	3	4	5	6	7
Mediator (mM)	60	60	60	60	60	50	90
								Buffer (mM)	75	75	75	75	75	100	112
FAD-GDH(U/μL)	3.00	3.00	3.00	3.00	3.00	1.25	3.75
								Polymer (HEC) (%)	0.38	0.38	0.38	0.38	0.38	0.63	0.60
MEGA 8(wt.％)		0.10	0.05				0.225
								Rhodasurf(wt.％)				0.10	0.05
Amphoteric detergent 312 (wt.%)						0.30
								Phospholan CS 131(wt.％)							0.10

Mediator 3- (2 ', 5' -disulfonic phenylimino) -3H-phenothiazine

Buffer (phosphate) except for TES used in formulation 6

TES ═ N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)

HEC ═ hydroxyethyl cellulose

Specifically, formulations 2 and 3 contained the surfactant MEGA 8, and formulations 4 and 5 contained the surfactant Rhodasurf. Formulation 6 contained surfactant amphoteric detergent and formulation 7 contained surfactant MEGA 8 and Phospholan (sulphate) CS131 d. Formulations 1-7 were stressed under two different conditions. Specifically, formulations 1-7 were stressed at a temperature of-20 ℃ for 2 weeks and at a temperature of 50 ℃ for 2 weeks.

Following stress under these conditions, formulations 1-7 were deposited on the electrodes of the test sensors. The sensors were tested in the vertical (90) position using 60-70% hematocrit whole blood. The sensor was videotaped during filling and the time was measured. The time required to fill the entire sensor reaction chamber with high hematocrit blood for formulations 1-7 is shown in fig. 5. For sensors stored at-20 ℃ and 50 ℃ for 2 weeks, the sensor-fill times for formulation 1 (without surfactant) were about 0.6 seconds and 0.7 seconds, respectively. In one aspect, the sensor-fill times in formulations 2-7 are all about 0.5 seconds or less for sensors stored at-20 ℃ and 50 ℃ for 2 weeks. For sensors stored at-20 ℃ and 50 ℃ for 2 weeks, the sensor-fill time for most of the formulations 2-7 was about 0.4 seconds or less, with a few being less than about 0.3 seconds. Thus, formulations 2-7 with surfactant greatly improved fill times compared to formulation 1 without surfactant.

Example 6

The reagents were tested to determine their maximum kinetic performance. The reagent comprises 40mM of 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine, 50mM of phosphate buffer, 2.00U/. mu.l of FAD-GDH, 0.25 wt.% of hydroxyethyl cellulose (HEC) and 0.20 wt.% of surfactant MEGA 8.

FIG. 6 shows the output signal of a test sensor with a blood sample having a glucose concentration of 400mg/dL and a hematocrit of 70%. The signal input to the sensor strip by the measurement device is a gated current pulse sequence comprising 8 pulses of excitation separated by 7 relaxations, such as described in U.S. patent application publication No. 2008/0173552. The duration of the excitation is less than 1 second. 3 output current values were recorded during each excitation.

To correlate the output current value from the input signal with the analyte concentration of the sample, the initial current value from the excitation is preferably greater than the value during decay. The output signal from the sensor strip of fig. 6 shows an initial high current value that decays about 2 seconds after the blood sample is introduced to the sensor strip. Thus, a first output current is observed in output current 110 with a high initial current value followed by a decay current value.

To correlate the output current values from the input signal with the analyte concentration of the sample, different sample analyte concentrations also preferably exhibit a substantially constant difference between the output signal current values. Preferably, the output current value related to the analyte concentration of the sample is also obtained from the decay of the current data comprising the maximum kinetic performance of the sensor strip. The kinetics of the redox reaction that produces the output current are affected by a number of factors. These factors may include the rate of rehydration of the reagent composition, the rate at which the enzyme system reacts with the analyte, the rate at which electrons are transferred by the enzyme system to the mediator, and the rate at which electrons are transferred by the mediator to the electrode. Of these and other kinetic factors that affect output current, the rate of rehydration of the reagent composition is believed to have the greatest effect on output current.

During the excitation of the gated current pulse sequence, a maximum kinetic performance of the sensor strip may be achieved when the initial current value of the excitation with decay current values is the maximum initial current value obtained from the plurality of excitations. This may also be referred to as sensor-peak time. Preferably, the maximum kinetic performance of the sensor strip is achieved when the end of current value obtained from the excitation with decay current value is the maximum end of current value obtained from the plurality of excitations. More preferably, the maximum kinetic performance of the sensor strip may be reached during a time period from when the initial current value of an excitation having a decay current value is the maximum initial current value obtained from a plurality of excitations to when the final current value obtained from the same excitation is the maximum final current value obtained from a plurality of excitations.

The maximum kinetic performance of the sensor strip is preferably less than about 3 seconds, more preferably less than about 2 seconds.

The gated current pulse sequence used to measure the maximum kinetic performance of the sensor strip included at least 7 duty cycles with an excitation duration of about 0.4 seconds and a relaxation duration of 1 second, including zero current through the sample and provided by an open circuit. At least 3 output current values are measured during each excitation. The potential applied to the sensor strip was maintained essentially constant at 250mV and the sample temperature was 23 ℃. A 400mV pulse was applied for 0.9 sec before the duty cycle.

The sensor strip of FIG. 6 containing 400mg/dL glucose reached maximum kinetic performance during the stimulated decay process, including output currents 120 and 125, between 3 and 4 seconds from the introduction of the sample to the strip. This is determined because there is both a maximum initial current value and a maximum current end value obtained from the excitation with decay current values in the cycle including the output currents 120 and 125. Initial output current 120 is compared to output currents 110, 130, 140, 150, 160, and 170, and output current final value 125 is also compared to output current final values 115, 135, 145, 155, 165, and 175. Thus, the sensor achieves maximum kinetic performance between 3 and 4 seconds even for a 70% hematocrit blood sample.

Example 7

Fig. 7-9 show that reagent formulations can also affect the maximum kinetic performance of blood with different hematocrit levels. Referring first to FIG. 7, the maximum kinetic performance using sensor-peak time increases with increasing hematocrit levels at a glucose concentration of 50 mg/dL. Mediators containing a smaller percentage of sulfate (5% sulfate) generally have faster peak times at comparable hematocrit levels and buffer concentrations. See, for example, 60% hematocrit levels when 50mM phosphate buffer is used (compare 3.5 seconds for a mediator using 5% sulfate to 7.5 seconds for a mediator using 20% sulfate). Reagent formulations, such as buffer strength and residual sulfate content of the mediator, have a large impact on the peak time of the sensor reaction, especially for high hematocrit (> 40%) samples. Thus, high inorganic salts (from the buffer or from the mediator, converted to sulfate concentration) in the sensor formulation can increase the sensor-peak time (i.e., slow the sensor reaction). FIG. 8 shows similar results using a glucose concentration of 100 mg/dL. Using a high glucose concentration of 400mg/dL, fig. 9 shows the maximum kinetic performance using sensor-peak times generally below about 3 or 3.5 seconds and some sensor-peak times of 2 seconds. At high hematocrit levels, mediators containing 5% sulfate and 100mM phosphate buffer had the greatest kinetic performance using a sensor-peak time of about 4.5 or 5 seconds.

Thus, to achieve rapid reagent rehydration and glucose response for high hematocrit samples, the salt content in the reagent formulation must be reduced, as shown in the 50mg/dL and 100mg/dL glucose concentrations.

Example 8

The maximum kinetic performance of both formulations was tested after the test sensors were stored under stressed conditions (-20 ℃ and 50 ℃/4 weeks). The formulation in figure 10a contains 50mM phosphate buffer and 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine with 5 wt.% sulfate. The formulation in figure 10b contains 100mM phosphate buffer and 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine with 20 wt.% sulfate. The gated current pulse sequence used in this example is similar to that described above in example 6.

As shown in fig. 10a, the maximum kinetic performance was about 2 seconds for the sample stressed at-20 ℃ for 4 weeks. For the sample stressed at 50 ℃ for 4 weeks, the maximum kinetic performance was about 3.5 seconds. Referring to FIG. 10b, the maximum kinetic performance was about 3.5 seconds for the sample stressed at-20 ℃ for 4 weeks and about 5 seconds for the sample stressed at 50 ℃ for 4 weeks. Thus, the formulation with lower inorganic salt content (e.g., fig. 10a) has improved maximum kinetic performance compared to the formulation with higher inorganic salt content (e.g., fig. 10 b).

In addition, the assay bias or% bias was tested for the formulation lot used in fig. 10a with a 2 second sensor-peak time. The% deviation of glucose concentration no greater than 100mg/dL is less than about +/-2%. The% deviation for a glucose concentration of 400mg/dL was about +/-4%. Assay bias was also tested for the formulation lot used in fig. 10b with 3-4 second sensor-peak time. The% deviation of glucose concentration no greater than 100mg/dL is less than about +/-3%. The% deviation for a glucose concentration of 400mg/dL was about +/-10%. Thus, the assay bias for batches with 3-4 second sensor-peak times is greater than for batches with 2 second sensor-peak times.

Example 9

Referring to FIG. 11, the coefficient of variation (CV%) for whole blood samples using 40% hematocrit at different glucose concentrations is shown. The concentration range of the glucose is 36-627 mg/dL. The reagent solution with the low salt content lot is compared with the reagent solution with the high salt content lot. The low salt batch contained 50mM phosphate buffer pH 6.5, 2U/. mu.l FAD-GDH, 40mM 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine with 5 wt.% sulfate, 0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8 surfactant. The high salt batch contained 100mM phosphate buffer pH 6.5, 2U/. mu.l FAD-GDH, 40mM 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine with 20 wt.% sulfate, 0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8 surfactant.

CV% was calculated by obtaining the average maximum kinetic performance using sensor-peak times and dividing by the standard deviation of these sensor-peak times. This result is multiplied by 100 to obtain CV%. A total of 40 samples were tested for the low salt reagent solution and the high salt reagent solution.

The low salt reagent solution used less than 3 seconds sensor-peak time to maximum kinetic performance, resulting in better CV% for the 40% hematocrit whole blood sample compared to the high salt reagent solution. For samples with lower glucose concentrations, the low salt reagent solution had better CV%.

While the examples provided herein relate to the in vitro use of test sensor reagents according to the invention, it is contemplated that these reagents may also be suitable for in vivo analyte monitoring by chemically immobilizing the mediator (e.g., by chemical reaction on one or more substituents of the aromatic ring) and incorporating the immobilized mediator into a device that may be subcutaneously implanted in a patient. The reagents of the embodiments described herein may also be used in continuous analyte monitoring systems.

Alternative embodiment A

A reagent for detecting an analyte, the reagent comprising:

a flavoprotein enzyme;

a mediator selected from the following formulae or a combination thereof,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹May be the same or different and is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, halogen, haloalkyl, carboxyl, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic ketonyl, aliphatic ketonyl, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfonic acid, dihydroxy boron, and combinations thereof;

at least one surfactant;

a polymer; and

a buffering agent.

Alternative embodiment B

The reagent of alternative embodiment A, wherein said flavoprotein is FAD-glucose dehydrogenase.

Alternative embodiment C

The reagent of alternative embodiment A, wherein the mediator comprises 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine.

Alternative embodiment D

The reagent of alternative embodiment a, wherein the surfactant comprises a saccharide surfactant or a phosphorylcholine-type surfactant.

Alternative embodiment E

The reagent of alternative embodiment a, wherein the polymer is a cellulosic polymer.

Alternative embodiment F

The reagent of alternative embodiment a, wherein the buffer comprises a phosphate buffer.

Alternative embodiment G

A reagent for detecting an analyte in a fluid sample, the reagent comprising:

FAD-glucose dehydrogenase having an activity of about 0.1 to 10U/. mu.L;

a 3- (2 ', 5' -disulfonic phenylimino) -3H-phenothiazine mediator at a concentration of about 5-120 mM;

a heptanoyl-N-methylglucamide surfactant at a concentration of about 0.05 to 0.5 wt.%; and

hydroxyethyl cellulose polymer at a concentration of about 0.1 to 4 wt.%.

Alternative embodiment H

The reagent of alternative embodiment G further comprising a phosphate buffer.

Alternative embodiment I

The reagent of alternative embodiment H, wherein the phosphate buffer is at a concentration of about 25 to 200mM and at a pH of about 4 to 8.

Alternative embodiment J

The reagent of alternative embodiment I, wherein the phosphate buffer is at a concentration of about 50 to 150mM and at a pH of about 6 to 7.

Alternative embodiment K

The reagent of alternative embodiment G wherein the reagent comprises FAD-glucose dehydrogenase having an activity of about 0.5 to 2.5U/. mu.L, a 3- (2 ', 5' -disulfonylphenylimino) -3H-phenothiazine mediator at a concentration of about 30 to 60mM, a heptanoyl-N-methylglucamide surfactant at a concentration of about 0.1 to 0.4 wt.%, and a hydroxyethylcellulose polymer at a concentration of about 0.2 to 0.5 wt.%.

Alternative embodiment L

An electrochemical test sensor, comprising:

a working electrode having a surface;

a counter electrode having a surface; and

a reagent coating at least a portion of a surface of the working electrode and at least a portion of a surface of the counter electrode, the reagent comprising a flavoprotein, a phenothiazine or phenoxazine mediator, a buffering agent, at least one surfactant, and a polymer.

Alternative embodiment M

The sensor of alternative embodiment L, wherein the flavoprotein comprises FAD-glucose dehydrogenase.

Alternative embodiment N

The sensor of alternative embodiment L, wherein the phenothiazine mediator includes 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine.

Alternative embodiment O

The sensor of alternative embodiment L, wherein the at least one surfactant comprises heptanoyl-N-methylglucamide.

Alternative embodiment P

The sensor of alternative embodiment L, wherein the polymer is a cellulosic polymer.

Alternative method Q

A method for detecting an analyte in a fluid sample, the analyte undergoing a chemical reaction, the method comprising the steps of:

providing an electrode surface;

causing the fluid sample to flow to the electrode surface using at least one surfactant;

catalyzing the chemical reaction with a flavoprotein enzyme;

producing a redox equivalent by the chemical reaction; and

transferring the redox equivalent to the electrode surface using a phenothiazine or phenoxazine mediator.

Alternative Process R

The method of alternative method Q, wherein the electrode surface comprises a working electrode and a counter electrode, the electrode surface comprising reagents comprising at least one surfactant, a flavoprotein enzyme, a phenothiazine mediator, and a buffer.

Optional method S

The method of alternative process R, wherein the reagent further comprises a polymer.

Alternative method T

The method of alternative method S, wherein the polymer is a cellulosic polymer.

Optional method U

The method of alternative process R, wherein the buffer comprises a phosphate buffer.

While the invention is susceptible to various modifications and alternative forms, specific embodiments and methods thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the invention is not intended to be limited to the particular forms or methods disclosed. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (9)

1. A reagent for detecting an analyte, the reagent comprising:

a flavoprotein enzyme;

a 3- (2 ', 5' -disulfonic phenylimino) -3H-phenothiazine mediator;

at least one surfactant;

a polymer; and

a buffering agent, a water-soluble polymer,

wherein at least one of the mediator and the buffer comprises an inorganic salt, the ratio of total inorganic salt to mediator being less than 3: 1.

2. the reagent of claim 1, wherein the ratio of total inorganic salt to mediator is less than 2: 1.

3. a reagent for detecting an analyte in a fluid sample, the reagent comprising:

FAD-glucose dehydrogenase with the activity of 0.1-10U/muL;

a 3- (2 ', 5' -disulfonic phenylimino) -3H-phenothiazine mediator at a concentration of 5-120 mM;

a surfactant at a concentration of 0.05 to 0.5 wt.%;

a hydroxyethyl cellulose polymer at a concentration of 0.1 to 4 wt.%; and

a buffering agent, a water-soluble polymer,

wherein at least one of the mediator and the buffer comprises an inorganic salt, the ratio of total inorganic salt to mediator being less than 2: 1.

4. the reagent according to claim 3, wherein the reagent comprises FAD-glucose dehydrogenase having an activity of 0.5 to 2.5U/. mu.L, a 3- (2 ', 5' -disulfonylphenylimino) -3H-phenothiazine mediator having a concentration of 30 to 60mM, a heptanoyl-N-methylglucamide surfactant having a concentration of 0.1 to 0.4 wt.%, and a hydroxyethyl cellulose polymer having a concentration of 0.2 to 0.5 wt.%.

5. An electrochemical test sensor, comprising:

a working electrode having a surface;

a counter electrode having a surface; and

a reagent coating at least a portion of the surface of the working electrode and at least a portion of the surface of the counter electrode, the reagent comprising a flavo-protease, a 3- (2 ', 5' -disulfonated phenylimino) -3H-phenothiazine mediator, a buffer, at least one surfactant, and a polymer, wherein at least one of the mediator and the buffer includes an inorganic salt, and wherein the ratio of total inorganic salt to mediator is less than 3: 1.

6. a test sensor for assisting in determining a concentration of at least one analyte in a fluid sample, the test sensor comprising:

a working electrode and a counter electrode;

a reagent, the reagent comprising:

a flavoprotein enzyme;

a 3- (2 ', 5' -disulfonic phenylimino) -3H-phenothiazine mediator;

at least one surfactant; and

a buffer, and

a capillary channel extending from the testing end of the test sensor to the reagent,

wherein the% deviation in analyte concentration determined using the test sensor is less than +/-10%, wherein at least one of the mediator and the buffer comprises an inorganic salt, and the ratio of total inorganic salt to mediator is less than 3: 1.

7. the test sensor of claim 6, wherein the reagent further comprises at least one polymer.

8. The test sensor of claim 6, wherein the% deviation in analyte concentration determined using the test sensor is less than +/-4%.

9. The test sensor of claim 6, wherein the% deviation in analyte concentration determined using the test sensor is less than +/-2%.