WO2018056762A1 - Reagent composition for measuring glycated albumin and method for measuring glycated albumin using same - Google Patents

Reagent composition for measuring glycated albumin and method for measuring glycated albumin using same Download PDF

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WO2018056762A1
WO2018056762A1 PCT/KR2017/010499 KR2017010499W WO2018056762A1 WO 2018056762 A1 WO2018056762 A1 WO 2018056762A1 KR 2017010499 W KR2017010499 W KR 2017010499W WO 2018056762 A1 WO2018056762 A1 WO 2018056762A1
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Prior art keywords
albumin
dye
glycated albumin
measuring
boronic acid
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PCT/KR2017/010499
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French (fr)
Korean (ko)
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이진우
전선아
박찬영
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주식회사 딕스젠
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Priority claimed from KR1020170122084A external-priority patent/KR102029798B1/en
Application filed by 주식회사 딕스젠 filed Critical 주식회사 딕스젠
Priority to US16/335,412 priority Critical patent/US20200018766A1/en
Priority to EP17853476.4A priority patent/EP3517963A4/en
Publication of WO2018056762A1 publication Critical patent/WO2018056762A1/en
Priority to US17/189,972 priority patent/US20210208152A1/en

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    • 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
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a reagent composition for measuring glycated albumin for diagnosing diabetes and a method for measuring glycated albumin using the same, and more specifically, a reagent for measuring glycated albumin including silica nanoparticle-boronic acid encapsulating dye. It relates to a composition and a method for measuring glycated albumin using the same.
  • Diabetes mellitus is a metabolic disease caused by an abnormality in insulin that plays a role in blood sugar control. Insulin production is insufficient or cannot be effectively used when insulin-producing cells are destroyed due to abnormal immune system. It is classified according to the cause of the onset, such as type 2 diabetes.
  • Diabetes is characterized by high blood glucose, which increases blood glucose levels, and failure to control blood glucose control can lead to complications such as diabetic retinopathy, kidney disease, and foot lesions. .
  • glucose oxidase which is used to measure glucose, is vulnerable to environmental effects such as pH or other interferences contained in blood, and may affect the activity of the enzyme as it generates hydrogen peroxide.
  • glycated hemoglobin (HbA1c) has been used as a biomarker for measuring glucose more accurately and more stably than glucose. Since glycated hemoglobin is stable until red blood cells disappear, it is used as an indicator of the average blood glucose level of 2 to 3 months. Therefore, it is used to investigate the progress of diagnosis and treatment of diabetes. However, glycated hemoglobin measurement is not suitable for some patients with abnormal blood glucose or patients with red blood cells, such as chronic renal failure.
  • Albumin is a protein that exists not only in the blood but also in major organs and body fluids. Depending on the concentration of glucose in the blood, glycated albumin is also formed. Albumin has a 10-fold higher binding rate of glucose than hemoglobin, and glycated albumin is more sensitive to glycemic changes than glycated hemoglobin, and albumin is 15-20 days shorter than red blood cell lifespan. The average value of blood glucose levels can be monitored. Therefore, it is useful as an important glycemic control indicator in diabetic patients with end-stage chronic renal failure patients, patients with iron deficiency anemia, and variants with hemoglobin.
  • U.S. Patent No. 77878989, U.S. Patent No. 6008006, U.S. Patent No. 8507223, China Patent Publication No. 104673878, and Chinese Patent Publication No. 104614459 relate to a conventional method for measuring glycated albumin and present in a sample.
  • Hydrogen peroxide (H 2 O 2 ) produced by the enzyme reaction of the glycosylated amino acid oxidase (EC 1.5.3) series that is specifically cleaved glycosylated protein to glycosylated amino acid or glycosylated peptide using protease
  • the glycosylated albumin enzymatic method was measured again using a peroxidase enzyme.
  • the glycated albumin enzyme method can test not only high selectivity and accuracy for albumin, but also faster than the immunoassay (10-30 minutes), but the activity of the enzyme is because the activity of the enzyme protein has a great effect on the efficiency of the glycated albumin assay. Strict attention is required to maintenance and storage.
  • the glycated albumin enzyme method must be preceded by a step for removing glycated amino acids and glycated peptides already present in the sample. Since glycated amino acid oxidase (EC 1.5.3) cannot use intact glycated proteins as a substrate, it is necessary to use proteolytic enzymes. It is a complex measurement consisting of a multi-step, such as the step of using a glycosylated protein to degrade glycated amino acids or peptides must be preceded. Therefore, there is a need to develop a simpler and faster method for measuring glycated albumin.
  • U.S. Pat.No.5223392, U.S. Pat.No.5908925, European Patent No.0657470, European Patent No.0257421 and Chinese Patent No.103554256 are albumin antibodies and peroxidase enzymes for detecting glycated albumin.
  • An enzyme immunoassay (ELISA) using a conjugated glycated albumin antibody was disclosed, and Ikeda et al.
  • an enzyme-boronic acid immunoassay (ELIBA) using an albumin antibody and peroxidase enzyme conjugated boronic acid. Ikeda et al, Clin Chem. 44 (2): 256-263, 1998.
  • US Patent No. 9128085, US Patent Publication No. 2006-0270060, US Patent Publication No. 2008-0227210, US Patent Publication No. 2010-0167306, US Patent No. 5470759 as Point of Care or Rapid Kit And US Patent No. 7659107 discloses a method using disposable strips and cassettes using antibody-based lateral flow immunochromatography to measure albumin and glycated albumin in samples of blood, saliva, and the like.
  • Patent No. 2014-0170766 discloses a method for preparing a rapid kit using lateral flow immunochromatography using albumin aptamer and glycated albumin aptamer derived from a nucleic acid, which acts similar to an antibody. Patent No.
  • 2014-0335630 discloses a measurement method using glycated albumin aptamer and surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • U.S. Patent No. 5631364, U.S. Patent No. 7374943, and International Patent No. 2014-033258 disclose that a dye-bonded boronic acid derivative is reacted with glycated hemoglobin in blood, loaded and washed in a cartridge composed of porous filter paper, A method of determining the ratio of two substances by measuring the reflectance (% Reflectance) of total hemoglobin and dye-bound glycated hemoglobin was disclosed.
  • U.S. Patent No.5589393 discloses a method of fixing a boronic acid derivative to agarose beads to improve safety, and measuring glycated hemoglobin by reacting it with glycated hemoglobin in blood using a disposable cartridge.
  • 8557590 and Korean Patent No. 1128037 disclose a method of measuring glycated hemoglobin by directly loading a reacted sample into a cartridge composed of porous filter paper.
  • silica nanoencapsulates (a) a dye that specifically binds to whole albumin and (b) a dye that is complementary to the dye that specifically binds to whole albumin.
  • boronic acid conjugated with the particles it was confirmed that the amount of albumin and glycated albumin can be measured quickly and accurately by a simple and stable method using an optical device, and the present invention was completed.
  • An object of the present invention is to provide a reagent composition for measuring glycated albumin and a method for measuring glycated albumin using the same, which can easily and accurately diagnose the presence or absence of diabetes.
  • the present invention provides a saccharification comprising (a) a dye that specifically binds to whole albumin and (b) “silica nanoparticle-boronic acid encapsulating dye” that specifically binds to glycated albumin.
  • a saccharification comprising (a) a dye that specifically binds to whole albumin and (b) “silica nanoparticle-boronic acid encapsulating dye” that specifically binds to glycated albumin.
  • a reagent composition for albumin measurement is provided.
  • the present invention also comprises the steps of (a) adding a blood or plasma solution to a reagent comprising a "silica encapsulated silica nanoparticles-boronic acid encapsulating specifically with glycated albumin; (b) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (c) measuring the amount of glycated albumin by measuring the optical reflectivity of the absorbent pad with an optical instrument; (d) adding blood or plasma solution to a reagent containing a dye that specifically binds to whole albumin and reacting the same; (e) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (f) measuring the optical reflectivity of the absorbent pad with optics to determine the total amount of albumin; (g) providing a method for measuring glycated albumin, comprising calculating a ratio of glycated albumin based on the measured amounts of glycated albumin and total albumin.
  • the dye specifically binding to the whole albumin is characterized in that bromocresol green (Bromocresol green) or bromocresol purple (Bromocresol purple).
  • the dye encapsulated in the silica nanoparticles is a yellow-based or red-based dye
  • the yellow-based dye has an absorption wavelength of 400 ⁇ 430nm
  • the red dye has an absorption wavelength of 500 ⁇ 530nm do.
  • the yellow dye is tarrazine (Tartrazine)
  • the red dye is characterized in that the red 80.
  • sica nanoparticles encapsulated with dye is prepared by adding dye and silica to water and surfactant mixture or water and organic solvent mixture and stirring, followed by addition of basic catalyst. It is characterized by combining as.
  • the diameter of the "silica nanoparticles encapsulated dye” is characterized in that 10 ⁇ 500nm.
  • the "silica encapsulated silica nanoparticles-boronic acid” is a 4-carboxyphenyl boronic acid (CPBA) after amination of the "silica nanoparticles encapsulated dye” Or conjugated with, or “carboxylated silica nanoparticles” and then conjugated with 3-aminophenyl boronic acid (APBA).
  • CPBA 4-carboxyphenyl boronic acid
  • APBA 3-aminophenyl boronic acid
  • the optical device is characterized by measuring the optical reflectance by irradiating a light source simultaneously with the wavelength of the dye specifically binding to the whole albumin and the specific wavelength of "silica nanoparticle-boronic acid encapsulated dye" do.
  • the method for measuring glycated albumin is characterized by diagnosing diabetes according to the ratio of glycated albumin.
  • the reagent composition for measuring saccharified albumin according to the present invention includes “silica nanoparticle-boronic acid encapsulated with dye”, the absorption wavelength of the dye is not affected by pH, and has excellent stability even when stored for a month or more.
  • the amount of light absorbed by one particle is greater than that of one dye molecule, thereby accurately measuring the amount of glycated albumin in the blood having a low detection limit.
  • FIG. 1 is an explanatory diagram showing a manufacturing method of "silica nanoparticles encapsulated dye" according to an embodiment of the present invention.
  • Figure 2 is an explanatory view showing the coupling reaction of "silica nanoparticles-boronic acid encapsulated dye” and glycated albumin according to an embodiment of the present invention.
  • FIG. 3 is a flowchart illustrating a method for measuring glycated albumin and total albumin using the reagent composition and optical apparatus for measuring glycated albumin of the present invention.
  • Figure 4 is a graph showing the absorbance according to the absorption wavelength of the "dye encapsulated silica nanoparticles-boronic acid" prepared according to an embodiment of the present invention and the dye used.
  • Figure 5 is analyzed using a shape image and (B) dynamic light scattering method measured using (A) scanning electron microscope of "dyed encapsulated silica nanoparticles-boronic acid" prepared according to an embodiment of the present invention This is a graph about one size.
  • Figure 6 is a graph showing the reflectance value of "silica encapsulated silica nanoparticles-boronic acid" appear according to the concentration of glycated albumin of the plasma samples known concentration.
  • silica nanoparticles encapsulated with boronic acid conjugated to The instrument was intended to confirm that the amount of albumin and glycated albumin can be accurately measured in a simple and stable manner.
  • die encapsulated silica nanoparticles-boronic acid that specifically binds to glycated albumin was prepared, and a dye that specifically binds to whole albumin was added to prepare a reagent composition for measuring glycated albumin.
  • blood or plasma samples containing albumin and glycated albumin were added to each of the prepared reagent compositions, reacted with an absorbent pad, and then washed with unreacted dyes and impurities combined with impurities.
  • Optical reflectance of the absorbent pad was also measured to determine the amount of albumin and glycated albumin. As a result, it was confirmed that diabetes can be diagnosed simply and quickly through the ratio of glycated albumin.
  • the yellow tartarazine dye is encapsulated in the silica nanoparticles, and the hydroxyl group (-OH) on the surface is substituted with a primary amine group, and then 4-carboxyl is a glycated albumin binding material.
  • Bromocresol green a dye that binds 4-carboxylicphenyl-boronic acid (CPBA) to the surface to produce “silica nanoparticles-boronic acid encapsulating tartrazine” and binds specifically to albumin.
  • CPBA 4-carboxylicphenyl-boronic acid
  • the plasma samples filtered through the silica nanoparticles-boronic acid and bromocresol green encapsulating tarrazine are reacted, administered to an absorbent pad, washed, and then the absorbent pad is optically applied.
  • Glycosylation by irradiation with red (430 nm) and blue (630 nm) light sources to measure the optical reflectivity of glycated albumin labeled with “silica nanoparticles-boronic acid encapsulating tartrazine” and total albumin labeled with bromocresolgreen, respectively. It was confirmed that the ratio of albumin can be measured simply and quickly.
  • the present invention relates to a reagent composition for measuring glycated albumin comprising a dye that specifically binds albumin and “silica nanoparticle-boronic acid encapsulated with dyes” that specifically binds to albumin. .
  • the dye that specifically binds to the total albumin can be used without particular limitation as long as it is a dye that specifically reacts with albumin and glycated albumin, bromo having a 620 nm absorption wavelength band in blue at physiological neutral pH. Examples thereof include bromocresol green and violet, and bromocresol purple having a 580 nm absorption wavelength band.
  • the dye encapsulated in the silica nanoparticles is characterized by using a yellow-based or red-based dye having a complementary color relationship with the dye specifically binding to the whole albumin.
  • the yellow dye has an absorption wavelength of about 400 ⁇ 430nm
  • the red dye has an absorption wavelength of about 500 ⁇ 530nm
  • yellow tar dye (Tartrazine, 425nm)
  • red dye red 80 (red 80, 528 nm) and the like, but are not limited thereto.
  • sica nanoparticles encapsulating dye may be prepared by adding a dye and silica to a water and a surfactant mixture or a water and an organic solvent mixture, followed by stirring, and then adding a basic catalyst.
  • the surfactant is not particularly limited, but in the present invention, tritone x-100 or n-hexane may be used, and the silica may be exemplified by tetraethyl orthosilicate or tetramethyl orthosilicate. can do.
  • the basic catalyst is to promote the silica precursor encapsulates the dye, it may promote the hydrolysis of the silica precursor with water.
  • the silica precursor in the ionized state thus reacts with each other to release water or alcohol (ROH) and is connected to each other to form a silica network.
  • ROH water or alcohol
  • the basic catalyst may be exemplified by ammonium hydroxide, tetrapropylammonium chloride, tetrapropylammonium hydroxide, tetrabutylammonium bromide, tetrabutylammonium chloride or tetrabutylammonium hydroxide.
  • “Dye-encapsulated silica nanoparticles” can increase stability and sensitivity because dyes do not spill out, and are less biotoxic and can easily change surface functional groups.
  • the diameter of the "silica nanoparticles encapsulated dye” may be 10 ⁇ 500nm, it is preferably 30 ⁇ 100nm to maintain the unique properties of the dye. If it is less than 10 nm, the operation is difficult, and if it is more than 500 nm, the thickness becomes thick and the dye may appear cloudy.
  • Boronic acid derivatives to impart selectivity to glycated albumin to “silica nanoparticles encapsulated with dyes” include 4-carboxylicphenyl boronic acid (CPBA) and 3-aminophenyl boronic acid (3- Aminophenyl boronic acid (APBA) is preferred, where CPBA is used to amine “dye encapsulated silica nanoparticles”, and APBA is used to carboxylate “dye encapsulated silica nanoparticles” Conjugation may be via carbodiimide cross coupling. It is preferable to use CPBA because 4-carboxylicphenyl boronic acid (CPBA) has relatively higher thermal stability than 3-aminophenyl boronic acid (APBA). Do.
  • CPBA 4-carboxylicphenyl boronic acid
  • APBA 3-aminophenyl boronic acid
  • “Dye-encapsulated silica nanoparticles-boronic acid” can react with cis-diol of glycated albumin, and several dye molecules are encapsulated in silica nanoparticles, so that one dye Since the absorption of light is higher than that of molecules, the detection limit of glycated albumin can be improved.
  • the blood or plasma solution was reacted with “dye-encapsulated silica nanoparticles-boronic acid” capable of labeling glycated albumin, and then added dropwise to the cartridge to wash and record the reflectance value of glycated albumin. Then, react with a bromocresolgreen (BCG) solution that can stain the whole albumin, drop it into a cartridge and wash it to record the total albumin reflectivity value.
  • BCG bromocresolgreen
  • the percentage of glycated albumin is compared to the total albumin reflectivity compared to the reflectivity of glycated albumin. Can be calculated At this time, the total albumin can be measured first, and then the glycated albumin can be measured, but the percentage of glycated albumin can be calculated.
  • the present invention in another aspect, the method comprising the steps of: (a) adding a blood or plasma solution to a reagent containing "silica nanoparticles-boronic acid encapsulated dye" that specifically binds to glycated albumin; (b) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (c) measuring the amount of glycated albumin by measuring the optical reflectivity of the absorbent pad with an optical instrument; (d) adding blood or plasma solution to a reagent containing a dye that specifically binds to whole albumin and reacting the same; (e) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (f) measuring the optical reflectivity of the absorbent pad with optics to determine the total amount of albumin; (g) calculating a ratio of glycated albumin based on the measured amounts of glycated albumin and total albumin.
  • the optical device can be used without particular limitation as long as it can measure the optical reflectivity using optical properties, and the wavelength of the dye (blue or purple) that specifically binds to the whole albumin and the "dye (yellow) Or red light-encapsulated silica nanoparticles-boronic acid ”using a light source (eg, blue (630 nm) or red (430 nm)) capable of generating a specific wavelength at the same time, and reflecting the optical signal to the photodiode detector.
  • a light source eg, blue (630 nm) or red (430 nm)
  • the amount of albumin and glycated albumin can be measured by optical signal converter.
  • the ratio of glycated albumin can be calculated by the relative amount of glycated albumin according to the total albumin amount by the following equation.
  • Glycosylated albumin ratio (%) Glycosylated albumin / total albumin
  • the ratio of the glycated albumin is 16% or more can be diagnosed as diabetes.
  • Example 1 Preparation of silica nanoparticle-boronic acid (YD @ SNP-CPBA) encapsulated yellow dye
  • YD @ SNP For crosslinking of the carboxyl groups of YD @ SNP and CPBA, the hydroxyl group (-OH) on the surface of YD @ SNP was substituted with a primary amine group. That is, 100 mg of YD @ SNP was added to 100 ml of ethanol, dispersed for 30 minutes using an ultrasonic disperser, and then 1 ml of APTES (3-Aminopropyltriethoxysilane) was added to a stirrer and reacted at room temperature for 2 hours. After the reaction, 4 times ethanol wash and 3 times DI wash at 3800 rpm and 15 minutes using a centrifuge were placed in an oven at 60 ° C. and dried to prepare aminated YD @ SNP (YD @ SNP-NH 2 ). .
  • APTES 3-Aminopropyltriethoxysilane
  • EDC 1-ethyl-3 [3-dimethylaminopropyl] carbodiimide hydrochloride
  • CPBA 4-carboxylicphenyl-boronic acid
  • silica nanoparticle-boronic acid (YD @ SNP-CPBA) encapsulated a yellow dye by washing with ethanol 4 times and DI DI 3 times at 3800rpm, 15 minutes using a centrifuge at room temperature or lyophilization (YD @ SNP-CPBA). ) was prepared.
  • Example 2 YD @ SNP-CPBA and Bromocresol green Containing Saccharification Determination of glycated albumin using reagent composition for albumin measurement
  • Example 2 200 ⁇ l of the reagent composition (ZnCl 2 , NaCl, MgCl 2 , Triton X-100, NaN 3 , glycine, HEPES, pH 8.1) prepared in Example 1 was placed in a brown tube, and the reference reagent was prepared. (Asahi Kasei GA-L) was added to 5 ⁇ l of a plasma sample whose% value of glycated albumin was measured by an Olympus AU400 analyzer, followed by reaction for 2 minutes.
  • the reagent composition ZnCl 2 , NaCl, MgCl 2 , Triton X-100, NaN 3 , glycine, HEPES, pH 8.1
  • reaction solution 25 ⁇ l was placed on the cartridge absorption pad of optics (Episode ® 616, Dixsen) for 15 seconds, absorbed and 25 ⁇ l of washing solution (Morpholine, NaCl, Triton X-100, Glycerol, and NaN 3 mixture) was added. Wash for 15 seconds. Next, a cartridge for an optical device (episodes ® 616, Dix Xen) was measured optical reflectivity of the glycated albumin in the yellow.
  • optics Episode ® 616, Dixsen
  • reagent composition (Succinic acid, pH 5.5) containing bromocresol green was placed in a brown tube, 5 ⁇ l of the same plasma sample was added, and the reaction was carried out for 2 minutes. 25 ⁇ l of the reaction solution was placed on the cartridge absorption pad of optics (Episode ® 616, Dixsen) for 15 seconds, absorbed and 25 ⁇ l of washing solution (Morpholine, NaCl, Triton X-100, Glycerol, and NaN 3 mixture) was added. Wash for 15 seconds. Next, in the cartridge optics (episodes ® 616, Dix Xen) it was measured optical reflectivity of the total albumin in the blue.
  • The% reflectivity of glycated albumin was determined by comparing the optical reflectivity of glycated albumin measured in 2-1 and the optical reflectivity of total albumin measured in 2-2.
  • The% reflectance (% R) measured from each wavelength was converted into K / S value, which is a quantitative indicator of how much colored material is on the surface, and% reflectance is converted into K / S value.
  • the formula is:
  • the% reflectance value obtained by investigating the yellow light source representing the amount of glycated albumin and the% reflectance value obtained from the blue light source representing the total amount of albumin are respectively substituted by K / S values, and then the ratio is calculated by saccharification.
  • the amount of albumin could be measured.
  • the reagent composition for measuring glycated albumin according to the present invention includes a dye which can distinguish the total albumin and the glycated albumin, respectively, and thus, the glycated albumin can be easily obtained by using an optical analyzer through administration of a washing solution to a measurement cartridge without a separate separation process. Because it can be measured, it can be widely used to diagnose diabetes.

Abstract

The present invention relates to a reagent composition for measuring glycated albumin to be able to diagnose the presence or absence of diabetes and a method for measuring glycated albumin using the same and, more specifically, to a reagent composition for measuring glycated albumin, the composition comprising a dye-encapsulated silica nanoparticle-boronic acid, and to a method for measuring glycated albumin using the same. The method for measuring glycated albumin according to the present invention comprises the steps of: (a) introducing blood or a plasma solution into a reagent comprising "dye-encapsulated silica nanoparticle-boronic acid" specifically binding to glycated albumin, followed by reaction; (b) injecting a reaction product to an absorbing pad of a cartridge, followed by washing with a washing liquid; (c) measuring the optical reflectance of the absorbing pad using an optical instrument to determine the amount of glycated albumin; (d) introducing blood or a plasma solution into a reagent comprising a dye specifically binding to total albumin, followed by reaction; (e) injecting a reaction product to an absorbing pad of a cartridge, followed by washing with a washing liquid; (f) measuring the optical reflectance of the absorbing pad using an optical instrument to determine the amount of total albumin; and (g) calculating the proportion of the glycated albumin on the basis of the measured amounts of glycated albumin and total albumin. In the reagent composition for measuring glycated albumin according to the present invention, the dye has been encapsulated inside silica nanoparticles, and thus the inherent absorption wavelength of the dye is not affected by pH, and the composition has excellent stability even when stored for one month or more.

Description

당화 알부민 측정용 시약 조성물 및 이를 이용한 당화 알부민의 측정방법Reagent composition for measuring glycated albumin and method for measuring glycated albumin using the same
본 발명은 당뇨병 유무를 진단할 수 있는 당화 알부민 측정용 시약 조성물 및 이를 이용한 당화 알부민의 측정방법에 관한 것으로, 더욱 상세하게는 염료를 캡슐화한 실리카 나노입자-보론산을 포함하는 당화 알부민 측정용 시약 조성물 및 이를 이용한 당화 알부민의 측정방법에 관한 것이다.The present invention relates to a reagent composition for measuring glycated albumin for diagnosing diabetes and a method for measuring glycated albumin using the same, and more specifically, a reagent for measuring glycated albumin including silica nanoparticle-boronic acid encapsulating dye. It relates to a composition and a method for measuring glycated albumin using the same.
당뇨병은 혈당조절역할을 하는 인슐린의 이상으로 인해 발생하는 대사질환으로, 인슐린을 생성하는 세포가 면역시스템의 이상으로 인해 파괴되어 부족할 때 발생되는 1형 당뇨와 인슐린의 분비량이 부족하거나 효과적으로 사용되지 못함에 따라 발생되는 2형 당뇨 등 발병 원인에 따라 분류된다. Diabetes mellitus is a metabolic disease caused by an abnormality in insulin that plays a role in blood sugar control. Insulin production is insufficient or cannot be effectively used when insulin-producing cells are destroyed due to abnormal immune system. It is classified according to the cause of the onset, such as type 2 diabetes.
당뇨병은 혈중 포도당의 농도가 높아지는 고혈당을 특징으로 하며, 혈당관리 조절을 실패할 경우 당뇨망막증, 신장질환, 족부병변 등의 합병증이 유발될 수 있기 때문에 당뇨병 환자들의 혈당 관리에 대한 중요성은 증가되고 있다.Diabetes is characterized by high blood glucose, which increases blood glucose levels, and failure to control blood glucose control can lead to complications such as diabetic retinopathy, kidney disease, and foot lesions. .
종래의 당뇨병 측정 마커는 포도당을 사용하였으나, 식전 식후의 혈당 값 변동이 심하기 때문에 측정하는 시간에 따른 오차 및 환자의 컨디션에 따른 변동이 직접적으로 나타날 수가 있다는 문제점이 있다. 또한 포도당 측정에 사용하는 포도당산화효소는 pH나 혈액 내에 포함된 다른 방해물질 등과 같은 측정하는 환경영향에 취약하며, 과산화수소를 발생시킴에 따라 효소의 활성에 영향을 줄 수 있다. Conventional diabetes measurement markers use glucose, but since there is a significant fluctuation in blood glucose values before and after meals, there is a problem in that errors due to measurement time and fluctuations depending on the condition of the patient may appear directly. In addition, glucose oxidase, which is used to measure glucose, is vulnerable to environmental effects such as pH or other interferences contained in blood, and may affect the activity of the enzyme as it generates hydrogen peroxide.
따라서, 최근에는 포도당보다 정확하게 측정할 수 있고, 보다 안정적인 혈당 측정용 바이오 마커로써 당화혈색소(glycated hemoglobin, HbA1c)를 이용하고 있다. 당화혈색소는 생성되면 적혈구가 소멸되기까지는 안정하므로 2~3개월의 평균혈당치를 나타내는 지표로 사용되므로, 실제 당뇨병의 진단 및 치료 경과 추이를 조사하는데 활용된다. 하지만 당화혈색소 측정법은 말기 만성신부전 등과 같이 혈당이 꾸준히 유지되지 못하는 일부의 질환자나 적혈구에 이상이 있는 환자에게는 적합하지 않다고 알려져 있다. Therefore, in recent years, glycated hemoglobin (HbA1c) has been used as a biomarker for measuring glucose more accurately and more stably than glucose. Since glycated hemoglobin is stable until red blood cells disappear, it is used as an indicator of the average blood glucose level of 2 to 3 months. Therefore, it is used to investigate the progress of diagnosis and treatment of diabetes. However, glycated hemoglobin measurement is not suitable for some patients with abnormal blood glucose or patients with red blood cells, such as chronic renal failure.
알부민은 혈액뿐 만 아니라 주요 장기와 체액에 존재하는 단백질로, 혈액의 포도당 농도에 따라 포도당이 결합된 당화 알부민이 형성되기도 한다. 알부민은 혈색소에 비해 포도당의 결합률이 10배 정도 높아서 당화 알부민은 당화혈색소보다 혈당 변화에 더욱 민감하게 반응하고, 적혈구 수명이 90일 정도인 것에 비하여 알부민은 15~20일 정도로 짧아서, 최근 2주 동안 혈중 포도당 수준의 평균치를 모니터링할 수 있다. 따라서, 혈당이 꾸준히 유지되지 않는 말기 만성신부전 환자, 철 결핍성 빈혈 환자, 변종 헤모글로빈을 가진 당뇨환자에서 중요한 혈당 컨트롤 지표로 유용하다. Albumin is a protein that exists not only in the blood but also in major organs and body fluids. Depending on the concentration of glucose in the blood, glycated albumin is also formed. Albumin has a 10-fold higher binding rate of glucose than hemoglobin, and glycated albumin is more sensitive to glycemic changes than glycated hemoglobin, and albumin is 15-20 days shorter than red blood cell lifespan. The average value of blood glucose levels can be monitored. Therefore, it is useful as an important glycemic control indicator in diabetic patients with end-stage chronic renal failure patients, patients with iron deficiency anemia, and variants with hemoglobin.
미국등록특허 제7871789호, 미국등록특허 제6008006호, 미국등록특허 제8507223호, 중국공개특허 제104673878호 및 중국공개특허 제104614459호는 종래의 당화 알부민을 측정하는 방법에 관한 것으로서, 시료에 존재하는 당화단백질을 단백질분해효소를 사용하여 당화 아미노산 또는 당화 펩타이드로 분해한 후 이와 특이적으로 반응하는 당화 아미노산 산화 효소 (EC 1.5.3) 계열의 효소 반응으로 생성된 과산화수소(H2O2)를 다시 페록시다아제 (peroxidase) 효소를 이용하여 측정하는 당화 알부민 효소법을 개시하였다.U.S. Patent No. 77878989, U.S. Patent No. 6008006, U.S. Patent No. 8507223, China Patent Publication No. 104673878, and Chinese Patent Publication No. 104614459 relate to a conventional method for measuring glycated albumin and present in a sample. Hydrogen peroxide (H 2 O 2 ) produced by the enzyme reaction of the glycosylated amino acid oxidase (EC 1.5.3) series that is specifically cleaved glycosylated protein to glycosylated amino acid or glycosylated peptide using protease The glycosylated albumin enzymatic method was measured again using a peroxidase enzyme.
당화 알부민 효소법은 알부민에 대한 높은 선택성 및 정확성뿐만 아니라, 면역법 보다 빠른 시간 내(10~30분) 검사가 가능하지만, 효소 단백질 특성상 그 활성이 당화 알부민 분석법 효율에 큰 영향을 미치기 때문에, 효소의 활성 유지 및 보관에 엄격한 주의가 요구된다. The glycated albumin enzyme method can test not only high selectivity and accuracy for albumin, but also faster than the immunoassay (10-30 minutes), but the activity of the enzyme is because the activity of the enzyme protein has a great effect on the efficiency of the glycated albumin assay. Strict attention is required to maintenance and storage.
당화 알부민 효소법은 시료 내에 이미 존재하는 당화 아미노산과 당화 펩타이드를 제거하기 위한 단계가 선행되어야 하며, 당화 아미노산 산화효소(EC 1.5.3)는 온전한 당화 단백질을 기질로 사용할 수 없기 때문에, 반드시 단백질 분해효소를 사용하여 당화 단백질을 당화 아미노산 또는 펩타이드로 분해하는 단계가 선행되어야 하는 등 다단계로 구성된 복잡한 측정법이다. 따라서 이보다 간단하며 신속하게 당화 알부민을 측정할 수 있는 방법의 개발이 필요하다.The glycated albumin enzyme method must be preceded by a step for removing glycated amino acids and glycated peptides already present in the sample. Since glycated amino acid oxidase (EC 1.5.3) cannot use intact glycated proteins as a substrate, it is necessary to use proteolytic enzymes. It is a complex measurement consisting of a multi-step, such as the step of using a glycosylated protein to degrade glycated amino acids or peptides must be preceded. Therefore, there is a need to develop a simpler and faster method for measuring glycated albumin.
또한 미국등록특허 제5223392호, 미국등록특허 제5908925호, 유럽공개특허 제0657470호, 유럽등록특허 제0257421호 및 중국공개특허 제103554256호는 당화 알부민을 검출하기 위하여 알부민 항체 및 페록시다아제 효소와 컨주게이션된 당화 알부민 항체를 이용한 효소 면역측정법(ELISA)을 개시하였고, Ikeda 등은 알부민 항체 및 페록시다아제 효소와 컨주게이션된 보론산을 이용한 효소-보론산 면역측정법(ELIBA)을 개시하였다(Ikeda et al, Clin Chem. 44(2):256-263, 1998). 이들 방법은 높은 선택성 및 정확성을 보였으나, 항체 및 효소의 보관 및 유지가 필요하고, 많은 시간(30~90분)이 소요되어 래피드 키트로의 개발에는 제한이 있다. In addition, U.S. Pat.No.5223392, U.S. Pat.No.5908925, European Patent No.0657470, European Patent No.0257421 and Chinese Patent No.103554256 are albumin antibodies and peroxidase enzymes for detecting glycated albumin. An enzyme immunoassay (ELISA) using a conjugated glycated albumin antibody was disclosed, and Ikeda et al. Disclosed an enzyme-boronic acid immunoassay (ELIBA) using an albumin antibody and peroxidase enzyme conjugated boronic acid. (Ikeda et al, Clin Chem. 44 (2): 256-263, 1998). These methods showed high selectivity and accuracy, but require the storage and maintenance of antibodies and enzymes, and require a lot of time (30-90 minutes) to limit their development into rapid kits.
POC(Point of care)나 래피드 키트로써 미국등록특허 제9128085호, 미국공개특허 제2006-0270060호, 미국공개특허 제2008-0227210호, 미국공개특허 제2010-0167306호, 미국등록특허 제5470759호 및 미국등록특허 제7659107호는 혈액, 침 등의 시료에서 알부민 및 당화 알부민을 측정을 위해 항체 기반의 측면유동면역발색법(lateral flow immunochromatography)을 이용한 일회용 스트립 및 카세트 이용한 방법을 개시하였고, 미국공개특허 제2014-0170766호는 항체와 유사한 작용을 하는 핵산 유래의 알부민 앱타머와 당화 알부민 앱타머를 이용한 측면유동면역발색법(lateral flow immunochromatography)을 적용시킨 래피드 키트의 제조방법을 개시하였으며, 미국공개특허 제2014-0335630호는 당화 알부민 앱타머와 SPR (surface plasmon resonance)을 이용한 측정법을 개시하였다. 그러나 항체 또는 핵산으로 구성된 앱타머 역시 항체와 같이 유지 및 보관에 주의가 필요하다. 따라서, 보다 안정적이고 신속하게 당화 알부민을 측정할 수 있는 진단 시약 및 방법의 개발이 필요하다.US Patent No. 9128085, US Patent Publication No. 2006-0270060, US Patent Publication No. 2008-0227210, US Patent Publication No. 2010-0167306, US Patent No. 5470759 as Point of Care or Rapid Kit And US Patent No. 7659107 discloses a method using disposable strips and cassettes using antibody-based lateral flow immunochromatography to measure albumin and glycated albumin in samples of blood, saliva, and the like. Patent No. 2014-0170766 discloses a method for preparing a rapid kit using lateral flow immunochromatography using albumin aptamer and glycated albumin aptamer derived from a nucleic acid, which acts similar to an antibody. Patent No. 2014-0335630 discloses a measurement method using glycated albumin aptamer and surface plasmon resonance (SPR). However, aptamers composed of antibodies or nucleic acids, like antibodies, require careful maintenance and storage. Therefore, there is a need for the development of diagnostic reagents and methods that can measure glycated albumin more stably and quickly.
한편, 당화혈색소 검출을 위하여 보론산 친화법이 널리 사용되고 있다. 미국등록특허 제5631364호, 미국등록특허 제7374943호 및 국제특허 제2014-033258호는 염료가 결합된 보로닌산 유도체를 혈액의 당화혈색소와 반응시키고, 다공성 여과지로 구성된 카트리지에 로딩 및 세척시킨 후, 전체 혈색소와 염료 결합 당화혈색소의 반사도(% Reflectance)를 측정하여 두 물질의 비율을 결정하는 방법을 개시하였다. Meanwhile, the boronic acid affinity method is widely used for the detection of glycated hemoglobin. U.S. Patent No. 5631364, U.S. Patent No. 7374943, and International Patent No. 2014-033258 disclose that a dye-bonded boronic acid derivative is reacted with glycated hemoglobin in blood, loaded and washed in a cartridge composed of porous filter paper, A method of determining the ratio of two substances by measuring the reflectance (% Reflectance) of total hemoglobin and dye-bound glycated hemoglobin was disclosed.
또한, 미국등록특허 제5589393호는 보론산 유도체를 아가로즈 비드에 고정하여 안전성을 향상시키고, 일회용 카트리지 이용하여 이를 혈액의 당화혈색소와 반응시켜 당화혈색소를 측정하는 방법을 개시하였고, 미국등록특허 제8557590호 및 한국등록특허 제1128037호는 반응된 시료를 다공성 여과지로 구성된 카트리지에 직접 로딩하여 당화혈색소를 측정하는 방법을 개시하였다. In addition, U.S. Patent No.5589393 discloses a method of fixing a boronic acid derivative to agarose beads to improve safety, and measuring glycated hemoglobin by reacting it with glycated hemoglobin in blood using a disposable cartridge. 8557590 and Korean Patent No. 1128037 disclose a method of measuring glycated hemoglobin by directly loading a reacted sample into a cartridge composed of porous filter paper.
보론산 친화법을 적용시킨 당화혈색소 래피드 키트는 연구가 되었으나, 보론산 친화법을 적용시킨 당화 알부민 래피드 키트는 아직까지 개발되지 못하였다. 이는 보론산 친화법을 당화 알부민 측정에 적용하기에는 제약사항이 있기 때문이다. 첫번째로, 당화혈색소 측정에 주로 사용되는 염료-보론산 유도체인 자일렌사이아놀-다폴-보론산(Xylene Cyanol-DAPOL-CPBA)는 알부민을 측정에 사용하는 브로모 크레졸 그린이나 브로모 크레졸 퍼플과 동일한 620nm 대 흡수파장을 가지고 있어서, 당화 알부민 측정에 적합하지 않다. 따라서, 당화 알부민 측정을 위해 브로모 크레졸 그린 또는 퍼플과 구별되는 염료와 결합한 보론산 유도체의 개발이 필요하다.Glycosylated hemoglobin rapid kits with boronic acid affinity have been studied, but glycated albumin rapid kits with boronic acid affinity have not been developed. This is because the boronic acid affinity method has limitations in applying the glycated albumin measurement. First, Xylene Cyanol-DAPOL-CPBA, a dye-boronic acid derivative mainly used for measuring glycated hemoglobin, is used for bromo cresol green or bromo cresol purple, which is used for measuring albumin. Having the same 620 nm band absorption wavelength, it is not suitable for glycated albumin measurement. Accordingly, there is a need for the development of boronic acid derivatives in combination with dyes that are distinguished from bromo cresol green or purple for the determination of glycated albumin.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 노력한 결과, (a) 전체 알부민과 특이적으로 결합하는 염료 및 (b) 전체 알부민과 특이적으로 결합하는 염료와 보색 관계에 있는 염료를 캡슐화한 실리카 나노입자와 컨쥬게이션된 보론산을 이용할 경우 광학기기로 알부민 및 당화 알부민의 양을 간단하고 안정된 방법으로 신속하고 정확하게 측정할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have tried to solve the above problems, and as a result, silica nanoencapsulates (a) a dye that specifically binds to whole albumin and (b) a dye that is complementary to the dye that specifically binds to whole albumin. When using boronic acid conjugated with the particles, it was confirmed that the amount of albumin and glycated albumin can be measured quickly and accurately by a simple and stable method using an optical device, and the present invention was completed.
본 발명의 목적은 당뇨병의 유무를 간단하고 정확하게 진단할 수 있는 당화 알부민 측정용 시약 조성물 및 이를 이용한 당화 알부민의 측정방법을 제공하는데 있다.Disclosure of Invention An object of the present invention is to provide a reagent composition for measuring glycated albumin and a method for measuring glycated albumin using the same, which can easily and accurately diagnose the presence or absence of diabetes.
상기 목적을 달성하기 위하여, 본 발명은 (a) 전체 알부민과 특이적으로 결합하는 염료 및 (b) 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 당화 알부민 측정용 시약 조성물을 제공한다.In order to achieve the above object, the present invention provides a saccharification comprising (a) a dye that specifically binds to whole albumin and (b) “silica nanoparticle-boronic acid encapsulating dye” that specifically binds to glycated albumin. Provided is a reagent composition for albumin measurement.
본 발명은 또한 (a) 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (b) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계; (c) 광학기기로 흡수 패드의 광학 반사도 측정하여 당화 알부민의 양을 측정하는 단계; (d) 전체 알부민과 특이적으로 결합하는 염료를 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (e) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계; (f) 광학기기로 흡수 패드의 광학 반사도를 측정하여 전체 알부민의 양을 측정하는 단계 및; (g) 측정된 당화 알부민 및 전체 알부민의 양을 기반으로 당화 알부민의 비율을 계산하는 단계를 포함하는 당화 알부민의 측정방법을 제공한다.The present invention also comprises the steps of (a) adding a blood or plasma solution to a reagent comprising a "silica encapsulated silica nanoparticles-boronic acid encapsulating specifically with glycated albumin; (b) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (c) measuring the amount of glycated albumin by measuring the optical reflectivity of the absorbent pad with an optical instrument; (d) adding blood or plasma solution to a reagent containing a dye that specifically binds to whole albumin and reacting the same; (e) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (f) measuring the optical reflectivity of the absorbent pad with optics to determine the total amount of albumin; (g) providing a method for measuring glycated albumin, comprising calculating a ratio of glycated albumin based on the measured amounts of glycated albumin and total albumin.
본 발명에 있어서, 상기 전체 알부민과 특이적으로 결합하는 염료는 브로모크레졸그린(Bromocresol green) 또는 브로모크레졸퍼플(Bromocresol purple)인 것을 특징으로 한다. In the present invention, the dye specifically binding to the whole albumin is characterized in that bromocresol green (Bromocresol green) or bromocresol purple (Bromocresol purple).
본 발명에 있어서, 상기 실리카 나노입자에 캡슐화된 염료는 황색 계열 또는 적색 계열 염료로서, 황색 계열 염료는 400~430nm의 흡수파장을 갖고, 적색 계열 염료는 500~530nm의 흡수파장을 갖는 것을 특징으로 한다.In the present invention, the dye encapsulated in the silica nanoparticles is a yellow-based or red-based dye, the yellow-based dye has an absorption wavelength of 400 ~ 430nm, the red dye has an absorption wavelength of 500 ~ 530nm do.
본 발명에 있어서, 상기 황색 계열 염료는 타르트라진(Tartrazine)이고, 적색 계열 염료는 레드80인 것을 특징으로 한다.In the present invention, the yellow dye is tarrazine (Tartrazine), the red dye is characterized in that the red 80.
본 발명에 있어서, “염료를 캡슐화한 실리카 나노입자”는 물과 계면활성제 혼합액 또는 물과 유기용매 혼합액에 염료 및 실리카를 첨가하고 교반시킨 후, 염기성 촉매제를 첨가하여 제조시킨 것으로서 당화 알부민과 특이적으로 결합하는 것을 특징으로 한다.In the present invention, “silica nanoparticles encapsulated with dye” is prepared by adding dye and silica to water and surfactant mixture or water and organic solvent mixture and stirring, followed by addition of basic catalyst. It is characterized by combining as.
본 발명에 있어서, “염료를 캡슐화한 실리카 나노입자”의 직경은 10~500nm인 것을 특징으로 한다.In the present invention, the diameter of the "silica nanoparticles encapsulated dye" is characterized in that 10 ~ 500nm.
본 발명에 있어서, 상기 “염료를 캡슐화한 실리카 나노입자-보론산”은 “염료를 캡슐화한 실리카 나노입자”를 아민화시킨 후 4-카르복실릭페닐 보론산(4-carboxylicphenyl boronic acid, CPBA)과 컨쥬게이션시키거나, “염료를 캡슐화한 실리카 나노입자”를 카르복실화시킨 후 3-아미노페닐 보론산(3-Aminophenyl boronic acid, APBA)과 컨쥬게이션시켜 제조된 것을 특징으로 한다.In the present invention, the "silica encapsulated silica nanoparticles-boronic acid" is a 4-carboxyphenyl boronic acid (CPBA) after amination of the "silica nanoparticles encapsulated dye" Or conjugated with, or “carboxylated silica nanoparticles” and then conjugated with 3-aminophenyl boronic acid (APBA).
본 발명에 있어서, 상기 광학기기는 전체 알부민과 특이적으로 결합하는 염료의 파장 및 “염료를 캡슐화한 실리카 나노입자-보론산”의 특이 파장을 동시에 광원을 조사하여 광학 반사도를 측정하는 것을 특징으로 한다.In the present invention, the optical device is characterized by measuring the optical reflectance by irradiating a light source simultaneously with the wavelength of the dye specifically binding to the whole albumin and the specific wavelength of "silica nanoparticle-boronic acid encapsulated dye" do.
본 발명에 있어서, 당화 알부민의 측정방법은 당화 알부민의 비율에 따라 당뇨병을 진단하는 것을 특징으로 한다.In the present invention, the method for measuring glycated albumin is characterized by diagnosing diabetes according to the ratio of glycated albumin.
본 발명에 따른 당화 알부민 측정용 시약 조성물은 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하기 때문에 염료 고유의 흡수파장이 pH에 영향을 받지 않고, 한달 이상 보관시에도 안정성이 우수한 장점이 있으며, 여러 개의 염료 분자를 하나의 실리카 나노입자 안에 캡슐화 함으로써, 입자 하나가 가지는 빛의 흡수량이 염료분자 1개의 흡수량보다 크기 때문에 낮은 검출한계를 갖는 혈중 당화 알부민 양을 정확하게 측정할 수 있다.Since the reagent composition for measuring saccharified albumin according to the present invention includes “silica nanoparticle-boronic acid encapsulated with dye”, the absorption wavelength of the dye is not affected by pH, and has excellent stability even when stored for a month or more. In addition, by encapsulating several dye molecules in one silica nanoparticle, the amount of light absorbed by one particle is greater than that of one dye molecule, thereby accurately measuring the amount of glycated albumin in the blood having a low detection limit.
도 1은 본 발명의 일 실시예에 따른 “염료를 캡슐화한 실리카 나노입자”의 제조방법을 나타낸 설명도이다.1 is an explanatory diagram showing a manufacturing method of "silica nanoparticles encapsulated dye" according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 “염료를 캡슐화한 실리카 나노입자-보론산”과 당화 알부민의 결합반응을 나타낸 설명도이다.Figure 2 is an explanatory view showing the coupling reaction of "silica nanoparticles-boronic acid encapsulated dye" and glycated albumin according to an embodiment of the present invention.
도 3은 본 발명의 당화 알부민 측정용 시약 조성물 및 광학기기를 이용하여 당화 알부민 및 전체 알부민을 측정방법을 나타낸 순서도이다. 3 is a flowchart illustrating a method for measuring glycated albumin and total albumin using the reagent composition and optical apparatus for measuring glycated albumin of the present invention.
도 4는 본 발명의 일 실시예에 따라 제조된 “염료를 캡슐화한 실리카 나노입자-보론산”의 흡수파장과 사용한 염료 각각의 흡수파장에 따른 흡광도를 나타내는 그래프이다.Figure 4 is a graph showing the absorbance according to the absorption wavelength of the "dye encapsulated silica nanoparticles-boronic acid" prepared according to an embodiment of the present invention and the dye used.
도 5는 본 발명의 일 실시예에 따라 제조된 “염료를 캡슐화한 실리카 나노입자-보론산”의 (A)주사전자현미경을 이용하여 측정한 형상 이미지 및 (B)동적광산란법을 이용하여 분석한 크기에 대한 그래프이다.Figure 5 is analyzed using a shape image and (B) dynamic light scattering method measured using (A) scanning electron microscope of "dyed encapsulated silica nanoparticles-boronic acid" prepared according to an embodiment of the present invention This is a graph about one size.
도 6은 농도를 알고있는 혈장 시료의 당화 알부민 농도에 따라 나타나는 “염료를 캡슐화한 실리카 나노입자-보론산”의 반사도 값을 나타낸 그래프이다.Figure 6 is a graph showing the reflectance value of "silica encapsulated silica nanoparticles-boronic acid" appear according to the concentration of glycated albumin of the plasma samples known concentration.
본 발명에서는 (a) 전체 알부민과 특이적으로 결합하는 염료 및 (b) 전체 알부민과 특이적으로 결합하는 염료와 보색 관계에 있는 염료를 캡슐화한 실리카 나노입자와 컨쥬게이션된 보론산을 이용할 경우 광학기기로 알부민 및 당화 알부민의 양을 간단하고 안정된 방법으로 정확하게 측정할 수 있다는 것을 확인하고자 하였다. According to the present invention, when (a) a dye specifically binding to total albumin and (b) a dye having a complementary relationship with a dye specifically binding to total albumin is used, silica nanoparticles encapsulated with boronic acid conjugated to The instrument was intended to confirm that the amount of albumin and glycated albumin can be accurately measured in a simple and stable manner.
본 발명에서는, 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 제조하고, 전체 알부민과 특이적으로 결합하는 염료를 추가하여 당화 알부민 측정을 위한 시약 조성물로 제조하였다. 다음으로, 제조된 시약 조성물 각각에 알부민 및 당화 알부민을 포함하는 혈액 또는 혈장샘플을 넣어 반응시키고, 흡수 패드에 투여한 후, 반응하지 않은 염료 및 불순물과 결합된 염료를 세척한 다음, 광학기기로 흡수 패드의 광학 반사도 측정하여 알부민 및 당화 알부민의 양을 측정하였다. 그 결과, 당화 알부민의 비율을 통하여 당뇨를 간단하고, 신속하게 진단할 수 있음을 확인하였다.In the present invention, "dye encapsulated silica nanoparticles-boronic acid" that specifically binds to glycated albumin was prepared, and a dye that specifically binds to whole albumin was added to prepare a reagent composition for measuring glycated albumin. . Next, blood or plasma samples containing albumin and glycated albumin were added to each of the prepared reagent compositions, reacted with an absorbent pad, and then washed with unreacted dyes and impurities combined with impurities. Optical reflectance of the absorbent pad was also measured to determine the amount of albumin and glycated albumin. As a result, it was confirmed that diabetes can be diagnosed simply and quickly through the ratio of glycated albumin.
즉, 본 발명의 일 실시예에서는 황색 계열의 타르트라진 염료를 실리카 나노입자에 캡슐화시키고, 표면에 있는 수산화 그룹(-OH)을 1차 아민기로 치환시킨 후, 당화 알부민 결합물질인 4-카르복실릭페닐보론산(4-carboxylicphenyl-boronic acid, CPBA)을 표면에 고정시켜 “타르트라진을 캡슐화한 실리카 나노입자-보론산”을 제조하고, 전체 알부민에 특이적으로 결합하는 염료인 브로모크레졸그린을 추가하여, 이들을 각각 포함하는 당화 알부민 측정을 위한 시약 조성물을 제조하였다. That is, in one embodiment of the present invention, the yellow tartarazine dye is encapsulated in the silica nanoparticles, and the hydroxyl group (-OH) on the surface is substituted with a primary amine group, and then 4-carboxyl is a glycated albumin binding material. Bromocresol green, a dye that binds 4-carboxylicphenyl-boronic acid (CPBA) to the surface to produce “silica nanoparticles-boronic acid encapsulating tartrazine” and binds specifically to albumin. In addition, a reagent composition for measuring glycated albumin, each containing them, was prepared.
다음으로 “타르트라진을 캡슐화한 실리카 나노입자-보론산”및 “브로모크레졸그린”각각에 걸러낸 혈장 샘플을 넣어 반응시키고, 흡수 패드에 투여한 후, 세척한 다음, 광학기기로 흡수 패드를 적색(430nm) 및 청색(630nm) 광원으로 조사하여 “타르트라진을 캡슐화한 실리카 나노입자-보론산”으로 표지된 당화 알부민 및 브로모크레졸그린으로 표지된 전체 알부민 각각의 광학 반사도를 측정함으로써, 당화 알부민의 비율을 간단하고 신속하게 측정할 수 있다는 것을 확인할 수 있었다.Next, the plasma samples filtered through the silica nanoparticles-boronic acid and bromocresol green encapsulating tarrazine are reacted, administered to an absorbent pad, washed, and then the absorbent pad is optically applied. Glycosylation by irradiation with red (430 nm) and blue (630 nm) light sources to measure the optical reflectivity of glycated albumin labeled with “silica nanoparticles-boronic acid encapsulating tartrazine” and total albumin labeled with bromocresolgreen, respectively. It was confirmed that the ratio of albumin can be measured simply and quickly.
따라서, 본 발명은 일 관점에서, 알부민과 특이적으로 결합하는 염료 및 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 당화 알부민 측정용 시약 조성물에 관한 것이다.Therefore, in one aspect, the present invention relates to a reagent composition for measuring glycated albumin comprising a dye that specifically binds albumin and “silica nanoparticle-boronic acid encapsulated with dyes” that specifically binds to albumin. .
본 발명에 있어서, 상기 전체 알부민과 특이적으로 결합하는 염료는 알부민 및 당화 알부민과 특이적으로 반응하는 염료라면 특별한 제한없이 사용할 수 있는데, 생리학적 중성 pH에서 청색이며, 620nm 흡수 파장대를 갖는 브로모크레졸그린(Bromocresol green)이나 자색이며, 580nm 흡수 파장대를 갖는 브로모크레졸퍼플(Bromocresol purple) 등을 예시할 수 있다.In the present invention, the dye that specifically binds to the total albumin can be used without particular limitation as long as it is a dye that specifically reacts with albumin and glycated albumin, bromo having a 620 nm absorption wavelength band in blue at physiological neutral pH. Examples thereof include bromocresol green and violet, and bromocresol purple having a 580 nm absorption wavelength band.
반면, 본 발명에 있어서, 실리카 나노입자에 캡슐화된 염료는 상기 전체 알부민과 특이적으로 결합하는 염료와 보색 관계에 있는 황색 계열 또는 적색 계열 염료를 사용하는 것을 특징으로 한다. 상기 황색 계열 염료는 약 400~430nm의 흡수파장을 갖고, 상기 적색 계열 염료는 약 500~530nm의 흡수파장을 갖는데, 황색 계열 염료로는 타르트라진(Tartrazine, 425nm), 적색 계열 염료로는 레드80(red 80, 528nm) 등을 예시할 수 있으나, 이에 한정되는 것은 아니다. On the other hand, in the present invention, the dye encapsulated in the silica nanoparticles is characterized by using a yellow-based or red-based dye having a complementary color relationship with the dye specifically binding to the whole albumin. The yellow dye has an absorption wavelength of about 400 ~ 430nm, the red dye has an absorption wavelength of about 500 ~ 530nm, yellow tar dye (Tartrazine, 425nm), red dye red 80 (red 80, 528 nm) and the like, but are not limited thereto.
도 1에 나타난 바와 같이, “염료를 캡슐화한 실리카 나노입자”는 물과 계면활성제 혼합액 또는 물과 유기용매 혼합액에 염료 및 실리카를 첨가하고 교반시킨 후, 염기성 촉매제를 첨가하여 제조할 수 있다. As shown in FIG. 1, "silica nanoparticles encapsulating dye" may be prepared by adding a dye and silica to a water and a surfactant mixture or a water and an organic solvent mixture, followed by stirring, and then adding a basic catalyst.
상기 계면활성제는 특별한 제한은 없으나, 본 발명에서는 트리톤 x-100 또는 n-헥산을 사용할 수 있으며, 상기 실리카는 테트라에틸오르소실리케이트(tetraethyl orthosilicate) 또는 테트라메틸오르소실리케이트(tetramethyl orthosilicate) 등을 예시할 수 있다.The surfactant is not particularly limited, but in the present invention, tritone x-100 or n-hexane may be used, and the silica may be exemplified by tetraethyl orthosilicate or tetramethyl orthosilicate. can do.
상기 염기성 촉매제는 실리카 전구체가 염료를 캡슐화시키는 것을 촉진하기 위한 것으로서, 물과 실리카 전구체의 가수분해를 촉진시킬 수 있다. 이렇게 생성된 이온화 상태의 실리카 전구체는 서로 반응하여 물 또는 알코올(ROH)을 배출하며 서로 연결되어 실리카 네트워크를 이루면서 커져가게 된다. The basic catalyst is to promote the silica precursor encapsulates the dye, it may promote the hydrolysis of the silica precursor with water. The silica precursor in the ionized state thus reacts with each other to release water or alcohol (ROH) and is connected to each other to form a silica network.
상기 염기성 촉매제는 암모늄 하이드록사이드, 테트라프로필암모늄 클로라이드, 테트라프로필암모늄 하이드록사이드, 테트라부틸암모늄 브로마이드, 테트라부틸암모늄 클로라이드 또는 테트라부틸암모늄 하이드록사이드 등을 예시할 수 있다.The basic catalyst may be exemplified by ammonium hydroxide, tetrapropylammonium chloride, tetrapropylammonium hydroxide, tetrabutylammonium bromide, tetrabutylammonium chloride or tetrabutylammonium hydroxide.
“염료를 캡슐화한 실리카 나노입자”는 염료가 외부로 유출되지 않기 때문에, 안정성 및 민감도를 증대시킬 수 있으며, 생체독성이 적고 손쉽게 표면의 작용기를 바꿀 수 있다. “Dye-encapsulated silica nanoparticles” can increase stability and sensitivity because dyes do not spill out, and are less biotoxic and can easily change surface functional groups.
상기 “염료를 캡슐화한 실리카 나노입자”의 직경은 10~500nm일 수 있는데, 염료 고유의 특성이 유지되는 30~100nm인 것이 바람직하다. 10nm 미만인 경우에는 조작이 어렵고, 500nm를 초과할 경우에는 두께가 두꺼워져 염료가 흐리게 보일 수 있다.The diameter of the "silica nanoparticles encapsulated dye" may be 10 ~ 500nm, it is preferably 30 ~ 100nm to maintain the unique properties of the dye. If it is less than 10 nm, the operation is difficult, and if it is more than 500 nm, the thickness becomes thick and the dye may appear cloudy.
“염료를 캡슐화한 실리카 나노입자”에 당화 알부민에 대한 선택성을 부여하기 위한 보론산 유도체는 4-카르복실릭페닐 보론산(4-carboxylicphenyl boronic acid, CPBA)과 3-아미노페닐 보론산(3-Aminophenyl boronic acid, APBA)를 사용하는 것이 바람직한데, CPBA를 사용할 경우 “염료를 캡슐화한 실리카 나노입자”를 아민화시키고, APBA를 사용할 경우 “염료를 캡슐화한 실리카 나노입자”를 카르복실화시킨 다음 카르보디이미드(Carbodiimide) 교차 커플링을 통하여 컨쥬게이션시킬 수 있다. 4-카르복실릭페닐 보론산(4-carboxylicphenyl boronic acid, CPBA)는 상대적으로 3-아미노페닐 보론산(3-Aminophenyl boronic acid, APBA)에 비하여 상대적으로 열적 안정성이 높기 때문에 CPBA를 사용하는 것이 바람직하다.Boronic acid derivatives to impart selectivity to glycated albumin to “silica nanoparticles encapsulated with dyes” include 4-carboxylicphenyl boronic acid (CPBA) and 3-aminophenyl boronic acid (3- Aminophenyl boronic acid (APBA) is preferred, where CPBA is used to amine “dye encapsulated silica nanoparticles”, and APBA is used to carboxylate “dye encapsulated silica nanoparticles” Conjugation may be via carbodiimide cross coupling. It is preferable to use CPBA because 4-carboxylicphenyl boronic acid (CPBA) has relatively higher thermal stability than 3-aminophenyl boronic acid (APBA). Do.
도 2에 도시된 바와 같이, “염료를 캡슐화한 실리카 나노입자-보론산”은 당화 알부민의 시스-다이올과 반응할 수 있으며, 여러 개의 염료분자가 실리카 나노입자내에 캡슐화되어 있어, 1개의 염료분자보다 빛의 흡수량이 높으므로, 당화 알부민의 검출한계를 향상시킬 수 있다. As shown in FIG. 2, “Dye-encapsulated silica nanoparticles-boronic acid” can react with cis-diol of glycated albumin, and several dye molecules are encapsulated in silica nanoparticles, so that one dye Since the absorption of light is higher than that of molecules, the detection limit of glycated albumin can be improved.
도 3에 도시된 바와 같이, 혈액 또는 혈장용액을 당화 알부민을 표지할 수 있는 “염료를 캡슐화한 실리카 나노입자-보론산”과 반응 시킨 후 카트리지에 적가 후 세척하여 당화 알부민의 반사도 값을 기록하고, 이후 전체 알부민을 염색할 수 있는 브로모크레졸그린(BCG) 용액과 반응시키고 카트리지에 적가 후 세척하여 전체 알부민의 반사도 값을 기록하여 당화 알부민의 반사도 대비 전체 알부민의 반사도를 통해 당화 알부민의 %를 계산할 수 있다. 이때 전체 알부민을 먼저 측정한 다음 당화 알부민을 측정하여도 당화 알부민의 %를 계산할 수 있다.As shown in FIG. 3, the blood or plasma solution was reacted with “dye-encapsulated silica nanoparticles-boronic acid” capable of labeling glycated albumin, and then added dropwise to the cartridge to wash and record the reflectance value of glycated albumin. Then, react with a bromocresolgreen (BCG) solution that can stain the whole albumin, drop it into a cartridge and wash it to record the total albumin reflectivity value.The percentage of glycated albumin is compared to the total albumin reflectivity compared to the reflectivity of glycated albumin. Can be calculated At this time, the total albumin can be measured first, and then the glycated albumin can be measured, but the percentage of glycated albumin can be calculated.
따라서, 본 발명은 다른 관점에서, (a) 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (b) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계; (c) 광학기기로 흡수 패드의 광학 반사도 측정하여 당화 알부민의 양을 측정하는 단계; (d) 전체 알부민과 특이적으로 결합하는 염료를 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (e) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계; (f) 광학기기로 흡수 패드의 광학 반사도를 측정하여 전체 알부민의 양을 측정하는 단계 및; (g) 측정된 당화 알부민 및 전체 알부민의 양을 기반으로 당화 알부민의 비율을 계산하는 단계를 포함하는 당화 알부민의 측정방법에 관한 것이다.Accordingly, the present invention in another aspect, the method comprising the steps of: (a) adding a blood or plasma solution to a reagent containing "silica nanoparticles-boronic acid encapsulated dye" that specifically binds to glycated albumin; (b) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (c) measuring the amount of glycated albumin by measuring the optical reflectivity of the absorbent pad with an optical instrument; (d) adding blood or plasma solution to a reagent containing a dye that specifically binds to whole albumin and reacting the same; (e) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution; (f) measuring the optical reflectivity of the absorbent pad with optics to determine the total amount of albumin; (g) calculating a ratio of glycated albumin based on the measured amounts of glycated albumin and total albumin.
본 발명에 있어서, 상기 광학기기는 광학 특성을 이용하여 광학 반사도를 측정할 수 있는 것이라면 특별한 제한없이 사용할 수 있으며, 전체 알부민과 특이적으로 결합하는 염료(청색 또는 자색)의 파장 및 “염료(황색 또는 적색)를 캡슐화한 실리카 나노입자-보론산”의 특이 파장을 동시에 특정 파장을 발생할 수 있는 광원(예: 청색(630nm), 적색(430nm))로 조사하고, 반사된 광신호를 포토다이오드 검출기(photodiode detector, PD)로 측정하여 광신호 변환기를 통해 알부민 및 당화 알부민의 양을 각각 측정할 수 있다. In the present invention, the optical device can be used without particular limitation as long as it can measure the optical reflectivity using optical properties, and the wavelength of the dye (blue or purple) that specifically binds to the whole albumin and the "dye (yellow) Or red light-encapsulated silica nanoparticles-boronic acid ”using a light source (eg, blue (630 nm) or red (430 nm)) capable of generating a specific wavelength at the same time, and reflecting the optical signal to the photodiode detector. By measuring with photodiode detector (PD), the amount of albumin and glycated albumin can be measured by optical signal converter.
당화 알부민의 비율은 전체 알부민 양에 따른 당화 알부민의 상대적인 양을 다음 식으로 계산할 수 있다.The ratio of glycated albumin can be calculated by the relative amount of glycated albumin according to the total albumin amount by the following equation.
당화 알부민 비율(%)=당화 알부민/전체 알부민Glycosylated albumin ratio (%) = Glycosylated albumin / total albumin
일반적으로 상기 당화 알부민의 비율이 16% 이상인 경우 당뇨병으로 진단할 수 있다.In general, when the ratio of the glycated albumin is 16% or more can be diagnosed as diabetes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 황색 염료를 캡슐화한 실리카 나노입자-보론산(YD@SNP-CPBA) 제조Example 1: Preparation of silica nanoparticle-boronic acid (YD @ SNP-CPBA) encapsulated yellow dye
1-1: 황색 염료를 캡슐화한 실리카 나노입자(YD@SNP) 합성1-1: Synthesis of Silica Nanoparticles (YD @ SNP) Encapsulated with Yellow Dye
사이클로헥세인(cyclohexane) 135.0㎖, 트리톤 X-100 31.8㎖, n-헥산올 (n-hexanol) 32.4㎖, 0.1M 타르트라진 6.12㎖, TEOS(tetraethyl orthosilicate) 2.7㎖을 1L 둥근 바닥 플라스크에 넣고 교반기를 이용하여 1시간 동안 균일하게 혼합시켰다. 25~30% 암모니아수(NH4OH) 1.08㎖를 넣고 상온에서 24시간 동안 반응시킨후 에탄올 200㎖을 첨가하여 반응을 종결하고, 원심분리기를 이용하여 3800rpm, 15분의 조건으로 4회 에탄올 세척과 3회 DI 세척한 후 60℃ 오븐에 넣고 건조시켰다. 135.0 ml of cyclohexane, 31.8 ml of Triton X-100, 32.4 ml of n-hexanol, 6.12 ml of 0.1 M tartrazine, and 2.7 ml of TEOS (tetraethyl orthosilicate) were placed in a 1 L round bottom flask and agitated. Was mixed uniformly for 1 hour. 1.08ml of 25 ~ 30% ammonia water (NH 4 OH) was added and reacted for 24 hours at room temperature. Then, 200ml of ethanol was added to terminate the reaction. The mixture was washed 4 times with 3800rpm and 15 minutes using a centrifuge. After washing DI three times, it was put in an oven at 60 ° C. and dried.
1-2: 황색 염료를 캡슐화한 실리카 나노입자(YD@SNP)의 아민화1-2: Amination of Silica Nanoparticles (YD @ SNP) Encapsulated with Yellow Dye
YD@SNP와 CPBA의 카르복실기의 교차 결합을 위하여, YD@SNP의 표면에 있는 수산화 그룹(-OH)을 1차 아민기로 치환하였다. 즉, YD@SNP 100㎎을 100㎖의 에탄올에 넣고, 초음파 분산기를 이용하여 30분간 분산시킨 후, APTES (3-Aminopropyltriethoxysilane) 1㎖을 교반기에 넣고 2시간 동안 상온에서 반응시켰다. 반응 후, 원심분리기를 이용하여 3800rpm, 15분의 조건으로 4회 에탄올 세척과 3회 DI 세척한 후 60℃ 오븐에 넣고 건조시켜 아민화된 YD@SNP (YD@SNP-NH2)를 제조하였다. For crosslinking of the carboxyl groups of YD @ SNP and CPBA, the hydroxyl group (-OH) on the surface of YD @ SNP was substituted with a primary amine group. That is, 100 mg of YD @ SNP was added to 100 ml of ethanol, dispersed for 30 minutes using an ultrasonic disperser, and then 1 ml of APTES (3-Aminopropyltriethoxysilane) was added to a stirrer and reacted at room temperature for 2 hours. After the reaction, 4 times ethanol wash and 3 times DI wash at 3800 rpm and 15 minutes using a centrifuge were placed in an oven at 60 ° C. and dried to prepare aminated YD @ SNP (YD @ SNP-NH 2 ). .
1-3: 아민화된 황색 염료를 캡슐화한 실리카 나노입자(YD@SNP-NH2) 및 CPBA의 접합1-3: Conjugation of CPBA with Silica Nanoparticles (YD @ SNP-NH 2 ) Encapsulating Aminated Yellow Dye
당화 알부민과의 결합능을 부여하기 위하여, 카르복실기와 1차 아민기를 연결하는 교차결합제인 1-에틸-3[3-디메틸 아미노프로필]카르보디이미드 하이드로클로라이드(EDC)를 이용한 카르보디이미드 교차 커플링 방법으로 당화 알부민 결합물질인 4-카르복실릭페닐보론산(4-carboxylicphenyl-boronic acid, CPBA)을 아민화된 황색 염료를 캡슐화한 실리카 나노입자(YD@SNP-NH2) 표면에 고정화시켰다. Carbodiimide cross-coupling method using 1-ethyl-3 [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) which is a crosslinking agent linking a carboxyl group with a primary amine group in order to impart binding ability with glycated albumin The glycosylated albumin-binding compound, 4-carboxylicphenyl-boronic acid (CPBA), was immobilized on the surface of silica nanoparticles (YD @ SNP-NH 2 ) encapsulating the aminated yellow dye.
즉, 빛이 차단된 환경에서, CPBA의 카르복실 작용기를 활성화시키기 위하여 3.48mM CPBA를 0.1M의 MES(2-(N-Morpholino)ethanesulfonic acid) 완충용액 (pH 6.0)에 녹이고 최종농도 1mM의 EDC를 넣고 30분간 교반하며 반응시킨 후, YD@SNP-NH2를 첨가한 후 10~20시간 동안 교반기에서 상온 반응시켰다. That is, in a light-blocked environment, 3.48 mM CPBA was dissolved in 0.1 M MES (2- (N-Morpholino) ethanesulfonic acid) buffer (pH 6.0) to activate the carboxyl functional groups of CPBA, and the final concentration was 1 mM EDC. After the reaction was stirred for 30 minutes, YD @ SNP-NH 2 was added, and the reaction was performed at room temperature for 10-20 hours in a stirrer.
반응이 완료된 후, 원심분리기를 이용하여 3800rpm, 15분의 조건으로 4회 에탄올 세척과 3회 DI 세척한 후 상온 또는 동결건조하여 황색 염료를 캡슐화한 실리카 나노입자-보론산(YD@SNP-CPBA)를 제조하였다. After the reaction was completed, silica nanoparticle-boronic acid (YD @ SNP-CPBA) encapsulated a yellow dye by washing with ethanol 4 times and DI DI 3 times at 3800rpm, 15 minutes using a centrifuge at room temperature or lyophilization (YD @ SNP-CPBA). ) Was prepared.
합성에 사용한 “타르트라진”염료와 “염료가 캡슐화된 실리카 나노입자-보론산”각각을 탈이온수(DI water)로 희석한 뒤 UV/Vis spectroscopy를 측정해 본 결과 도 4에 도시된 바와 같이 “염료가 캡슐화된 실리카 나노입자-보론산"은 합성 이후에도 황색 염료 고유의 흡수파장을 가지고 있음을 알 수 있으며, 이를 주사전자현미경과 동적광산란법을 이용하여 분석한 결과, 도 5에 도시된 바와 같이 입자의 형태가 균일하고, 크기가 약 35~45nm인 나노입자가 합성된 것을 확인할 수 있었다.After diluting each of the “tartrazine” dye and “dye-encapsulated silica nanoparticles-boronic acid” used in the synthesis with DI water, UV / Vis spectroscopy was measured. As shown in FIG. The dye encapsulated silica nanoparticles-boronic acid "can be seen that even after synthesis has a unique absorption wavelength of yellow dye, and analyzed using a scanning electron microscope and dynamic light scattering method, as shown in Figure 5 It was confirmed that nanoparticles having a uniform particle shape and a size of about 35 to 45 nm were synthesized.
실시예Example 2: YD@SNP-CPBA 및  2: YD @ SNP-CPBA and 브로모크레졸그린을Bromocresol green 포함하는  Containing 당화Saccharification 알부민 측정용 시약 조성물을 이용한 당화 알부민 측정  Determination of glycated albumin using reagent composition for albumin measurement
2-1: 당화 알부민 측정2-1: glycated albumin measurement
실시예 1에서 제조된 YD@SNP-CPBA을 포함하는 시약 조성물 (ZnCl2, NaCl, MgCl2, Triton X-100, NaN3, glycine, HEPES, pH 8.1) 200㎕을 갈색 튜브에 넣고, 기준시약(아사히 가세이 GA-L)을 이용하여 올림푸스 AU400 분석기로 당화알부민의 %값을 측정한 혈장 샘플 5㎕을 첨가한 후, 2분간 반응시켰다. 반응액 25㎕을 광학기기(에피소드® 616, 딕스젠)의 카트리지 흡수 패드에 올려 15초 동안 흡수시키고, 세척 용액(Morpholine, NaCl, Triton X-100, Glycerol, 및 NaN3 혼합액) 25㎕을 넣고 15초 동안 세척시켰다. 다음으로, 카트리지를 광학기기(에피소드®616, 딕스젠)에서 황색의 당화 알부민의 광학 반사도를 측정하였다.200 μl of the reagent composition (ZnCl 2 , NaCl, MgCl 2 , Triton X-100, NaN 3 , glycine, HEPES, pH 8.1) prepared in Example 1 was placed in a brown tube, and the reference reagent was prepared. (Asahi Kasei GA-L) was added to 5 μl of a plasma sample whose% value of glycated albumin was measured by an Olympus AU400 analyzer, followed by reaction for 2 minutes. 25 μl of the reaction solution was placed on the cartridge absorption pad of optics (Episode ® 616, Dixsen) for 15 seconds, absorbed and 25 μl of washing solution (Morpholine, NaCl, Triton X-100, Glycerol, and NaN 3 mixture) was added. Wash for 15 seconds. Next, a cartridge for an optical device (episodes ® 616, Dix Xen) was measured optical reflectivity of the glycated albumin in the yellow.
2-2: 전체 알부민 측정2-2: Total Albumin Determination
브로모크레졸그린을 포함하는 시약 조성물 (Succinic acid, pH 5.5) 200㎕을 갈색 튜브에 넣고, 동일한 혈장샘플 5㎕을 첨가한 후, 2분간 반응시켰다. 반응액 25㎕을 광학기기(에피소드® 616, 딕스젠)의 카트리지 흡수 패드에 올려 15초 동안 흡수시키고, 세척 용액(Morpholine, NaCl, Triton X-100, Glycerol, 및 NaN3 혼합액) 25㎕을 넣고 15초 동안 세척시켰다. 다음으로, 카트리지를 광학기기(에피소드®616, 딕스젠)에서 청색의 전체 알부민의 광학 반사도를 측정하였다. 200 µl of the reagent composition (Succinic acid, pH 5.5) containing bromocresol green was placed in a brown tube, 5 µl of the same plasma sample was added, and the reaction was carried out for 2 minutes. 25 μl of the reaction solution was placed on the cartridge absorption pad of optics (Episode ® 616, Dixsen) for 15 seconds, absorbed and 25 μl of washing solution (Morpholine, NaCl, Triton X-100, Glycerol, and NaN 3 mixture) was added. Wash for 15 seconds. Next, in the cartridge optics (episodes ® 616, Dix Xen) it was measured optical reflectivity of the total albumin in the blue.
2-3: K/S값을 이용한 당화 알부민 측정2-3: Determination of glycated albumin using K / S value
2-1에서 측정된 당화 알부민의 광학 반사도 및 2-2에서 측정된 전체 알부민의 광학 반사도를 비교하여 당화 알부민의 %값을 결정하였다. 각각의 파장으로부터 측정된 %반사도(%R)는 얼마만큼의 색을 띠는 물질이 해당 표면 있는지에 대한 정량지표인 K/S 값으로 변환하여 사용하였으며, %반사도를 K/S 값으로 변환하는 공식은 아래와 같다.The% reflectivity of glycated albumin was determined by comparing the optical reflectivity of glycated albumin measured in 2-1 and the optical reflectivity of total albumin measured in 2-2. The% reflectance (% R) measured from each wavelength was converted into K / S value, which is a quantitative indicator of how much colored material is on the surface, and% reflectance is converted into K / S value. The formula is:
Figure PCTKR2017010499-appb-I000001
(K=흡수계수, S= 산란계수)
Figure PCTKR2017010499-appb-I000001
(K = absorption coefficient, S = scattering coefficient)
따라서, 당화 알부민의 양을 대변하는 황색광원을 조사하여 얻은 %반사도값과 전체 알부민의 양을 대변하는 청색광원으로부터 얻은 %반사도값을 각각 K/S값으로 치환한 뒤 이들의 비율을 계산함으로써 당화 알부민의 양을 측정할 수 있었다. Therefore, the% reflectance value obtained by investigating the yellow light source representing the amount of glycated albumin and the% reflectance value obtained from the blue light source representing the total amount of albumin are respectively substituted by K / S values, and then the ratio is calculated by saccharification. The amount of albumin could be measured.
도 6에 도시된 바와 같이, 혈액 내에 포함된 당화 알부민의 비율이 증가함에 따라 상대적으로 결합한 “염료가 캡슐화된 실리카 나노입자-보론산”의 양(K/S값)이 높아지는 것을 알 수 있었다. 따라서, 본 발명에 따른 당화알부민의 측정방법은 당뇨병 진단에 널리 활용될 수 있다는 것을 확인하였다.As shown in FIG. 6, as the ratio of glycated albumin contained in the blood was increased, the amount (K / S value) of the relatively bound “dye-encapsulated silica nanoparticles-boronic acid” increased. Therefore, it was confirmed that the method for measuring glycated albumin according to the present invention can be widely used for diagnosing diabetes.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 당화 알부민 측정용 시약 조성물은 전체 알부민과 당화 알부민을 각각 표지하여 구별할 수 있는 염료를 포함하고 있어, 별도의 분리과정 없이 측정 카트리지에 세척액 투여만을 통해 광학분석기로 간단하게 당화 알부민을 측정할 수 있으므로 당뇨병 진단에 널리 이용될 수 있다.The reagent composition for measuring glycated albumin according to the present invention includes a dye which can distinguish the total albumin and the glycated albumin, respectively, and thus, the glycated albumin can be easily obtained by using an optical analyzer through administration of a washing solution to a measurement cartridge without a separate separation process. Because it can be measured, it can be widely used to diagnose diabetes.

Claims (11)

  1. (a) 전체 알부민과 특이적으로 결합하는 염료 및 (b) 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 당화 알부민 측정용 시약 조성물.A reagent composition for measuring glycated albumin comprising (a) a dye specifically binding to total albumin and (b) “silica nanoparticle-boronic acid encapsulated with dye” specifically binding to glycated albumin.
  2. 제1항에 있어서, 상기 전체 알부민과 특이적으로 결합하는 염료는 브로모크레졸그린(Bromocresol green) 또는 브로모크레졸퍼플(Bromocresol purple)인 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The reagent composition for measuring glycated albumin according to claim 1, wherein the dye that specifically binds to the whole albumin is bromocresol green or bromocresol purple.
  3. 제1항에 있어서, 실리카 나노입자에 캡슐화된 염료는 황색 계열 또는 적색 계열 염료인 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The reagent composition for measuring glycated albumin according to claim 1, wherein the dye encapsulated in the silica nanoparticles is a yellow or red dye.
  4. 제3항에 있어서, 상기 황색 계열 염료는 400~430nm의 흡수파장을 갖고, 상기 적색 계열 염료는 500~530nm의 흡수파장을 갖는 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The reagent composition for measuring glycated albumin according to claim 3, wherein the yellow dye has an absorption wavelength of 400 to 430 nm, and the red dye has an absorption wavelength of 500 to 530 nm.
  5. 제3항에 있어서, 상기 황색 계열 염료는 타르트라진(Tartrazine)이고, 적색 계열 염료는 레드80인 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The reagent composition for measuring glycated albumin according to claim 3, wherein the yellow dye is tarrazine, and the red dye is red 80.
  6. 제1항에 있어서, “염료를 캡슐화한 실리카 나노입자”는 물과 계면활성제 혼합액 또는 물과 유기용매 혼합액에 염료 및 실리카를 첨가하고 교반시킨 후, 염기성 촉매제를 첨가하여 제조시킨 것으로서 당화 알부민과 특이적으로 결합하는 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The method of claim 1, wherein the "silica nanoparticles encapsulated with dye" is prepared by adding dye and silica to water and surfactant mixture or water and organic solvent mixture and stirring, followed by addition of basic catalyst. Reagent composition for the measurement of glycated albumin, characterized in that coupled to the enemy.
  7. 제1항에 있어서, “염료를 캡슐화한 실리카 나노입자”의 직경은 10~500nm인 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The reagent composition for measuring glycated albumin according to claim 1, wherein the diameter of the “silica nanoparticles encapsulating the dye” is 10 to 500 nm.
  8. 제1항에 있어서, 상기 “염료를 캡슐화한 실리카 나노입자-보론산”은 “염료를 캡슐화한 실리카 나노입자”를 아민화시킨 후 4-카르복실릭페닐 보론산(4-carboxylicphenyl boronic acid, CPBA)과 컨쥬게이션시키거나, “염료를 캡슐화한 실리카 나노입자”를 카르복실화시킨 후 3-아미노페닐 보론산(3-Aminophenyl boronic acid, APBA)과 컨쥬게이션시켜 제조된 것을 특징으로 하는 당화 알부민 측정용 시약 조성물.The method of claim 1, wherein the “silica encapsulated silica nanoparticles-boronic acid” is a 4-carboxylicphenyl boronic acid (CPBA) after amination of the “silica encapsulated silica nanoparticles” ) Or glycated albumin, characterized in that it is prepared by conjugating with 3-aminophenyl boronic acid (APBA) after carboxylating “silica nanoparticles encapsulated with dye”. Reagent composition.
  9. (a) 당화 알부민과 특이적으로 결합하는 “염료를 캡슐화한 실리카 나노입자-보론산”을 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (a) adding blood or plasma solution to a reagent comprising “silica encapsulated silica nanoparticles-boronic acid” that specifically binds to glycated albumin;
    (b) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계;(b) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution;
    (c) 광학기기로 흡수 패드의 광학 반사도 측정하여 당화 알부민의 양을 측정하는 단계; (c) measuring the amount of glycated albumin by measuring the optical reflectivity of the absorbent pad with an optical instrument;
    (d) 전체 알부민과 특이적으로 결합하는 염료를 포함하는 시약에 혈액 또는 혈장용액을 넣고 반응시키는 단계; (d) adding blood or plasma solution to a reagent containing a dye that specifically binds to whole albumin and reacting the same;
    (e) 반응물을 카트리지의 흡수 패드에 투여한 후 세척액으로 세척하는 단계; (e) administering the reactants to an absorption pad of the cartridge followed by washing with a wash solution;
    (f) 광학기기로 흡수 패드의 광학 반사도를 측정하여 전체 알부민의 양을 측정하는 단계 및;(f) measuring the optical reflectivity of the absorbent pad with optics to determine the total amount of albumin;
    (g) 측정된 당화 알부민 및 전체 알부민의 양을 기반으로 당화 알부민의 비율을 계산하는 단계를 포함하는 당화 알부민의 측정방법.(g) calculating the ratio of glycated albumin based on the measured amounts of glycated albumin and total albumin.
  10. 제9항에 있어서, 상기 광학기기는 전체 알부민과 특이적으로 결합하는 염료의 파장 및 “염료를 캡슐화한 실리카 나노입자-보론산”의 특이 파장을 동시에 광원으로 조사하여 광학 반사도를 측정하는 것을 특징으로 하는 당화 알부민의 측정방법.10. The optical apparatus of claim 9, wherein the optical device measures optical reflectivity by simultaneously irradiating a wavelength of a dye specifically binding to total albumin and a specific wavelength of “silica nanoparticle-boronic acid encapsulating dye” with a light source. Method for measuring saccharified albumin
  11. 제9항에 있어서, 상기 당화 알부민의 비율에 따라 당뇨병을 진단하는 것을 특징으로 하는 당화 알부민의 측정방법.The method of measuring glycated albumin according to claim 9, wherein diabetes is diagnosed according to the ratio of glycated albumin.
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