HK1116220A

HK1116220A - Stabilizing the activity of pqq-dependent glucose dehydrogenase in electrochemical biosensors

Info

Publication number: HK1116220A
Application number: HK08105443.4A
Authority: HK
Inventors: A．H．查; H．G．斯普拉德林
Original assignee: 拜尔保健有限公司
Priority date: 2005-03-04
Filing date: 2006-03-03
Publication date: 2008-12-19

Description

Stabilizing PQQ-dependent glucose dehydrogenase activity in electrochemical biosensor

Technical Field

The present invention is generally directed to the field of medical devices for analyzing biological fluids.

Background

More particularly, the present invention relates to biosensors for measuring the amount of an analyte in a body fluid, in particular measuring glucose in a whole blood sample. Optical methods are commonly used to make such measurements, but the present invention relates to improvements in electrochemical sensors.

Although the methods of the invention to be described herein may be applied to the measurement of other analytes, the measurement of glucose in whole blood samples is of particular interest. The invention also relates to an electrochemical instrument, wherein a constant or varying potential is applied to an electrode contacting a blood sample, and the resulting current is measured after a short period of time. The measured current is correlated with the amount of analyte in the sample. Such instruments are known as galvanometers.

Glucose biosensors used in amperometric instruments may employ a number of reagent systems that react an enzyme with glucose in a sample and produce a measurable current by oxidation of a redox compound, referred to as a mediator, in the following general sequence of steps:

glucose + E_{Oxidation by oxygen}→E_{Reduction of}+ oxidized glucose (gluconolactone)

E_{Reduction of}+n Med_{Oxidation by oxygen}→n Med_{Reduction of}+E_{Oxidation by oxygen}

n Med_{Reduction of}→Med_{Oxidation by oxygen}+ne^-

Wherein E_{Oxidation by oxygen}And E_{Reduction of}Is an enzyme redox centre in both oxidised and reduced form, and Med_{Oxidation by oxygen}And Med_{Reduction of}Are mediators of both the oxidized and reduced forms. Glucose oxidase has been used as an enzyme in electrochemical biosensors, but recently, glucose dehydrogenase has been introduced. These enzymes are used with coenzymes or cofactors, such as NAD, FAD, and PQQ. Among others familiar to those skilled in the art, the mediator may be a ferricyanide or tetrazolium salt.

Glucose Dehydrogenase (GDH), its co-factor and a mediator are combined in a formulation that is applied to an electrode pair, described as a working and counter electrode. When a potential is applied across the electrodes, the enzyme/cofactor oxidizes glucose (the analyte) and the mediator is reduced as it reoxidizes the enzyme. The reduced mediator migrates to the working electrode where it is reoxidized and in the process releases electrons that move to the counter electrode and establish a current that is proportional to the amount of glucose present in the sample.

Because a new sensor is used each time a patient tests their blood glucose level, the reagent formulation should provide consistent performance from one biosensor to the next. It is clear that it is important that the results are reliable, as the user will adjust his diet or medication according to his test results. Thus, among other requirements, the enzyme should maintain its activity throughout its useful shelf life. The present invention is particularly directed to limiting or preventing loss of activity of an enzyme/co-factor system [ i.e., glucose dehydrogenase-pyrroloquinoline quinone (GDH-PQQ) ] used in electrochemical biosensors.

One method for maintaining the activity of GDH-PQQ is proposed in U.S. Pat. No. 6,656,702, which teaches the addition of sugars, particularly trehalose containing GDH-PQQ, to a reagent formulation. In one example, a hydrophilic polymer, carboxymethyl cellulose, is deposited on the electrode and dried. Then, a reagent mixture comprising GDH-PQQ, trehalose, and potassium ferricyanide as an electron acceptor (mediator) was deposited on the dried carboxymethyl cellulose layer by "dropping" and then dried to complete a reagent layer on the electrode. The patent also suggests that hydrophilic polymers may also be added to the agent-containing layer. The' 702 patent is believed to be indicated by reference to the "dripping" of reagent layers, which are deposited by dispensing reagent droplets into holes surrounding the bare electrodes.

Another patent (U.S. Pat. No. 6,270,637) describes a formulation for an electrochemical biosensor that contains GDH-PQQ and also contains hydroxyethyl cellulose. The patent emphasizes the value of including polyethylene oxide having a molecular weight of 100-900 kilodaltons (kDa). The method of applying its formulation to the electrodes is not provided in detail, but it is believed that this can be accomplished by dispensing the reagent with a pumping system.

Screen printing of reagent layers is used in the fields of U.S. Pat. nos. 5,708,247; 5,951,836, respectively; and 6,241,862 by the methods described in the specification. Glucose oxidase is used as the enzyme and the reagent composition further comprises hydroxyethyl cellulose and a treated silica selected to have a balance of hydrophobicity and hydrophilicity, thereby forming a two-dimensional network excluding red blood cells.

The reagent formulation may be deposited by various methods including dipping, stripe coating, ink jet printing or microdeposition using a syringe pump which may include the addition of a micro solenoid valve droplet ejection device. Screen printing is a particularly interesting method because it is efficient and well-adapted to mass production of biosensors. It requires that the formulation (i.e., ink) applied to the electrode have certain physical properties in order to be successfully applied. In particular, the ink applied by screen printing should have the following properties: adsorptivity with the substrate, cohesion, thixotropy (shear thinning), and optimum rheology with respect to viscosity and flow.

The present inventors found that the composition for screen printing when glucose oxidase was used as the enzyme could not be used when the enzyme was changed to GDH-PQQ because the new enzyme lost activity quickly. After the study, it was determined that some of the components used to provide the physical properties necessary for screen printing resulted in loss of enzyme activity. Therefore, it is necessary to find a component that does not cause premature inactivation of GDH-PQQ and satisfies the screen printing requirements. Those reagent formulations are described below.

Summary of The Invention

The invention in one aspect is a reagent composition for application to an electrode in an electrochemical biosensor. The novel compositions can be screen printed and avoid premature loss of enzyme-cofactor system (GDH-PQQ) activity for oxidizing glucose in a glucose biosensor. In a preferred embodiment, GDH-PQQ is combined with the following in a buffer solution maintained at a pH of 4.5-6.5: a hydrophilic polymer, preferably hydroxyethyl cellulose, and amorphous hydrophilic silica powder, a surfactant, and a mediator, preferably ferricyanide.

In one embodiment, the invention is an electrochemical biosensor in which the above-described reaction composition is screen printed onto the working and counter electrodes.

In another aspect, the invention is a method for maintaining the activity of GDH-PQQ in a screen printed reagent composition.

Brief Description of Drawings

Fig. 1a and b are exploded views of a typical electrochemical sensor.

FIG. 2 is a graph of reagent background current versus different buffers.

FIG. 3 is a histogram showing the effect of certain components in a reagent formulation on enzyme activity.

FIG. 4 is a histogram showing the effect of reagent compositions of the present invention on enzyme activity.

FIG. 5 is a graph of measured glucose versus a standard method.

Fig. 6 is a graph of the deviation in the measurement results of fig. 5.

Description of the preferred embodiments

Measurement of blood glucose content

The glucose contained in blood can be measured by various methods. Of particular interest are methods used by diabetic patients at home. These methods include optical and electrochemical methods, both of which can function by oxidizing glucose with an enzyme. The amount of glucose present is determined by measuring the color developed via the indicator or by measuring the current generated via oxidation of the redox mediator when a potential is applied across the electrode pair. The present invention relates to the latter method for measuring the glucose content of whole blood, and more particularly to an electrochemical biosensor using Glucose Dehydrogenase (GDH) and the cofactor pyrroloquinoline quinone (PQQ). The GDH-PQQ is combined with other components including mediators, polymers, surfactants, buffers, and thickeners to produce a composition that is deposited on the electrode pair. When a voltage is applied across the electrodes, the reaction of the GDH-PQQ with glucose and the redox reaction of the mediator generate a current proportional to the amount of glucose in the blood sample that has been contacted with the reaction composition.

The present invention is not limited to a particular biosensor design among many that have been disclosed in the art. One example of a biosensor that can be used is described in U.S. Pat. No. 6,531,040, which is illustrated in FIGS. 1 a-b.

An exploded view of the biosensor 10 is shown in fig. 1. It comprises an insulating base 12 on which are printed (typically by screen printing techniques) in sequence an electrical conductor pattern (pattern)14, an electrode pattern (portions 16 and 18), an insulating (dielectric) pattern 20, and a reactive layer 22. The biosensor is completed by adding a cover layer 28. A capillary 30 formed between the cover layer 28 and the reagent layer 22 provides a flow path for the fluid test sample.

The reagent in reagent layer 22 reacts with the analyte in the fluid test sample (e.g., glucose in blood) and generates a current that is measured and correlated to the amount of analyte present. The reaction layer 22 generally contains an enzyme and an electron acceptor. The enzyme reacts with the analyte to generate electrons, which are carried to the surface of the working electrode by an electron acceptor or mediator, which is reduced in response to the reaction between the analyte and the enzyme. In the present invention, the enzyme is Glucose Dehydrogenase (GDH), and pyrroloquinoline quinone (PQQ) which is a co-factor thereof, and the mediator is a ferricyanide salt.

The two portions 16, 18 of the electrode pattern provide the respective working and counter electrodes required to electrochemically determine the analyte concentration. A feature of the design shown is that the working and counter electrodes are configured such that a major portion of the counter electrode is located downstream (in terms of the direction of fluid flow along the flow channel) of the exposed portion of the working electrode 16 a.

However, counter electrode sub-element 18a is positioned upstream of working electrode upper element 16a such that when a test fluid sample volume (e.g., a whole blood sample) that is insufficient to completely cover the working electrode enters the capillary space, an electrical connection is made between counter electrode sub-element 18a and the exposed portion of working electrode 16a due to the conductivity of the whole blood sample. However, the available counter electrode area for contact by a whole blood sample is so small that only a very weak current can pass between the electrodes and thus through the current detector. By arranging for the current detector to give an error signal when the received signal is below a certain predetermined level, the sensor device informs the user that insufficient blood has entered the sensor chamber and that another test should be performed or that more blood should be added. Although the specific dimensions of the electrode are not critical, the area of the counter electrode sub-element 18a is typically about 10% less than the working electrode, and more specifically about 6% less. This element should be made as small as possible.

It is also contemplated in U.S. Pat. No. 6,531,040 that the reactive layer 22 may not be in contact with the counter electrode sub-element 18a by producing a screen that does not print reagent ink on the counter electrode sub-element 18 a. This will starve the subelement of reagent, thereby not allowing it to function as a proper counter electrode, so that an error condition is reached when the test fluid sample cannot contact the body of the counter electrode 18. Although the tube element 18a is shown as being physically connected to part of the counter electrode 18, 18a may be physically separated from the rest of the counter electrode, provided that it has its own connector and the sensor is provided with a third contact to the detector.

The working and counter electrodes are typically printed using an electrode ink, which is typically about 14 μm (0.00055 ") thick and typically contains electrochemically activated carbon. The composition of the conductive ink may be a mixture of carbon and silver selected to provide a low chemical resistance path between the electrode and the meter to which the electrode is connected via contact with the conductive pattern at the fish tail end 26 of the sensor. The counter electrode may comprise silver/silver chloride, although carbon is preferred. To enhance the repeatability of the meter readings, the dielectric pattern insulates the electrodes from the fluid test sample except for a defined area near the center of the electrode pattern 24. The defined area is important in this type of electrochemical assay because the measured current depends not only on the analyte concentration and the area of the reaction layer 22, but also on the area of the working electrode 16a exposed to the test sample containing the analyte.

A typical dielectric layer 20 comprises a UV-cured acrylate-modified monomer, oligomer, or polymer and is about 10 μm (0.0004 ") thick. The dielectric layer may also be moisture curable or thermally curable. The cover or cover 28 is adapted to mate with the base to form a space for receiving the fluid test sample in which the counter and working electrodes are disposed. The cover 28 provides a concave space 30 and is typically formed by embossing a flat sheet of deformable material. The lid 28 is perforated to provide a vent 32 and is connected to the base 12 by a sealing operation. The lid and base may be sealed together by sonic welding in which the base 12 and lid 28 are first aligned and then pressed together between the vibrating heat seal members or corners and the fixed jaws, with only contact being made with the flat, unembossed areas of the lid. The embossed cover and base may also be attached by using an adhesive material under the cover. A method of attaching the cover and base is more fully described in U.S. patent No. 5,798,031.

Suitable materials for the insulative base 12 include polycarbonate, polyethylene terephthalate, vinyl and acrylic polymers that are spatially stable, and polymer blends such as polycarbonate/polyethylene terephthalate, and metal foil structures (e.g., nylon/aluminum/polyvinyl chloride foil). The lid is typically made from a deformable polymeric sheet material such as polycarbonate or an embossable grade of polyethylene terephthalate, glycol modified polyethylene terephthalate, or a metal foil composition (e.g., an aluminum foil structure).

Other electrochemical sensors may be used in the present invention. An example of an electrochemical sensor that can be used to measure glucose concentration is in Bayer HealthCare's Ascensia^TMAUTODISC ® and Ascensia^TMThose used in the ELITE ® system. More details on such electrochemical sensors can be found in U.S. Pat. nos. 5,120,420 and 5,320,732. Other electrochemical sensors are available from the Matsushita Electric Industrial Company. Can be powered onFurther examples of electrochemical sensors used in flow assay monitoring systems are disclosed in U.S. Pat. No. 5,429,735.

The electrochemical sensor may be located in a blood glucose sensor dispensing instrument loaded with a plurality of sensors. One example of a sensor pack (pack) loaded in a sensor-dispensing instrument is disclosed in U.S. Pat. No. 5,660,791.

When the electrochemical sensor is placed in a suitable measuring instrument and a blood sample is introduced, a voltage is applied across the electrodes and the current associated with the glucose content of the sample is measured and reported to the user.

The amperometric sensor may apply a fixed voltage across the electrodes and measure the resulting current over a predetermined period of time, which may be very short, say 5-10 seconds, in order to correct for deviations that may exist due to premature reduction of mediator. That period is referred to as the "burn period". The current rises to a peak and then decreases while the sample rehydrates the reagent layer so that oxidation and reduction reactions occur. After this combustion period, the applied potential is removed or at least reduced during a quiescent period that allows the reaction to occur. The potential is then reapplied and the current measured for a predetermined "reading" period (e.g., 10 seconds). Since reduced mediator occurs due to concomitant oxidation of the enzyme, the current generated is high initially, but then decreases asymptotically and approaches a steady state. The current recorded at the end of the short "reading" period is used to determine the glucose content of the blood sample by means of the previously obtained correlation between the current at the end of the reading period and the glucose contained in the test sample having a known concentration.

Reactive composition

The composition that reacts with the blood sample in the electrochemical biosensor must provide a consistent response from one sensor to the next. A consistent response is important to users who rely on the results to adjust their diet or medication. It follows this: the change in activity of the agent over the lifetime of the biosensor should be as small as possible, but at least predictable, so that adjustments in the results can be made. The present invention solves the problems found when using enzyme-cofactor GDH-PQQ in an electrochemical biosensor in which the reaction composition is screen printed on the electrodes of the sensor. The present inventors found that GDH-PQQ among the components generally used in screen printing causes GDH-PQQ to lose activity.

A mixture of GDH-PQQ and potassium ferricyanide as a redox mediator was prepared in a series of buffers. While GDH-PQQ is known to be preferred at neutral pH, buffers that provide a pH of about 4.5 to about 6.5, more particularly, about 5.0 to about 6.0, including acetate, citrate, and succinate buffers, have been found to be more desirable because the mediator ferricyanide is more stable at acidic pH (see fig. 2).

It was found that certain polymeric materials also showed a loss of activity even when low pH buffers were used. The two polymers used to form the carrier for the enzyme and redox mediator were tested with 75mM calcium acetate and 75mM sodium succinate buffer. Potassium ferricyanide is not included. The results are shown in FIG. 3, which shows the initial effect on enzyme activity, and compared to the effect after 2 weeks at-20 ℃. As can be seen, when the reagent ink (reagent ink) containing 8.8 units of GDH-PQQ/mg and 10mM CaCl₂In the presence of only succinate and acetate buffers (but no ferricyanide), the buffers did not result in significant loss of activity after 2 weeks at-20 ℃. Using Beckman SYNCHRON CX 4^®Delta instruments measure enzyme activity by measuring the rate of formation of dimethyl (diformazan) by reduction of Nitrotetrazolium Blue (NBT) with Phenazine Methosulfate (PMS) at 37 ℃ at 560 nm. However, a significant loss of activity was seen when additives previously used to prepare screen-printing inks were included. Bentonite, a clay thickener, supplied by RHEOX, inc. was included at 1.3 wt% in the buffer solution. The results shown in FIG. 3 indicate that bentonite results in a loss of about 10% of the enzyme activity in acetate buffer, whereas in succinate buffer, it is newAbout 90% of the enzyme activity in fresh reagents and also from reagents stored at-20 ℃ for 2 weeks. When 7.4 wt% polyethylene oxide was added to the buffer solution, the activity was also lost, although the difference in the effect of the buffer was significant. It can be concluded that reagent formulations comprising GDH-PQQ in combination with bentonite or polyethylene oxide show lower enzyme activity and are therefore less desirable thickening components in screen printable reagent inks.

Investigation of the impact of other thickeners that may be suitable as replacements for bentonite and polyethylene oxide led to the discovery that certain materials produced reagent inks that were suitable for screen printing and did not cause loss of GDH-PQQ activity. The improved results are illustrated in fig. 4. In the test reported in FIG. 4, 4-8u/mg GDH-PQQ, 1.6 wt% CAB-O-SILM5 was added to acetate buffer solution^®Untreated fumed silica, 4.5-6.5 wt% hydroxyethyl cellulose (HEC), and several surfactants. The enzyme activity was measured using PMS/NBT enzyme assay on a Beckman analyzer at 37 ℃. CAB-O-SILM5^®Suitable for combination with HEC. It is clear that CAB-O-SILM5^®The combination with HEC did not result in loss of enzyme activity over the test period.

As has been shown, hydroxyethyl cellulose can be used as a matrix component in a reagent layer in electrochemical biosensors using GDH-PQQ without causing undue loss of enzyme activity. Other related hydrophilic polymers may also be used to make the viscosity of the composition suitable for screen printing. Other cellulose derivatives include, but are not limited to, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, or hydroxypropyl methyl cellulose. Other water-soluble polymers that may be useful include xanthan gum, guar gum, locust bean gum, carrageenan, agarose, and synthetic polymers including polyvinyl alcohol, polyvinyl pyrrolidone, and the like.

CAB-O-SILM5^®Is amorphous untreated fumed silica which has been shown to provide desirable physical properties to the inks used in screen printing of the reagent layers of the present invention. Such untreated parentThe aqueous silica is different from the treated silica taught in several earlier patents for balancing hydrophobic and hydrophilic properties. Alternative water-insoluble thickeners include, but are not limited to, talc, mica, diatomaceous earth, natural and modified clays such as bentonite (bentonitcelays), hectorite (hectorite clays) (e.g., Optigel sH from IMV Nevada), sepiolite (e.g., Sepiogel F from IMV Nevada), montmorillonite (e.g., IGB clay from IMV Nevada), saponite (saponate) (e.g., Sepiogel F and IMVITE 1016 from IMV Nevada, and Laponite from southern products), and the like.

As shown in FIG. 4, the choice of surfactant can also affect the activity of GDH-PQQ. Triton x-100 (an alkylaryl polyether alcohol) supplied by Sigma is very suitable. However, it is believed that other surfactants, including FC 170C (product of 3M), Surfynol 485 (product of Air Products and Chemicals, Inc.) and Pluronic L62D (product of BASF), may be used in the formulations of the present invention.

Preferred reagent compositions for screen printing biosensors according to the present invention include:

about 30 to about 200mM, and more particularly, about 50 to about 150mM of a buffer, preferably calcium acetate;

about 5 to about 50mM CaCl₂；

Up to about 0.5 wt% surfactant, based on the total weight of the composition;

from about 2 to about 10% by weight, based on the total weight of the composition, of a cellulose derivative, preferably hydroxyethyl cellulose;

from about 1 to about 6 weight percent, based on the total weight of the composition, of amorphous untreated silica;

from about 10 to about 20 weight percent, based on the total weight of the composition, of potassium ferricyanide as a mediator;

about 1 to about 8 units of GDH-PQQ enzyme-co-factor per mg total weight of the composition;

a viscosity of about 60,000 to about 180,000cps (mPas).

Example 1

A75 mM calcium acetate buffer, pH5.3, was prepared by adding calcium acetate and glacial acetic acid. To the buffer solution was added the following compounds: 10mM CaCl₂0.05 wt.% TritonX-100 surfactant, 1-3 wt.% Cabosil M5 amorphous untreated fumed silica powder, and 4-8 wt.% hydroxyethyl cellulose. After allowing the silica and cellulose to hydrate for 16 hours, 15-20 wt% potassium ferricyanide and 3-8 units of GDH-PQQ were added per mg of reagent ink. The reagent ink was mixed with a blade mixer at 600rpm for about 10-20 minutes, resulting in a reagent composition having a viscosity of about 80,000-140,000 cps.

The reagent composition was screen printed on a substrate containing electrodes and dried at 45-50 ℃ for 5 minutes.

Example 2

A glucose sensor was prepared using the reagent composition of example 1 and tested with a blood sample containing a known glucose content and having a hematocrit of 40%. A potential of 200-400 mv was applied to the electrodes and the current measured 10 seconds after the potential was applied was used to determine the glucose content of the sample. The results of these tests are shown in FIG. 5, where Ascensia over a 10 second test time would be planned^TMResponses of the AUTODISC ® glucose meter were plotted against glucose content measured by an industry standard YSI glucose meter. It can be seen that a linear response is obtained which is relatively insensitive to enzyme-cofactor concentrations in the range studied.

The sensor also exhibited a good dose response when compared to the results obtained from YSI instrumentation (industry standard). The deviation from YSI instrument results are shown in figure 6. For samples containing 400mg/dL glucose or less, the deviation is within + -5%, and for samples containing 680mg/dL glucose, the deviation is within + -10%.

Alternative embodiment A

A reagent composition for screen printing an electrochemical biosensor comprising:

(a) glucose Dehydrogenase (GDH) and co-factor pyrroloquinoline quinone (PQQ) for oxidizing glucose in a biological sample;

(b) a hydrophilic polymer selected from the group consisting of cellulose derivatives, natural gums and gels, and water-soluble synthetic polymers;

(c) a thickener selected from the group consisting of amorphous untreated silica powder, talc, mica, diatomaceous earth, and natural and modified clays;

(d) a buffer sufficient to maintain a pH of about 4.5 to about 6.5 to 6.0;

(e) a surfactant; and

(f) a mediator.

Alternative embodiment B

The reagent composition of alternative embodiment a wherein the buffer is an acetate, citrate or succinate buffer.

Alternative embodiment C

The reagent composition of alternative embodiment a wherein the surfactant is an alkylaryl polyether alcohol.

Alternative embodiment D

The reagent composition of alternative embodiment a, wherein the mediator is potassium ferricyanide.

Alternative embodiment E

The reagent composition of alternative embodiment a wherein the buffer is 5.0-6.0.

Alternative embodiment F

The reagent composition of alternative embodiment E wherein the composition has a viscosity of from about 60,000 to about 180,000cps (mPa-s).

Alternative embodiment G

The reagent composition of alternative embodiment a wherein the hydrophilic polymer is a cellulose derivative selected from the group consisting of sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose.

Alternative embodiment H

The reagent composition of alternative embodiment G wherein the hydrophilic polymer is hydroxyethyl cellulose.

Alternative embodiment I

The reagent composition of alternative embodiment a wherein the thickening agent is an amorphous untreated silica powder.

Alternative embodiment J

The reagent composition of alternative embodiment I wherein the amorphous untreated silica powder is hydrophilic.

Alternative embodiment K

The reagent composition of alternative embodiment A wherein 1-8 units of said GDH-PQQ are present per milligram of the total weight of the composition.

Alternative embodiment L

The reagent composition of alternative embodiment B, wherein the buffer is present at about 30 to about 200 mM.

AlternativesEmbodiment M of

The reagent composition of alternative embodiment C wherein up to about 0.5 weight percent of said polyether alcohol is present based on the total weight of said composition.

Alternative embodiment N

The reagent composition of alternative embodiment D wherein said potassium ferricyanide is present in an amount of about 10 to about 20 weight percent based on the total weight of the composition.

Alternative embodiment O

The reagent composition of alternative embodiment G wherein the cellulose derivative is present in an amount of about 2 to about 10 weight percent, based on the total weight of the composition.

Alternative embodiment P

The reagent composition of alternative embodiment I wherein said amorphous untreated silica powder is present in an amount of from about 1 to about 6 weight percent based on the total weight of said composition.

Alternative embodiment Q

The reagent composition of alternative embodiment a, further comprising about 5 to about 50mM of calcium chloride.

Alternative embodiment R

An electrochemical biosensor, comprising:

(a) a non-porous substrate;

(b) working and counter electrodes disposed on the substrate;

(c) a reagent composition screen-printed on the electrode, the composition comprising:

(1) about 1-8 units of Glucose Dehydrogenase (GDH) and the co-factor pyrroloquinoline quinone (PQQ) for oxidizing glucose in a biological sample per milligram of the total weight of the composition;

(2) a hydrophilic polymer selected from the group consisting of cellulose derivatives, natural gums and gels, and water-soluble synthetic polymers;

(3) a thickener selected from the group consisting of amorphous untreated silica powder, talc, mica, diatomaceous earth, and natural and modified clays;

(4) a buffer sufficient to maintain a pH of about 4.5 to about 6.5;

(5) a surfactant; and

(6) a mediator; and

(d) a shield for the electrode and the reagent composition.

Alternative embodiment S

The electrochemical biosensor of alternative embodiment R, wherein the buffer is about 30 to about 200mM acetate, citrate, or succinate buffer.

Alternative embodiment T

The electrochemical biosensor of alternative embodiment R, wherein the surfactant is up to about 0.5 wt% of an alkyl aryl polyether alcohol, based on the total weight of the composition.

Alternative embodiment U

The electrochemical biosensor of alternative embodiment R, wherein the mediator is from about 10 to about 20 wt% potassium ferricyanide, based on the total weight of the composition.

Alternative embodiment V

The electrochemical biosensor of alternative embodiment R, wherein components (c) (2) and (c) (3) are included in sufficient amounts to provide a composition suitable for screen printing.

Alternative embodiment W

The electrochemical biosensor of alternative embodiment R, wherein the composition has a viscosity of about 60,000 to about 180,000cps (mPa-s).

Alternative embodiment X

The electrochemical biosensor of alternative embodiment R, wherein the hydrophilic polymer is about 2 to about 10 wt% of a cellulose derivative selected from the group consisting of sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose, based on the total weight of the composition.

Alternative embodiment Y

The electrochemical biosensor of alternative embodiment X, wherein the hydrophilic polymer is hydroxyethyl cellulose.

Alternative embodiment Z

The electrochemical biosensor of alternative embodiment R, wherein the thickener is about 1 to about 6 wt% of amorphous untreated silica powder based on the total weight of the composition.

Alternative embodiment AA

The electrochemical biosensor of alternative embodiment Z, wherein the amorphous untreated silica powder is hydrophilic.

Alternative embodiment BB

The electrochemical biosensor of alternative embodiment R, wherein the reagent composition comprises about 5 to about 50mM calcium chloride.

Alternative procedure CC

A method of maintaining Glucose Dehydrogenase (GDH) and the cofactor pyrroloquinoline quinone (PQQ) activity in a screen-printed reagent composition for use in an electrochemical biosensor, comprising screen-printing the reagent composition, said method comprising the action of:

(d) a buffer sufficient to maintain a pH of about 4.5 to about 6.5;

(e) a surfactant; and

(f) a mediator.

Alternative procedure DD

The method of alternative process CC, wherein the hydrophilic polymer is a cellulose derivative selected from the group consisting of sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

Alternative Process EE

The method of alternative process CC, wherein the thickening agent is an amorphous untreated silica powder.

Alternative process FF

An alternative process CC method wherein the pH is maintained at 5.0-6.0.

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. It should be understood, however, that there is no intention to limit the invention to the specific forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A reagent composition for screen printing an electrochemical biosensor comprising

(d) a buffer sufficient to maintain a pH of about 4.5 to about 6.5 to 6.0;

(e) a surfactant; and

(f) a mediator.

2. The reagent composition of claim 1, wherein the buffer is an acetate, citrate, or succinate buffer.

3. The reagent composition of claim 1, wherein the surfactant is an alkyl aryl polyether alcohol.

4. The reagent composition of claim 1, wherein the mediator is potassium ferricyanide.

5. The reagent composition of claim 1, wherein the buffer is 5.0-6.0.

6. The reagent composition of claim 5 wherein the composition has a viscosity of from about 60,000 to about 180,000cps (mPa-s).

7. The reagent composition of claim 1, wherein said hydrophilic polymer is a cellulose derivative selected from the group consisting of sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

8. The reagent composition of claim 7, wherein the hydrophilic polymer is hydroxyethyl cellulose.

9. The reagent composition of claim 1 wherein the thickening agent is an amorphous untreated silica powder.

10. The reagent composition of claim 9, wherein the amorphous untreated silica powder is hydrophilic.

11. The reagent composition of claim 1 wherein said GDH-PQQ is present in an amount of 1 to 8 units per milligram total weight of the composition.

12. The reagent composition of claim 2, wherein the buffer is present at about 30 to about 200 mM.

13. The reagent composition of claim 3, wherein up to about 0.5 weight percent of said polyether alcohol is present based on the total weight of said composition.

14. The reagent composition of claim 4, wherein said potassium ferricyanide is present in an amount of about 10 to about 20 weight percent, based on the total weight of the composition.

15. The reagent composition of claim 7 wherein said cellulose derivative is present in an amount of about 2 to about 10 weight percent, based on the total weight of said composition.

16. The reagent composition of claim 9, wherein the amorphous untreated silica powder is present in an amount of about 1 to about 6 weight percent, based on the total weight of the composition.

17. The reagent composition of claim 1, further comprising about 5 to about 50mM calcium chloride.

18. An electrochemical biosensor, comprising:

(a) a non-porous substrate;

(b) working and counter electrodes disposed on the substrate;

(4) a buffer sufficient to maintain a pH of about 4.5 to about 6.5;

(5) a surfactant; and

(6) a mediator; and

(d) a shield for the electrode and the reagent composition.

19. The electrochemical biosensor of claim 18, wherein the buffer is about 30 to about 200mM acetate, citrate, or succinate buffer.

20. The electrochemical biosensor of claim 18, wherein said surfactant is up to about 0.5% by weight of an alkyl aryl polyether alcohol, based on the total weight of said composition.

21. The electrochemical biosensor of claim 18, wherein the mediator is from about 10 to about 20 wt% potassium ferricyanide, based on the total weight of the composition.

22. The electrochemical biosensor of claim 18, wherein components (c) (2) and (c) (3) are included in sufficient amounts to provide a composition suitable for screen printing.

23. The electrochemical biosensor of claim 18, wherein the composition has a viscosity of about 60,000 to about 180,000cps (mPa-s).

24. The electrochemical biosensor of claim 18, wherein said hydrophilic polymer is about 2 to about 10% by weight, based on the total weight of said composition, of a cellulose derivative selected from the group consisting of sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

25. The electrochemical biosensor of claim 24, wherein the hydrophilic polymer is hydroxyethyl cellulose.

26. The electrochemical biosensor of claim 18, wherein said thickener is about 1 to about 6 weight percent amorphous untreated silica powder based on the total weight of said composition.

27. The electrochemical biosensor of claim 26, wherein the amorphous untreated silica powder is hydrophilic.

28. The electrochemical biosensor of claim 18, wherein the reagent composition comprises about 5 to about 50mM calcium chloride.

29. A method of maintaining Glucose Dehydrogenase (GDH) and the cofactor pyrroloquinoline quinone (PQQ) activity in a screen-printed reagent composition for use in an electrochemical biosensor, comprising screen-printing the reagent composition, said method comprising the action of:

(d) a buffer sufficient to maintain a pH of about 4.5 to about 6.5;

(e) a surfactant; and

(f) a mediator.

30. The method of claim 29, wherein the hydrophilic polymer is a cellulose derivative selected from the group consisting of sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

31. The method of claim 29 wherein said thickener is an amorphous untreated silica powder.

32. The method of claim 29, wherein the pH is maintained between 5.0 and 6.0.