KR101494542B1 - Gloucose sensor - Google Patents

Abstract

The present invention relates to a glucose sensor and, more specifically, to a glucose sensor having excellent sensitivity capable of detecting glucose even in a sample having a low concentration of 0.02 mM of glucose, and detecting glucose from the sample without a special device. The glucose sensor comprises: a liquid crystal copolymer; and glucose oxidase.

Description

Glucose sensor

The present invention relates to a glucose sensor, more particularly, to a glucose sensor capable of detecting glucose in a sample even without a specific device as well as being excellent in sensitivity to detect glucose even in a sample containing glucose having a low concentration of 0.02 mM will be.

We humans have many sensory organs and sense various sensations from the outside such as pain, temperature sensation as well as the five senses. These functions are called sensory organs in living organisms, and machines or instruments are called sensors. Ultimately, a biosensor is a system that uses biological elements or imitates a biological system to convert it into recognizable signals such as color, fluorescence, and electrical signals when obtaining information from a measurement object. A biological element fixed to a sensor, a signal converter, and the like. Various physical and chemical techniques such as electrochemical, thermal, optical, and mechanical methods are used as the signal conversion method of the signal converter.

The first biosensor was known as a glucose sensor made by Clark in 1962 using a dialysis membrane for measuring glucose. In the early days, most of the biosensors were made by fixing an enzyme to a signal conversion device. Recently, however, Sensors made using monoclonal antibodies or antibody-enzyme conjugates have been developed and used. In addition, development studies on chip sensors such as DNA chip and protein chip for processing a large amount of genetic information at high speed are vigorous, and efforts have been made to develop advanced sensors in which molecular biology technology, nanotechnology and information communication technology are combined It is concentrated.

Such a biosensor is intended to quantify or qualify a physical or chemical reaction depending on the presence or concentration of a target substance by an electrical or optical method. The biosensor is used for the detection of environmental related substances such as environmental hormones, BOD of waste water, heavy metals, pesticides, residual pesticides contained in foods, antibiotics, pathogens , Food safety tests used for the detection of harmful substances such as heavy metals, military uses to detect biochemical weapons such as sarin and anthrax, control of microbial growth conditions in fermentation processes, chemical / petrochemical, pharmaceutical, Monitoring of specific chemical substances occurring in food processing, etc., and researches such as kinetics analysis of binding between biomaterials.

However, the biosensor technology applied so far requires a large amount of samples to recognize the biomaterial to be detected. In addition, in order to analyze a sample, there is a complicated process such as an input step of an analyte, a signal generating step, a signal amplifying step, and a complicated analysis result interpretation step, and it takes a very complicated process.

Recently, simple and inexpensive biosensors with very small amounts of recognition have been actively studied, which do not require complicated steps, and biosensors using liquid crystals are being studied. Detecting chemicals or biomaterials with liquid crystals has attracted much attention due to their high detection sensitivity, low cost, small size, and easy-to-read advantages.

In order to apply the advantage of liquid crystal to a biosensor, an attempt has been made to apply the biosensor to a biosensor by using adsorption of a biosensor at a liquid crystal / water interface. However, a biosensor using a liquid crystal / water interface has a low reactivity with a biosensor There is a problem that it is difficult to recognize the biomolecule to be detected and thus the recognition is not transmitted to the liquid crystal.

Accordingly, it is necessary to functionalize the interface with the liquid crystal / water in order to react rapidly to various stimuli. Conventionally, the liquid crystal / water interface is controlled by using an surfactant to orient the liquid crystal. However, emulsifiers do not bond with biomaterials (DNA, RNA, proteins, etc.) and have a problem in that they are not suitable for functionalizing the liquid crystal / water interface because they are not only capable of binding to liquid crystals,

In addition, diabetes mellitus is a serious disease accompanied by various acute and chronic complications resulting from an increase in blood glucose due to abnormality in the production or utilization of insulin. In the United States, 9.6% (over 20 million people) of people aged 20 or older have diabetes and more than 50 million people with pre-diabetes who are at high risk of developing the disease (2005 national diabetes fact sheet). In 2002, $ 132 billion was spent on direct and indirect medical expenses related to diabetes.

According to the National Health and Nutrition Examination Survey conducted by Korea Centers for Disease Control and Prevention in 2005, 9.0% of males over 30 years old and 7.2% of females were diabetic. According to the 2007 Korean Diabetes Research Report published by the Korean Diabetes Association, the prevalence of diabetes is about 8%, new patients are getting 10% every year, and diabetes related medical expenses account for about 20% (about 3 trillion won) Respectively. Considering the recent rapid increase in the number of diabetic patients currently estimated at 4 million to 5 million, it could reach 10 million in 10 to 20 years.

As such, the prevalence of diabetes mellitus, which is a problem in the world, is expected to increase further due to the increase in the elderly population and the living environment factors, and the social and economic problems are seriously arising. As the number of patients increases, the demand for self - monitoring blood glucose meter, which is mainly used for monitoring blood glucose level, is increasing and the frequency of use is gradually increasing. Several kinds of self-monitoring blood glucose meters have already been introduced in Korea, and research and development have been actively carried out.

However, since the measurement limit of the conventional self-glucose meters is 0.05 mM, glucose having a low concentration of 0.05 mM or less can not be measured. Therefore, it is necessary to research and develop a self-glucose meter capable of detecting glucose with high sensitivity and small amount It is true.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a glucose sensor capable of promptly recognizing a response to glucose in response to glucose, Glucose sensor. Further, it is intended to provide a biosensor capable of detecting a trace amount of glucose.

Accordingly, the present invention relates to a liquid crystal copolymer represented by the following formula (1): And a glucose sensor comprising glucose oxidase:

또는

이고, A는 탄소수 1 ~ 3인 알킬기이고, R₄ 는 -CN 또는 -R₆-CN 이고, R₆ 은 탄소수 1 ~ 6인 직쇄형 알킬기, 탄소수 3 ~ 6인 분쇄형 알킬기, 탄소수 5 ~ 6인 사이클릭 알킬기 또는 탄소수 6 ~ 10인 방향족 탄화수소이다.However, n is an integer from 1 to 2000 in the general formula 1, m is an integer of from 1 to 2000, and P is an integer of 1-50, R ₁ is _{-COOR 5, -C (CH 3)} 2 Ph , or - is _{CH (CH 3) Ph, R} 5 is a hydrogen atom, a straight-chain alkyl group having 1 to 6 carbon atoms or 3 to 6 grinding alkyl group, R ₂ is an aromatic hydrocarbon having 6 to 10, R ₃ is

or

R is an alkyl group having 1 to 3 carbon atoms, R ₄ is -CN or -R ₆ -CN, R ₆ is a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, An aromatic cyclic alkyl group or an aromatic hydrocarbon having 6 to 10 carbon atoms.

According to a preferred embodiment of the present invention, the liquid crystal copolymer may be a block copolymer.

According to another preferred embodiment of the present invention, the liquid crystal copolymer and the glucose oxidase can be covalently bonded.

According to another preferred embodiment of the present invention, the glucose sensor includes a substrate including a hydrophobic material layer; A liquid portion including a liquid crystal monomer on an upper portion of the substrate portion; And a sensor unit including the liquid crystal copolymer and the glucose oxidase on the liquid unit; . ≪ / RTI >

According to another preferred embodiment of the present invention, the hydrophobic material layer is made of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) and octadecyltrichlorosilane and octadecyltrichlorosilane (OTS).

According to another preferred embodiment of the present invention, the liquid crystal monomer is a mixture of cyclohexane-fluorinated biphenyl and fluorinated terphenyls (TL ₂ 0 ₅ ), 4- 4-cyano-4'-cyano-biphenyl (8CB) and 4-cyano-4'-pentylbiphenyl (5CB) And the like.

According to another preferred embodiment of the present invention, the glucose sensor comprises: a body portion including an aqueous solution containing glucose; An inlet for introducing the aqueous solution; And a discharge unit for discharging the aqueous solution.

According to another preferred embodiment of the present invention, when glucose is bound to the sensor unit, the change in birefringence according to the orientation change of the liquid crystal monomer in the liquid crystal unit is measured to qualitatively analyze glucose, and the qualitative analysis is performed under physiological conditions It can be seen that a bright image is obtained when a polarizing microscope is measured at a pH lower than pH 7, and a tendency that a dark image is displayed when a polarizing microscope is measured at a pH higher than a pH 7, which is a physiological condition.

According to another preferred embodiment of the present invention, the qualitative analysis can be performed at pH 2-12.

According to another preferred embodiment of the present invention, the glucose sensor may include glucose oxidase in an amount of 200 to 30,000 parts by weight based on 100 parts by weight of the liquid crystal copolymer.

According to another preferred embodiment of the present invention, the liquid crystal copolymer may be in the form of a monomolecular film.

According to another preferred embodiment of the present invention, glucose detection may be possible in a sample containing a glucose concentration of 0.02 mM or more.

Hereinafter, terms of the present invention will be described.

The term "HP alignment" of the present invention means that the liquid crystal of the glucose sensor changes its orientation from a homeotropic state to a horizontal orientation, and conversely, the PH orientation is oriented from a horizontal orientation to a homeotropic orientation .

The present invention can detect glucose in a sample containing glucose at a low concentration of 0.02 mM, and it is possible to immediately detect the presence or absence of the detection without any special equipment.

In addition, the present invention can instantly and simply initialize glucose to a pre-recognition state after the sensor recognizes it, thereby facilitating the reuse of the glucose sensor.

1 is a schematic cross-sectional view of a glucose sensor according to an embodiment of the present invention.
2 is a schematic cross-sectional view of a glucose sensor according to another embodiment of the present invention.
3 is a cross-sectional view of a glucose sensor according to another embodiment of the present invention.
Fig. 4 shows the density of GOx fixed per area measured in Experimental Example 1. Fig.
5 is a POM image measured in Experimental Example 2. FIG.
6 is a POM image measured in Experimental Example 3;

Hereinafter, the present invention will be described in more detail.

As described above, conventionally, there is a problem that the bonding force between glucose and liquid crystal is not so large, and it is difficult to functionalize a plurality of stimuli, so that it is not suitable for functionalizing the liquid crystal / water interface. In addition, since the conventional glucose sensor has a measurement limit of 0.05 mM, there is a problem that glucose at a low concentration of 0.05 mM or less can not be measured.

Accordingly, the present invention relates to a liquid crystal copolymer represented by the following formula (1): And a glucose sensor including a glucose oxidase, thereby solving the above-mentioned problem.

[Chemical Formula 1]

또는

이고, A는 탄소수 1 ~ 3인 알킬기이고, R₄ 는 -CN 또는 -R₆-CN 이고, R₆ 은 탄소수 1 ~ 6인 직쇄형 알킬기, 탄소수 3 ~ 6인 분쇄형 알킬기, 탄소수 5 ~ 6인 사이클릭 알킬기 또는 탄소수 6 ~ 10인 방향족 탄화수소이다.)(Wherein n is an integer of 1 to 2000, m is an integer of 1 to 2000, P is an integer of 1 to 50, R ₁ is -COOR ₅ , -C (CH ₃ ) ₂ Ph or and _{-CH (CH 3) Ph, R} 5 is a hydrogen atom, a straight-chain alkyl group having 1 to 6 carbon atoms or 3 to 6 grinding alkyl group, R ₂ is an aromatic hydrocarbon having 6 to 10, R ₃ is

or

R is an alkyl group having 1 to 3 carbon atoms, R ₄ is -CN or -R ₆ -CN, R ₆ is a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, An aromatic cyclic alkyl group or an aromatic hydrocarbon having 6 to 10 carbon atoms.

This provides a glucose sensor with increased sensitivity by enhancing the binding of glucose to liquid crystals and by using compounds that are not sensitive to a number of stimuli and are therefore suitable for functionalizing the liquid crystal / water interface. Also provided is a glucose sensor capable of detecting glucose at a low concentration of less than 0.05 mM and a minimum of 0.02 mM, which is the measurement limit of conventional glucose sensors. Furthermore, the glucose sensor of the present invention can instantly and simply initialize the glucose precursor state after the sensor recognizes the glucose, thereby facilitating the reuse of the glucose sensor.

FIG. 1 is a schematic cross-sectional view of a glucose sensor 100 according to an embodiment of the present invention. Referring to FIG. 1, a substrate 101 includes a hydrophobic material layer. A liquid portion 102 including a liquid crystal monomer on the substrate portion; And a sensor unit 103 including a liquid crystal copolymer and glucose oxidase on the liquid phase.

Specifically, the description unit 101 will be described first.

The substrate may be coated with a hydrophobic material layer 105 on the upper side of the substrate 104, and the substrate may be any substrate that can be used as the substrate, preferably a glass substrate.

The hydrophobic material layer serves to vertically align the liquid crystal of the liquid crystal unit and is not particularly limited as long as it can be added to the liquid crystal of the biosensor. Preferably, octadecyldimethyl (3-trimethoxysilylpropyl) And may include at least one member selected from the group consisting of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) and octadecyltrichlorosilanem (OTS), and more preferably octadecyltrichlorosilane .

Next, the description will be made as to the solution unit 102. Fig.

In the present invention, the liquid crystal part plays a role in detecting glucose through orientation and is not particularly limited as long as it can be added to the liquid crystal of a biosensor. Preferably, the liquid crystal part is a mixture of cyclohexane- a mixture of terphenyls (cyclohexane-fluorinated mixture of biphenyls and terphenyls fluorinated; TL ₂ 0 _5), 4-cyano-4'-oxyl Novi phenyl (4-n-octyl-4' -cyano-biphenyl, 8CB) and 4- Cyano-4'-pentylbiphenyl, 5CB), and more preferably 4-cyano-4'-pentylbiphenyl (5CB) .

According to a preferred embodiment of the present invention, the liquid crystal portion may be formed by accumulating the liquid crystal monomers.

Next, the sensor unit 103 will be described.

The sensor unit of the present invention plays a role of recognizing the presence or absence of glucose by binding with glucose, and includes a liquid crystal copolymer represented by the following formula (1); And glucose oxidase.

[Chemical Formula 1]

또는

일 수 있고, 바람직하게는

일 수 있다. 나아가, A는 탄소수 1 ~ 3인 알킬기일 수 있고, R₄ 는 -CN 또는 -R₆-CN 일 수 있고, 바람직하게는 -CN일 수 있다. 더불어, R₆ 은 탄소수 1 ~ 6인 직쇄형 알킬기, 탄소수 3 ~ 6인 분쇄형 알킬기, 탄소수 5 ~ 6인 사이클릭 알킬기 또는 탄소수 6 ~ 10인 방향족 탄화수소일 수 있으며, 바람직하게는 탄소수 6 ~ 10인 ?항족 탄화수소일 수 있다.In Formula 1, n may be an integer of 1 to 2000, preferably an integer of 100 to 1000, and more preferably an integer of 200 to 500. In addition, m may be an integer of 1 to 2000, preferably an integer of 10 to 300, and more preferably an integer of 15 to 100. Furthermore, P may be an integer of 1 to 50, preferably an integer of 5 to 20, and more preferably an integer of 10 to 12. In addition, R ₁ may be -COOR ₅ , -C (CH ₃ ) ₂ Ph or -CH (CH ₃ ) Ph, preferably -COOR ₅ , and R ₅ is a hydrogen atom, A linear alkyl group or a branched alkyl group having 3 to 6 carbon atoms, preferably a hydrogen atom. R < ₂ > may be an aromatic hydrocarbon having 6 to 10 carbon atoms, and R < ₃ >

or

, And preferably

Lt; / RTI > Furthermore, A may be an alkyl group having 1 to 3 carbon atoms, and R ₄ may be -CN or -R ₆ -CN, preferably -CN. In addition, R ₆ may be a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, a cyclic alkyl group having 5 to 6 carbon atoms, or an aromatic hydrocarbon having 6 to 10 carbon atoms, It may be an aliphatic hydrocarbon.

The content of the sensor part is not particularly limited as long as it includes a liquid crystal copolymer and a glucose oxidase, but preferably 200 to 30,000 parts by weight of glucose oxidase may be contained in 100 parts by weight of the liquid crystal copolymer.

If the amount of the glucose oxidase is less than 200 parts by weight based on 100 parts by weight of the liquid crystal copolymer, the amount of the glucose oxidase may be too low to cause a slow reaction between the glucose and the glucose oxidase, If the glucose oxidase is contained in an amount of more than 30,000 parts by weight based on 100 parts by weight of the liquid crystal copolymer, the excess glucose oxidase may not be used for the reaction with glucose, resulting in waste of material.

The liquid crystal copolymer is not particularly limited as long as it is in a form capable of being bonded to the liquid portion, but preferably the liquid crystal copolymer is adsorbed on the liquid portion and / or the liquid crystal copolymer forms a film and is bonded onto the liquid portion And more preferably the liquid crystal copolymer may be in the form of forming a film and being bonded onto the liquid portion.

In one embodiment of the present invention, the liquid crystal copolymer is formed as a film and bonded to the liquid portion.

As described above, when a liquid crystal copolymer film is prepared and used, the thickness is not particularly limited as long as it is a typical thickness of the polymer film, but it may preferably be a monomolecular film.

If the liquid crystal copolymer film is not a monomolecular film, a problem may arise that a part of the liquid crystal copolymer hardly bonds with the liquid crystal.

The liquid crystal copolymer includes a portion having affinity with the liquid portion and a portion having affinity with glucose. The portion having affinity with the liquid portion is a functional group of "-COO (CH ₂ ) PR ₃ ", and the portion having affinity with the glucose is a functional group of "-COOH".

According to one embodiment of the present invention, the functional group of "-COO (CH ₂ ) PR ₃ " and the functional group of "-COOH" of the liquid crystal copolymer may be included in the liquid component and may be included in the liquid component Some of the functional groups of "-COOH" that are not capable of binding to glucose oxidase.

According to another embodiment of the present invention, the liquid crystal copolymer and the glucose oxidase can form a covalent bond. The covalent bond may be formed by bonding a part of the functional group of -COOH with glucose oxidase.

Due to the covalent bonding between the liquid crystal copolymer and glucose oxidase, the glucose sensor of the present invention showed excellent sensitivity to detect glucose even in an aqueous solution containing at least 0.02 mM of glucose. This is much lower than the detection limit of 0.05 mM of conventional biosensors.

The glucose oxidase is an enzyme that generates glucose (D-gluconic acid) by oxidizing glucose (? -D-glucose) with oxygen and is not particularly limited as long as it can be usually sold and / or synthesized, Lt; RTI ID = 0.0 > Penicillium & notatum , and the like found in fungi or honey.

According to another embodiment of the present invention, the liquid crystal copolymer may be a block copolymer, and if the liquid crystal copolymer is a block copolymer, a layer may be formed at the interface between the liquid crystal and the water .

According to another embodiment of the present invention, when the glucose unit is bonded to the sensor unit 103, the birefringence change according to the orientation change of the liquid crystal monomer in the liquid unit 102 is measured to qualitatively analyze the glucose.

As can be seen from FIG. 5, the qualitative analysis shows a bright image at the measurement of the polarizing microscope at pH 5, which is lower than the pH 7, which is the physiological condition, and a dark image at the measurement of the polarizing microscope at the pH higher than the physiological condition, Respectively.

In addition, the qualitative analysis is not particularly limited as long as the biosensor is used, but it is preferably performed at a pH of 2 to 12.

According to another aspect of the present invention, there is provided a glucose sensor comprising: a body including an aqueous solution containing glucose; An inlet for introducing the aqueous solution; And a discharge unit for discharging the aqueous solution.

2 is a cross-sectional view of a glucose sensor according to another preferred embodiment of the present invention. Referring to FIG. 2, a base portion 101, a liquid portion 102, and a sensor portion 103 And the counterpart substrate 205 is disposed so as to be spaced apart from the sensor section 103 and fixed with the silicone rubber 206 therebetween. A body 201 including an aqueous solution containing glucose is formed in a space between the organic substrate 204 and the counter substrate 205. The body 201 is formed on the side of the silicone rubber 206 so as to change the composition of the body aqueous solution There are provided an inlet portion and an outlet portion 202, 203, respectively.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.

[ Example ]

Preparation Example . Synthesis of liquid crystal copolymer

Preparation Example  1: polymer synthesis

19.380 mg (0.0641 mmol) of s-methoxycarbonylphenylmethyl dithiobenzoate and 5.469 mg (0.0192 mmol) of 2,2'-azobisisobutyronitrile (2,2'- azobisisobutyronitrile (AIBN) were placed in 1.5 ml of dimethylformamide, mixed in a closed flask, and then the gas formed in the flask was removed using a pump. Then, a mixed solution of 1.456 ml (10 mmol) of Tert-butyl acrylate (98% by Aldrich) and 1.463 ml of DMF (manufactured by Aldrich) was injected into the flask, And allowed to stand in an oil bath for 18.5 hours.

After the standing, the reaction product formed in the flask was poured into 500 ml of methanol (manufactured by Aldrich) / water mixture (1/1, v / v) to precipitate. Then, the precipitated reaction product (poly (tBA) -CTA (PtBA-CTA)) was purified by using 10 ml of THF (Aldrich), 500 ml of a methanol / water mixture (1/1, v / v) To obtain a precipitate. This was dried at 25 < 0 > C for 12 hours to give 0.90 g (52%) of polymer. The polymerization process of the polymer (PtBA) is shown in the following reaction formula (1).

[Reaction Scheme 1]

The polymer had a number average molecular weight (Mn) of 2.65 x 10 ⁴ , and a weight average molecular weight / number average molecular weight (Mw / Mn) of 1.21.

Preparation Example  2: Preparation of liquid crystal copolymer precursor

0.868 mg (0.0042 mmol) of 2,2'-azobisisobutyronitrile (AIBN) and 83.85 mg (0.20 mmol) of 4-cyanobiphenyl-4-oxyndecyl acrylate 4-cyanobiphenyl-4-oxyundecylacrylate; LC11) and 110.9 mg (0.0042 mmol) of the polymer obtained in Preparation Example 1 were placed in a sealed flask and mixed. Subsequently, the gas formed in the flask was removed using a pump, DMF (0.32 ml) was added to the flask, and the flask was left to stand in a 70 占 폚 oil chamber for 40 hours. Then, the reaction product formed in the flask was precipitated by adding 500 ml of a methanol / water mixture (1/1, v / v), and precipitated precipitate was obtained. After drying at 25 ° C for 12 hours, 125 mg (52% Of a liquid crystal copolymer precursor (PtBA-b-LCP). The process for synthesizing the liquid crystal copolymer precursor is shown in the following Reaction Scheme 2.

[Reaction Scheme 2]

이다.)(Reaction Scheme 2)

to be.)

The liquid crystal copolymer precursor had a number average molecular weight (Mn) of 3.27 x 10 ⁴ and a weight average molecular weight / number average molecular weight (Mw / Mn) of 1.27.

Preparation Example  3: Preparation of liquid crystal copolymer

0.1 g of the liquid crystal copolymer precursor obtained in Step 2 of Preparation Example and 1 ml of trifluoroacetic acid (TFA, manufactured by Aldrich, 99%) were dissolved in 5.0 ml of dichloromethane (DCM, manufactured by Aldrich) , And the mixture was reacted by stirring at 25 DEG C for 24 hours. Then 500 ml of ethyl ether was added A precipitate was obtained, and the precipitate was vacuum-dried at 25 캜 for 24 hours to prepare a liquid crystal copolymer. The process for preparing the liquid crystal copolymer is shown in the following reaction formula (3).

[Reaction Scheme 3]

이다.)(Reaction Scheme 3)

to be.)

The weight average molecular weight / number average molecular weight (Mw / Mn) of the liquid crystal copolymer was 1.14.

Preparation Example  4: Production of liquid crystal copolymer monomolecular film

2 mg of the liquid crystal copolymer prepared in Preparation Example 3 was dissolved in 1.2 ml of dioxane and maintained at 60 ° C for 2 days. To the solution was added 0.8 ml of toluene to obtain a solution having a final concentration of 1 mg / ml .

The solution was applied to a Langmuir-Blodgett (LB) KSV Layer builder (model AAA100178 from KSV Instruments Ltd., Finland) and a KSV minimicro trough to prepare a monolayer. The monomolecular film was fabricated with 17 cm x 5 cm (width x length), and the surface pressure of the air / liquid on the surface of the monomolecular film was measured with a wilhelmy plate equipped with a micro balance.

When the surface pressure of the air / liquid reached 0 as measured by a KSV layer builder, 100 μl of the solution was applied to prepare a liquid crystal copolymer monolayer.

Example  One. Glucose  Sensor Manufacturing

Example  1-1: The substrate portion  Produce

Slide glass (product of Duran group, Germany) having a size of 12 mm x 8 mm (width x length) was washed with acetone and dried. 150 μl of octadecyltrichlorosilane (hereinafter, OTS) was added to 150 ml of toluene to prepare a mixed solution. The slide glass was coated with a mixed solution and allowed to react at 50 ° C for 1 hour. The OTS-coated glass was sequentially washed with toluene, acetone, ethanol and deionized water, and then dried under nitrogen to prepare a substrate (OTS-coated glass).

Example  1-2: [0043]  Produce

The base material prepared in Example 1-1 was placed on an ordinary glass and bonded with epoxy.

The copper grid used was a product of Ted Pella having a hole width of 285 μm, a hole spacing of 340 μm, a bar thickness of 55 μm, a grid size of 3.05 mm, and a grid thickness of 18 μm.

The copper grid was washed with DCM, ethanol and methanol and placed on the substrate. To the copper grid was added 1 μl of 4-cyano-4'-pentylbiphenyl (hereinafter abbreviated as 5CB) And 5CB which did not enter the copper grid was removed with a capillary tube.

Example  1-3: The sensor unit  Produce

The liquid crystal copolymer monomolecular film prepared in Preparation Example 4 was placed on a copper grid of the liquid crystal portion prepared in Example 1-2. Thereafter, a 2 mm thick silicon spacer (80 mm x 30 mm, width x length) was placed on both ends of the ordinary glass, and the counter substrate was placed on top of the silicon spacer. The glass (glass substrate and counter substrate) was fixed with a clip. Then, two injection needles for solution injection were inserted into both ends of the silicon spacer to form an injection port and an exhaust port, thereby completing the body part. A sectional view of the main body is shown in Fig.

Then, a glucose oxidase (GOx) was immobilized on the liquid crystal copolymer monolayer to prepare a sensor unit.

The GOx is a product of Sigma Aldrich obtained from Aspergillus niger and has a specific activity of more than 100,000 units / g and a molecular weight of 1.6 × 10 ⁵ g / mol.

(3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC.HCl, manufactured by Sigma Aldrich) and 0.1 M of N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride N-hydroxysulfosuccinimide sodium salt (NHS, manufactured by Sigma Aldrich) was injected through the injection part of the body part and left for 1 hour. Then, 6 ml of GOx solution was further added and reacted for 12 hours to prepare a sensor portion including a glucose oxidase and a liquid crystal copolymer. The process of manufacturing the sensor part is shown in the following reaction formula (4).

[Reaction Scheme 4]

The GOx solution was prepared by adding 0.1 g of glucose oxidase to 1000 ml of distilled water and adjusting the concentration to 0.63 μmol.

Thus, the glucose sensor of the present invention was prepared.

Example  2.

A glucose sensor was prepared in the same manner as in Example 1 except that 0.5 g of glucose oxidase was added to 1000 ml of distilled water and a GOx solution having a concentration of 3.13 占 퐉 ol was used.

Example  3.

A glucose sensor was prepared in the same manner as in Example 1 except that 0.75 g of glucose oxidase was added to 1000 ml of distilled water and a GOx solution having a concentration of 4.69 占 퐉 ol was used.

Example  4.

A glucose sensor was prepared in the same manner as in Example 1 except that 1.0 g of glucose oxidase was added to 1000 ml of distilled water and a GOx solution having a concentration of 6.25 탆 ol was used.

Example  5.

A glucose sensor was prepared in the same manner as in Example 1 except that 2.0 g of glucose oxidase was added to 1000 ml of distilled water and a GOx solution having a concentration of 12.50 占 퐉 ol was used.

Example  6.

A glucose sensor was prepared in the same manner as in Example 1, except that 2.5 g of glucose oxidase was added to 1000 ml of distilled water and a GOx solution having a concentration of 15.63 占 퐉 ol was used.

Example  7.

A glucose sensor was prepared in the same manner as in Example 1, except that 3.0 g of glucose oxidase was added to 1000 ml of distilled water, and a GOx solution having a concentration of 18.75 μmol was used.

Comparative Example  One.

A glucose sensor was prepared in the same manner as in Example 1, except that the glucose oxidase (GOx) was not added or fixed to the sensor portion.

Experimental Example  One. The sensor GOx  Fixed quantity measurement

The amount of GOx immobilized on the liquid crystal copolymer monolayer of the sensor part was measured spectrophotometrically by comparison with the value of deionized water.

The spectrophotometric method was measured by UV-vis spectroscopy. The density of the fixed GOx per area was calculated by the following equation (1).

Where C _imb is the mass of the fixed GOx, Na is the Avogadro's number, A is the area of the copper grid (7.31 x 10 ¹² nm ² ), and M _GOx is the molecular weight of GOx.

The density of the fixed GOx per measured area is shown in FIG.

As seen on Figure 4, was that the concentration (C _g) is the density of GOx (N _GOx) fixed by 12.5 μmol of GOx solution increase, N _GOx is 1.3 molecules / ㎚ when the C _g is 15.63 μmol and 18.75 μmol ² no longer increased.

Therefore, it was confirmed that the most suitable concentration was 12.5 μmol of GOx solution.

Experimental Example  2. Glucose  detection

At pH 7, 0.4 ml of 3 mM glucose (Sigma D (+) glucose) solution was injected through the inlet of the glucose sensor prepared in Example 5, and a POM image was obtained using a polarizing microscope. The POM image is shown in FIG. 5 (a).

The POM image was photographed using a POM (Polarizing Microscope of Model ANA-006, manufactured by Leitz, Germany) using a CCD camera (product name: STC-TC83USB manufactured by Korea Samwon Company) as a polarizing plate.

The POM image was obtained by injecting 0.4 ml of 1 mM acetic acid through the inlet of the glucose sensor prepared in Example 5. The obtained POM image was shown in Figure 5 (b), and through the inlet of the glucose sensor prepared in Comparative Example 1 0.4 ml of a 3 mM glucose solution was injected and the obtained POM image was shown in Fig. 5 (c). Further, 0.4 ml of 3 mM of galactose (D (+) galactose, manufactured by Sigma) was injected through the inlet of the glucose sensor prepared in Example 5, and the obtained POM image was shown in FIG. 5 (d).

As can be seen from FIG. 5, in (a), it can be seen that the homeotropic orientation is changed from the homeotropic orientation to the horizontal orientation (planar).

Further, in (b), the change of the glucose sensor at pH 5 was measured by adding 1 mM of acetic acid. As a result, a change in the HP orientation was observed. This was caused by a low pH change, and the glucose sensor of the present invention was pH- Sensor.

Further, in (c), it was confirmed that in the sensor not immobilized with glucose oxidase, the H-P orientation did not change even though glucose was injected at the same concentration as in (a). Therefore, it was found that the glucose sensor including the glucose oxidase of the present invention is more suitable as a glucose sensor than the glucose sensor including the glucose oxidase of the present invention.

In addition, in (d), it can be seen that no change occurred in the initial vertical orientation. Thus, it was confirmed that the glucose sensor of the present invention can selectively detect only glucose among monosaccharides.

As can be seen from the above Experimental Example, the glucose sensor of the present invention includes glucose oxidase, and glucose can be selectively detected even among monosaccharides, and thus it can be confirmed that glucose sensor is very suitable as a glucose sensor.

Experimental Example  3. Glucose  Sensitivity to concentration

0.4 ml of a glucose solution of 0.015 mM, 0.02 mM, 0.05 mM, 0.5 mM, 3 mM or 6 mM was injected through the inlet of the glucose sensor prepared in Example 5, and a POM image was obtained in the same manner as in Experimental Example 2 . The obtained POM image is shown in FIG. (C) is a glucose solution of 0.05 mM, (d) is a glucose solution of 0.5 mM, (e) is a glucose solution of 3 mM glucose Solution (f) is the result of using 6 mM glucose solution.

As can be seen from Fig. 6, in (a), it was found that no change occurred in the initial vertical alignment. Further, in (b) to (f), it was confirmed that the glucose was first detected by the first vertical orientation being changed to the horizontal orientation.

As a result, it was confirmed that the glucose sensor of the present invention is able to detect glucose at a low concentration of 0.02 mM, which is very suitable as a glucose sensor. Furthermore, the glucose sensor of the present invention was able to detect glucose at a surprisingly lower concentration than the detection limit of conventional biosensors of 0.058 mM.

As can be seen from the above Examples, Comparative Examples and Experimental Examples, the glucose sensor of the present invention can detect 0.02 mM of glucose, which is lower than the detection limit of the conventional biosensors, , It was found that the detection of glucose can be confirmed immediately without any special equipment.

In addition, the glucose sensor of the present invention can initialize the glucose precursor state immediately and easily after the sensor recognizes the glucose, thereby facilitating the reuse of the glucose sensor.

100: glucose sensor 101: base unit 102:
102: sensor part 201: main body part 202: inflow part
203: discharging portion 204: glass substrate 205:
206: Silicone rubber

Claims (12)

A liquid crystal copolymer represented by the following formula (1); And a glucose sensor comprising glucose oxidase:
[Chemical Formula 1]

Wherein n is an integer of 1 to 2000, m is an integer of 1 to 2000, P is an integer of 1 to 50, R ₁ is -COOR ₅ , -C (CH ₃ ) ₂ Ph or -CH CH ₃₎ Ph, and, R ₅ is a hydrogen atom, a straight alkyl group having 1 to 6 carbon atoms or having from 3 to 6 grinding alkyl group, R ₂ is is a 6 to 10 carbon atoms in an aromatic hydrocarbon, R ₃

or

R is an alkyl group having 1 to 3 carbon atoms, R ₄ is -CN or -R ₆ -CN, R ₆ is a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, An aromatic cyclic alkyl group or an aromatic hydrocarbon having 6 to 10 carbon atoms. The glucose sensor according to claim 1, wherein the liquid crystal copolymer is a block copolymer. The glucose sensor according to claim 1, wherein the liquid crystal copolymer and the glucose oxidase are covalently bonded. The method according to claim 1,
A substrate portion including a hydrophobic material layer;
A liquid portion including a liquid crystal monomer on an upper portion of the substrate portion; And
A sensor part including the liquid crystal copolymer and the glucose oxidase on the liquid part;
And a glucose sensor. 5. The method of claim 4, wherein the hydrophobic material layer is formed from octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) and octadecyltrichlorosilanem (OTS) Wherein the glucose sensor comprises at least one of the following groups. The method of claim 4, wherein the liquid crystal monomer is cyclohexane-Flory nitro-biphenyl and flat Metalurgs nitro emitter mixture of phenyl (cyclohexane-fluorinated biphenyls and a mixture of _{fluorinated terphenyls; TL 2 0 5)} , 4- cyano-4'-oxyl At least one group selected from the group consisting of 4-n-octyl-4'-cyano-biphenyl (8CB) and 4-cyano-4'-pentylbiphenyl And a glucose sensor. 5. The method of claim 4,
A body portion including an aqueous solution containing glucose;
An inlet for introducing the aqueous solution; And
And a discharge unit for discharging the aqueous solution. 5. The method according to claim 4, wherein, when glucose is bound to the sensor unit, the change in birefringence according to the orientation change of the liquid crystal monomer in the liquid crystal unit is measured,
The qualitative analysis is performed by analyzing a tendency of showing a bright image when a polarizing microscope is measured at a pH lower than a pH 7 which is a physiological condition and a tendency of showing a dark image at the time of measuring a polarizing microscope at a pH higher than a pH 7 which is a physiological condition Lt; / RTI > The glucose sensor according to claim 8, wherein the qualitative analysis is performed at a pH of 2 to 12. The glucose sensor according to claim 1, wherein the glucose sensor comprises 200 to 30,000 parts by weight of glucose oxidase based on 100 parts by weight of the liquid crystal copolymer. The glucose sensor according to claim 1, wherein the liquid crystal copolymer is in the form of a monomolecular film. 12. The glucose sensor according to any one of claims 1 to 11, wherein glucose can be detected in a sample containing glucose at a concentration of 0.02 mM or more.

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