REAGENT COMPOSITION FOR MEASURING GLYCATED ALBUMIN AND METHOD FOR MEASURING GLYCATED ALBUMIN USING SAME

Abstract

Provided is a reagent composition for measuring glycated albumin to diagnose the presence or absence of diabetes and a method of measuring glycated albumin using the same, and more particularly is a reagent composition for measuring glycated albumin, the composition including a dye-encapsulated silica nanoparticle-boronic acid, and to a method of measuring glycated albumin using the same. In the reagent composition for measuring glycated albumin, since a dye is encapsulated in silica nanoparticles, 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.

Claims

1. A method of measuring glycated albumin, the method comprising: (a) introducing blood or a plasma solution into a reagent including a dye-encapsulated silica nanoparticle-boronic acid specifically binding to the glycated albumin, followed by reaction; (b) injecting a reactant into an absorption pad of a cartridge, followed by washing with a washing liquid; (c) measuring an optical reflectance of the absorption pad using an optical instrument to measure an amount of the glycated albumin; (d) introducing the blood or the plasma solution into a reagent including a dye specifically binding to total albumin, followed by reaction; (e) injecting a reactant into the absorption pad of the cartridge, followed by washing with the washing liquid; (f) measuring an optical reflectance of the absorption pad using the optical instrument to measure an amount of the total albumin; and (g) calculating a ratio of the glycated albumin on a basis of measured amounts of the glycated albumin and the total albumin.

2. The method of claim 1, wherein the optical instrument simultaneously radiates a wavelength of the dye specifically binding to the total albumin and a specific wavelength of the dye-encapsulated silica nanoparticle-boronic acid as light sources, thus measuring the optical reflectance.

3. The method of claim 1, wherein diabetes is diagnosed according to the ratio of the glycated albumin.

Description

DESCRIPTION OF DRAWINGS

[0029] FIG. 1 is a view showing a method of manufacturing dye-encapsulated silica nanoparticles according to an embodiment of the present invention;

[0030] FIG. 2 is a view showing a binding reaction of a dye-encapsulated silica nanoparticle-boronic acid and glycated albumin according to the embodiment of the present invention;

[0031] FIG. 3 is a flowchart showing a method of measuring glycated albumin and total albumin using a reagent composition for measuring glycated albumin of the present invention and an optical instrument;

[0032] FIG. 4 is a graph showing the absorbance depending on the absorption wavelength of the dye-encapsulated silica nanoparticle-boronic acid manufactured according to the embodiment of the present invention and the absorption wavelength of each of the dyes that are used;

[0033] FIG. 5A shows the shape image of the dye-encapsulated silica nanoparticle-boronic acid manufactured according to the embodiment of the present invention, measured using a scanning electron microscope, and FIG. 5B is a graph showing the size thereof, analyzed using a dynamic light-scattering method; and

[0034] FIG. 6 is a graph showing the reflectance value of the dye-encapsulated silica nanoparticle-boronic acid depending on the glycated albumin concentration of a plasma sample, the concentration of which is known.

BEST MODE

[0035] In the present invention, the intention is to confirm that when using boronic acid conjugated with dye-encapsulated silica nanoparticles including (a) a dye specifically binding to total albumin and (b) a dye that is complementary in color to the dye specifically binding to total albumin, the amounts of albumin and glycated albumin are capable of being measured accurately in a simple and stable manner using an optical instrument.

[0036] In the present invention, a dye-encapsulated silica nanoparticle-boronic acid specifically binding to glycated albumin was manufactured, and a dye specifically binding to total albumin was added thereto, thus manufacturing a reagent composition for measuring glycated albumin. Next, blood or a plasma sample containing albumin and glycated albumin was added to the manufactured reagent composition to perform reaction, and was then injected into an absorption pad, followed by washing of the unreacted dye and the dye binding to impurities. The optical reflectance of the absorption pad was then measured using the optical instrument in order to measure the amounts of albumin and glycated albumin. As a result, it was confirmed that it was possible to diagnose diabetes simply and quickly using the ratio of glycated albumin.

[0037] That is, in an embodiment of the present invention, a yellow-colored tartrazine dye was encapsulated in silica nanoparticles, the hydroxyl group (OH) on the surface thereof was substituted with a primary amine group, and 4-carboxylicphenyl-boronic acid (CPBA), which is a glycated albumin-binding material, was fixed to the surface thereof, thus manufacturing a tartrazine-encapsulated silica nanoparticle-boronic acid. Bromocresol green, which is a dye specifically binding to total albumin, was added thereto, thus manufacturing a reagent composition for measuring glycated albumin, the composition including the same.

[0038] Next, the filtered plasma sample was added to the tartrazine-encapsulated silica nanoparticle-boronic acid and bromocresol green to perform reaction, and was then injected into the absorption pad, followed by washing. Red (430 nm) and blue (630 nm) light sources were radiated on the absorption pad using the optical instrument to thus measure the optical reflectance of each of glycated albumin labeled with the tartrazine-encapsulated silica nanoparticle-boronic acid and total albumin labeled with bromocresol green. Thereby, it could be confirmed that the ratio of glycated albumin was capable of being measured simply and quickly.

[0039] Therefore, in an aspect, the present invention relates to a reagent composition for measuring glycated albumin, the composition including a dye specifically binding to albumin and a dye-encapsulated silica nanoparticle-boronic acid specifically binding to glycated albumin.

[0040] In the present invention, any dye specifically binding to total albumin may be used without particular limitation, as long as the dye is a dye that specifically reacts with albumin and glycated albumin. Examples thereof may include bromocresol green, which is blue and has an absorption wavelength band of 620 nm, or bromocresol purple, which is purple and has an absorption wavelength band of 580 nm at a physiologically neutral pH.

[0041] On the other hand, in the present invention, as the dye encapsulated in the silica nanoparticles, a yellow-colored dye or a red-colored dye that is complementary in color to the dye specifically binding to total albumin is used. The yellow-colored dye has an absorption wavelength of about 400 to 430 nm, and the red-colored dye has an absorption wavelength of about 500 to 530 nm. Examples of the yellow-colored dye may include tartrazine (425 nm), and examples of the red-colored dye may include red 80 (528 nm), without being limited thereto.

[0042] As shown in FIG. 1, the dye-encapsulated silica nanoparticles may be manufactured by adding a dye and silica to a mixture solution of water and a surfactant or a mixture solution of water and an organic solvent, followed by agitation and then addition of a basic catalyst. The surfactant is not particularly limited, but triton x-100 or n-hexane may be used in the present invention. Examples of the silica may include tetraethyl orthosilicate or tetramethyl orthosilicate.

[0043] The basic catalyst is to promote the encapsulation of the dye by a silica precursor, which may promote the hydrolysis of water and the silica precursor. The ionized silica precursors react with each other to thus produce water and alcohol (ROH), which are connected to each other to thus form a silica network and grow.

[0044] Examples of the basic catalyst may include ammonium hydroxide, tetrapropylammonium chloride, tetrapropylammonium hydroxide, tetrabutylammonium bromide, tetrabutylammonium chloride, or tetrabutylammonium hydroxide.

[0045] In the case of the dye-encapsulated silica nanoparticles, since the dye does not leak to the outside, stability and sensitivity may be increased, bio-toxicity may be low, and the functional groups on the surface thereof may be easily changed.

[0046] The diameter of the dye-encapsulated silica nanoparticles may be 10 to 500 nm, and preferably 30 to 100 nm, which makes it possible to maintain the inherent properties of the dye. When the diameter is less than 10 nm, it is difficult to perform the operation. When the diameter is more than 500 nm, since the thickness thereof is increased, the dye may appear cloudy.

[0047] As boronic acid derivatives for imparting selectivity for glycated albumin to the dye-encapsulated silica nanoparticles, it is preferable to use 4-carboxylicphenyl boronic acid (CPBA) and 3-aminophenyl boronic acid (APBA). When the CPBA is used, the dye-encapsulated silica nanoparticles may be aminated. When the APBA is used, after the dye-encapsulated silica nanoparticles are carboxylated, conjugation may be performed via carbodiimide cross-coupling. Since the 4-carboxylicphenyl boronic acid (CPBA) has relatively higher thermal stability than the 3-aminophenyl boronic acid (APBA), it is preferable to use the CPBA.

[0048] As shown in FIG. 2, the dye-encapsulated silica nanoparticle-boronic acid may react with the cis-diol of glycated albumin. A plurality of dye molecules is encapsulated in the silica nanoparticles, so that the amount of light absorbed by one particle is larger than that absorbed by one dye molecule. Accordingly, the detection limit of glycated albumin may be improved.

[0049] As shown in FIG. 3, blood or the plasma solution may be reacted with a dye-encapsulated silica nanoparticle-boronic acid capable of labeling glycated albumin, dropped on the cartridge, and washed, and the reflectance value of glycated albumin may then be recorded. Thereafter, the resultant substance may be reacted with a bromocresol green (BCG) solution capable of dyeing total albumin, dropped on the cartridge, and washed, and the reflectance value of total albumin may then be recorded. Thereby, the % value of glycated albumin may be calculated using the reflectance of total albumin relative to the reflectance of glycated albumin. After total albumin is measured, glycated albumin may be measured in order to calculate the % value of glycated albumin.

[0050] Accordingly, in another aspect, the present invention relates to a method of measuring glycated albumin. The method includes (a) introducing blood or a plasma solution into a reagent including a dye-encapsulated silica nanoparticle-boronic acid specifically binding to glycated albumin, followed by reaction, (b) injecting a reactant into an absorption pad of a cartridge, followed by washing with a washing liquid, (c) measuring the optical reflectance of the absorption pad using an optical instrument to measure the amount of glycated albumin, (d) introducing blood or a plasma solution into a reagent including a dye specifically binding to total albumin, followed by reaction, (e) injecting a reactant into an absorption pad of a cartridge, followed by washing with a washing liquid, (f) measuring the optical reflectance of the absorption pad using the optical instrument to measure the amount of total albumin, and (g) calculating a ratio of glycated albumin on the basis of the measured amounts of glycated albumin and total albumin.

[0051] In the present invention, any optical instrument may be used without particular limitation as long as the optical instrument can measure optical reflectance using optical properties. The optical instrument may radiate a wavelength of a dye (blue or purple) specifically binding to total albumin and a specific wavelength of the dye(yellow or red)-encapsulated silica nanoparticle-boronic acid as light sources (e.g. blue (630 nm) and red (430 nm)) that can simultaneously emit predetermined wavelengths, and may measure the reflected optical signal using a photodiode detector (PD), thereby measuring the amounts of albumin and glycated albumin using an optical signal converter.

[0052] The ratio of glycated albumin may be obtained by calculating the amount of glycated albumin relative to the amount of total albumin using the following equation.

Ratio (%) of glycated albumin=glycated albumin/total albumin

[0053] Generally, in the case when the ratio of glycated albumin is 16% or more, the case may be diagnosed as diabetes.

Mode for Invention

[0054] Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these Examples are only for illustrating the present invention and that the scope of the present invention is not to be construed as being limited by these Examples.

EXAMPLE 1

Manufacture of Yellow Dye-Encapsulated Silica Nanoparticle-Boronic Acid (YD@SNPCPBA)

[0055] 1-1: Synthesis of Yellow Dye-Encapsulated Silica Nanoparticles (YD@SNP)

[0056] 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 added to a 1 L round bottom flask, and uniformly mixed for 1 hour using an agitator. 1.08 ml of 2530% aqueous ammonia (NH.sub.4OH) was added thereto and reacted at room temperature for 24 hours. 200 ml of ethanol was then added to terminate the reaction. Ethanol washing and DI washing were respectively performed four times and three times using a centrifuge at 3800 rpm for 15 minutes, followed by drying in an oven at 60 C.

[0057] 1-2: Amination of Yellow Dye-Encapsulated Silica Nanoparticles (YD@SNP)

[0058] In order to perform cross-coupling 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 and dispersed for 30 minutes using an ultrasonic disperser. Then, 1 ml of APTES (3-aminopropyltriethoxysilane) was added to an agitator, followed by reaction at room temperature for 2 hours. After the reaction, ethanol washing and DI washing were respectively performed four times and three times using a centrifuge at 3800 rpm for 15 minutes, followed by drying in an oven at 60 C., thus manufacturing aminated YD@SNP (YD@SNPNH.sub.2).

[0059] 1-3: Joining of Aminated Yellow Dye-Encapsulated Silica Nanoparticles (YD@SNPNH.sub.2) and CPBA

[0060] In order to provide binding ability to glycated albumin, according to a carbodiimide cross-coupling method using 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), which is a cross-coupling agent connecting a carboxyl group and a primary amine group, 4-carboxylicphenyl-boronic acid (CPBA), which is a glycated albumin-binding material, was fixed on the surface of the aminated yellow dye-encapsulated silica nanoparticles (YD@SNPNH.sub.2).

[0061] That is, in order to activate the carboxyl functional group of CPBA in an environment from which light was blocked, 3.48 mM CPBA was dissolved in a 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer solution (pH 6.0), EDC having a final concentration of 1 mM was added thereto, and the reaction was allowed to progress for 30 minutes with agitation. Then, YD@SNPNH.sub.2 was added, followed by reaction in an agitator at room temperature for 10 to 20 hours.

[0062] After completion of the reaction, ethanol washing and DI washing were respectively performed four times and three times using a centrifuge at 3800 rpm for 15 minutes, followed by drying at room temperature or freeze-drying, thus manufacturing a yellow dye-encapsulated silica nanoparticle-boronic acid (YD@SNPCPBA).

[0063] Each of the tartrazine dye used in the synthesis and the dye-encapsulated silica nanoparticle-boronic acid was diluted with deionized water (DI water), and measurement with UV/Vis spectroscopy was then performed. As a result, as shown in FIG. 4, it can be seen that the dye-encapsulated silica nanoparticle-boronic acid has an inherent absorption wavelength of the yellow dye even after the synthesis. With respect to this, analysis was performed using a scanning electron microscope and a dynamic light-scattering method. As a result, as shown in FIGS. 5A and 5B, it could be confirmed that nanoparticles having uniform morphology and a size of about 35 to 45 nm were synthesized.

EXAMPLE 2

Measurement of Glycated Albumin Using a Reagent Composition for Measuring Glycated Albumin Containing YD@SNPCPBA and Bromocresol Green

[0064] 2-1: Measurement of Glycated Albumin

[0065] 200 l of a reagent composition containing YD@SNPCPBA manufactured in Example 1 (ZnCl.sub.2, NaCl, MgCl.sub.2, Triton X-100, NaN.sub.3, glycine, HEPES, pH 8.1) was placed in a brown tube, and 5 l of a plasma sample in which the % value of glycated albumin was measured using an Olympus AU 400 analyzer and a reference reagent (Asahi Kasei GA-L) was added thereto, followed by reaction for 2 minutes. 25 l of the reaction solution was put on an absorption pad of a cartridge of an optical instrument (Epithod616, DxGen) to be absorbed for 15 seconds, and 25 l of a washing solution (morpholine, NaCl, Triton X-100, glycerol, and NaN.sub.3 mixture solution) was added thereto, followed by washing for 15 seconds. Next, the optical reflectance of yellow glycated albumin on the cartridge was measured in the optical instrument (Epithod616, DxGen).

[0066] 2-2: Measurement of Total Albumin

[0067] 200 l of a reagent composition containing bromocresol green (Succinic acid, pH 5.5) was placed in a brown tube, and 5 l of the same plasma sample was added thereto, followed by reaction for 2 minutes. 25 l of the reaction solution was put on an absorption pad of a cartridge of an optical instrument Epithod616, DxGen) to be absorbed for 15 seconds, and 25 l of a washing solution (morpholine, NaCl, Triton X-100, glycerol, and NaN.sub.3 mixture solution) was added thereto, followed by washing for 15 seconds. Next, the optical reflectance of blue total albumin on the cartridge was measured in the optical instrument (Epithod616, DxGen).

[0068] 2-3: Measurement of Glycated Albumin Using K/S Value

[0069] The % value of glycated albumin was determined by comparing the optical reflectance of glycated albumin measured in 2-1 and the optical reflectance of total albumin measured in 2-2. The % reflectance (% R) measured for each wavelength was converted into a K/S value, which is a quantitative index of how much of the coloring material is present on the surface thereof in use, and the formula for converting the % reflectance into the K/S value is as follows.

[00001]$K/S=\frac{{\left(1-\%.Math..Math.R\right)}^2}{2\%.Math..Math.R}$$\left(K=\mathrm{absorption}.Math..Math.\mathrm{coefficient},S=\mathrm{scattering}.Math..Math.\mathrm{coefficient}\right)$

[0070] Therefore, after the % reflectance value obtained by radiating the yellow light source representing the amount of glycated albumin and the % reflectance value obtained from the blue light source representing the amount of the total albumin were each substituted with the K/S value, the ratios thereof were calculated, thereby measuring the amount of glycated albumin.

[0071] As shown in FIG. 6, it could be seen that, as the ratio of glycated albumin contained in the blood is increased, the relative amount (K/S value) of the dye-encapsulated silica nanoparticle-boronic acid binding thereto is increased. Therefore, it was confirmed that the method of measuring glycated albumin according to the present invention is capable of being widely used to diagnose diabetes.

[0072] Although specific portions of the present invention have been described in detail above, those skilled in the art will appreciate that this specific description is only a preferred embodiment, and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

INDUSTRIAL APPLICABILITY

[0073] The reagent composition for measuring glycated albumin according to the present invention contains a dye that is used to distinguish total albumin and glycated albumin by labeling, so that glycated albumin may be measured in a simple manner using an optical analyzer merely by injecting a washing liquid into a measurement cartridge without any separation process. Accordingly, the reagent composition is capable of being widely used to diagnose diabetes.