KR101782949B1 - Liquid crystal-based glucose biosensor functionalized with mixed PAA and QP4VP brushes - Google Patents

Liquid crystal-based glucose biosensor functionalized with mixed PAA and QP4VP brushes Download PDF

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KR101782949B1
KR101782949B1 KR1020150060804A KR20150060804A KR101782949B1 KR 101782949 B1 KR101782949 B1 KR 101782949B1 KR 1020150060804 A KR1020150060804 A KR 1020150060804A KR 20150060804 A KR20150060804 A KR 20150060804A KR 101782949 B1 KR101782949 B1 KR 101782949B1
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glucose
liquid crystal
carbon atoms
alkyl group
biosensor
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KR20160128820A (en
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박수영
마슈크 칸
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경북대학교 산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/23—Bi-refringence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0092—Polarisation microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour 
    • G02F1/13—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells

Abstract

The invention PAA-QP4VBVP relates to a liquid crystal-based glucose biosensor functionalized with a brush, for bio-glucose sensor according to the present invention, a monomolecular film composed of the liquid crystal liquid crystal copolymer mixture of PAA- -LCP b and b QP4VP- -LCP And the glucose oxidase enzyme is electrostatically immobilized on the liquid crystal / water interface, so that it can be manufactured quickly, precisely, easily, reliably, and cost-effectively. In addition, the glucose biosensor according to the present invention is capable of detecting a small amount of glucose in an aqueous medium and a complex mixture, has a small Michaelis-Menten constant (K m ) of 1.67 mM, is stable for a period of 16 days or more, Reproducibility, it can be usefully used instead of the glucose biosensor used conventionally.

Description

[0001] The present invention relates to a liquid crystal-based glucose biosensor functionalized with a PAA-QP4VBVP brush and a liquid crystal-based glucose biosensor functionalized with mixed PAA and QP4VP brushes.

The present invention relates to a glucose biosensor, more particularly, to a glucose biosensor having a low concentration of 0.5 mM, which is excellent in sensitivity to detect glucose, and can be easily manufactured at low cost, To a glucose sensor capable of detecting glucose in a sample without any special device.

Diabetes mellitus is a serious disease with various acute and chronic complications resulting from an increase in blood sugar 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 the self-monitoring blood glucose meter, which is mainly used for monitoring the blood glucose level, is increasing and the frequency of use is gradually increasing.

On the other hand, the biosensor can be applied to various fields such as medical use, environmental use, food use, military use, and industrial use. 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 analyte injecting step, a signal generating step, a signal amplifying step, and a complicated analysis result interpreting step, and it is very expensive to apply to real life.

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, enzyme immobilization is an important factor in developing an efficient biosensor due to characteristics such as storage stability, high sensitivity, high selectivity, short reaction time, and high reproducibility. The immobilized enzyme must not disappear during the detection of the substance and must maintain its inherent structure or function. In addition, ideal biosensors should be available for long periods of time. Therefore, the fixation method affects the function and stability of the enzyme biosensor, and the factors such as measurement accuracy, reproducibility, and action time are significantly influenced by the stability of the enzyme.

Many methods of fixing such as entrapment, physical adsorption, crosslinking, affinity, and covalent coupling have been developed to produce a high-efficiency biosensor, And the reaction takes place through the initial activation of the coupling agent. However, when a toxic coupling agent is used, methods such as a specific active group on the surface of the support, activation of the surface group, replacement of the active group with the enzyme, and modification of the enzyme may be required. In addition, long-term stability of biosensors remains a challenge.

Accordingly, the present inventors paid attention to this point and used a PAA-QP4VBVP mixed brush as a liquid crystal copolymer capable of functionalizing the liquid crystal / water interface, and found that the above liquid crystal copolymer was used for glucose detection in which glucose oxidase enzyme was electrostatically immobilized The inventors of the present invention discovered that the glucose biosensor has advantages such as low manufacturing cost, simple enzyme fixation, high enzyme sensitivity and stability, and can easily detect the glucose level in the body using an optical microscope, .

In order to solve the above problems, the present invention provides a biosensor for detecting glucose in which a glucose oxidase enzyme is electrostatically immobilized on the liquid crystal copolymer, using a PAA-QP4VBVP mixed brush as a liquid crystal copolymer. We will do it.

In order to solve the above problems, the present invention provides, as one aspect,

A substrate portion including a hydrophobic material layer; A liquid portion including a liquid crystal monomer on an upper portion of the substrate portion; A sensor unit coated with a mixture of a liquid crystal copolymer of Formula 1 and a liquid crystal copolymer of Formula 2 on the liquid crystal portion and glucose oxidase is electrostatically fixed on the liquid crystal copolymer of Formula 2; The present invention also provides a biosensor for detecting glucose.

[Chemical Formula 1]

Figure 112015042009049-pat00001

(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

Figure 112015042009049-pat00002
or
Figure 112015042009049-pat00003
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.

(2)

Figure 112015042009049-pat00004

(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, and R < 3 >

Figure 112015042009049-pat00005
or
Figure 112015042009049-pat00006
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.

More preferably, the biosensor for detecting glucose 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.

More preferably, the hydrophobic material layer is made of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) and octadecyltrichlorosilane (OTS). And at least one of the groups.

More preferably, the liquid crystal monomer is a mixture of cyclohexane-fluorinated biphenyl and flurinitethenyl (mixture of cyclohexane-fluorinated biphenyls and fluorinated terphenyls; TL205), 4-oxyl-4'-cyanobiphenyl 4-cyano-4'-cyano-biphenyl, 8CB) and 4-cyano-4'-pentylbiphenyl (5CB) .

More preferably, the biosensor for measuring glucose comprises 200 to 30,000 parts by weight of glucose oxidase per 100 parts by weight of the liquid crystal copolymer mixture.

More preferably, the sensor portion is characterized in that the mixing ratio of the liquid crystal copolymer of Formula (1) and the liquid crystal copolymer of Formula (2) is 20: 3 to 20: 5 and the amount of salt is 1M.

More preferably, the liquid crystal copolymer mixture is coated in the form of a monomolecular film.

More preferably, 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 the glucose, and the qualitative analysis is performed at a pH lower than the physiological condition pH 7 And a tendency of showing a dark image when a polarizing microscope is measured at a pH higher than a pH 7, which is a physiological condition, is analyzed and performed.

More preferably, when combined with the glucose to the sensor unit, by measuring the birefringence change in the orientation change of the liquid crystal portion liquid crystal monomer, a known glucose infusion concentration (C 0) birefringence change (Δn) / glucose infusion concentration (C for 0 ) is used to quantitatively analyze the concentration of glucose in a mixed solution containing an unknown amount of glucose.

More preferably, the qualitative or quantitative analysis is performed at pH 2-12.

More preferably, the biosensor is characterized in that glucose can be detected in a sample containing a glucose concentration of 0.5 mM or more.

The biosensor for measuring glucose according to the present invention is characterized in that a monomolecular film composed of a mixture of a liquid crystal copolymer of PAA- b- LCP of formula (1) and QP4VP- b -LCP of formula (2) is coated on a liquid crystal and glucose oxy The enzyme is electrostatically immobilized, making it fast, precise, easily manufacturable, reliable, and cost effective. In addition, the glucose biosensor according to the present invention is capable of detecting a small amount of glucose in an aqueous medium and a complex mixture, has a small Michaelis-Menten constant (K m ) of 1.67 mM, is stable for a period of 16 days or more, Reproducibility, it can be usefully used instead of the glucose biosensor used conventionally.

1 is a schematic cross-sectional view of a biosensor for measuring glucose according to an embodiment of the present invention.
2 is a schematic cross-sectional view of a biosensor for measuring glucose according to another embodiment of the present invention.
3 is a cross-sectional schematic view of a biosensor for measuring glucose according to another embodiment of the present invention and a photograph showing an actual flow cell.
((a) cross-sectional schematic view, (b) flow cell including TEM grid)
FIG. 4 shows a POM image of a TEM grid with respect to the concentration of liquid crystal copolymers and the concentration of an electrolyte according to an experimental example of the present invention.
(NaCl concentration (C NaCl = 1M) and PAA-b-LCP amount of solution (v 1 = the volume (v 2) of QP4VP-b-LCP solution in 200 μL) (a) 0, (b) 10, ( c) 20, (d) 30 , (e) 40, (f) 50, (g) 60, and (h) is changed by 80 μL measured; and fixed to v 2 = 50 μL, NaCl concentration (C NaCl) (1) TEM grid (TEM PAA -b-LCP solution) coated with only PAA-b-LCP solution in 1.5M NaCl without QP4VP-b- ) POM image measurement)
5 is a graph showing the amount (GOx imb ) of the fixed glucose oxidase relative to the amount (v 2 ) of the liquid crystal copolymer QP4VP-b-LCP solution according to an experimental example of the present invention.
FIG. 6 shows changes in POM image and fluorescence microscope image of TEM grid after fixation of glucose oxidase according to an experimental example of the present invention.
(POM image measurement of the TEM grid (TEM mixed ) by varying the amount (v 2 ) of the QP4VP-b-LCP solution in the GOx solution of 20 μM to (i) 40, (ii) 50, and (iii) ; v 2 = 50 μL of labeled GOx after electrostatic fixation (iv) TEM mixed And (v) Fluorescence microscope measurement of TEM PAA )
7 is a POM image of a TEM grid showing glucose detection results of the biosensor according to an example of the present invention.
((a), 8 mM glucose solution, (b) pH = 6 the TEM mixed in an aqueous acidic solution - POM image of GOx; (c) a TEM image of a POM mixed in a glucose solution without fixing GOx)
8 is a POM image of a TEM grid showing the glucose detection result of the biosensor with respect to the concentration of the injected glucose solution according to an experimental example of the present invention.
(C) 1, (d) 3, (e) 5, (f) 8, (g) 11 and (h) 16 mM )
9 shows the glucose detection reaction rate of a biosensor according to an experimental example of the present invention.
(a) normalized GI as a function of time (s), (b) Δn as a function of C 0 and (c) graph of C 0 / Δn vs. C 0 )
10 is a graph showing the stability and reproducibility of a biosensor according to an experimental example of the present invention.
(a) the fluorescence intensity ratio (I / I 0 ) as a percentage of time as a function of time, (b) the average GI over the cycle when the biosensor is washed and the glucose solution injected cycle is repeated a number of times)
11 is a POM image of a TEM grid showing glucose detection results according to glucose content in a mixed solution according to an experimental example of the present invention.
(C) 0.5, (d) 2 mM, and (e) an unknown concentration of glucose)

Hereinafter, terms of the present invention will be described.

Herein, "HP change" means that the liquid crystal of the glucose sensor changes its orientation from a homeotropic state to a horizontal orientation state, while conversely, "PH change" means a homeotropic state ). ≪ / RTI >

In the present specification, the term "mixing brush" means that a mixture of two or more liquid crystal copolymers is bonded on a liquid crystal so that a side chain of the liquid crystal copolymer has a brush shape, and "liquid crystal copolymer blending brush" May be used interchangeably.

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, the problem raised by the enzyme fixing method using covalent bond and the long-term stability of the biosensor still remain as a problem to be solved.

Accordingly, the present invention provides a magnetic recording medium comprising: 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 mixture of a liquid crystal copolymer (PAA) of formula (1) and a liquid crystal copolymer (QP4VP) of formula (2) is coated on the liquid crystal part and a glucose sensor is electrostatically immobilized on the liquid crystal copolymer of formula part; The present invention provides a biosensor for detecting glucose.

Figure 112015042009049-pat00007

(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

Figure 112015042009049-pat00008
or
Figure 112015042009049-pat00009
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.

Figure 112015042009049-pat00010

(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, and R < 3 >

Figure 112015042009049-pat00011
or
Figure 112015042009049-pat00012
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 biosensor according to the present invention is a biosensor in which a mixing brush in which two liquid crystal copolymers (polymer electrolytes) of QP4VP and PAA are mixed is used for enzyme fixing and pH-dependent glucose detection by electrostatic interaction, , It is possible to fix the structure of the enzyme for a long time without any chemical modification and to fix it on the brush easily. Thus, it can be manufactured at low cost and has long-term stability since it consumes less time and does not require use of other expensive reagent. In addition, since glucose can be detected from the HP orientation caused by the deprotonation of the PAA chain, it is possible to easily detect the glucose level in the body using an optical microscope.

1-3 are schematic cross-sectional views of a glucose sensor 100 according to an embodiment of the present invention. Referring to FIG. 1, a substrate 101 including a hydrophobic material layer is illustrated. 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 level.

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' -cyanobiphenyl, 8CB) and 4-cyano (4-cyano-4'-pentylbiphenyl, 5CB), more preferably 4-cyano-4'-pentylbiphenyl .

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 of glucose by binding with glucose and is coated with a mixture of the liquid crystal copolymer (PAA) of Formula 1 and the liquid crystal copolymer (QP4VP) of Formula 2, And glucose oxidase (GOx) electrostatically immobilized on the liquid crystal copolymer of Formula 2.

In the present invention, QP4VP (strong polyelectrolyte (PE)) is a cation regardless of pH, while PAA (weak PE) is an anion at a pH higher than its pKa (~ 4.7). Thus, QP4VP and PAA in water can have positive and negative charges, respectively, on the liquid crystal at pH = 7. The GOx enzyme has a PI of 5.2, which means that GOx has a net negative charge at pH = 7, so that electrostatic interactions of QP4VP with GOx at pH = 7 can be expected, The enzyme can be immobilized.

The content of the liquid crystal copolymer mixture and glucose oxidase in the sensor part is not particularly limited, but preferably 200 to 30,000 parts by weight, based on 100 parts by weight of the liquid crystal copolymer, of glucose oxidase.

If the amount of glucose oxidase is less than 200 parts by weight based on 100 parts by weight of the liquid crystal copolymer mixture, the amount of glucose oxidase is too small to cause a slow reaction between glucose and glucose oxidase, If 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 in the reaction with glucose, resulting in waste of material .

In the sensor portion, the mixing ratio of the liquid crystal copolymer of Formula 1 and the liquid crystal copolymer of Formula 2 is preferably 20: 3 to 20: 5. If it is out of the above range, there is a problem of not being vertically aligned.

In this sensor part, the amount of salt must be controlled since the relative salt to the liquid crystal copolymer in solution greatly affects the orientation of the liquid crystal in the TEM grid.

In the sensor portion, the amount of the salt is preferably 1 M, and if the amount is out of the above range, there is a problem that vertical alignment is not performed.

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 mixture is coated with a film and bonded onto 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 glucose oxidase is an enzyme which generates glucose (D-gluconic acid) by oxidizing glucose (? -D-glucose) with oxygen, and is not particularly limited as long as it can be sold and / May include those found in fungi or honey, such as Penicillium notatum.

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 change in birefringence according to the orientation change of the liquid crystal monomer in the liquid unit 102 can be measured to qualitatively analyze the glucose , A linear function of the birefringence change (? N) versus the known glucose injection concentration (C 0 ) / glucose injection concentration (C 0 ) can be used to quantitatively analyze the glucose concentration in a mixed solution containing an unknown amount of glucose .

As shown in FIG. 7, the qualitative analysis showed a bright image at a pH lower than pH 7, which is a physiological condition, and a dark image at a pH higher than pH 7, which is a physiological condition .

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

2 and 3 are sectional views of a glucose sensor according to another preferred embodiment of the present invention. Referring to FIG. 2 and FIG. 3, a substrate 101, a liquid portion 102, And the counter substrate 205 is disposed so as to be spaced apart from the sensor unit 103 and is 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.

material

Microscope glass slides (Duran group, Germany) were washed with piranha solution to remove organic contaminants, then rinsed with distilled water and dried under nitrogen.

A copper TEM sample grid (G75, grid hole width 285 m, pitch 340 m, bar width 55 m, 3.05 mm, thickness 18 m) was purchased from Ted Pella.

Aspergillus niger species-derived GOx (EC 1.1.3.4) was purchased from Sigma-Aldrich in the form of a salt-free lyophilized powder having a species activity of> 100,000 units per gram and a molecular size of 1.6 × 10 5 g / mol.

D (+) - glucose (Sigma), N - (3- dimethylaminopropyl) -N- ethyl kaboyiyi imide hydrochloride (N - (3-dimethylaminopropyl) - N -ethylcarbodiimide hydrochloride; EDCHCl hereinafter referred to, Sigma-Aldrich) , N - hydroxyl facilities posuk god imide sodium salt (N -hydroxysulfosuccinimide sodium salt; hereinafter referred to as NHS, Sigma-Aldrich), sodium chloride (Aldrich), trifluoroacetic acid (TFA, Aldrich, 99%) , 4- cyano- Octadecyltrichlorosilane (hereinafter referred to as OTS, Sigma-Aldrich), methanol (Aldrich), and dichloro (4-cyano-4-pentylbiphenyl Methane (Aldrich), yeast extract (Sigma), ascorbic acid (Fluka), and human hemoglobin (Sigma) were used as supplied.

Device

Image and video images of the TEM grid cell were obtained under a polarizing microscope POM (Leitz, ANA-006, Germany) and the cross-polarization state was recorded using a CCD camera (Samwon, STC-TC83USB, Korea). The UV-visible spectrum was measured using an ultraviolet-visible spectrophotometer (Shimadzu, 2401, Japan). GOx rhd6G on the TEM grid was measured by fluoerscence microscopy (Nikon Eclipse, E600POL, Japan) and fluorescence spectra were recorded using a high resolution spectrometer (HR4000, Ocean Optics, Inc., Japan) attached to a fluorescence microscope Respectively. HPLC analysis was carried out using an HPLC apparatus (Waters Co. Model 600E) equipped with a column (Sugar-Pak 1 column (6.5 x 300 mm)) and a refractive index detector (RI, Model 410). Ca-EDTA buffer (50 mg Ca-EDTA / 1 L dH 2 O) was used as the mobile phase at a flow rate of 0.5 mL / min at 90 ° C in the column. The gray scale intensity (GI) of the video frame was calculated using Adobe Photoshop CS5 software. The optical birefringence (Δn) was measured at a 50% intensity of the total illumination using a tilting compensator (2073 K, equipped with a calcite compensator plate, Leitz, Germany). At the 0 position of the corrector, the crystal axis was parallel to the polarizer axis. The optical birefringence Δn is defined as Γ / d, where Γ is the phase difference and d is the sample thickness (18,000 nm). Γ was measured from the tilt angle (2i) using the following equation (1).

Figure 112015042009049-pat00013

(Where f (2i) is tabulated in the device manual,

c is the correction constant (4.54 x 10 4 ).

The measurement was repeated three times, and the error bars indicate the standard deviation (SD).

< Manufacturing example  1> liquid crystal copolymer PAA - b - LCP Synthesis of

Figure 112015042009049-pat00014

(In the above scheme, LC is

Figure 112015042009049-pat00015
to be.)

<1-1> PtBA - b - LCP Manufacturing

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 polymer PtBA (110.9 mg, 0.0042 mmol) 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 PtBA- b- LCP was obtained.

The number average molecular weight (Mn) of the PtBA- b -LCP was 3.27 x 10 4 ,

The weight average molecular weight / number average molecular weight (Mw / Mn) was 1.27.

<1-2> PAA - b - LCP Manufacturing

0.1 g of the PtBA- b -LCP obtained in the above <1-1> and 1 ml of trifluoroacetic acid (TFA, manufactured by Aldrich, 99%) were dissolved in dichloromethane (DCM, ), And the mixture was reacted by stirring at 25 DEG C for 24 hours. Thereafter, 500 ml of ethyl ether was added to obtain a precipitate. The precipitate was vacuum-dried at 25 DEG C for 24 hours to prepare a liquid crystal copolymer PAA- b- LCP.

The weight average molecular weight / number average molecular weight (Mw / Mn) of the PAA- b -LCP was 1.14.

< Manufacturing example  2> liquid crystal copolymer QP4VP - b - LCP Synthesis of

Figure 112015042009049-pat00016

<2-1> P4VP - MCPDB Synthesis of

To the vial with graduations was added 4-vinylpyridine (4-VP) (1.068 mL), MCPDB (CTA) (5.49 mg, 0.018 mmol) and DMF (1.115 mL) and the mixture was bubbled with dry nitrogen for 60 minutes Respectively.

A Schlenk flask containing AIBN (0.596 mg, 0.00363 mmol) was allowed to vacuum for 60 minutes and then the previously prepared 4-VP and CTA mixture solution in DMF was introduced into the flask using a syringe needle Then purged with N 2 . The flask was left in an oil bath at 80 &lt; 0 &gt; C for 4 hours for the RAFT reaction. The resulting macroinitiator, P4VP-MCPDB, was precipitated in diethyl ether. The number average molecular weight (Mn) and the polydispersity index (PDI) of the homogeneous polymer were 47.6 K and 1.24, respectively. The volatiles were removed in vacuo at 40 &lt; 0 &gt; C to give a dried pink powder. The origin of the pink is due to the terminal group of MCPDB.

<2-2> QP4VP - b - LCP Synthesis of

AIBN (0.34 mg, 0.0021 mmol), LCP (88.04 mg, 0.2099 mmol) and P4VP-MCPDB (199.84 mg, 0.0042 mmol) were added to a Schlenk flask to synthesize P4VP- b -LCP via the RAFT method. A toluene / ethanol mixed solvent (4/6, v / v, 3.037 mL) was poured into the Schlenk flask and the solution was degassed by bubbling with nitrogen gas for 60 minutes. The flask was placed in an oil bath set at 65 DEG C for 6 hours. After the reaction was completed, the viable material was precipitated in diethyl ether. The volatiles were removed in vacuo at 40 &lt; 0 &gt; C to give a yellowish powder. Mn and PDI of P4VP- b -LCP were 53.8 and 1.23 K, respectively.

P4VP- b- LCP (0.062 g) was dissolved in DMF (5 mL) and CH 3 I (0.186 mL) was added dropwise to the polymer solution at 0 ° C in the dark. The reaction was continued at 20 &lt; 0 &gt; C for 16 hours with stirring. At the end of the reaction, the mixture was added dropwise to excess ethyl ether to precipitate the product, which was filtered and dried in a vacuum oven at 40 ° C to completely remove unwanted volatile components.

< Example  1> Glucose  Manufacture of biosensors for measurement

Example  1-1: The substrate portion  Produce

Slide glass (Slide glass, product of Duran group, Germany) having a size of 12 mm × 8 mm (width × 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 (glass coated with OTS).

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

1. Monolayer formation

In this example, the monolayer was formed using a Langmuir Blodgett (LB) KSV layer builder (AAA100178, KSV Instruments, Ltd., Finland) connected to a KSV mini-micro trough. The monomolecular film was fabricated at 17 × 5 cm 2 , and the surface pressure of the monomolecular film at the gas / liquid interface of the monomolecular film was measured using a Wilhelmy plate attached to the microammetric plate. All experiments were performed at room temperature.

Specifically, PAA- b- LCP was dissolved in dioxane and left at 60 ° C for two days. The toluene was then added to the dioxane solution so that the dioxane / toluene was 6/4 (v / v), and QP4VP was dissolved in distilled water (1 mg / ml) to a final concentration of 1 mg / ml.

A fixed volume of PAA- b- LCP solution (v 1 = 200 μL) was dropped to a 1 M NaCl subphase when the surface pressure (π) of the vapor / liquid reached 0, as measured by a KSV layer builder, of QP4VP- b -LCP solution (v 2) dropped immediately to form a monomolecular film. One hour after the formation of the monomolecular film, the barrier of the layer builder moved, compressing and expanding the monomolecular film at the interface, and then compressing it again at a speed of 3 mm / min to form an equilibrium. The monomolecular film was set to have an areal density of 36 mm 2 (= 45 mN / m).

2. The body portion  Produce

The above-prepared liquid crystal copolymer monomolecular film was placed on the copper grid of the liquid crystal part 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 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.

3. GOx Cover and Fix

Then, a glucose oxidase (GOx) was immobilized on the liquid crystal copolymer monomolecular film 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.

The GOx was dissolved in phosphate buffered saline (PBS) buffer (pH = 7) in a reaction vessel to obtain a 20 M solution. The coupling reagents EDCHCl and NHS were added and reacted for 4 hrs. The carboxyl group was activated. Then, 1 mg of Rhodamin 6G was added for labeling, and then the mixture was stirred at room temperature for 12 hours. A saturated aqueous ammonium sulfate solution (0.5 g / mL) was added dropwise to the reaction. The labeled GOx (GOx rhd6G ) precipitated and it was centrifuged twice at 5000 rpm for 20 minutes. The supernatant was removed by filtration, and the filtered GOx rhd6G was dried under vacuum. The UV-vis spectra of the GOx rhd6G solution showed absorption peaks at 526 and 276 nm , which are attributed to Rhodamin 6G and GOx, respectively, indicating that GOx was successfully labeled.

 Electrostatic fixation of GOx to the QP4VP chain was performed on 5CB in the TEM grid by injecting 3 mL of 20 M GOx solution into the flow cell.

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

< Experimental Example  1> The sensor GOx The optimal conditions

To determine the optimum conditions for fixing GOx, the amount of PAA polymer of formula ( 1 ) was kept constant at 200 μL (v 1 ) in the production of the sensor part, and the amount (v 2 ) of QP4VP was controlled. The reference orientation should be vertical, because the reaction of glucose with GOx induces an orientation change from vertical to planar.

Previously reported studies have shown that QP4VP-coated 5CB in the TEM grid exhibits vertical orientation in the range of pH 2-12 (Omer and Park, 2014). This result was due to the large amount of charge generated by QP4VP, which generates an electric field at the CB / aqueous interface.

Figures 4a-h show POM images (under crossed polarizers) of a TEM mixed grid of the present invention at constant v C 2 with constant C NaCl at 1M.

Observations, v 2 ≤30 μL (Fig. 4a-d), whereas the plane of alignment of the liquid crystal 5CB observed, v 2 in ≥40 μL was observed (Fig. 4e-h), vertical alignment. These results indicate that the vertical orientation is due to the high charge density exhibited by the QP4VP polyelectrolyte (PE) chain at the 5CB / aqueous interface and that the weak PE, PAA, did not significantly affect the vertical orientation under the experimental conditions.

The relative salt to PE greatly affects the orientation of the 5CB in the TEM grid, so the amount of salt must be controlled. Figure 4i-k shows POM images of TEM mixed at constant v 2 and various values of C NaCl at 50 μL. v 2 = 50 μL, C NaCl = 1M, vertical orientation was shown as shown in FIG. 4f. C NaCl = 0.75M (Fig. 4i), both the planar and dark portions due to sporadic vertical orientation were observed together. At C NaCl = 0.5M and 0.2M (Fig. 4j-k), the dark areas decreased and a clear planar orientation was observed. These results suggest that sufficient salt is necessary to maintain the vertical orientation of 5CB in the TEM grid. However, at v 2 = 0 μL, 5CB (TEM PAA ) without QP4VP on the TEM mixed grid showed planar alignment even at high C NaCl (1.5M) (Fig. Thus, to obtain the reference vertical orientation, a sufficient QP4VP brush must be coated on 5CB in the TEM grid and there must be a sufficient amount of NaCl.

Another way to create planar orientation is to include NaCl in the aqueous phase which can lead to the formation of an electrical double layer through the division of ions into the 5CB from the aqueous phase at the 5CB / . This results in a vertical orientation of 5CB in the TEM mixed grid through the electric field induced by the double layer. In the present invention, unless otherwise noted, C NaCl was fixed at 1 M to form a single layer film.

< Experimental Example  2> The sensor GOx  Fixed quantity measurement

To determine the amount of GOx immobilized on the liquid crystal copolymer monolayer of the sensor part, the amount of immobilized GOx was measured by spectrophotometrically comparing with the value of deionized water.

The amount of GOx immobilized on the TEM grid (GOx imb ) can be determined from the optical density of the ultraviolet-visible light spectrophotometry.

FIG. 5 shows a graph of GOx imb according to the amount (v 2 ) of QP4VP after 20 μM GOx solution is injected. As shown in Fig. 5, v 2 is increased more GOx imb were increased, which indicates that successfully combine the QP4VP chain while having a possible link density GOx is adjusted.

Figure 6 is v 2 = 40, 50, the TEM mixed produced in 60 μL of 20 μM after injection GOx solution to the cell - represents a POM image of GOx. As discussed above, prior to implanting the GOx solution, the POM image shows a vertical orientation.

As a result, the vertical alignment was maintained at v 2 = 40 and 50 μL (FIGS. 6i and 6ii), whereas the plane orientation was observed at v 2 = 60 μL (FIG. Observation of the planar orientation at high v 2 will be due to the large amount of immobilized GOx, which induces planar orientation through electrostatic complex formation and leads to a reduction of the electric field due to the neutrality of the complex. Previously, when a PE-coated 5CB cell in a TEM grid was subjected to application of a protein or enzyme solution onto its PI, a similar HP change was observed (Omer and Park, 2014). In order to use this surface for glucose detection, a vertical orientation is initially required because the GOx oxidation of glucose releases a proton (H + ) and the protonation of PAA causes the planar orientation of the 5CB. In previous studies, 2M NaCl solution was used to obtain the initial vertical orientation after GOx fixation (Khan and Park, 2013), which may affect the long-term stability of the biosensor. However, in the present invention, 1 M NaCl was not used during enzyme immobilization and glucose detection, and 1 M NaCl was used only during monolayer formation to avoid interactions between polymer brushes with opposite charges during biosensor operation. Thus, the system of the present invention will have high stability and low interference. Unless otherwise stated, v 2 = TEM mixed with 50 μL - GOx (GOx imb = 0.78 M) were used in all experiments.

Figure 6iv shows v 2 = Fluorescence image of TEM mixed after fixation of GOx rhd6G at 50 μL. The inside of the TEM grid was evenly distributed, whereas the outside of the TEM grid was mostly black, confirming successful fixation of GOx. For comparison, TEM PAA was used under the same conditions as TEM mixed . After washing, a black image was observed (FIG. 6v), suggesting that GOx rhd6G was not anchored to the PAA chain. These results suggest that GOx is due to electrostatic attraction between the fixed GOx and QP4VP chains. GOx (PI = 5.2) has anionic charge at pH = 7 (test pH), so GOx can only attach to cationic QP4VP, not to PAA.

< Experimental Example  3> Glucose  detection

8 mM glucose (Sigma D (+) glucose) solution was injected through the inlet of the glucose sensor prepared in Example 1 to obtain a POM image of a TEM mixed- GOx grid cell at pH = 7 under an intersecting polarizer . The POM image is shown in Fig.

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 initial vertical orientation changed to planar orientation. This HP change would be due to the release of H + ions during the enzymatic oxidation of glucose, which reduces the pH in the cell and causes protonation and contraction of the PAA chain. As reported elsewhere, the pH-dependent shrinkage of PAA at the interface can change the orientation of the 5CB, which is strongly immobilized within the TEM grid cell (Lee et al., 2010). The 5CB coated with poly (ethyleneimine) -bN- [3- (dimethylamino) -propyl] acrylamide as the amphiphilic block copolymer at the 5CB / aqueous interface exhibited reversible change in light appearance upon exposure to solutions of pH 5 and 9 , Which is due to the protonation and deprotonation of the hydrophilic chain (Kinsinger et al., 2007).

In addition, the various charge densities on the PAA chain lead to changes in the 5CB orientation (Lee et al., 2010). An acidic aqueous solution (pH = 6) was introduced to determine if HP changes as the pH was lowered. Similar HP changes were observed (Fig. 7b), confirming that the HP change due to the addition of glucose to TEM mixed - GOx grid cells was actually due to the lowered pH due to the enzymatic reaction.

To confirm that the HP change was due to the enzymatic reaction of GOx, 8 mM glucose solution was injected under the same conditions except for a flow cell (TEM mixed ) in which no other conditions were identified and fixed GOx. As a result, the initial vertical orientation was maintained (Fig. 7c), suggesting that this HP change was due to glucose and GOx reaction.

8 is a TEM mixed with glucose solutions of different concentration (C o) - represents a POM image of GOx grid cell. In C o = 0.4 mM (Fig. 8A), the initial vertical alignment did not change. In C o = 0.5 mM (FIG. 8b), a slightly bright portion appeared in the TEM mixed - GOx grid cell, and as the C o increased (FIG. 8c-h), the PH change became more visible. Thus, TEM mixed according to the invention - GOx grid sensor can detect glucose in a C o = 0.5 mM. The detection limits have similar ranges to those reported in many other reports. For example, a nanocomposite electrode consisting of MWCNT trapped between chitosan layers has been reported to have a detection limit of 0.96 mM (Monosik et al., 2012) and on CNTs combined with gold (Au) and platinum Fixed GOx (Che et al., 2007) and CNTs with plasma-polymerized films (Hoshino et al., 2012) were observed to have detection limits of 0.4 mM.

However, electrostatic clamping of the GOx of the present invention is simpler and more cost effective, since it is performed without chemical reaction.

< Experimental Example  4> Glucose  Reaction rate of detection

The color of the TEM mixed-GOx observed by the POM under the polarizer crossed according to the Michel-Levy chart represents the specific level of retardation. Vertical and planar orientations each appeared as dark and bright images, thus increasing GI as HP changes progressed. The difference in light appearance with increasing C o can be an indicator of the glucose level in the media. An image frame represents an image in a short period (4 seconds), and a GI of each scene represents an orientation state.

Figure 9a shows the GI over time at different C o . At high C o (8 and 5 mM), GI rapidly increased to saturation level. The time required to reach half of the saturation level was respectively 180, 120, and 75 sec at C o = 0.5, 3 and 8mM. The saturation level also increased with increasing C o .

By taking the GI measurement as time, it has been found that the rate of change of light appearance and its final saturation value depend on the amount of glucose present in the cell. Therefore, precise measurement of birefringence ( n ) can be very important for quantitative measurement of glucose levels. Δ n value of TEM mixed-GOx was determined for different C o in the range of 0.5 to 42 mM. Figure 9b shows the change in Δ n value as a function of C o . The value of n increased with increasing C o up to C o = 11 mM, and saturates to about 0.08. The linear kinetic range of the TEM mixed - GOx glucose sensor (up to 11 mM) was reported to be 3 mM (Ho et al., 2014), 6 mM (Hwa and Subramani, 2014), 8 mM (Jing-Juan and Hong (Yang, 2000), 5 mM (Liu and Lin, 2006) and 6.5 mM (Zhai et al., 2013) and 2 mM (Khan and Park, 2013) reported previously by the present inventor.

The mechanism of the GOx-catalyzed reaction was studied using a linear Michaelis-Menten model as shown in Scheme 1 below.

Figure 112015042009049-pat00017

Where n max and K m are the maximum birefringence values and Michaelis-Menten constants obtained at the saturation level of the substrate, respectively.

From the slice and slope of the C o / Δ n versus C o plot (FIG. 9c), K m and Δn max were 1.67 and 0.084, respectively. The K m value was 1.67, which was smaller than the reported values of 3.94 mM (Jing-Juan and Hong-Yuan, 2000), 14 mM (Malitesta et al., 1990) and 4.3 mM (Zhang et al., 2005) All. However, this is higher than the previously reported K m value of 0.32 mM, which seems to be due to the fixed density of GOx. Small K m values suggest that the change in light output from glucose oxidation by GOx immobilized on the QP4VP chain in TEM mixed-GOx is more sensitive than other studies. Δn max is obtained when the top of 5CB in contact with the aqueous medium is in a fully parallel orientation and depends on the hydrophobicity of the OTS coated glass, the composition of the brush density and PE, and the density of the fixed GOx.

< Experimental Example  5> Glucose  Stability and Reproducibility of Measurement Biosensor

10A shows the fluorescence emission intensity ratio ( I / I 0 , I 0 = initial intensity) at 551 nm as a percentage of time as a function of time. At pH = 3 (below PI of GOx), fluorescence intensity was observed to decrease by about 75% after 4 days. However, at pH 7 and 9 (above PI of GOx), only about 8% reduction was observed over 4 days and no significant decrease occurred over 16 days. These results indicate that electrostatic fixation results in high GOx stability for the QP4VP chain over a long period of time.

To test the reproducibility, the TEM mixed-GOx sensor of the present invention was exposed to a glucose solution and then washed with about 10 mL of each PBS buffer (pH = 7) and distilled water to obtain a baseline vertical state again. FIG. 10B shows the reference vertical state after cleaning, C 0 = Flat state in a 8 mM glucose solution, a reference vertical state after washing again, and C 0 Of the present invention having a period of a flat state at = 3mM glucose solution mixed TEM - GOx The average GI value of the glucose sensor is shown. In each cycle, the GI values, after washing, had a low value of 143 due to the dark image of the vertical orientation and increased to 841 and 542 respectively in the 8 mM and 3 mM glucose solutions. These results indicate that the TEM mixed-GOx sensor cell can be used for glucose detection many times at different concentrations. Previous TEM sensors could only be used once for glucose detection due to the difficulty of obtaining a 5CB reference vertical orientation again. Therefore, the TEM mixed-GOx sensor according to the present invention has high stability and activity and can be used many times through electrostatic fixing of the enzyme (GOx).

< Experimental Example  6> Glucose  Actual analysis of measurement biosensor

(YE) (1 mg / ml), hemoglobin (5 mM), ascorbic acid (AA) (0.1 mM) to test the sugar detection of the TEM mixed - GOx grid sensor of Example 1 according to the present invention, , And glucose (0.3, 0.5, 2 mM) were prepared. The measured results of TEM mixed - GOx sensor were compared with high performance liquid chromatography (HPLC).

YE is a term commonly used in yeast products and is used primarily for practical analysis because it contains all the components of the blood and includes, for example, saturated, unsaturated fatty acids, sodium, potassium, magnesium , Chromium, carbohydrate sugars, proteins, vitamin C-complexes (Anderson, 1997) and hemoglobin and ascorbic acid, it is possible to mimic an actual blood sample by adding glucose.

Since the chromatogram of YE does not have a peak indicating the glucose at a retention time (RT) = 9.75 bun, YE / glucose mixture indicates the peak due to glucose in RT = 9.75 bun, YE / glucose mixture is TEM mixed - GOX It can be used as a standard sample when conducting actual analysis of the sensor.

It shows an image of a POM GOx - 11 is mixed with a TEM YE / glucose mixture under crossed polarizers. The initial vertical orientation (Figure 11a) was maintained when the aqueous medium in the sensor was replaced with a YE / glucose (0.3 mM) mixture (Figure 11b). However, when YE / glucose (0.5 mM) mixed solution was injected into the cell, HP changes occurred (FIG. 11C). N value was obtained from Figure 11c is 0.03380.0003, which C 0 = 0.03340.0004 (Figure 7b) from a pure glucose solution at 0.5 mM. As the content of glucose in the mixed solution became higher, the planar orientation became more visible (FIG. 11D).

FIG. 11E shows the HP transition of the TEM mixed - GOx sensor cell of the present invention when an unknown amount of glucose is used in the mixed solution. The Δn value was 0.0550.0003, corresponding to a glucose concentration of 4.7 mM according to the TEM mixed - GOx linear range in FIG. 9b. To check the exact amount of glucose in the YE / glucose mixture, a standard curve in the range of C 0 = 2 to 11 mM was obtained using HPLC technology. The unknown glucose content in the YE / glucose mixed solution from HPLC was 4.62 mM, which was similar to the value obtained by? N. Thus, TEM mixed according to the invention - even good specificity and GOx is a complex mixture of glucose detection provides a quantitative measurement.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

100: Biosensor for glucose measurement
101:
102:
103:
201:
202:
203:
204: glass substrate
205:
206: Silicone rubber

Claims (11)

A substrate portion including a hydrophobic material layer; A liquid portion including a liquid crystal monomer on an upper portion of the substrate portion; A sensor unit coated with a mixture of a liquid crystal copolymer of Formula 1 and a liquid crystal copolymer of Formula 2 on the liquid crystal portion and glucose oxidase is electrostatically fixed on the liquid crystal copolymer of Formula 2; A biosensor for detecting glucose, comprising:
The mixture of the liquid crystal copolymer of Formula 1 and the liquid crystal copolymer of Formula 2 has a mixing ratio of 20: 4 to 20: 5,
The mixture of the liquid crystal copolymer of the formula (1) and the liquid crystal copolymer of the formula (2) contains a salt of 1 M concentration,
Wherein the biosensor is capable of recovering a reference vertical orientation of the liquid crystal monomer after being exposed to glucose and reusable.
[Chemical Formula 1]
Figure 112017033113902-pat00018

(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
Figure 112017033113902-pat00019
or
Figure 112017033113902-pat00020
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.
(2)
Figure 112017033113902-pat00021

(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, and R &lt; 3 &gt;
Figure 112017033113902-pat00022
or
Figure 112017033113902-pat00023
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 method according to claim 1,
Wherein the biosensor 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. &Lt; Desc / Clms Page number 19 &gt;
The method according to claim 1,
The hydrophobic material layer may be at least one of the group consisting of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) and octadecyltrichlorosilane (OTS) And a biosensor for detecting glucose.
The method according to claim 1,
The liquid crystal monomer may be a mixture of cyclohexane-fluorinated biphenyl and fluorinated terphenyls (TL205), 4-oxyl-4'-cyanobiphenyl (4-n-octyl- 4'-cyano-biphenyl, 8CB), and 4-cyano-4'-pentylbiphenyl (5CB). For example.
The method according to claim 1,
Wherein the sensor unit comprises 200 to 30,000 parts by weight of glucose oxidase per 100 parts by weight of the liquid crystal copolymer mixture.
delete The method according to claim 1,
Wherein the liquid crystal copolymer mixture is coated in the form of a monomolecular film.
The method according to claim 1,
When the glucose is bound to the sensor unit, the birefringence change according to the orientation change of the liquid crystal monomer in the liquid crystal unit is measured to qualitatively analyze the glucose. The qualitative analysis is performed at a pH lower than the physiological condition pH 7, And a tendency of showing a dark image when a polarizing microscope is measured at a pH higher than pH 7, which is a physiological condition, is analyzed and performed.
The method according to claim 1,
When a glucose coupled to the sensor, by measuring the birefringence change in the orientation change of the liquid crystal portion liquid crystal monomer, a linear function of the double refraction change (Δn) / glucose infusion concentration (C 0) for a known glucose infusion concentration (C 0) Wherein the concentration of the glucose is quantitatively analyzed in a mixed solution containing an unknown amount of glucose by using the glucose concentration sensor.
9. The method of claim 8,
Characterized in that the qualitative or quantitative analysis is carried out at a pH of 2 to 12.
The method according to claim 1,
Wherein the biosensor is capable of detecting glucose in a sample containing a glucose concentration of 0.5 mM or more.
KR1020150060804A 2015-04-29 2015-04-29 Liquid crystal-based glucose biosensor functionalized with mixed PAA and QP4VP brushes KR101782949B1 (en)

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