WO2009104836A1

WO2009104836A1 - A glucose sensor comprising glucose oxidase variant

Info

Publication number: WO2009104836A1
Application number: PCT/KR2008/002213
Authority: WO
Inventors: Bong Hyun Chung; Chang Soo Lee; Hye Jung Park; Jeong Min Lee
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2008-02-22
Filing date: 2008-04-18
Publication date: 2009-08-27
Also published as: KR100964026B1; KR20090090804A

Abstract

The present invention relates to a glucose sensor comprising an enzyme variant that uses glucose as a substrate, in particular, a blood glucose-measuring strip sensor having a high sensitivity with a trace amount of enzyme and a high detection rate, in which the glucose oxidase commonly used in glucose sensors is genetically engineered to produce an enzyme variant tagged with a positively or negatively charged peptide or protein, and then the enzyme variant is linked to an oppositely charged self-assembled monolayer that is formed on a gold, silver or copper thin layer, and a method for manufacturing the same.

Description

[DESCRIPTION] [Invention Title]

A GLUCOSE SENSOR COMPRISING GLUCOSE OXIDASE VARIANT [Technical Field] The present invention relates to a glucose sensor comprising an enzyme variant that uses glucose as a substrate, in particular, a blood glucose-measuring strip sensor having a high sensitivity with a trace amount of enzyme and a high detection rate, in which the glucose oxidase commonly used in glucose sensors is genetically engineered to produce an enzyme variant tagged with a positively or negatively charged peptide or protein, and then the enzyme variant is linked to an oppositely charged self-assembled monolayer that is formed on a gold, silver or copper thin layer, and a method for manufacturing the same.

[Background Art]

Since the development of glucose sensors to measure glucose levels, much focus has been placed on the development of biosensors owing to their promising applications in various fields . Glucose biosensors, currently occupying a dominant part of the medical market, have received a great deal of attention worldwide, as well as in Korea.

Currently marketed glucose sensor strips are generally constructed with a carbon electrode or a carbon/silver multilayer electrode. The carbon electrode is advantageous in that it can be simply produced at low cost, thereby being widely used. However, there are disadvantages that it has low conductivity and enzyme stability in the hydrophobic carbon paste environment should be considered. Meanwhile, in the conventional blood glucose sensors, the blended substrate and enzyme are dried on a reaction portion in a large amount, leading to much loss of the substrate and enzyme tobeusedin one strip sensor. In addition, when blood is injected to the sensor, considerable time is required to dissolve a large amount of the dried substrate and enzyme, so as to extend the measurement time. Thus, an enzyme dispersing agent is additionally needed.

In connection with the biosensors for measuring blood glucose levels, studies have focused on minimizing the amount of blood sample as well as immobilization of enzyme onto the strip to improve their sensitivity and shorten measurement time (Jun-Min Qian et al., Clinical Biochemistry, Vol. 37, p 155, 2004) .

[Disclosure] [Technical Problem]

Considering the above problems, the present inventors have studied intensively to develop a glucose sensor comprising a glucose oxidase variant, in which a gold, silver or copper thin layer electrode is used to minimize the problems of carbon electrode and improve the performance of measuring blood glucose, a commonly used enzyme is genetically engineered to produce a glucose oxidase variant tagged with a positively or negatively charged amino acid, and then the enzyme variant is immobilized on a reaction portion of the electrode at a high density to improve accuracy of the electric signals, compared to the known electrodes made of carbon or palladium, and moreover, chemical treatment on the surface of the gold, silver or copper thin layer is performed to provide various functional groups, so that the enzyme is immobilized by chemical covalent or ionic bonding, not physical bonding, thereby completing the present invention.

[Technical Solution]

It is an object of the present invention to provide a blood glucose-measuring strip sensor comprising an enzyme variant that uses glucose as a substrate, in which the sensor has a higher sensitivity and a higher detection rate than the known sensors.

It is another object of the present invention to provide a method for manufacturing the blood glucose-measuring strip sensor comprising the enzyme variant, which has a higher sensitivity and a higher detection rate than the known sensors.

It is still another object of the present invention to provide a glucose oxidase variant that is prepared by tagging the glucose oxidase used in the blood glucose-measuring strip sensor with a positively or negatively charged peptide, and a method for preparing the same. [Advantageous Effects]

According to the present invention, an enzyme is genetically engineered to produce an enzyme variant that is tagged with a peptide or protein consisting of basic or acidic amino acids, and then the enzyme variant is covalently or ionically linked to an oppositely charged self-assembled monolayer that is formed on a gold, silver or copper thin layer, thereby providing a glucose sensor having improved effects such as a high sensitivity with a trace amount of enzyme and a high detection rate.

[Description of Drawings]

FIG. 1 is a diagram illustrating the blood glucose-measuring strip sensor according to one embodiment of the present invention; FIG. 2 illustrates the construction of arginine-tagged glucose oxidase recombinant protein-expressing vector, in which

(a) is a schematic diagram illustrating the construction of a gene fragment encoding the arginine-tagged glucose oxidase, amplified by PCR, and (b) illustrates a map of the expression vector pGAL10-α-AmySS-GOD-6R that is prepared by inserting the gene fragment;

FIG. 3 is a photograph showing the result of electrophoresis, after expressing the arginine-tagged glucose oxidase variant in yeast according to one embodiment of the present invention; FIG. 4 is a schematic diagram illustrating a structure of the strip sensor according to one embodiment of the present invention, in which the arginine-tagged glucose oxidase is immobilized onto the self-assembled monolayer formed at the reaction portion of the strip sensor; and FIG. 5 is a graph showing the result of measuring real time current according to glucose concentration in the strip sensor according to one embodiment of the present invention.

[Best Mode] To achieve the above objects, in accordance with one aspect, the present invention provides a blood glucose-measuring strip sensor, comprising a non-conductive substrate, a reaction portion that is provided with a negatively or positively charged self-assembled monolayer on the surface, an enzyme that uses glucose as a substrate and is tagged with a peptide or protein being oppositely charged to that of the self-assembled monolayer, and an electron transfer mediator generating electrons by reacting with the enzyme, and an electrode portion that is provided with a working electrode and a reference electrode, each end of which is connected to the reaction portion.

The self-assembled monolayer of the blood glucose-measuring strip sensor according to the present invention can be formed by coating with a compound having a negatively or positively charged functional group at its end, more preferably, formed by coating with a mixture of the compound having a negatively or positively charged functional group at its end and a compound having a hydroxyl group at its end.

In the present invention, examples of the compound having an amine group as a functional group at its end may include mercaptoalkylamine, wherein the alkyl group preferably has 2 to 11 carbon atoms, but are not limited thereto. In the present invention, examples of the compound having an amidine group as a functional group at its end may include mercaptoalkylamidine, wherein the alkyl group preferably has 2 to 11 carbon atoms, but are not limited thereto.

In the present invention, examples of the compound having a guanidine group as a functional group at its end may include mercaptoalkylguanidine, wherein the alkyl group preferably has 2 to 11 carbon atoms, but are not limited thereto.

Since the compound having a hydroxyl group (-0H) has a hydroxyl group (-0H) at its end, it does not react with arginine. Examples of the compound having a hydroxyl group (-0H) may include 2-mercaptoethanol, 4-mercaptophenol, 3-mercaptopropanol, 4-mercaptobutanol, and β-mercaptohexanol, but are not limited thereto. Among these compounds, the most preferred compound is 2-mercaptoethanol . In the present invention, the most preferred compound that is used to form the self-assembled monolayer is a mixture of 11-mercaptoundecanoic acid and 2-mercaptoethanol.

In accordance with the preferred embodiment, the compound having a negatively charged functional group at its end may be a compound having a carboxyl group (-COOH) , a phosphate group (-PO₃H₂) , or a sulfonic acid group (-SO₃H) at its end, and the compound having a positively charged functional group at its end may be a compound having an amine group (-NR₁R₂R₃) , an amidine group [-C (NR₁) (NR₂R₃) ] , or a guanidine group [ (R₁R₂N) (R₃R₄N) C=N-R₅] at its end.

Examples of the compound having a carboxyl group at its end may include 11-mercaptoundecanoic acid, 4-mercapto phenylacetic acid, 4-mercapto benzoic acid, 3-mercapto propanoic acid, 2-thiomalic acid, 2-thiolactic acid, 12-mercaptododecanoicacid, and mixtures thereof, but are not limited thereto.

Examples of the compound having a phosphate group at its end may include (aminomethyl) phosphonic acid, 2-aminoethylphosphonic acid, 3-aminopropylphosphonic acid, and mixtures thereof, but are not limited thereto.

Examples of the compound having a sulfonic acid group at its end may include aminomethanesulfonic acid, aminoethanesulfonic acid, 4-aminobenzenesulfonic acid, and mixtures thereof, but are not limited thereto. The compound having an amine group at its end may be mercaptoalkylamine, the compound having an amidine group at its end may be mercaptoalkylamidine, and the compound having a guanidine group at its end may be mercaptoalkylguanidine, but is not limited thereto. In addition, examples of the compound having a hydroxyl group at its end may include 2-mercaptoethanol, 4-mercaptophenol, 3-mercaptopropanol, 4-mercaptobutanol, β-mercaptohexanol, and mixtures thereof, but are not limited thereto. Most preferably, the compound having a hydroxyl group at its end is 2-mercaptoethanol, and more preferably, the self-assembled monolayer may be coated with a mixture of 11-mercaptoundecanoic acid and 2-mercaptoethanol .

The enzyme that is tagged with a peptide or protein being oppositely charged to that of the self-assembled monolayer may be preferably prepared by combination of 3 to 100 basic or acidic amino acids, but is not limited thereto. Preferably, the enzyme may be glucose oxidase, but is not limited thereto. In addition, any enzyme may be used to manufacture the blood glucose-measuring strip sensor according to the present invention, as long as it uses blood glucose as a substrate, such as cholesterol oxidase, glutamic oxaloacetic transaminase (GOT) , and glutamic pyruvic transaminase (GPT) .

Preferably, the basic amino acidmay be selected fromarginine, lysine, histidine and combinations thereof, and more preferably the basic amino acid may be 3 to 10 arginines.

Preferably, the acidic amino acid may be selected from aspartic acid, glutamic acid, and combinations thereof.

In the present invention, the peptide or protein tag composed of the acidic or basic amino acids may be preferably prepared by linking to a N- or C-terminus of the enzyme using a genetic engineering method.

Examples of the electron transfer mediator reacting with the enzyme variant may include typically used ferrocene, ferrocene derivatives, quinone, quinone derivatives, organic conducting salts, and viologen, and more preferably, potassium hexacyanoferrate (III), potassium ferricyanide, potassium ferrocyanide, and hexaammineruthenium (III) chloride.

The electron transfer mediator reacts with metabolites, and is reduced via oxidation-reduction reaction with reduced enzyme. The reduced electron transfer mediator is diffused to the surface of an electrode. At the surface of the electrode, measured is the current generated when the oxidation potential of the reduced electron transfer mediator is applied.

In accordance with still another embodiment, the present invention provides a method for manufacturing the blood glucose-measuring strip sensor, comprising the steps of:

(a) preparing an enzyme variant by tagging an enzyme using glucose as a substrate with a peptide or protein composed of basic amino acids ;

(b) treating the reaction portion of the sensor with a compound having a negatively charged functional group at its end, so that a self-assembled monolayer having a negatively charged functional group is formed to allow covalent or ionic bonding with the tag of the enzyme; and

(c) immobilizing the enzyme variant onto the self-assembled monolayer at the reaction portion.

The present invention further provides a method for manufacturing the blood glucose-measuring strip sensor, comprising the steps of:

(a) preparing an enzyme variant by tagging an enzyme using glucose as a substrate with a peptide or protein composed of acidic amino acids;

(b) treating the reaction portion of the sensor with a compound having a positively charged functional group at its end, so that a self-assembled monolayer having a positively charged functional group is formed to allow covalent or ionic bonding with the tag of the enzyme; and

Amethod for determining blood glucose levels using the blood glucose-measuring strip sensor manufactured by the above method may include the steps of (a) treating the reaction portion with a substrate; and (b) applying a predetermined voltage between working electrode and reference electrode to cause cycling reaction at the reaction portion, and then reading a stationary current value.

In the specific Example of the present invention, a gene encoding a hexa arginine-tagged glucose oxidase was inserted into an expression vector by a genetic engineering method, and then the expression vector was expressed in yeast or E.coli, so as to prepare the enzyme variant used in the blood glucose-measuring strip sensor. The arginine-tagged glucose oxidase may be prepared by linking an arginine-encoding base sequence to the C-terminus of the glucose oxidase-encoding gene . Preferably, to facilitate isolation and purification of the arginine-tagged glucose oxidase protein, the alpha-amylase signal sequence of Aspergillus niger is linked to the N-terminus of the glucose oxidase-encoding gene for extracellular secretion of the produced recombinant protein by yeast or E.coli. In the specific Example of the present invention, to express the arginine-tagged glucose oxidase, an expression vector pGALlO, which is regulated by the GALlO promoter and GAL7 terminator, may be used.

In the preferred Example of the present invention, a gene fragment, which is linked to the gene encoding the glucose oxidase variant having an α-amylase signal sequence at the N-terminus and 6 arginines at the C-terminus, was inserted into the pGALlO vector to prepare an expression vector, pGAL10-α-amylase-GOD-6R. Saccharomyces cerevisiae was transformed with the prepared expression vector pGAL10-α-amylase-GOD-6R to prepare an arginine-tagged glucose oxidase-producing recombinant strain, Saccharomyces cerevisiae/pGAL10-α-amylase-GOD-6R, which was deposited in Genetic Resources Center, Korean Research Institute of Bioscience and Biotechnology (#52, Oun-dong, Yusong-ku, Taejon) with Accession No. KCTC 11225BP on Oct. 24, 2007.

In still another Example of the present invention, Escherichia coli DH5 was transformed with the prepared pGAL10-α-amylase-GOD-6R vector that expresses the hexa arginine-tagged glucose oxidase variant, so as to prepare an arginine-tagged glucose oxidase-producing recombinant strain, Escherichia coli DH5α/pGAL10-α-amylase-GOD-6R, which was deposited in Genetic Resources Center, Korean Research Institute of Bioscience and Biotechnology with Accession No. KCTC 11224BP on Oct. 24, 2007. In the preferred embodiment of the present invention, the strip sensor comprises a reaction portion and an electrode portion. Onto the reaction portion, immobilized is the enzyme tagged with a peptide or protein composed of basic or acidic amino acids, which reacts with blood glucose, and an electron transfer mediator that reacts with the enzyme to generate electrons. Each end of the electrode portion is connected to the reaction portion, and is provided with a working electrode and a reference electrode, where electrons generated from the reaction portion flow.

In the reaction portion, the electrode to immobilize the enzyme that is tagged with a peptide or protein composed of basic or acidic amino acids can be made of one selected from the group consisting of gold, silver, and copper, and most preferably, gold in consideration of accuracy of the electrical signals.

In the present invention, to immobilize the enzyme that is tagged with a peptide or protein composed of basic or acidic amino acids onto the reaction portion, chemical treatment can be performed to form an oppositely charged self-assembled monolayer capable of reacting with the tag.

In the present invention, upon immobilizing an enzyme that is tagged with a peptide or protein composed of basic amino acids onto the reaction portion, chemical treatment can be performed using a compound having a negatively charged functional group at its end, so as to form the self-assembled monolayer, upon immobilizing an enzyme that is tagged with a peptide or protein composed of acidic amino acids onto the reaction portion, chemical treatment can be performed using a compound having a positively charged functional group at its end, so as to form the self-assembled monolayer.

When the chemical treatment is performed using a mixture of the compound having a positively or negatively charged functional group and a compound having a hydroxyl group, the orientation of the self-assembled monolayer is improved, thereby improving the immobilization of the positively or negatively charged enzyme. The enzyme immobilization onto the reaction portion can be more improved by the self-assembled monolayer that is formed by the chemical treatment. Therefore, since a glucose sensor can be manufactured by using a much smaller amount of enzyme, production costs can be reduced and its sensitivity can be also improved.

In the present invention, in order to react the self-assembled monolayer formed on the reaction portion with glucose oxidase that is tagged with a peptide or protein composed of basic or acidic amino acids, the reaction portion and the glucose oxidase variant are contacted with each other at l~10°C for 2-5 hrs, preferably at 4⁰C for 3 hrs, thereby improving immobilization and stabilization of the enzyme variant on the reaction portion.

In the preferred embodiment of the present invention, a sensor comprising the glucose oxidase variant are only described, but those skilled in the art will readily appreciate that a blood glucose-measuring strip sensor can be manufactured by using variants of cholesterol oxidase, glutamic oxaloacetic transaminase (GOT) , or glutamic pyruvic transaminase (GPT) in the same manner as mentioned above. 6he above objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. Hereinbelow, the preparation of the glucose oxidase variant and the method for manufacturing a blood glucose-measuring strip sensor according to the preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings.

The method for manufacturing a blood glucose-measuring strip sensor according to the present invention will be described as follows .

FIG. 1 is a diagram illustrating the blood glucose-measuring strip sensor according to one embodiment of the pres^~ent invention, in which the strip sensor (10) comprises a non-conductive substrate (11) , a reaction portion (12) , and an electrode portion (13) that is provided with a working electrode (14) and a reference electrode (15) .

FIG. 2 (a) shows a gene fragment amplified by PCR in order to prepare the arginine-tagged glucose oxidase recombinant protein according to the present invention, and (b) illustrates a map of the recombinant expression vector pGAL10-α-amylase-GOD-6R that is prepared by inserting the gene fragment into the expression vector pGAL10.

FIG. 3 is a photograph showing the result of electrophoresis of the protein that is produced by transforming yeast with the expression vector, in which corresponding lanes are as follows; Lane M: protein size marker, Lane 1 : intracellular protein before expression, Lane 2 : intracellular protein after expression, Lane 3: extracellular protein before expression, Lane 4 : extracellular protein after expression, Lane 5: extracellular protein after dialysis, Lane 6: extracellular protein solution concentrated by ultrafiltration (cut-off M.W.= 10,000) .

FIG. 4 illustrates a structure of the strip sensor of the present invention, in which the arginine-tagged glucose oxidase (33) is immobilized onto the self-assembled monolayer (30) formed at the reaction portion (12) of the strip sensor (10) via -COOH groups of the compounds having the -COOH group (32) that constitutes the self-assembled monolayer (30) .

In the preferred Example of the present invention, each of 4-mercaptoethanol (31) and 11-mercaptoundecanoic acid (32) was diluted in ethanol at a concentration of 20 mM, and then they were mixed with each other in a volume ratio of 2:1 to 6:1. The reaction portion (12) of the strip sensor was coated with the mixed solution to form the self-assembled monolayer (30) . The mixed solution was used to provide the self-assembled monolayer (30) with better orientation, thereby improving immobilization of the arginine-tagged glucose oxidase (33) .

Meanwhile, since the enzyme is immobilized onto the reaction portion (12) by covalent or ionic bonding of arginine with the terminal carboxyl group of 11-mercaptoundecanoic acid, thereby forming the monolayer, a glucose sensor can be manufactured by using a much smaller amount of enzyme than that used in the known glucose sensors . Therefore, there are advantages that production costs can be reduced and its sensitivity can be also improved. FIG. 5 is a cyclic voltammogram, which shows immobilization of the self-assembled monolayer (30) and the arginine-tagged glucose oxidase (33) onto the reaction portion (12) of the strip sensor (10) . A buffer for measurement was dropped to the non-treated reaction portion (12), a voltage of 0.4 v was applied to the working electrode (14), and the reference electrode (15) was connected to ground, so as to obtain a graph (40) of current versus voltage at a rate of 50 mV/sec. Then, the self-assembled monolayer (30) was formed on the reaction portion (12), and the arginine-tagged glucose oxidase (33) was immobilized thereto. In the same manner as above, each graph (41), (42) of current versus voltage was obtained. In general, enzymes have a characteristic of inhibiting current flowing in the working electrode (14) and the reference electrode (15) . Thus, FIG. 5 indicates that the arginine-tagged glucose oxidase (33) is immobilized onto the self-assembledmonolayer (30) of the reaction portion ( 12) .

Thereafter, the electron transfer mediator to react with the immobilized enzyme was mixed with a stabilizer for enzyme stabilization, and the mixture was applied on the reaction portion (12) of the strip sensor. FIG. 6 is a graph of measuring real time current according to glucose concentration, after the reaction portion (12) of the strip sensor was reacted with a suitable amount of glucose. In this regard, a voltage of 0.4 V was applied to the working electrode (14), and the glucose concentration was within the range of 10 μM to 100 mM. Current values against the enzyme, glucose, and electron transfer mediator were found to depend on the glucose concentration. In addition, the minimum glucose concentration that can be detected by a small amount of enzyme was 10 μM, indicating that the blood glucose-measuring strip sensor according to the present invention is of high sensitivity.

FIG. 7 shows the relationship between current and glucose concentration on the basis of the graph in FIG. 6. High reliability was obtained at a glucose concentration of 10 μM to 100 mM. FIG. 8 shows data of the linear region in the graph of FIG.

7, in which glucose was reacted at a concentration of 1 mM to 8 mM with an interval of 1 mM, and real time current was measured to confirm a suitable measurement time. In accordance with FIG.

8, it is possible to measure the glucose level at 3 sec, and high reliability can be obtained at 5 sec.

[Mode for Invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for the illustrative purpose only, and the invention is not intended to be limited by these Examples.

Example 1: Construction of gene encoding arginine-tagged glucose oxidase variant In order to prepare the glucose oxidase variant having an α-amylase signal sequence at the N-terminus and 6 arginines at the C-terminus, the following four primers represented by SEQ ID NOs. 1-4 were prepared. PCR was performed using the primers to obtain a PCR product that encodes the glucose oxidase variant having an α-amylase signal sequence and 6 arginines at the C-terminus. The PCT products were linked to prepare a gene fragment to be inserted into an expression vector, as shown in FIG.2 (a) . In order to insert the gene fragment into the expression vector pGALlO, the EcoRI restriction site was introduced at the N-terminal primer (SEQ ID NO. 1), and the HindIII restriction site was introduced at the C-terminal primer (SEQ ID NO. 4) . The gene fragment was inserted into the pGALlO vector digested with EcoRI and HindIII restriction enzymes, so as to construct a pGAL10-α-amylase-GOD-6R vector (FIG. 2) . The expression vector expressed Met at the N-terminus.

Primer 1: α-amylase sense (SEQ ID NO. 1)

5- GGC GGC GAA TTC AAA AAT GGT CGC GTG GTG GTC T-3

Primer 6 pG:AαL-1a0m-yαl-aasmeylaanstei-sGeOnDs-e6R(SEQ ID NO. 2) 5-GGC CAA AGC AGG TGC CGC GAC CTG-3

Primer 1: GOD-6R sense (SEQ ID NO. 3) 5- AGC AAT GGC ATT GAA GCC AGC CTC-3

Primer 6: GOD-6R antisense (SEQ ID NO. 4)

5-GGT ATC GATAAG CTTTCA GCG GCG GCG GCG GCG GCG CTG CAT GGA AGC ATA ATC-3

Example 2: Expression of arginine-tagged glucose oxidase variant

Saccharomyces cerevisiae L3262Δpmrl was transformed with the pGALlO-α-amylase-GOD-βR vector prepared in Example 1 according to the lithium acetate method (Gietz et al 1995) . The transformed Saccharomyces cerevisiae strain was deposited in Genetic Resources Center, Korean Research Institute of Bioscience and Biotechnology with Accession No. KCTC 11225BP on Oct. 24, 2007.

In order to induce protein expression, Saccharomyces cerevisiae L3262Δpmrl that was transformed with the pGAL10-α-amylase-GOD-6R vector was cultured in a protein expression-inducing medium, YPDG (1% Yeast extract, 2% peptone, 1% glucose, 1% galactose) at 30⁰C for 48 hrs.

After 48 hrs, centrifugation was performed to obtain a cell pellet and supernatant , and then an extracellular protein solution was obtained. The supernatant was dialyzed using a buffer solution ( 1OmM Tris-HCl, pH 8.0) , and then protein was concentrated by ultrafiltration (cut-off M.W.= 10,000) , and freeze-dried, so as to obtain a highly concentrated extracellular protein solution. For digestion of the yeast cell wall, lysozyme and zymolyase were added to the cell pellet, followed by sonication (Branson, Sonifier450, 3 KHz, 3 W, 5 min) . Then, centrifugation was performed to obtain an intracellular protein solution.

Each protein solution obtained by the above method was mixed with a buffer solution (12 mM Tris-Cl, pH 6.8, 5% glycerol, 2.88 mM mercaptoethanol, 0.4% SDS, 0.02% bromphenol blue) , and heated at 100⁰C for 5 min. Then, the solution was loaded on a polyacrylamide gel having a 5% stacking gel (pH 6.8, width 10 cm, length 12.0 cm) on a 12% separation gel (1 mm thick, pH 8.8, width 20 cm, length 10 cm) , and electrophoresis was performed at 200-100 V and 25 mA for 1 hr. The gel was stained with Coomassie dye to detect the recombinant protein.

FIG. 3 is a photograph showing the result of electrophoresis . In FIG. 3, corresponding lanes are as follows; Lane M: protein size marker, Lane 1: intracellular protein before expression, Lane 2: intracellular protein after expression, Lane 3: extracellular protein before expression, Lane 4: extracellular protein after expression, Lane 5: extracellular protein after dialysis, Lane 6: extracellular protein solution concentrated by ultrafiltration (cut-off M.W.= 10,000) . Example 3: Formation of self-assembled monolayer and manufacture of glucose sensor by enzyme immobilization

A gold thin layer strip for the measurement of blood glucose was sonicated in ethanol for 10 min, and then washed with fresh ethanol three times, and dried.

Each of 4-mercaptoethanol and 11-mercaptoundecanoic acid was diluted in ethanol at a concentration of 20 mM, and then they were mixed with each other in a volume ratio of 4:1 to prepare a mixed solution.

In order to react only one surface of the strip with the solution, two strips were stacked facing each other, and then put in a 1 ml tube. 300 μl of the prepared mixed solution was added to the tube, so as to treat only the reaction portion of the strip sensor for 20 hrs. The strip sensor having the formed self-assembledmonolayer was washed with fresh ethanol five times, and then dried. The arginine-tagged glucose oxidase was diluted with Tris buffer (50 mM, pH 8.0) at a concentration of 5 mg/ml, and 5 μl thereof was spotted in the dried reaction portion of the strip sensor, left for reaction at 4⁰C for 3 hrs. By the reaction, arginine was covalently or ionically linked to the carboxyl group of 11-mercaptoundecanoic acid to form a monolayer on the gold thin layer. After completing the reaction, the strip sensor was washed with distilled water five times, and then dried using nitrogen gas to manufacture the glucose sensor of the present invention.

Example 4: Determination of glucose level using glucose sensor of the present invention To analyze sensitivity of the glucose sensor of the present invention, the glucose strip sensor comprising the arginine-tagged glucose oxidase, manufactured in Example 3, was used to measure the current change according to time at different glucose concentrations. Upon the determination of glucose level, 0.1 μg/ml BSA (bovine serum albumin, Sigma Aldrich) dissolved in Tris buffer was used as a stabilizer for enzyme stabilization. Potassium hexacyanoferrate III (Fluka) was dissolved in Tris buffer at a concentration of 10 mM, and used as an electron transfer mediator. To determine the glucose level, the BSA solution and the electron transfer mediator solution were first mixed, and 3 μl of the mixture was spotted on the reaction portion of the strip sensor. A vacuum pump was used to dry the spotted solution for 20 mins. Then, the strip sensor was measured using KEITHLY SCS-4200 system for the current change according to time at different glucose (D-glucose, Sigma Aldrich) concentrations. The glucose was diluted in Tris buffer at a concentration of 10 μM~100 mM, and then dropped in the strip sensor to measure current values. In this regard, a voltage of 0.4 V was applied, total measurement time was 20 sec, and detection time was within 5 sec. As a result, it was found that the current increased with increasing glucose concentration (FIG. 6) .

The preferred embodiments have been described in detail, hereinabove. It will be apparent to those skilled in the art that the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments.

Therefore, the scope of the present invention should be determined with references to the appended claims and includes the full scope of equivalents to which such claims are entitled.

[Industrial Applicability]

As described above, an enzyme variant that is tagged with a positively or negatively charged peptide or protein is covalently or ionically linked to an oppositely charged self-assembled monolayer that is formed on a reaction portion of the glucose sensor, thereby providing a blood glucose-measuring strip sensor having a high sensitivity with a trace amount of enzyme and a high detection rate, and a method for manufacturing the same.

Claims

[CLAIMS]

[Claim 1]

A blood glucose-measuring strip sensor, comprising a non-conductive substrate, a reaction portion that is provided with a negatively or positively charged self-assembled monolayer on the surface, an enzyme that uses glucose as a substrate and is tagged with a peptide or protein being oppositely charged to that of the self-assembled monolayer, and an electron transfer mediator generating electrons by reacting with the enzyme, and an electrode portion that is provided with one or more working electrodes and a reference electrode, each end of which are connected to the reaction portion.

[Claim 2] The blood glucose-measuring strip sensor according to claim

1, wherein the self-assembled monolayer is formed by coating with a compound having a negatively or positively charged functional group at its end.

[Claim 3] The blood glucose-measuring strip 'sensor according to claim

2, wherein the self-assembled monolayer is formed by coating with a mixture of the compound having a negatively or positively charged functional group at its end and a compound having a hydroxyl group at its end.

[Claim 4] The blood glucose-measuring strip sensor according to claim 2 or 3, wherein the compound having a negatively charged functional group at its end is a compound having a carboxyl group (-COOH) , a phosphate group (-PO₃H₂) , or a sulfonic acid group (-SO₃H) at its end.

[Claim 5]

The blood glucose-measuring strip sensor according to claim 2 or 3, wherein the compound having a positively charged functional group at its end is a compound having an amine group (-NRiR₂R₃) , an amidine group [-C(NRi) (NR₂R₃)], or a guanidine group [(RiR₂N) (R₃R₄N)C=N-R₅] at its end.

[Claim 6]

The blood glucose-measuring strip sensor according to claim

4, wherein the compound having a carboxyl group at its end is 11-mercaptoundecanoic acid, 4-mercapto phenylacetic acid,

4-mercapto benzoic acid, 3-mercapto propanoic acid, 2-thiomalic acid, 2-thiolactic acid, or 12-mercapto dodecanoic acid.

[Claim 7]

The blood glucose-measuring strip sensor according to claim 4, wherein the compound having a phosphate group at its end is (aminomethyl) phosphonic acid, 2-aminoethylphosphonic acid or 3-aminopropylphosphonic acid.

[Claim 8]

The blood glucose-measuring strip sensor according to claim 4, wherein the compound having a sulfonic acid group at its end is aminomethane sulfonic acid, aminoethane sulfonic acid or 4-aminobenzene sulfonic acid.

[Claim 9]

The blood glucose-measuring strip sensor according to claim 5, wherein the compound having an amine group at its end is mercaptoalkylamine .

[Claim 10]

The blood glucose-measuring strip sensor according to claim 5, wherein the compound having an amidine group at its end is mercaptoalkylamidine .

[Claim 11]

The blood glucose-measuring strip sensor according to claim 5, wherein the compound having a guanidine group at its end is mercaptoalkylguanidine .

[Claim 12]

The blood glucose-measuring strip sensor according to claim 3, wherein the compound having a hydroxyl group at its end is 2-mercaptoethanol, 4-mercaptophenol, 3-mercaptopropanol, 4-mercaptobutanol or 6-mercaptohexanol .

[Claim 13]

The blood glucose-measuring strip sensor according to claim 12, wherein the compound having a hydroxyl group at its end is 2-mercaptoethanol .

[Claim 14] The blood glucose-measuring strip sensor according to claim 3, wherein the mixture is a mixture of 11-mercaptoundecanoic acid and 2-mercaptoethanol .

[Claim 15]

The blood glucose-measuring strip sensor according to claim 1, wherein the tag is prepared by combination of 3 to 100 basic or acidic amino acids.

[Claim 16]

The blood glucose-measuring strip sensor according to claim

15, wherein the basic amino acid is selected from arginine, lysine, histidine, and combinations thereof.

[Claim 17]

The blood glucose-measuring strip sensor according to claim

16, wherein the basic amino acid is composed of 3 to 10 arginines.

[Claim 18] The blood glucose-measuring strip sensor according to claim 15, wherein the acidic amino acid is selected from aspartic acid, glutamic acid, and combinations thereof.

[Claim 19]

The blood glucose-measuring strip sensor according to claim 1, wherein the electron transfer mediator is selected from the group consisting of potassium hexacyanoferrate (III) , potassium ferricyanide, potassium ferrocyanide, and hexaammineruthenium (III) chloride.

[Claim 20] The blood glucose-measuring strip sensor according to claim 1, wherein the electrode is made of gold, silver or copper.

[Claim 21]

The blood glucose-measuring strip sensor according to claim 1, wherein the enzyme is glucose oxidase, cholesterol oxidase, glutamic oxaloacetic transaminase (GOT) , or glutamic pyruvic transaminase (GPT) .

[Claim 22]

A recombinant strain for producing an arginine-tagged glucose oxidase, which is transformed with a gene encoding a glucose oxidase variant that has an α-amylase signal sequence at the N-terminus and 6 arginines at the C-terminus.

[Claim 23]

The recombinant strain for producing an arginine-tagged glucose oxidase according to claim 22, wherein the recombinant strain is identified by Accession No. KCTC 11225BP.

[Claim 24]

The recombinant strain for producing an arginine-tagged glucose oxidase according to claim 22, wherein the recombinant strain is identified by Accession No. KCTC 11224BP.