KR101552041B1 - Biosensor for urea measurement - Google Patents

Abstract

The present invention relates to a bio-sensor for measuring urea and, more specifically, to a bio-sensor for measuring urea effective for diagnosing uremia. It is possible to detect urea contained in a specimen in a short period of time without using a high-priced measuring instrument and also to detect urea even in a specimen comprising 5 mM of urea which is less concentrated than a conventional determining standard of uremia which is 7 mM.

Description

[0001] The present invention relates to a biosensor for urea measurement,

The present invention relates to a biosensor for element measurement, and more particularly, it is capable of detecting an element contained in a sample in a short time without an expensive measuring device, and is capable of detecting a component of 5 mM The present invention relates to a biosensor for element measurement, which is effective for discriminating uremia.

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,

On the other hand, urea is also called carbamide, or diaminomethanal, and ammonia is produced when proteins are decomposed in the body. However, since ammonia is toxic, it is a harmful substance to stay in the body for a long time, so mammals and amphibians convert the ammonia into urea through the ornithine in the liver and send it to the excretory organ for storage for some time and then to the outside of the body.

However, the symptoms that occur when the waste (uremia) to be discharged into the urine can not be discharged are collectively referred to as uremia in general, and the uremia is one of very dangerous diseases because it affects the whole body in blood. In addition, blood is responsible for systemic health, so it is not an exaggeration to circulate the waste through the circulation of all the waste and oxygen / nutrition / water supply to supply each cell and function to maintain the role , Which is possible because of height. The kidneys have the role of circulating blood through the recovered material and discharging it out of the body, resulting in a clear state of blood. Therefore, the problem of uraemia is related to urine, and most of all, it is caused by a decrease in kidney function.

On the other hand, renal failure is defined as the failure of renal failure in any cause. Renal insufficiency is known to cause more severe uremic symptoms when oliguria lasts for about 1 week. However, chronic kidney failure It is known that the gradual decline in urine volume due to the breakdown of urine is a gradual uremia followed by progression of kidney failure even if it is not oliguria.

However, the chronic renal failure and / or uremia test as described above usually proceeds by a blood test and / or a urine test. Since there is a problem that a special device and / or time is required to confirm the result, It is necessary to research and develop a self -

On the other hand, Korean Patent Registration No. 0141779 (published on March 22, 1996) discloses a biosensor for element measurement having improved sensitivity of element measurement by measuring ammonium ion and bicarbonate ion produced as a result of urease hydrolysis reaction and And a manufacturing method thereof is described. However, when the element is measured using the biosensor of the above-mentioned patent, a measuring instrument or an analyzer capable of confirming the element measurement result is required, and it takes time to check the element content of the sample.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide a biosensor for element measurement, which comprises a sample containing 5 mM of element lower than 7 mM, which is a reference concentration for discriminating chronic renal failure and / A liquid crystal / water interfacial functionalized biosensor capable of confirming the response to the liquid crystal / water reaction in a short period of time without any equipment,

The present invention relates to a liquid crystal copolymer represented by the following formula (1): And a urease. The biosensor for element measurement comprises:

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 -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.

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

In another preferred embodiment of the present invention, the liquid crystal copolymer and the urease may be covalently bonded.

In yet another preferred embodiment of the present invention, the biosensor for element measurement 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 part including the liquid crystal copolymer and urease on the liquid part; As shown in FIG.

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

Another preferred embodiment of the present invention 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- oxyl- 4-cyano-4'-cyano-biphenyl, 8CB) and 4-cyano-4'-pentylbiphenyl (5CB) ≪ / RTI >

In still another preferred embodiment of the present invention, the biosensor for element measurement includes a body portion to which a sample for element measurement is added; An inlet for introducing the sample for elemental measurement; And

A discharging portion for discharging the sample for element measurement; As shown in FIG.

In another preferred embodiment of the present invention, the element is subjected to qualitative analysis by measuring a birefringence change according to the orientation change of the liquid crystal monomer in the liquid crystal unit when the element is coupled to the sensor unit, and the qualitative analysis is performed at pH 7 It can be performed by analyzing the tendency of showing a dark image when a polarizing microscope is measured at a pH higher than the physiological condition pH 7, which is a bright image when a polarizing microscope is measured at a low pH.

In another preferred embodiment of the present invention, the qualitative analysis can be performed at pH 6-8.

In another preferred embodiment of the present invention, the biosensor for elemental measurement may have an amount of urease of 10-20 in a unit area of a liquid crystal copolymer represented by the following formula (1): < EMI ID =

In the above formula (1), N _urease is the amount of urease contained per unit area of the liquid crystal copolymer, C _imb is the amount of fixed urease, Na is the number of Avogadro, 6.02 × 10 ²³ , A is the amount of urea (Nm ² ), and M _urease is an integer of 4.8 × 10 ⁵ to 5.5 × 10 ⁵ (g / mol), which is the molecular weight of urease.

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

In still another preferred embodiment of the present invention, the biosensor for elemental measurement may be capable of detecting an element in a sample containing an urea concentration of 5 mM or more.

In addition, the present invention provides a uremia diagnostic method using the biosensor for element measurement as described above.

Furthermore, the present invention provides a method for diagnosing renal failure using the biosensor for element measurement as described above.

Hereinafter, terms of the present invention will be described.

The term "HP alignment" of the present invention means that the orientation of the liquid crystal of the biosensor for elemental measurement changes from a homeotropic state to a horizontal orientation. On the contrary, the PH orientation is a homeotropic state in a planar state ). ≪ / RTI >

The biosensor for elemental measurement of the present invention is sensitive to urea in a sample containing 5 mM urea lower than 7 mM, which is a reference concentration for determining chronic renal failure and / or uremia, There is an effect of providing a biosensor for element measurement which can be confirmed by human eyes.

In addition, the present invention provides an element measuring biosensor capable of instantly and simply initializing a biosensor for element measurement after the element is recognized by the sensor, and for facilitating the reuse of the sensor have.

Furthermore, the present invention provides a method for diagnosing uremia and / or renal failure in a short time using a biosensor for element measurement as described above.

1 is a schematic cross-sectional view of a biosensor for element measurement according to an embodiment of the present invention.
2 is a schematic cross-sectional view of a biosensor for element measurement according to another embodiment of the present invention.
3 is a cross-sectional view of a biosensor for element measurement according to another embodiment of the present invention.
4 is a calibration curve of the urease solution used for the measurement of the amount of ureaase fixation in Experimental Example 1. FIG.
5 is the density of the fixed urease per unit area of the liquid crystal copolymer measured in Experimental Example 1. Fig.
6 is an infrared absorption spectrum of the urease solution, urease, and rhodamine 123 labeled with the red dye measured in Example 8. Fig.
7 is a POM image of the biosensor of Example 8 or Comparative Example 1 measured in Experimental Example 2. FIG.
8 is a POM (polarized light microscope) image measured for the evaluation of the urea detection ability in Experimental Example 3. Fig.
9 is a POM image measured in Experimental Example 4 for the measurement and evaluation of the element detection sensitivity of the biosensor of the present invention.
10 is a POM image obtained by evaluating the element detection capability of the biosensor of the present invention using actual blood in Experimental Example 5. FIG.
11 is a graph illustrating stability of the biosensor of the present invention measured over time according to Experimental Example 6. FIG.
12 is a POM image after a certain time after injecting a solution containing 8 mM of urea in Experimental Example 7. FIG.
13 is a graph showing the average gray scale intensity change according to the concentration of the urea solution injected in Experimental Example 7. FIG.

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

As described above, conventionally, in order to measure an element in a sample, equipment for confirming the detection result is required, and it is difficult to confirm the detection result in a short time.

Accordingly, the present invention relates to a liquid crystal copolymer represented by the following formula (1): And a urease. The present invention has been made to solve the above-mentioned problems by providing a biosensor for element measurement.

[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 allows sensitive samples to react with urea in a sample containing 5 mM lower than the reference concentration of 7 mM, which is used to identify chronic renal failure and / or uremia, It has an effect of providing a biosensor for element measurement. After the element is recognized by the sensor, the biosensor for element measurement can be instantly and simply initialized before the element recognition, There is an effect of providing a biosensor.

FIG. 1 is a schematic cross-sectional view of a biosensor 100 for element measurement according to an embodiment of the present invention. Referring to FIG. 1, the biosensor 100 includes a substrate 101 including 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 an urease on the liquid crystal unit.

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.

The liquid crystal unit in the present invention plays a role in enabling element detection through orientation and is not particularly limited as long as it can be added to liquid crystals of a biosensor. Preferably, the liquid crystal unit is composed of cyclohexane-flurinite biphenyl and flurinite 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 is capable of recognizing the presence or absence of an element in combination with the element, and includes a liquid crystal copolymer represented by the following formula (1); And urease. ≪ / RTI >

[Chemical Formula 1]

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.

Also, 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.

또는

일 수 있고, 바람직하게는

일 수 있다. 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 straight-chain alkyl group having 1 to 6 carbon atoms may be any one of methane, ethane, n-propane, n-butane, n-pentane, (n-haxane).

The branched alkyl group having 3 to 6 carbon atoms is preferably selected from the group consisting of propane, iso-butane, iso-pentane, neo-pentane, 2-methylpentane 3-methylpentane, 2,3-dimethylbutane, or 2,2-dimethylbutane.

The cyclic alkyl group having 5 to 6 carbon atoms includes cyclopentane, methylcyclopentane, or cyclohexane.

The aromatic hydrocarbons having 6 to 10 carbon atoms include, for example, benzene, toluene, ethylbenzene, ortho-xylene, meta-xylene, para - para-xylene, 1,2,3-trimethylbenzene, n-propylbenzene, cumene, 1-methyl- Benzene, 1-methyl-2-ethylbenzene, 1-methyl-3-ethylbenzene and 1-methyl- ethyl-benzene, tetramethylbenzene, diethylbenzene, t-buthylbenzene or n-buthylbenzene.

The content of the urease is not particularly limited as long as it contains a liquid crystal copolymer and an urease, but preferably the amount of the urease per unit area of the liquid crystal copolymer represented by the following formula (1) And more preferably the amount of the urease to be contained per unit area of the liquid crystal copolymer represented by the following formula (1) may be 13 to 18.

[Equation 1]

( _Wherein , N _urease is the amount of urease which is contained per unit area of the liquid crystal copolymer, C _imb is the amount of fixed urease, Na is the number of Avogadro, 6.02 × 10 ²³ , A is (㎚ ² ) of the liquid crystal copolymer, and _Murease is an integer of 4.8 × 10 ⁵ to 5.5 × 10 ⁵ (g / mol), which is the molecular weight of the urease.

As can be seen from FIG. 5, even when the concentration of the urease to be added is increased, it can be understood that the urease enzyme contained per unit area of the liquid crystal copolymer represented by the formula 1 can not exceed 0.2.

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 the element. 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 element 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" which can not be bound to urease can be combined.

According to another embodiment of the present invention, the liquid crystal copolymer and the urease can form a covalent bond. The covalent bond may be formed by a combination of a functional group of "-COOH" and an urease.

Due to the covalent bonding between the liquid crystal copolymer and the urease, the biosensor for elemental measurement of the present invention showed sensitivity to detect the element in samples containing 5 mM of urea.

The urease is an enzyme involved in the reaction of hydrolyzing an urea to produce ammonia and carbon dioxide, and is not particularly limited as long as it can be commercially sold and / or synthesized.

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, the birefringence change according to the orientation change of the liquid crystal monomer in the liquid portion 102 is measured at the time of element coupling to the sensor portion 103, and the element is qualitatively analyzed.

As can be seen from FIG. 8, the qualitative analysis showed a bright image at the measurement of the polarizing microscope at pH 7, which is a physiological condition, and a dark image at the measurement of the polarization microscope at pH 10, which is higher than the physiological condition, pH 7 .

In addition, the qualitative analysis is not particularly limited as long as the biosensor is used, but it is preferably performed at pH 6 to 8.

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

2 is a cross-sectional view of an element sensor according to another preferred embodiment of the present invention. Referring to FIG. 2, a substrate part 101, a liquid part 102, and a sensor part 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 an element 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 added to a solution containing 1.2 ml of dioxane and 0.8 ml of toluene and dissolved at 60 ° C for 2 days to give a final concentration of 1 mg / Solution.

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 with a KSV Layer builder, 100 μl of the solution was applied and allowed to stand for about 1 hour to prepare a liquid crystal copolymer monolayer. Then, the monomolecular layer of the liquid crystal copolymer was immersed in water, and then the barrier of the layer builder was moved to compress the monolayer at the interface and adjusted to a density of 35 mN / m.

Preparation Example  5

(Urea), N- (3-dimethylaminopropyl) -N-ethyl carbodiimide hydrochloride (EDC.HCl), N-hydroxysuccinimide hydrochloride N-hydroxysulfosuccinimide sodium salt (NN), dimethylformamide (NN), ascorbic acid, uric acid, trifluoroacetic acid (TFA, 99% ), Octadecyltrichlorosilane (OTS), methanol, dichloromethane (DCM) and urease, type III, EC 3.5.1.5, mass average molecular weight 4.8 × 10 ⁵ g / mol ) Were purchased from Sigma-Aldrich. Further, 4-cyano-4-pentylbiphenyl (5CB, 100%) was purchased from TCI (Japan) and used.

Example  1. Biosensor manufacturing for element measurement

Example  1-1: The substrate portion  Produce

(Piranha solution, mixed solution of H ₂ SO ₄ and H ₂ O ₂ in a volume ratio of 7: 3) of a slide glass (Slide glass, manufactured by Duran group, Germany) having a size of 12 mm × 8 mm , Further washed with distilled water and dried with nitrogen. 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 reacted 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 an epoxy adhesive.

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 acetic acid, ethanol and acetone, and then placed on the substrate. To the copper grid, 1 μl of 4-cyano-4'-pentylbiphenyl 5CB) was dropped, and 5CB which did not enter the copper grid was removed with a capillary.

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. The internal volume of the body was 0.4 ml.

Then, the urease was fixed to the liquid crystal copolymer monolayer to prepare a sensor unit.

(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 allowed to stand at 4 캜 for 1 hour. Then, 6 ml of urease solution was further added and reacted for 12 hours to prepare a sensor portion including urease and liquid crystal copolymer. The process of manufacturing the sensor part is shown in the following reaction formula (4).

[Reaction Scheme 4]

The urease solution was prepared by adding 0.96 g of urease to 1000 ㎖ of distilled water to make a concentration of 2 μM.

Example  2 .

A biosensor for urea measurement was prepared in the same manner as in Example 1, except that 1.92 g of urease was added to 1000 ml of distilled water and 4 탆 ol of urea solution was used.

Example  3.

A biosensor for urea measurement was prepared in the same manner as in Example 1, except that 2.88 g of urease was added to 1000 ml of distilled water and 6 탆 ol of urea solution was used.

Example  4.

A biosensor for urea measurement was prepared in the same manner as in Example 1, except that 5.28 g of urease was added to 1000 ml of distilled water and 11 탆 ol of urea solution was used.

Example  5.

A biosensor for urea measurement was prepared in the same manner as in Example 1 except that 10.0 g of urease was added to 1000 mL of distilled water and a 21 탆 ol urea solution of urea was used.

Example  6.

A biosensor for urea measurement was prepared in the same manner as in Example 1, except that 20.1 g of urease was added to 1000 ml of distilled water and 42 탆 ol of urea solution was used.

Example  7.

A biosensor for urea measurement was prepared in the same manner as in Example 1, except that 30.2 g of urease was added to 1000 ml of distilled water and that a urea solution having a concentration of 63 탆 ol was used.

Experimental Example  One. The sensor  Determination of urea enzyme-fixed amount

Optical density was measured using a spectrophotometer (UV-vis spectrometer) to measure the amount of urease enzyme immobilized on the liquid crystal copolymer monolayer of the sensor part of the biosensor for element measurement prepared in Examples 1 to 7.

Specifically, the density of the urease enzyme fixed per unit area of the liquid crystal copolymer was calculated using the following equation (1).

[Equation 1]

In the above formula (1), N _urease is the amount of urease contained per unit area of the liquid crystal copolymer, C _imb is the amount of fixed urease, Na is the number of Avogadro, 6.02 × 10 ²³ , A is the amount of urea (㎚ ² ), and M _urease is 4.9 × 10 ⁵ g / mol, the molecular weight of the urease used.

The above C _imb indicates the absorbance of the urease solution before and after the injection of the urease solution for the production of the sensor part of Examples 1 to 7 by measuring the maximum absorbance at a wavelength of 276 ㎚, The concentration of the urease solution was calculated using the calibration curve of the solution before and after the injection. A graph of N _urease and C _ureas (concentration of used urease solution), which is a calculation result, is shown in FIG.

As shown in FIG. 5, it was found that the amount of ureaase fixed per unit area of the liquid crystal copolymer was increased as the concentration of the urease solution was increased. However, when the concentration of the used urease solution was more than 21 M M, the amount of urease was not increased compared with the concentration of the used urease solution, which was not economical.

Comparative Example  One.

A biosensor was prepared in the same manner as in Example 5, except that the liquid crystal copolymer monomolecular film prepared in Preparation Example 4 was not used.

Example  8.

0.4 M EDC.HCl and 0.1 M NHS were added to phosphate buffered saline (PBS buffer, pH 7), and 20 μM urease was added to the PBS solution at 4 ° C. for 1 hour. Then, 1 mg of Rhodamin 123 was added and stirred at 25 ° C and 400 rpm for 12 hours to prepare an urease solution labeled with a red dye. Then, the infrared absorption spectrum was measured using a urea enzyme solution labeled with a red dye, urease, and a UV-vis spectrometer of Rhodamine 123. The measurement results are shown in Fig.

As can be seen in FIG. 6, rhodamine 123 showed strong absorbance at 500 nm, and urease showed strong absorbance at 276 nm. In addition, the urease _Rhodamin labeled with red dye showed that the maximum absorption peak of urease, 276 ㎚, was shifted to 263 ㎚ by the amide bond between rhodamine 123 and urease, 123 showed a strong absorbance at 500 nm which is the maximum absorption peak, indicating that it was successfully labeled with a red dye.

Then, a biosensor was prepared in the same manner as in Example 5, except that the urease enzyme labeled with red dye was used as described above.

Experimental Example  2. urease immobilization

Fluorescence images of the biosensor prepared in Example 8 or Comparative Example 1 were measured to confirm immobilized urease.

Specifically, fluorescence images of the biosensor prepared in Example 8 or Comparative Example 1 were observed by irradiating 450 to 490 nm using a fluorescence microscope (Nikon eclipse, E600p02, japan). The fluorescence image of the biosensor of Example 8 or Comparative Example 1 as an observation result is shown in FIG. FIG. 7A is a fluorescence image of the biosensor of Embodiment 8, and FIG. 7B is a fluorescence image of the biosensor of Comparative Example 1. FIG.

7, unlike the black image of the biosensor of Comparative Example 1, the biosensor of Example 8 was able to confirm the PAA chain in green, which indicated that the urease was immobilized on the copper grid there was.

Comparative Example  2.

A biosensor was prepared in the same manner as in Example 1 except that the urease solution was not used.

Experimental Example  3. Element detection

DI water containing distilled water (pH 7), phosphate buffer solution (PBS buffer) of pH 10 or 20 mM urea was flowed through the inlet of the biosensor prepared in Example 5 or Comparative Example 2, and a polarized light microscope image The element detection ability was evaluated.

Specifically, a polarized light microscope image was obtained by using a CCD camera (Samwon, STC-TC83USB, Korea) under crossed polarizers using a polarized optical microscope (POM, Leitz, ANA-006, Germany) Respectively. An observed polarized light microscope image is shown in FIG.

8A is an image obtained by injecting distilled water of pH 7 into the biosensor of Example 5, FIG. 8B is an image of 20 mM of urea solution injected into the biosensor of Example 5, FIG. 8C is an image of phosphate buffer FIG. 8D is an image obtained by injecting 20 mM urea solution into the biosensor of Comparative Example 2. FIG.

As can be seen from FIG. 8, in (a), the horizontal alignment of the liquid crystal by the distilled water of pH 7 was confirmed. As a result, it was found that the liquid was horizontally aligned at a pH of 7, which is a normal blood.

On the other hand, in (c), it was confirmed that the vertical orientation was maintained by the phosphate buffer solution of pH 10 because the pH was increased by the enzyme reaction.

In comparison of (b) and (d) in which the urea solution of the same concentration was added, the biosensor of Example 5 in which the urease was immobilized had a vertical orientation It can be confirmed that it is suitable for element detection. However, the biosensor of Comparative Example 2, in which the urease was not immobilized, could not confirm the horizontal-vertical orientation (PH) change in the horizontal orientation even when the urea solution was injected.

Experimental Example  4. Detection sensitivity according to urea concentration

DI water containing 2 mM, 3 mM, 5 mM, 8 mM, 20 mM or 80 mM of urea was injected into the injection part of the biosensor for element measurement prepared in Example 5, The element detection ability was evaluated through an optical microscope image. 9A is a 2 mM urea solution injected, FIG. 9B is a 3 mM urea solution injected, FIG. 9C is a 5 mM urea solution injected, and FIG. 9d is the injection of 8 mM urea solution, FIG. 9e is injection of 20 mM urea solution, and FIG. 9f is injection of 80 mM urea solution.

As can be seen in FIG. 9, the initial horizontal alignment (Planer) was not changed in the urea solution of 2 mM or less, and it was confirmed that when the 3 mM urea solution was injected, the liquid crystal became slightly dark 9a and 9b).

In addition, it was confirmed that when the concentration of the injected urea solution was increased to 5 mM, the horizontal alignment of the liquid crystal completely changed to the vertical alignment (see FIG. 9C). It can be seen from Figs. 9d to 9f that the same result is obtained when the urea solution having a concentration higher than 5 mM is injected.

Therefore, it was confirmed that the biosensor for element measurement of the present invention is suitable for measuring the element in a sample containing a concentration higher than 5 mM. Which is lower than 7 mM, which is a standard for diagnosing uraemia patients, indicating that the biosensor of the present invention is suitable for the diagnosis of uremia and / or renal failure.

Experimental Example  5. Element detection using blood samples

The blood used in the experiment was provided by a healthy donor. Ultraviolet rays of 365 nm wavelength were treated for 15 minutes and then 1 hour later to exclude the blood microbial activity.

The blood (hereinafter referred to as "normal blood") treated as described above was added to the injection unit of the biosensor for elemental measurement of Example 5, supernatant blood or 40 mM hemoglobin supplemented with 5 mM urea, 0.2 mM ascorbic acid and 5 mM urea was injected to evaluate the urea detection ability through a polarized light microscope image in the same manner as in Experimental Example 3. [ 10B is a graph showing the result of injection of blood and supernatant. FIG. 10C is a graph showing the results of a fluorescence microscope image obtained by adding hemoglobin, ascorbic acid (vitamin C) and urea mixture solution (FIG. 40 mM hemoglobin, 0.2 mM ascorbic acid, and 5 mM urea).

As can be seen from FIG. 10, in (a), it was found that the orientation of the liquid crystal did not change in the normal blood containing the element lower than the minimum detection concentration of the biosensor for elemental measurement of the present invention. In (b), it was confirmed that the orientation of the liquid crystal was changed from the horizontal orientation to the vertical orientation (change in the PH orientation) including the minimum detection concentration of 5 mM of the biosensor for element measurement of the present invention.

Furthermore, blood is known to contain hemoglobin and ascorbic acid (AA). However, it has been known that ascorbic acid has electro-activity, causing interference in an electrochemical element sensor, thereby causing a P-H orientation change. Therefore, in order to demonstrate the effect of the P-H orientation change of hemoglobin and ascorbic acid in the biosensor for elemental measurement of the present invention, experiments were performed using DI water containing 40 mM hemoglobin and 0.2 mM ascorbic acid. The concentration of hemoglobin and ascorbic acid was the maximum concentration that could be contained in blood.

As can be seen in FIG. 10C, it was found that the horizontal alignment of the liquid crystal changed to the vertical alignment similar to the result of (b) in which the hyperfractionated blood was injected. This result confirms that the biosensor for element measurement of the present invention has substantially no electrochemical interference due to hemoglobin and ascorbic acid. It was confirmed that the biosensor for element measurement of the present invention is more suitable for element measurement than the electrochemical element sensor of the prior art Results.

Experimental Example  6. Stability measurement of biosensor

Since the stability of the biosensor containing the enzyme is an important factor, the stability of the biosensor for elemental measurement prepared in Example 5 was measured.

Specifically, the average gray scale intensity of the element measuring biosensor prepared in Example 5 at 25 ° C and pH 7 was changed to black and white, and the average RGB value was measured. The measurement results are shown in FIG. 11 . A 75 mesh TEM grid cell was used.

11, it was found that there was no change in the average gray scale intensity for 10 days. After 10 days, a solution containing 5 mM of element was injected in the same manner as in Experimental Example 4 to measure the element detection ability As a result, the results were the same as 10 days before the start of storage (results not shown).

Thus, it was confirmed that the biosensor for elemental measurement of the present invention exhibits high stability.

Experimental Example  7. Detection speed measurement

In order to measure the element detection rate of the biosensor for element measurement, it was confirmed that the element containing the elements of 2 mM, 3 mM, 5 mM, 8 mM, 20 mM or 80 mM DI water was injected and the time for the vertical orientation of the biosensor liquid crystal to change to the horizontal orientation was measured using a polarized light microscope in the same manner as in Experimental Example 3. [

FIG. 12 shows the result of injecting DI water containing 8 mM of the above concentration. FIG. 12 (i) shows the results after 5 seconds, FIG. 12 After 12 seconds, 100 seconds after the injection of the solution, after 120 seconds after the injection of the solution, and after 120 seconds after the injection of the solution, the image of the biosensor liquid crystal was taken by the polarized light microscope.

The average grayscale intensity over time at different concentrations as described above was measured after changing the image to black and white, and the average RGB value was measured. The measurement results are shown in FIG. Figure 13 (iii) is a solution containing 3 mM urea, Figure 13 (ii) is a solution containing 5 mM urea, Figure 13 (iii) is a solution containing 8 mM urea, Figure 13 Is a result of a biosensor in which a solution containing 20 mM urea is injected, and Fig. 13 vi is a solution containing 80 mM urea.

As can be seen in FIG. 12, it was confirmed that the vertical alignment of the liquid crystal gradually changed from the edge of the biosensor to the horizontal orientation after the solution containing the element was injected. Especially, it was confirmed that most of the liquid crystals of the biosensor were horizontally oriented after 5 minutes of solution injection.

As can be seen in FIG. 13, it can be seen that as the concentration of the injected urea solution increases, the liquid crystals in the vertical alignment are horizontally aligned at a high speed. In addition, the time (t _half ) to half the saturation value of the concentration of the used solution is 250 seconds for 5 mM urea solution, 160 seconds for 8 mM urea solution, 63 seconds for 20 mM urea solution, The solution appeared 24 seconds. The decrease in t _{half as} the urea concentration of the used solution increased was a result of confirming that the urease enzyme immobilized on the biosensor effectively decomposed the urea.

As can be seen from the above Examples, Comparative Examples and Experimental Examples, the biosensor for element measurement of the present invention can detect urea above 5 mM, which was difficult to measure with conventional biosensors, It is very suitable to apply, and the effect of detecting element can be confirmed immediately without special equipment.

In addition, the biosensor for element measurement of the present invention can instantaneously and simply initialize (inject the distilled water at pH 7) into the pre-element recognition state after the element is recognized by the sensor, thereby facilitating the reuse of the biosensor for element measurement I was able to confirm the merit.

100: Biosensor for element measurement 101: Base material part 102: Liquid part
102: sensor part 201: main body part 202: inflow part
203: discharging portion 204: glass substrate 205:
206: Silicone rubber

Claims (14)

A liquid crystal copolymer represented by the following formula (1); And an urease,
The liquid crystal copolymer and the urease are covalently bonded,
The amount of urease to be contained per unit area of the liquid crystal copolymer represented by the following formula (1) is 10 to 20,
A biosensor for element measurement, characterized by being able to detect an element in a sample containing an urea concentration of 5 mM to 7 mM;
[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.
[Equation 1]

In the above formula (1), N _urease is the amount of urease contained per unit area of the liquid crystal copolymer, C _imb is the amount of fixed urease, Na is the number of Avogadro, 6.02 × 10 ²³ , A is the amount of urea (Nm ² ), and M _urease is an integer of 4.8 × 10 ⁵ to 5.5 × 10 ⁵ (g / mol), which is the molecular weight of urease.
The biosensor for elemental measurement according to claim 1, wherein the liquid crystal copolymer is a block copolymer. delete The biosensor 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 urease on the liquid part;
Further comprising a biosensor for measuring an element. 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 biosensor comprises at least one member selected from the group consisting of: 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 biosensor for measuring an element. 5. The biosensor according to claim 4, wherein the element measuring biosensor
A main body portion to which a sample for element measurement is added;
An inlet for introducing the sample for elemental measurement; And
A discharging portion for discharging the sample for element measurement;
Further comprising a biosensor for measuring an element. 5. The liquid crystal display device according to claim 4, wherein, when the element is coupled to the sensor unit, the birefringence change 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 Biosensor for element measurement. 9. The biosensor for element measurement according to claim 8, wherein the qualitative analysis is performed at a pH of 6-8. delete The biosensor for element measurement according to claim 1, wherein the liquid crystal copolymer is in the form of a monomolecular film. delete delete delete

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