KR102231944B1 - Method for measuring glucose concentration using complexes containing aminophenylboronic acid, biosensor using the same and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a method for measuring glucose concentration, and more particularly, relates to a method for measuring glucose concentration using a photonic IPN complex including a photonic crystal structure, a polyacrylic acid (PAA) hydrogel, and aminophenylboronic acid (APBA), and a biosensor using the same, and a preparation method thereof. The glucose concentration measuring method of the present invention can measure the concentration of added glucose according to the reflection wavelength of the complex observed with the naked eye, and the reaction proceeds independently of the change in pH, and the biosensor using the same can measure the wavelength of the optical bandgap. Moreover, the glucose concentration can be measured by visually detecting the color of the photonic crystal structure due to the change. In addition, the biosensor preparation method of the present invention can control the color spectrum of the photonic crystal structure by adjusting the ratio of the chiral dopant to the mesogen, and it is possible to secure stability by forming a complete solid photonic crystal structure by extracting the chiral dopant.

Description

Method for measuring glucose concentration using complexes containing aminophenylboronic acid, biosensor using the same and method for manufacturing the same}

The present invention relates to a method for measuring glucose concentration, and in particular, a method for measuring glucose concentration using a photonic IPN complex including a photonic crystal structure, polyacrylic acid (PAA) hydrogel, and aminophenylboronic acid (APBA), and a biosensor using the same And it relates to a method of manufacturing the same.

Diabetes is a serious disease that increases blood sugar due to an abnormality in the production or use of insulin, resulting in various acute and chronic complications. Insulin deficiency or hyperglycemia leads to diabetes, increasing the risk of cardiovascular, nerve, kidney, ocular and peripheral vascular diseases. As described above, diabetes, which is a global problem, is predicted to increase in the rate of invention further due to an increase in the elderly population and environmental factors, and social and economic problems are seriously emerging.

Proper monitoring of blood sugar levels is important to maintaining a healthy body. Glucose biosensors occupy about 85% of the total biosensor market, and research activities on the development of new glucose biosensors that are highly sensitive and easy to use are very active.

Meanwhile, biosensors for glucose detection can be largely classified into enzyme-based and non-enzyme-based biosensors. Most conventional biosensors for glucose detection are enzyme-based biosensors using glucose oxidase (GOx). Such an enzyme-based biosensor exhibits high sensitivity and enables precise detection of glucose, but has disadvantages such as inhibiting the activity of enzymes due to the presence of pH, temperature, humidity, and other chemicals, and poor stability and reproducibility. Has.

In order to overcome the limitations of this enzyme-based glucose biosensor, studies on non-enzyme-based glucose biosensors have been actively conducted. As in Korean Patent Laid-Open No. 10-2015-0061720, a non-enzyme-based glucose biosensor was developed in which a polymer film that minimizes the action of organic substances was developed or a transition metal oxide, copper nanoparticles, or gold nanoparticles were deposited on the surface of the cathode , Preparation of such an electrode has problems such as the process is complicated, time consuming, and requires sophisticated technology.

Republic of Korea Patent Publication No. 10-2015-0061720

An object of the present invention is to provide a method for measuring glucose concentration using a photonic crystal structure, a polyacrylic acid (PAA) hydrogel, and a photonic IPN complex including aminophenylboronic acid (APBA) in order to solve the above problems.

In addition, an object of the present invention is to provide a biosensor using the above measurement method and a method of manufacturing the same.

In order to achieve the above object, the present invention,

A first step of adding a glucose solution to a complex including a photonic crystal structure, a polyacrylic acid (PAA) hydrogel, and an aminophenylboronic acid (APBA);

A second step of swelling the polyacrylic acid hydrogel due to the complex formed by the aminophenylboronic acid and the glucose; And

A third step of changing the color of the photonic crystal structure due to the swelling of the hydrogel; Including,

There is provided a method for measuring glucose concentration in which the concentration of glucose is measured according to different colors indicated by the concentration of glucose independently of a change in pH.

In order to achieve the above other object, the present invention,

It provides a biosensor for measuring glucose concentration using a photonic crystal structure, a polyacrylic acid (PAA) hydrogel penetrating into the inner space of the photonic crystal structure, and a complex including aminophenylboronic acid (APBA) fixed to the polyacrylic acid.

In order to achieve the above another object, the present invention,

A first step of mixing and curing a chiral dopant and mesogen to form a helical cholesteric liquid crystal;

A second step of forming a solid photonic crystal structure by removing a chiral dopant from the cholesteric liquid crystal;

A third step of infiltrating polyacrylic acid (PAA) into the space from which the chiral dopant has been removed; And

Including; a fourth step of fixing aminophenylboronic acid (APBA) to the polyacrylic acid,

The photonic crystal structure forms an interwined polymer network (IPN) structure with the polyacrylic acid, and has a different color according to the concentration of glucose, providing a method of manufacturing a biosensor for measuring glucose concentration.

The glucose concentration measurement method of the present invention has the effect of measuring the concentration of added glucose according to the reflection wavelength of the complex observed with the naked eye, and measuring the concentration of glucose by performing a reaction independently of a change in pH. .

In addition, the biosensor of the present invention causes contraction and expansion of the hydrogel depending on the glucose concentration, thereby inducing contraction and expansion of the photonic crystal structure, and by visually detecting the color of the photonic crystal structure due to the change in the wavelength of the optical band gap. There is an effect that can measure the concentration.

In addition, the biosensor manufacturing method of the present invention can adjust the color spectrum of the photonic crystal structure by adjusting the ratio of chiral dopant and mesogen, and secure stability by forming a completely solid photonic crystal structure according to chiral dopant extraction. There is an effect of detecting a change in color according to the contraction and expansion of the hydrogel by using the hydrogel penetrated into the space from which the water has been removed.

1 is a diagram showing a process in which a hydrogel is swollen by binding glucose to a complex according to an embodiment of the present invention.
2 is a view showing a method of manufacturing a biosensor for detecting glucose according to the present invention.
3 is a UV-Vis spectrum before and after UV curing according to an embodiment of the present invention.
4 is an FTIR spectrum of each step structure according to an embodiment of the present invention.
5 is a UV-Vis spectrum of _{a CLC solid} film and a photonic IPN composite according to an embodiment of the present invention.
6 is a photograph of an array using a photonic IPN composite according to an embodiment of the present invention.
7 is a cross-sectional scanning microscope (SEM) image of a CLCsolid film and a photonic IPN film according to an embodiment of the present invention.
8 is a side view of a photonic IPN array to which APBAs of different concentrations are fixed according to an embodiment of the present invention.
9 is a UV-Vis spectrum of a photonic IPN dot region to which APBAs of different concentrations are fixed according to an embodiment of the present invention.
10 is a photograph of a photonic IPN dot area to which APBAs of different concentrations are fixed according to an embodiment of the present invention.
11 is a photograph illustrating a color change of a photonic IPN array for various glucose concentrations according to an embodiment of the present invention.
12 is a UV-Vis spectrum of a photonic IPN array for various glucose concentrations according to an embodiment of the present invention.
13 is a graph showing a _{photonic band gap (λ PBG} ) of a photonic IPN array for various concentrations of glucose according to an embodiment of the present invention.
14 is a photograph showing a color change of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention.
15 is a UV-Vis spectrum of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention.
16 is a graph showing a _{photonic band gap (λ PBG} ) of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention.
17 is a photonic IPN array according to an embodiment of the present invention with an interference substance added thereto, and a UV-Vis spectrum of dots for each substance.
18 is a graph showing the difference (Δλ) of the _{photonic band gap (λ PBG} ) for each interfering material of the photonic IPN array according to an embodiment of the present invention.
19 is a photograph showing a color change of metal ions in a photonic IPN array according to an embodiment of the present invention.

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

According to an aspect of the present invention, a first step of adding a glucose solution to a complex comprising a photonic crystal structure, polyacrylic acid (PAA) hydrogel, and aminophenylboronic acid (APBA); A second step of swelling the polyacrylic acid hydrogel due to the complex formed by the aminophenylboronic acid and the glucose; And a third step of changing the color of the photonic crystal structure due to swelling of the hydrogel; and measuring the concentration of glucose according to different colors indicated by the concentration of glucose independently of the pH change. do.

First, a first step of adding a glucose solution to the complex will be described.

In the present invention, the composite includes a photonic crystal structure, a polyacrylic acid (PAA) hydrogel penetrated into the interior space of the photonic crystal structure, and aminophenylboronic acid (APBA) fixed to the polyacrylic acid, and the photonic crystal structure includes polyacrylic acid and IPN (Interwined Polymer Network). ) Structure can be formed.

The photonic crystal structure may be solid, and may be prepared by mixing and curing a chiral dopant and a reactive mesogen. Through the curing process, a complete solid photonic crystal structure can be obtained, and a unique spiral structure of a cholesteric liquid crystal can be maintained while extracting and removing a chiral dopant. As the photonic crystal structure is maintained in a solid form, it can have improved effects in terms of stability and durability.

In the process of removing the chiral dopant, the polyacrylic acid (PAA) hydrogel may penetrate into the interior space of the photonic crystal structure. The photonic crystal structure in which the polyacrylic acid hydrogel is infiltrated may form an IPN polymer interposed between photons by combining the reaction isotropic properties of the hydrogel with the anisotropy and helical molecular order of the photonic crystal structure.

Since polyacrylic acid contains a carboxyl group, it can form a covalent bond with aminophenylboronic acid having an amine group. Polyacrylic acid and aminophenylboronic acid can be bonded using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling. Accordingly, it is possible to form a complex in which aminophenylboronic acid is bound to the hydrogel forming the photonic crystal structure and the IPN structure.

Aminophenylboronic acid has the property of being capable of reversibly binding to glucose, and through this, it serves to detect glucose. The aminophenylboronic acid may be any one selected from the group consisting of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, and mixtures thereof. Preferably, 3-aminophenylboronic acid can be used.

The glucose solution added to the complex may be human serum. Compared to the case of adding an aqueous glucose solution, there is no difference in function and effectiveness, and may have high selectivity and sensitivity without interference with other substances contained in human serum.

The first step in which glucose is added to the complex can be done in a pH buffer. The pH buffer is a composition that is not affected by small changes in the concentration of acidic and/or basic components and maintains a fixed pH range, and may be a solution.

In the present invention, the glucose solution is prepared in a carbonate buffer pH = 8.5, which is a pH buffer, so that the reaction can proceed independently of a change in pH. Protons are generated during the reversible condensation reaction of aminophenylboronic acid and glucose, but when glucose is added under pH buffer conditions, the reaction can proceed without being affected by pH.

Next, a second step in which the polyacrylic acid hydrogel swells due to the complex formed by aminophenylboronic acid and glucose will be described.

The aminophenyl boronic acid in the complex can specifically bind to the added glucose to form a complex having a charged ionic boron group. By combining with a diol of aminophenylboronic acid glucose, a boronate complex having a trigonal or tetragonal structure can be formed. The following Chemical Formula 1 shows the binding reaction of aminophenylboronic acid and glucose fixed to the hydrogel according to an embodiment of the present invention.

[Formula 1]

Boron groups are negatively charged by bonding with glucose, and as they exist in an ionic form, a charge imbalance inside and outside the hydrogel can be formed. Donnan osmotic pressure is induced due to charge imbalance due to the difference in ion concentration, resulting in swelling of the hydrogel.

Finally, a third step in which the color of the photonic crystal structure changes due to the swelling of the hydrogel will be described.

1 is a diagram showing a process in which a hydrogel is swollen by binding glucose to a complex according to an embodiment of the present invention. Referring to FIG. 1, the degree of swelling of the hydrogel varies according to the glucose concentration, and the color of the photonic crystal structure may vary according to the degree of swelling of the hydrogel. The contraction and expansion of the hydrogel induces contraction and expansion of the photonic crystal structure, and the color of the photonic crystal structure may change by changing the wavelength range of the optical band gap of the photonic crystal structure.

The glucose concentration measurement method according to the present invention can measure the concentration of added glucose according to the reflected wavelength of the complex observed with the naked eye, and the added glucose solution is prepared in a carbonate buffer pH = 8.5, which is a pH buffer. The reaction can be carried out independently to measure the concentration of glucose.

According to another aspect of the present invention, using a photonic crystal structure, a polyacrylic acid (PAA) hydrogel penetrating into the interior space of the photonic crystal structure, and using a complex containing aminophenyl boronic acid (APBA) fixed to the polyacrylic acid, glucose concentration Provides a biosensor for measurement.

The biosensor according to the present invention may be manufactured in the form of an array by forming a composite including a photonic crystal structure, a polyacrylic acid hydrogel, and aminophenylboronic acid, and then selectively curing it through photolithography. The shape of the array is preferably a dot, and since it is manufactured in an array shape, cross-contamination does not occur.

In the present invention, the photonic crystal structure of the composite included in the biosensor may form an interwined polymer network (IPN) structure with polyacrylic acid. According to the contraction and expansion of the polyacrylic acid hydrogel, the spiral pitch of the photonic crystal structure may also contract and expand, causing a change in color.

The photonic crystal structure may be solid, and may be prepared by mixing and curing a chiral dopant and a reactive mesogen. Through the curing process, a complete solid photonic crystal structure can be obtained, and a unique spiral structure of a cholesteric liquid crystal can be maintained while extracting and removing a chiral dopant. As the photonic crystal structure is maintained in a solid form, it may have improved effects in terms of stability and durability.

Since polyacrylic acid contains a carboxyl group, it can form a covalent bond with aminophenylboronic acid having an amine group. Polyacrylic acid and aminophenylboronic acid can be bonded using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling. Accordingly, it is possible to form a complex in which aminophenylboronic acid is bound to the hydrogel forming the photonic crystal structure and the IPN structure.

Aminophenylboronic acid has the property of being capable of reversibly binding to glucose, and through this, it serves to detect glucose. The aminophenylboronic acid may be any one selected from the group consisting of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, and mixtures thereof. Preferably, 3-aminophenylboronic acid can be used.

The process of adding glucose to the biosensor according to the present invention may be performed in a pH buffer. In the present invention, the glucose solution is prepared in a carbonate buffer pH = 8.5, which is a pH buffer, so that the reaction can proceed independently of a change in pH.

The aminophenyl boronic acid of the biosensor according to the present invention may specifically bind to added glucose to form a complex having a charged ionic boron group. It is possible to form a square complex (boronate complex) having a trigonal or pentagonal structure by binding with a diol of aminophenylboronic acid glucose.

By binding to glucose, the boron group has a negative charge, and as it exists in an ionic form, a charge imbalance in and out of the hydrogel can be formed. Donnan osmotic pressure is induced due to charge imbalance due to the difference in ion concentration, resulting in swelling of the hydrogel. That is, the degree of swelling of the hydrogel varies depending on the glucose concentration, and the color of the photonic crystal structure may vary depending on the degree of swelling of the hydrogel.

The biosensor may be configured including a substrate. The material used as the substrate is not particularly limited, and a glass substrate may be preferably used.

The biosensor according to the present invention causes contraction and expansion of the hydrogel depending on the glucose concentration, thereby inducing contraction and expansion of the photonic crystal structure, and visually detecting the color of the photonic crystal structure due to the change in the wavelength range of the optical band gap. Thus, the glucose concentration can be measured.

According to another aspect of the present invention, a first step of mixing and curing a chiral dopant and mesogen to form a helical cholesteric liquid crystal; A second step of forming a solid photonic crystal structure by removing the chiral dopant from the cholesteric liquid crystal; A third step of infiltrating polyacrylic acid (PAA) into the space from which the chiral dopant has been removed; And a fourth step of fixing aminophenylboronic acid (APBA) to polyacrylic acid, wherein the photonic crystal structure forms an interwined polymer network (IPN) structure with polyacrylic acid, and has a different color depending on the concentration of glucose. It provides a method of manufacturing a biosensor for measuring concentration.

First, a first step of forming a spiral-shaped cholesteric liquid crystal will be described. Among photonic crystals, Cholesteric Liquid Crystal (CLC) exhibits a spirally twisted molecular orientation that imparts specific optical properties, and has an advantage in that it is easy to fabricate a one-dimensional photonic structure. The CLC can be used as a sensor by using a pitch varying from an external stimulus because the lesseric optical material exhibits a unique reflection pattern as it exhibits selective light reflection by the pitch of the helix.

The helical structure formed according to the ratio of the chiral dopant mixed with the mesogen and the wavelength of the optical band gap may be changed accordingly. Since the color of the reflected light changes according to the change in the wavelength of the optical band gap, chiral dopant and mesogen are 20 to 40% by weight and 80 to 60% by weight, respectively, for the manufacture of a photonic crystal structure having a wavelength of visible light that is visible to the naked eye. It is preferred to be mixed. Accordingly, it is preferable that the optical band gap wavelength range of the photonic crystal structure is 350 to 650 nm.

Chiral dopants are non-reactive chiral dopants, C15, CB15, CM21, R/S-811, CM44, CM45, CM47, R/S-2011, R/S-3011, R/S-4011, R/S-5011 And it is characterized in that any one dopant selected from the group consisting of R/S-1011, and preferably CB15 ((S)-4-cyano-4'-(2-methylbutyl)biphenyl) may be used.

Mesogen is a reactive nematic mesogen, characterized in that it is any one mesogen selected from the group consisting of RM 82, RM 257, RM308 and RMM727, and preferably RMM727 may be used. RMM727 is a mixture containing acryloyloxy group, 1,6-hexamethylenediol diacrylate, 2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one, and reactive acryloyloxy mesogen APBMP as a material containing the acryloyloxy group, reactive acryloyloxymesogen AHBCP, reactive acryloyloxy mesogen AHBMP and reactive acryloyloxy mesogen AHBPCHP can be used.

Next, a second step of forming a solid photonic crystal structure will be described.

As conventional CLCs are manufactured as liquid droplets, there is a limit to maintaining stability for a long period of time, and in order to overcome this, a chiral dopant may be removed to obtain a solid photonic crystal structure. Although the chiral dopant is removed, the helical structure formed by the chiral dopant is maintained as it is, while the helical structure formed by the chiral dopant is removed, and it can be manufactured as a photonic crystal structure exhibiting cholesteric liquid crystal characteristics, and it is manufactured in a completely solid state, resulting in stability. It can be improved.

Next, a third step of infiltrating polyacrylic acid (PAA) will be described.

As the chiral dopant is removed, a space is generated in the photonic crystal structure, and acrylic acid (AA) is penetrated into the hydrogel to form an IPN structure with the photonic crystal structure. As the hydrogel contracts and expands, the photonic crystal structure may also form a structure in which the spiral pitch changes and the reflected color changes as the photonic crystal structure contracts and expands.

In the process of removing the chiral dopant, the polyacrylic acid (PAA) hydrogel penetrates the interior space of the photonic crystal structure, and the photonic crystal structure in which the polyacrylic acid hydrogel is infiltrated combines the reaction isotropic properties of the hydrogel with the anisotropy and helical molecular order of the photonic crystal structure To form an IPN polymer interposed between photons.

Next, a fourth step of fixing aminophenylboronic acid (APBA) to polyacrylic acid will be described.

Since polyacrylic acid contains a carboxyl group, it can form a covalent bond with aminophenylboronic acid having an amine group. Polyacrylic acid and aminophenylboronic acid can be bonded using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling. Accordingly, it is possible to form a complex in which aminophenylboronic acid is bound to the hydrogel forming the photonic crystal structure and the IPN structure.

Aminophenylboronic acid has the property of being capable of reversibly binding to glucose, and through this, it serves to detect glucose. The aminophenylboronic acid may be any one selected from the group consisting of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, and mixtures thereof. Preferably, 3-aminophenylboronic acid can be used.

The biosensor manufacturing method according to the present invention can adjust the color spectrum of the photonic crystal structure by adjusting the ratio of chiral dopant and mesogen, and secure stability by forming a completely solid photonic crystal structure according to chiral dopant extraction, and A biosensor capable of detecting a change in color according to contraction and expansion of the hydrogel by using the hydrogel penetrated into the space from which the hydrogel has been removed can be manufactured.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. On the other hand, the illustrations and detailed descriptions of configurations, actions and effects thereof, which can be easily recognized by those of ordinary skill in the art, will be simplified or omitted, and will be described in detail centering on parts related to the present invention.

<Example>

material

Reactive liquid crystal mixture (RMM727; Merck UK); CB15 (Sinton, Germany); AA from Junsei, Japan; Photoinitiators (Irgacure 500; Ciba Inc., Switzerland); APBA (Silgard 184; Dow Corning, USA); TPGDA, TMSPMA (98%), trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (PFOTS, 97%), NHS, EDC HCl, octadecyltrichlorosilane (OTS), glucose and human Serum (Sigma-Aldrich, USA); Micro Pearl (Sekisui, Japan); Acetone, ethanol and PBS. Unless otherwise noted, deionized water (DI) used in all experiments was purified using a reverse osmosis system (μPure RO; Romax, Korea). In all experiments, glucose solutions were prepared in carbonate buffer pH = 8.5.

Example 1-Biosensor for glucose detection

2 is a view showing a method of manufacturing a biosensor for measuring glucose concentration according to the present invention. Hereinafter, a method of manufacturing a biosensor for measuring glucose concentration will be described with reference to FIG. 2.

(1) Glass substrate preparation

The glass substrate was sonicated in ethanol for 15 minutes, washed with deionized water, and dried under air pressure. The TMSPMA solution was spin-coated (for 45 seconds at 3000 rpm) on the activated surface prepared in advance using plasma and then cured at 100° C. for 10 minutes. Another glass substrate was coated with PFOTS by chemical vapor deposition at 70° C. for 1 hour in a closed chamber.

(2) formation of CLC film

RMM727 (74 wt%) and CB15 (26 wt%) were mixed for 30 minutes at 85° C. under continuous magnetic stirring.

The mixture was thoroughly stirred and cooled to 25° C. to prepare a milky, transparent CLC mixed solution. Separated by a 6 μm-thick spacer, a CLC mixed solution (20 μl) was permeated between two glass substrates coated with TMSPMA and PFOTS on the surface. After infiltrating the mixed solution, photopolymerization was performed at 365 nm and 20° C. for 10 minutes using a UV curing machine (Inocure 100N; Lichtzen, Korea). After UV curing, the glass substrate coated with PFOTS was removed and washed 10 times with acetone to remove and extract unreacted CB15.

(3) Preparation of photonic (photonic) IPN film and array

The monomer mixture (AA / TPGDA / Irgacure 500, 98.5: 0.5: 1 wt%) was dropped on the CLC film and allowed to soak for 30 minutes. No noticeable color change was seen, and the uninfiltrated excess was removed prior to UV-curing. Thereafter, the film was selectively photopolymerized for 10 minutes using a photo mask located 6 cm from the film.

(4) Fabrication of photonic IPN array functionalized with APBA

Then, 30mM APBA was fixed to individual dots, followed by EDC·HCl:NHS (1:2M, pH = 7) coupling for 2 hours and dried in air. Accordingly, a photonic IPN composite and an array using the same were prepared.

<Experimental Example>

General information

Cross-sectional images of the photonic IPN array were obtained using a field emission SEM microscope (SU8220; Hitachi, Japan) operating at 15 kV. The surface of the sample was prepared by coating with platinum.

The attenuated total reflection FTIR spectrum was obtained by collecting an average of 64 scans with an FTIR spectrometer (FT / IR-4100; Jasco, Japan) in the range of ^{600-4000 cm -1} at a resolution of ^{4 cm -1.}

The UV-Vis spectrum of the CLC film in the range of 300-900 nm was obtained using a tabletop UV spectrophotometer (Ocean Optics, DH-2000-BAL) under the condition that the film was oriented perpendicular to the beam.

Experimental Example 1-Structure confirmation

(1) Confirmation of formation of solid spiral photonic crystal structure

The CLC mixed solution was infiltrated into the gap between the two glass substrates by capillary force and then UV cured to prepare a solid photonic crystal structure. 3 is a UV-Vis spectrum before and after UV curing according to an embodiment of the present invention. _Referring to FIG. 3, the reflection band before UV curing showed a red reflection band at λ PBG = 650 nm. Where λ _PBG is the wavelength in the wide band gap measured from the peak of the UV-Vis spectrum. After UV curing, the resulting CLC film showed a band of orange/red reflection band at _{λ PBG = 615 nm.}

In addition, it was possible to confirm the photonic crystal structure before and after UV curing and whether or not it was formed through the presence or absence of an acrylate peak identified by Fourier transform infrared (FTIR) spectrum. 4 is an FTIR spectrum of each step structure according to an embodiment of the present invention. Referring to (a) of FIG. 4, as the RMM727 is used after photopolymerization according to UV curing, the peaks of the acrylate double bonds (C = C) ^{appearing at 1635 cm -1} and 810 cm ^{-1 disappear.} It was possible to confirm the formation of a photonic crystal structure according to UV curing.

The wavelength of the CLC film was controlled by the helical pitch appearing according to the ratio of the chiral dopant in the CLC mixed solution, so that a structure having an optical band gap wavelength consistent with the wavelength band of visible light could be obtained.

(2) Confirmation of IPN complex formation

The CLC film is solidified according to UV curing, has stability, and maintains a helical pitch, thereby having a function as a photonic crystal structure. When the unreacted chiral dopant is removed, a space can be created inside a solid one-dimensional photonic crystal structure, and AA, one of the functional materials, can penetrate into the space to prepare a photonic IPN composite.

After formation of the photonic crystal structure by UV curing, remove the glass coated with PFOTS and wash the solidified CLC film 10 times with acetone. Phenyl (CB15) was extracted. 5 is a UV-Vis spectrum of _{a CLC solid} film and a photonic IPN composite according to an embodiment of the present invention. Referring to FIG. 5, the CLC _solid film from which CB15 was extracted exhibited a reflection band at _{λ PBG = 460 nm.} CB15 removal could be further confirmed by FTIR analysis. Referring to FIG. 4B, it was confirmed that CB15 was removed through the disappearance of the peak at ^{2220 cm -1} representing the characteristic cyano group of CB15.

The IPN complex was formed by infiltrating acrylic acid (AA) into the space from which CB15 was extracted. When the acrylic acid (AA)-tripropylene glycol diacrylate (TPGDA) mixture was impregnated into the CLCsolid film, the color changed from blue to green, and it was confirmed that film swelling due to the impregnated acrylic monomer appeared.

The array obtained by selectively (dot-shaped) photopolymerization of a CLCsolid film impregnated with acrylic acid using a photomask, and washing the unreacted acrylic acid monomer outside the area using a phosphate-buffered saline (PBS) solution, The outside area appeared in a different color. Referring to FIG. 5, it can be seen that the reflection band of the dot region in which the photonic IPN composite formed by infiltration of acrylic acid is formed _{appears at λ PBG = 520 nm.}

6 is a photograph of an array using a photonic IPN composite according to an embodiment of the present invention. _Referring to FIG. 6, it can be seen that the dot region is green (λ PBG = 520 nm), while the other regions are blue (λ _{PBG = 460 nm).} In addition, the formation of the IPN complex can be confirmed by FTIR analysis. Referring to (c) of FIG. 4, the FTIR spectrum of the dot region peaks at ^{3500, 3000, 1750 and 1408 cm -1} due to the non-hydrogen bond -OH stretch of the carboxylic acid group, the NH stretch, and the symmetric and asymmetric stretch bands. Was able to confirm. It can be seen that poly(acrylic acid) (PAA) is infiltrated to form an IPN complex as a result of the presence of a carboxylic acid group and a carboxylate ion.

7 is a cross-sectional scanning microscope (SEM) image of a CLCsolid film and a photonic IPN film according to an embodiment of the present invention. Referring to (a) of FIG. 7, dark and light layers that are periodically alternating can be seen. This indicates the formation of a photonic crystal structure in a solid state. This periodicity (245 nm) represents p/2, where p is the helix pitch. Referring to (b) of FIG. 7, an increased periodicity (260 nm) was confirmed according to the IPN structure formed of impregnated acrylic acid. The wavelength at the photonic bandgap was calculated by p×n, where n is the refractive index. The values of the CLCsolid and photonic IPN films were found to be 460 nm and 520 nm, respectively, which could be confirmed to be consistent with the corresponding observed colors (blue and green).

(3) APBA immobilization and optimal concentration confirmation

For the detection of glucose, the present invention fixed APBA (Aminophenylboronic acid) to the photonic IPN complex. Photo by using N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide HCl:N-hydroxy succinimide (EDC HCl: NHS, molar ratio 2: 1) as a coupling agent for fixing APBA. Nick IPN complex was activated. The fixation of APBA was confirmed by FTIR analysis. Referring to (d) of Figure 4, in _{the FTIR spectrum of the APBA-immobilized CLC AAc-APBA} , B-OH stretching vibration, C = C aromatic vibration, amide-I formation and C = O of the covalently immobilized APBA, and -OH / NH _{2 It} was confirmed that peaks appeared at 1253, 1555, 1631, 1708 and 3400 cm ^{-1 in response to elongation.}

When the photonic IPN-APBA complex forms a square complex with glucose at an alkaline pH, Donnan osmotic pressure is induced, resulting in a change in the volume of the matrix. 8 is a side view of a photonic IPN array to which APBAs of different concentrations are fixed according to an embodiment of the present invention. Referring to FIG. 8, it was confirmed that droplets were formed without cross contamination due to the hydrophilic and hydrophobic properties of the dots and background regions. In addition, even after the glucose aqueous solution was contained in the photonic IPN dot region, cross contamination between the dot region and the background region did not appear.

In order to confirm the optimal amount of _APBA , a photonic IPN array was prepared by fixing different concentrations of APBA (C _{APBA) to individual photonic IPN complexes (IPN APBA).} In addition, the photonic IPN array in which different concentrations of APBA were fixed was immersed in a 5 mM glucose solution to confirm the optimal APBA concentration.

9 is a UV-Vis spectrum of a photonic IPN dot region to which APBAs of different concentrations are fixed according to an embodiment of the present invention. 10 is a photograph of a photonic IPN dot area to which APBAs of different concentrations are fixed according to an embodiment of the present invention. The number at the bottom of each dot is the APBA concentration (mM). Referring to FIGS. 9 and 10, when the concentration of APBA is low (5 to 10 mM), λ _PBG slightly shifted red from 520 nm to 534 nm, which appears when there is no glucose, but the green of the initial dot was maintained. When the concentration of APBA increased to 15 mM, a little yellow (568 nm) appeared, and when the concentration of APBA increased to 20 nm and 25 nm, yellow-orange (593 nm) and red (607 nm) appeared, respectively. I did. When the concentration of APBA was 30 mM, red (645 nm) was clearly observed, and it was confirmed that the concentration of APBA was increased with increasing concentration.

Accordingly, a photonic IPN array was fabricated using 30 mM APBA in which color change was clearly observed, and the following experiment was conducted.

Experimental Example 2-Measurement of sensitivity and reaction time

In order to confirm the change in sensitivity and color of the photonic IPN array according to the present invention, an experiment was conducted by adding glucose of various concentrations to the photonic IPN array.

11 is a photograph illustrating a color change of a photonic IPN array for various glucose concentrations according to an embodiment of the present invention. The number at the bottom of each clay is the concentration of glucose (mM). 12 is a UV-Vis spectrum of a photonic IPN array for various glucose concentrations according to an embodiment of the present invention.

Referring to FIGS. 11 and 12, when the concentration of glucose is low (C _g = 0.5 mM, hypoglycemic level), a slight red shift is observed from 520 nm to 526 nm when _{λ PBG does not have glucose, but the dot is} Maintained the initial green color. As the concentration of glucose (C _g ) increased to 2 mM (hypoglycemic) and 5 mM (normal), the color of the dots appeared corresponding to the colors of emerald green (534 nm) and yellow (570 nm). Orange-red (590 nm) and red (627 nm) were observed at the concentration of 5.2 to 6.8 mM (normal) and 9 mM (hyperglycemia), respectively, and when the concentration of glucose exceeded 10 mM (diabetic range) It _{was confirmed that λ PBG} reached 653 nm and was saturated. That is, it was confirmed that the photonic IPN array according to the present invention can perform different analysis for each dot at the same time, and detect each glucose concentration through visually recognizable colors.

_{The λ PBG} plot was confirmed with respect to the limit of detection (LOD). 13 is a graph showing a _{photonic band gap (λ PBG} ) of a photonic IPN array for various concentrations of glucose according to an embodiment of the present invention. Referring to FIG. 13, it can be seen that _{λ PBG} is saturated at a concentration of 12 mM glucose. Using this plot, a linear range was obtained and the detection limit was calculated. The limit of detection (LOD) was calculated using LOD = 3.3 × (SD / s). Where SD is the standard deviation of the blank sample and s is the slope of the graph. The calculated LOD was 0.35 mM in the linear range of 1-12 mM. Through this, it was confirmed that the photonic IPN array according to the present invention can detect _{up to 0.35 mM glucose according to the λ PBG value.} However, before the addition of glucose, the color of the photonic IPN array, the green wavelength band, was wide, so that the color change (emerald green) could be detected with the naked eye from the time when glucose of 2mM or more was added.

In addition, in relation to the reaction time, when 4 μL of a 10 mM glucose solution was dropped into the photonic IPN array of the present invention, it was confirmed that the green dot turned red after 12 minutes. That is, it was confirmed that the photonic IPN array according to the present invention can be conveniently used because the reaction time is short in glucose detection.

Experimental Example 3-Human Serum Subject

For proper monitoring of blood glucose levels, it is necessary to confirm whether glucose in human serum can be selectively detected. In order to confirm the sensitivity and selectivity of detection of glucose in human serum, human serum containing different concentrations of glucose was dropped onto the photonic IPN array according to the present invention, and a change in color was observed. Human serum samples used in the experiment were prepared by diluting a sample with an initial glucose concentration of 5.32 mM with carbonate buffer (pH = 8.5) and adding glucose to display various glucose concentrations.

(1) sensitivity

14 is a photograph illustrating a color change of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention. The number at the bottom of each dot is the concentration of glucose (mM). 15 is a UV-Vis spectrum of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention.

Referring to FIGS. 14 and 15, it was observed that the human serum sample changes from green to red as the _{glucose concentration (C g) increases.} The color of the dots that appeared green (526 nm) when the glucose concentration is 0.5 mM is emerald green when the glucose concentration is 2 mM (hypoglycemia), yellow when the glucose concentration is 2 mM (hypoglycemia), and orange when the glucose concentration is between 5.2 and 6.8 mM (normal). -Red, 9 mM (diabetes level) or more, it was confirmed that it turns red.

Regarding the limit of detection (LOD), the λ _PBG plot was confirmed as in Experimental Example 2 detected in an aqueous solution. 16 is a graph showing a _{photonic band gap (λ PBG} ) of a photonic IPN array for human serum having various glucose concentrations according to an embodiment of the present invention. Referring to FIG. 16, it can be seen that _{λ PBG} is saturated at a concentration of 12 mM glucose. The LOD calculated as in Experimental Example 2 was 0.35 mM in the linear range of 1 to 12 mM, and it was confirmed that glucose in human serum could be detected with almost the same sensitivity as that detected in the aqueous solution (FIG. 13). . That is, it means that different glucose levels ranging from hypoglycemia to diabetes can be quantitatively characterized using the photonic IPN array according to the present invention.

(2) selectivity

Since human serum contains a variety of substances other than glucose, it is important to have a selectivity that can characteristically recognize glucose in the serum. Representative interfering substances in human serum include hemoglobin, urea, dopamine, fructose, and galactose. Accordingly, the reactivity was confirmed by adding each interfering material to the photonic IPN array according to the present invention.

17 is a photograph of adding an interfering substance to a photonic IPN array according to an embodiment of the present invention and a UV-Vis spectrum of dots for each substance. 18 is a graph showing the difference (Δλ) of the _{photonic band gap (λ PBG} ) for each interfering material of the photonic IPN array according to an embodiment of the present invention.

Referring to FIGS. 17 and 18, it was confirmed that a slight red shift appears in the absorption band for all materials. _{Δλ PBG} values for hemoglobin, urea and dopamine were 7, 12 and 15 nm, respectively, and sugars such as fructose and galactose exhibited larger Δλ (20 nm and 31 nm, respectively) due to structural similarity with glucose molecules. However, it was confirmed that 103 nm of Δλ was overwhelmingly high with respect to glucose, and red color appeared clearly. This means that it can be detected independently from other interfering substances in human serum, and it was confirmed that the photonic IPN array according to the present invention has high selectivity for glucose.

Regarding the bonding of sugars and boronic acid (BA), the bonding strength is determined by the orientation and relative position of the hydroxyl groups. Boronic acid can distinguish between structurally similar sugar molecules.

Monoboronic acid is known to have intrinsic selectivity to other monosaccharides, particularly fructose. In the case of fructose, boronic acid bonding is possible because it has a pair of hydroxyl groups located on the same plane (syn-periplanar). The combination of monoboronic acid and fructose is shown in Formula 2 below.

[Formula 2]

When the binding affinity between phenylboronic acid and monosaccharide is compared, it appears as fructose, lactose, mannose, and glucose. The structure of each monosaccharide is shown in Formula 3 below.

[Formula 3]

Considering the three-dimensional structure of glucose according to Formula 3, in the case of monoboronic acid, since the binding affinity to glucose is relatively low, it is preferable to use multi-boronic acid to improve binding selectivity.

Multi-boronic acid has one or more binding sites, and forms two or three B-O-C linkages to stabilize the bond between boronic acid and diol. The selectivity of a boronic acid-based glucose sensor can be improved by using a plurality of boronic acid groups that are appropriately arranged for glucose.

In the case of detecting glucose using the glucose sensor according to the present invention, it is preferable to use diboronic acid which forms a cyclic boronate ester group with glucose one to one. The two boronic acids can bind to the alcohol groups at the 1,2- and 3,5,6-positions of glucose. The bond between diboronic acid and glucose is shown in Formula 4 below.

[Formula 4]

The presence of two binding sites between diboronic acid and glucose can form multiple bonds, and selectivity for glucose can be improved according to the two bonds (Multivalent diboronic acid-glucose interaction). Even in the case of fructose, if there is a very high concentration of boronic acid and an excessive amount of fructose, it can combine with two boronic acids to form a β-pyranose form, but this is physiologically impossible. It can be detected selectively.

On the other hand, there are various metal ions in human serum, and it is necessary to confirm whether the photonic IPN array of the present invention is not affected and can independently detect glucose with respect to the metal ions.

19 is a photograph showing a color change of metal ions in a photonic IPN array according to an embodiment of the present invention. Referring to FIG. 19, ^{the physiological concentrations of Ca 2+} , Mg ²⁺ , K ⁺ , and Na ⁺ ions in human serum are 2.2 to 2.8, 0.8 to 1.2, 3.5 to 5, and 136 to 145 mM, respectively. It was confirmed that the photonic IPN array according to the present invention maintained the initial green color even when the metal ions of the chemical concentration were treated. That is, it was confirmed that the ionic strength of the physiological concentration did not affect the optical response of the photonic IPN array of the present invention.

The photonic IPN array according to the present invention was prepared by binding APBA to an IPN composite composed of a solid CLC film and a PAA network. The photonic IPN array according to the present invention selectively detects glucose, and color change can be confirmed with the naked eye according to the concentration of glucose. In addition, the solid CLC film and APBA, which is a non-enzyme material, can be used to fundamentally block instability due to inactivation and denaturation of enzymes, which have been pointed out as problems based on existing enzymes. It can be used as an improved glucose biosensor because it does not require the use of.

The foregoing has been somewhat broadly described in terms of features and technical advantages of the present invention in order to better understand the claims of the invention to be described later. Those of ordinary skill in the art to which the present invention pertains will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. The scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (11)

A first step of adding a glucose solution to a complex including a photonic crystal structure, a polyacrylic acid (PAA) hydrogel, and a multi-boronic acid;
A second step of swelling the polyacrylic acid hydrogel due to the complex formed by the multi-boronic acid and the glucose; And
A third step of changing the color of the photonic crystal structure due to the swelling of the hydrogel; Including,
A method for measuring glucose concentration in which the concentration of glucose is measured according to different colors indicated by the concentration of glucose independently of a change in pH.
The method of claim 1,
The composite is a photonic crystal structure;
Polyacrylic acid (PAA) hydrogel penetrating into the interior space of the photonic crystal structure; And
Including; multi-boronic acid fixed to the polyacrylic acid,
The photonic crystal structure is characterized in that the polyacrylic acid and IPN (Interwined polymer network) to form a structure, glucose concentration measurement method.
The method of claim 1,
The method for measuring glucose concentration, wherein the photonic crystal structure is in a solid state.
The method of claim 1,
The multi-boronic acid is a method for measuring glucose concentration, characterized in that diboronic acid (Diboronic acid).
The method of claim 1,
The glucose solution is characterized in that the human serum, glucose concentration measurement method.
The method of claim 1,
The first step is characterized in that it is carried out in a pH buffer (buffer), the glucose concentration measurement method.
A biosensor for measuring glucose concentration using a photonic crystal structure, a polyacrylic acid (PAA) hydrogel penetrating into the inner space of the photonic crystal structure, and a composite including multi boronic acid fixed to the polyacrylic acid.
The method of claim 7,
The photonic crystal structure is characterized in that the polyacrylic acid and IPN (Interwined polymer network) to form a structure, glucose concentration measurement biosensor.
The method of claim 7,
The photonic crystal structure is characterized in that the solid phase, glucose concentration measurement biosensor.
The method of claim 7,
The multi-boronic acid is a biosensor for measuring glucose concentration, characterized in that the diboronic acid (Diboronic acid).
A first step of mixing and curing a chiral dopant and mesogen to form a helical cholesteric liquid crystal;
A second step of forming a solid photonic crystal structure by removing a chiral dopant from the cholesteric liquid crystal;
A third step of infiltrating polyacrylic acid (PAA) into the space from which the chiral dopant has been removed; And
Including; a fourth step of fixing multi boronic acid to the polyacrylic acid,
The photonic crystal structure forms an interwined polymer network (IPN) structure with the polyacrylic acid, and has a different color according to the concentration of glucose, a biosensor manufacturing method for measuring glucose concentration.

Images