KR102332618B1 - High sensitivity detection method for glycated albumin using urchin-like platinum nanoparticles - Google Patents

Abstract

The present invention relates to a method for measuring glycated albumin with high sensitivity using sea urchin-shaped platinum nanoparticles, and the present invention uses sea urchin-shaped platinum nanoparticles having a peroxidase-mimicking catalytic activity to more sensitively and accurately quantify the concentration of glycated albumin in plasma By providing a method that can do this, it is expected that it will be usefully used in the diagnosis and tracking of diabetes.

Description

High sensitivity detection method for glycated albumin using urchin-like platinum nanoparticles}

The present invention relates to a method for measuring glycated albumin with high sensitivity using sea urchin-shaped platinum nanoparticles.

Diabetes Mellitus (DM) is a chronic metabolic disease with heterogeneous dystrophy due to insulin resistance and/or insulin deficiency. Diabetes mellitus is characterized by high blood sugar in which the concentration of glucose in the blood rises. Failure to control blood sugar can lead to complications such as diabetic retinopathy, kidney disease, and foot lesions, so the importance of blood sugar management in diabetic patients is increasing. .

As indicators used for blood glucose monitoring, there are various examples such as 1,5-anhydro-D-glucitol (1,5-AG), glycated hemoglobin (HbA1c), and glycated albumin (GA). Unlike glycated hemoglobin, which indicates the average blood glucose level for the past 2 to 3 months, glycated albumin sensitively reflects the average blood glucose level for the past 2 to 4 weeks, making it easier for patients with type 1 diabetes, chronic kidney disease, or pregnant women. It is attracting attention as a useful blood sugar indicator that can stably provide a change in blood sugar level.

As a method for measuring glycated albumin, affinity chromatography, high performance liquid chromatography, enzymatic method, chromaticity detection method, etc. are commonly used. However, these methods are not widely used for biosensor applications because they take a lot of time, require complicated procedures and specialized manipulation techniques. On the other hand, electrochemical measurement methods are easy to use for many applications because they provide quick and simple results with high sensitivity.

On the other hand, when a natural enzyme is introduced into an electrochemical measurement method for high-sensitivity detection of glycated albumin, there is a disadvantage in that reliability and stability problems occur due to changes in activity caused by the surrounding environment such as pH or temperature. As an alternative to this, if nanozyme, an artificial enzyme that has recently been attracting attention, is used as a substitute for natural enzymes, it can be used for stable signal detection at temperature, pH, and the like.

The present inventors completed the present invention as a result of intensive research for the development of a nanozyme capable of quickly and simply measuring the concentration of glycated albumin with high sensitivity.

KR 10-2011-0002293

An object of the present invention is to provide a method for measuring glycated albumin using sea urchin-shaped platinum nanoparticles and a kit for measuring glycated albumin including the same.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a method comprising: (1) inducing the formation of a first complex of a glycosylated albumin-bead conjugate by reacting a plasma sample with a bead conjugate to which a glycated albumin binding material is bound; (2) centrifuging the reaction product after step (1) and removing the supernatant to obtain a first complex; (3) inducing the formation of a second complex of the first complex-nanozyme by reacting the first complex with urchin-like Pt nanoparticles modified with a glycosylated albumin binding material; and (4) centrifuging the reaction product after step (3) and removing the supernatant to recover the second complex.

In one embodiment of the present invention, the measuring method is 3,3',5,5'-tetramethylbenzidine (3,3',5,5) in the recovered second complex (a) after step (4). After mixing '-tetramethylbenzidine, TMB) and hydrogen peroxide, the step of confirming the color change of the mixed sample may be further included.

As another embodiment of the present invention, the measuring method may further include quantifying the concentration of glycated albumin by measuring the absorbance intensity at 652 nm in the UV-vis absorbance spectrum after step (a).

As another embodiment of the present invention, in step (a), 0.1 to 1.6 mM of TMB and 10 to 140 mM of hydrogen peroxide may be mixed with the recovered second complex, but preferably 0.5 mM of TMB and 100 mM of hydrogen peroxide can be mixed.

As another embodiment of the present invention, the measuring method is after the step (4), (b) after mixing thionine and hydrogen peroxide in the recovered second complex, the electrochemical properties of the mixed sample ITO ( The method may further include quantifying the concentration of glycated albumin by measuring by differential pulse voltammetry with an indium tin oxide) film electrode.

In addition, the present invention provides a kit for measuring glycated albumin, comprising a bead conjugate to which a glycated albumin binding material is bound and urchin-like Pt nanoparticles modified with a glycated albumin binding substance.

In one embodiment of the present invention, the kit may further include hydrogen peroxide.

In another embodiment of the present invention, the kit may further comprise TMB and/or thionine.

In another embodiment of the present invention, the beads are agarose, cellulose, sepharose, polystyrene, polymethyl methacrylate, polyvinyltoluene. , latex beads, and may be at least one selected from the group consisting of glass beads, preferably may be agarose beads.

In another embodiment of the present invention, the glycated albumin binding material bound to the beads and modified to the nanoparticles is from the group consisting of boronic acid, concanavalin A, and an antibody, respectively. It may be one or more selected, preferably, the glycosylated albumin binding material bound to the beads may be boronic acid, and the binding material modified to the nanoparticles may be an antibody that specifically binds to the glycosylated albumin.

In addition, the present invention may provide a method for diagnosing diabetes using the measured value of glycated albumin in plasma obtained through the method for measuring glycated albumin.

The present invention is expected to be useful in diagnosing and tracking diabetes by providing a method that can more sensitively and accurately quantify the concentration of glycated albumin in plasma using sea urchin-shaped platinum nanoparticles having peroxidase-mimicking catalytic activity.

1 is a transmission electron microscope (TEM) image of sea urchin-shaped platinum nanoparticles weaved from a platinum seed (Pt seed) of 5 nm or less synthesized using platinum ions (H ₂ PtCl _{6 ).}
Figure 2 visually confirms the color change of each sample in which (a) TMB + hydrogen peroxide, (b) Pt + hydrogen peroxide, (c) Pt + TMB, and (d) Pt + TMB + hydrogen peroxide is mixed (A), It is a figure confirmed through the UV-vis absorbance spectrum (B).
3 is a view confirming the peroxidase mimic catalytic activity of sea urchin-shaped platinum nanoparticles through enzyme kinetic analysis. Specifically, the absorbance (A) and oxidation rate (C) of oxidized TMB according to the change in the concentration of hydrogen peroxide (10 to 140 mM) in the presence of 0.5 mM TMB were confirmed, and the change in the concentration of TMB in the presence of 100 mM hydrogen peroxide (0.1 to 1.6 mM) The absorbance (B) and oxidation rate (D) of oxidized TMB according to ).
4 is a first complex (bead-BA-GA complex), which is a binding structure of agarose beads bound to boronic acid and glycated albumin, and a second complex (bead-), which is a binding structure of the first complex and platinum nanoparticles modified with an antibody. It is a schematic diagram of the formation process of the BA-GA-Ab-Pt complex.
Figure 5 shows the sample according to the chromaticity quantification measurement method after obtaining a second complex (agarose bead-BA-GA-Ab-PtNP complex) according to the measurement method of the present invention using a sample having a glycated albumin concentration of 0.02 to 10% It is a drawing confirming the color change (A: Experimental schematic diagram, B: Color change result photo showing different glycated albumin concentration, C: UV-vis absorbance spectrum according to glycated albumin concentration, D: calibration curve for UV-vis absorbance spectrum graph).
Figure 6 confirms the difference in the size of the reduction current according to the substrate (hydroquinone, ferrocene, 2-aminophenol, TMB, Thionine) in the use of the electrochemical measurement method for measuring the concentration of glycated albumin (A), Thionine showing the most distinct current It is a diagram confirming the largest current flow in three types of mixtures of thionine, hydrogen peroxide and platinum nanoparticles by measuring the magnitude of the reduction current through various mixing of hydrogen peroxide and platinum nanoparticles (B).
7 shows electrochemical reactions using thionine as a substrate after obtaining a second complex (agarose bead-BA-GA-Ab-PtNP complex) according to the measurement method of the present invention using a sample having a glycated albumin concentration of 0.01 to 20%. It is a drawing of the result of the quantitative measurement method (A: experimental schematic diagram, B: reduction current measurement result, C: calibration curve graph of the measurement current).
8a and 8b are views confirming the oxidation activity of TMB according to the shape of the platinum nanoparticles.

The present inventors have intensively studied a method for providing more accurate result data by increasing the sensitivity and accuracy in measuring the glycated albumin concentration for the diagnosis and tracking of diabetes. To confirm the effect, and to provide a method for measuring the concentration of glycated albumin using the nanozyme.

The present invention provides a blood glucose measurement method and a blood glucose measurement kit for diabetic patients with improved sensitivity by accurately measuring the concentration of glycated albumin in plasma.

Nanozyme is an enzyme analog that catalyzes a reaction based on redox activity, such as peroxidase or oxidase. It refers to nanoparticles with peroxidase mimic activity that promotes and catalyzes.

The present inventors prepared sea urchin-like platinum nanoparticles (urchin-like PtNP) with platinum seeds of 5 nm or less using platinum ions through specific examples, and confirmed the peroxidase-mimicking catalytic activity of the sea urchin-like platinum nanoparticles. It was confirmed that the sea urchin-shaped platinum nanoparticles of the present invention function as nanoparticles by catalyzing the oxidation of TMB in the presence of hydrogen peroxide (see Examples 1 and 2).

Next, the present inventors modified the antibody that specifically binds glycated albumin to the prepared sea urchin-shaped nanoparticles, and coated the outside of the antibody-modified area with PVP to block the reaction with substances other than glycated albumin, so that the concentration of glycated albumin in plasma was intended to measure. Boronic acid forms a cis-diol bond with glycated albumin, and glycated albumin (GA) and boronic acid-agarose beads (bead-BA) are reacted with agarose beads bound to boronic acid (BA) with plasma. The first complex and the sea urchin-shaped nanoparticles are formed by binding the antibody to glycosylated albumin by treating the conjugate with the prepared antibody-modified sea urchin-shaped nanoparticles (Ab-PtNP). It induced the formation of a bound second complex (see Example 3-1).

The formed second complex was able to provide a specific concentration of glycated albumin in plasma as a numerical value using chromaticity quantification and electrochemical quantification. In particular, the present inventors found that in electrochemical quantification of glycated albumin, thionine It was confirmed that it can act as a substrate showing remarkably excellent sensitivity in the ITO electrode, and the reduction current was measured by applying a voltage in the presence of thionine and hydrogen peroxide. Detection results having limitations and high sensitivity were confirmed (see Examples 3-2 and 3-3).

In addition, the present inventors conducted a comparative experiment with spherical general platinum nanoparticles (PtNP) in order to confirm the excellent effect by the morphological characteristics of the sea urchin-like platinum nanoparticles (urchin-like PtNP). As a result, hydrogen peroxide and TMB were present. After mixing each of the nanoparticles in the solution, a peak at 652 nm was confirmed when the optical signal of the solution was detected. As a result of comparing the absorbance at , it was found that the sea urchin-shaped platinum nanoparticles catalyze the oxidation of a large amount of TMB faster than the spherical platinum nanoparticles (see Example 4).

Accordingly, the present inventors can provide sea urchin-shaped platinum nanoparticles to be used for accurate detection and concentration measurement of glycated albumin in plasma. As used herein, the term 'nanoparticles' means 'sea urchin-shaped nanoparticles' and 'platinum nanoparticles' unless the shape is specifically described otherwise.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Example]

Example 1. Preparation of antibody-modified sea urchin-shaped platinum nanoparticles

After synthesizing Pt seeds with a diameter of 5 nm or less using platinum ions, urchin-like PtNPs with peroxidase mimic activity were synthesized using the seeds, and a transmission electron microscope image was taken. (Fig. 2).

For the production of urchin-like PtNPs, the diffusion control of Pt ions was controlled by controlling the temperature, the concentration of precursor ions, the concentrations of stabilizers and reduction agents, and the rate of addition, and thus the reduction rate of Pt ions was controlled to shape the shape of the sea urchin. It can be synthesized with

More specifically, after synthesizing a platinum seed of 5 nm or less, 1% sodium citric acid and 1.25% L-ascorbic as a stabilizer and reduction agent in an aqueous solution in which the platinum seed and the precursor ion, 0.4M chloroplatinic acid (H ₂ PtCl _{6 ) are mixed.} The acid was added dropwise every 3 seconds, dropwise. At this time, the temperature of the mixed aqueous solution was gradually increased from 60°C to 80°C.

The urchin-like PtNPs were modified with an antibody that specifically binds to glycosylated albumin, and the surface of the nanozyme unmodified with the antibody was coated with polyvinylpyrrolidone (PVP) to prevent adsorption of unspecified substances.

Example 2. Enzyme kinetic analysis of platinum nanoparticles as nanozymes

2-1. Confirmation of peroxidase-mimicking catalytic activity of platinum nanoparticles

In order to confirm the peroxidase-mimicking catalytic activity of the urchin-like PtNPs prepared in Example 1, the platinum nanoparticles were mixed in an aqueous solution containing 0.5 mM TMB in the presence of 80 mM hydrogen peroxide, and the color change of the solution was checked and the Changes in UV absorbance were confirmed.

As a result, as can be seen in A of FIG. 2, the aqueous solution of (a) TMB + hydrogen peroxide, (b) Pt + hydrogen peroxide had no color change at all, and (c) the mixed solution of Pt + TMB had a slight color. change could be observed. On the other hand, in the case of (d) a mixed solution of Pt + TMB + hydrogen peroxide, a clear blue change was observed within 1 minute after Pt mixing. The color change of the solution means that the TMB is sufficiently oxidized by the nanoparticles.

In addition, as can be seen in FIG. 2B , from the UB-vis absorption spectrum results of each of the mixed solutions (a) to (d), the oxidation of TMB is catalyzed by urchin-like PtNP when hydrogen peroxide is present. It can be seen that the absorption intensity at 652 nm corresponding to the TMB is large (blue graph curve). From this, it can be seen that the platinum nanoparticles prepared in Example 1 are nanozymes that catalyze the oxidation of TMB in the presence of hydrogen peroxide, similar to peroxidase.

2-2. Enzyme Kinetic Analysis of Platinum Nanoparticles

Next, the enzyme kinetics analysis of nanozyme with respect to hydrogen peroxide and TMB was performed. Specifically, the peroxidase-mimicking catalytic activity of platinum nanoparticles according to the change in the concentration of hydrogen peroxide (10 to 140 mM) at a fixed TMB concentration (0.5 mM) and the change in the concentration of TMB under the fixed hydrogen peroxide concentration (100 mM) (.01 to 1.6 mM) ) was measured by checking the absorbance at 652 nm (absorption wavelength of oxidized TMB (TMBox)) over time.

_{The initial velocity of TMB oxidation (V 0} ), Michaelis-Menten constant (K _m ), and maximum rate of TMB oxidation (V _max ) according to the concentration of the substrate were determined by the Michaelis-Menten equation graph for _{V 0 and the Lineweaver-Burk} It was calculated through the formula obtained by plot and the following formulas 1 to 4.

[Equation 1]

[Equation 2]

[Equation 3]

[Equation 4]

( C : concentration of oxidized TMB, : extinction coefficient of oxidized TMB (3.9×10 ⁴ M ⁻¹ cm ⁻¹ ), L : optical path length of the measuring cuvette (1 cm), V _max : maximum rate of TMB oxidation , [S] : concentration of substrate (hydrogen peroxide or TMB), K _m : Michaelis-Menten constant)

As a result, as can be seen in FIG. 3 , the absorbance of TMB oxidized by nanozyme increased rapidly as the concentrations of hydrogen peroxide and TMB were increased with respect to the fixed TMB and hydrogen peroxide concentrations, respectively ( FIGS. 3A and 3B ). Reference). In addition, as the concentrations of hydrogen peroxide and TMB were increased, the initial rate of TMB oxidation gradually increased and then saturated from a certain point (see C and D in FIG. 3 ), and the reciprocal of the concentrations of hydrogen peroxide and TMB and the inverse of the initial rate of TMB oxidation. There is a linear correlation between (see E and F in Fig. 3), and the Michaelis-Menten constant ( K _m ) and the maximum rate of TMB oxidation ( V _max ) for hydrogen peroxide and TMB of urchin-like PtNPs nanozymes were respectively It was found that 82.69mM, 1.77μMs ^-1 and 0.174mM, 1.01μMs ^-1 were sequentially obtained. This means that the nanozyme prepared in Example 1 has a catalytic activity on a substrate about 50 times higher than that of a natural enzyme, horseradish peroxidase (HRP).

Example 3. Detection of glycated albumin concentration using nanozyme

3-1. Induction of bead-BA-GA-Ab-Pt complex formation

When plasma is reacted with agarose beads bound with boronic acid, the boronic acid (BA) of the beads specifically binds with glycated albumin (GA) through a cis-diol bond to form a first complex (bead-BA-GA complex) And, when platinum nanoparticles modified with an antibody that specifically binds to glycated albumin are treated, a sandwich-shaped second complex (bead-BA-GA-Ab-PtNP complex) is formed with glycated albumin interposed therebetween ( Fig. 4).

Therefore, the agarose beads bound with boronic acid are mixed with total human serum albumin (tHSA) and centrifuged after sufficient time has elapsed so that the glycated albumin and boronic acid can react to remove unglycosylated normal albumin (HSA). After removal, only the first complex, which is a complex of agarose beads-boronic acid-glycated albumin, was recovered. Next, the first complex is mixed with sea urchin-shaped nanoparticles modified with an antibody that specifically binds to glycosylated albumin, and centrifuged after a sufficient time elapses to react, agarose beads-boronic acid-glycosylated albumin-antibody- A second complex, which is a complex of platinum nanoparticles, was recovered.

3-2. Detecting the glycated albumin concentration using a colorimetric quantification method

The concentration of glycosylated albumin was detected according to the principle of colorimetric quantification according to the concentration of glycosylated albumin using TMB oxidation catalyzed by urchin-like PtNPs nanozyme in the presence of hydrogen peroxide shown in FIG. 5A .

Specifically, the second complex was recovered by the method of Example 3-1 from a sample containing glycated albumin at various concentrations (0.02 to 10%), mixed with an aqueous solution containing 0.5 mM TMB and 100 mM hydrogen peroxide, and then mixed solution Color change and absorbance were confirmed.

As a result, as the concentration of glycated albumin present in 50 mg/mL total albumin increased, the amount of oxidized TMB increased and the blue color became darker (FIG. 5B), and as the concentration of glycated albumin increased, the solution at 652 nm It was confirmed that the absorbance was increased (FIG. 5C). In addition, from the calibration curve for the absorbance graph, it was confirmed that the linear range was 0.02 - 10 % (10 µg/mL - 5 mg/mL), the detection limit was 0.0184 % (9.2 µg/mL), and the R ² value was 0.978 (Fig. 5 d).

3-3. Detection of glycated albumin concentration using electrochemical quantification method

(1) substrate selection

In order to perform high-sensitivity electrochemical quantitation of glycated albumin using urchin-like PtNPs nanozyme, it was attempted to select a substrate with excellent sensitivity in the ITO film electrode.

Specifically, the activities of hydroquinone (HQ), ferrocene, 2-aminophenol, TMB, and thionine, known as electrochemical substrates of HRP, were compared. After minutes, a current curve was obtained in the range of -0.8 ~ 0.9V using cyclic voltammetry (CV, Cyclic Voltammetry) with an ITO film electrode.

As a result, as shown in FIG. 6A , the substrate showing the greatest reduction current was thionine, and then, the peroxidase-mimicking catalytic activity of nanozymes for thionine was confirmed through an electrochemical method.

Specifically, to electrochemically confirm the peroxidase-mimicking catalytic activity of urchin-like PtNPs nanozymes with respect to thionine, the substrate with the best electrochemical activity, (1) hydrogen peroxide + Pt, (2) thionine + Pt, (3) Current curves were obtained by cyclic voltammetry in three mixed solutions: thionine + hydrogen peroxide + Pt.

As a result, as shown in FIG. 6B, the reduction current of the current curve was greatest in the mixed solution of (3) thionine + hydrogen peroxide + Pt, which catalyzes the oxidation of thionine as a substrate in the presence of hydrogen peroxide. suggests that it was That is, it can be seen that the urchin-like PtNPs nanozyme has a peroxidase-mimicking catalytic activity.

(2) Electrochemical quantification of glycated albumin concentration

Next, according to the principle of electrochemical quantification according to the concentration of glycosylated albumin using thionine oxidation catalyzed by nanozyme in the presence of hydrogen peroxide shown in FIG.

Specifically, for the electrochemical quantification measurement, the second complex was recovered by the method of Example 3-1 from a sample containing glycated albumin at various concentrations (0.01 to 20%), and 0.5 mM Thionine and 100 mM hydrogen peroxide were mixed in aqueous solution. Thionine oxidized on the nanozyme surface was reduced again by differential pulse voltammetry (DPV) on the ITO film electrode, and the current generated at that time was measured to quantify the concentration of glycated albumin.

As a result, as the concentration of glycated albumin present in 50 mg/mL total albumin increased, the amount of oxidized thionine increased, and the current value obtained by reducing it again at the working electrode increased (FIG. 7B), and the reduction current size From the calibration curve for , it was confirmed that the linear range was 0.01 - 20 % (5 μg/mL - 10 mg/mL), the detection limit was 0.0076 % (3.8 μg/mL), and the R ² value was 0.984 ( FIG. 7C ). From the above results, it can be seen that the urchin-like PtNPs nanozyme-based glycated albumin detection can provide accurate values by electrochemical quantification due to the linear relationship between the glycated albumin concentration and the current. In addition, it can be seen that the nanozyme of the present invention can provide the concentration of glycated albumin with a wider detection range, lower detection limit, and higher sensitivity than in the case of chromaticity quantification when using the electrochemical quantification method.

Example 4. Comparison of effects of sea urchin-shaped platinum nanoparticles and platinum nanoparticles

A comparative experiment with spherical platinum nanoparticles (PtNP) was conducted to confirm the detection efficiency and sensitivity of glycated albumin of sea urchin-like platinum nanoparticles (urchin-like PtNP). More specifically, after mixing PtNP and urchin-like PtNP at the same concentration in a solution in which 0.5 mM TMB and 100 mM hydrogen peroxide are present, the absorbance of the mixed solution over time was measured.

As a result, as can be seen in FIG. 8a , both PtNP and urchin-like PtNP signal peaks appeared at 652 nm at 90 seconds after mixing the platinum nanoparticles, and the signal of urchin-like PtNP was more than doubled. appear. More specifically, as a result of confirming the optical signal with the passage of time of 652 nm, which is the absorption wavelength of the TMBox over time in FIG. 8b, it was found that urchin-like PtNP oxidized a large amount of TMB significantly faster than PtNP. . From the above results, it can be seen that urchin-like PtNP has exceptionally superior peroxidase mimic activity than PtNP.

In addition to spherical platinum nanoparticles, the glycated albumin measurement efficiency of enzymes and nanozymes with known peroxidase activity and sea urchin-shaped platinum nanoparticles were compared and summarized in Table 1 below.

As can be seen in Table 1, when the k _cat value was compared with other metal nanoparticles, platinum nanoparticles had superior peroxidase-like activity than other metal nanoparticles such as iron. In particular, urchin-like PtNP compared with spherical PtNP. Therefore, it can be seen that urchin-like PtNP has the best activity as a nanozyme from the fact that it exhibits about 2 times better activity against TMB and about 5 times better activity against hydrogen peroxide.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A method for measuring glycated albumin comprising the steps of:
(1) inducing the formation of a first complex of the glycated albumin-bead conjugate by reacting the plasma sample with the bead conjugate to which the glycated albumin binding material is bound;
(2) centrifuging the reaction product after step (1) and removing the supernatant to obtain a first complex;
(3) inducing the formation of a second complex of the first complex-nanozyme by reacting the first complex with urchin-like Pt nanoparticles modified with a glycosylated albumin binding material; and
(4) centrifuging the reaction product after step (3) and removing the supernatant to recover the second complex. According to claim 1,
The beads of step (1) are agarose, cellulose, sepharose, polystyrene, polymethyl methacrylate, polyvinyl toluene, latex beads , and a measuring method, characterized in that at least one selected from the group consisting of glass beads. According to claim 1,
The glycosylated albumin-binding material in steps (1) and (3) is at least one selected from the group consisting of boronic acid, concanavalin A, and an antibody, respectively. Measurement, Way. 4. The method of claim 3,
The measuring method, characterized in that the glycosylated albumin-binding material in step (1) is boronic acid, and the glycosylated albumin-binding material in step (3) is an antibody. According to claim 1,
The sea urchin-shaped platinum nanoparticles of step (3) synthesized a platinum seed (Pt seed) of 5 nm or less, and then added 1% citric acid and 1.25 to a mixed solution of the platinum seed and 0.4M chloroplatinic acid. % L-ascorbic acid (L-ascorbic acid) is prepared by adding one drop every 3 seconds, except for the surface on which the glycosylated albumin binding material is modified, the surface is coated with polyvinylpyrrolidone (PVP) A measurement method, characterized in that. According to claim 1,
The measuring method is 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and hydrogen peroxide in (a) the recovered second complex after step (4). The measuring method, characterized in that it further comprises the step of confirming the color change of the mixed sample after mixing. 7. The method of claim 6,
In the step (a), TMB is 0.5mM, hydrogen peroxide is 100mM, characterized in that the mixing, the measuring method. According to claim 1,
In the measurement method, after step (4), (b) after mixing hydrogen peroxide with the recovered second complex, thionine and hydrogen peroxide were mixed, and then the electrochemical properties of the mixed sample were measured using indium tin oxide (ITO). Measurement method, characterized in that it further comprises the step of measuring using a cyclic voltammetry as an electrode. A glycated albumin measurement kit comprising a bead conjugate to which a glycosylated albumin binding material is bound and a sea urchin-like Pt nanoparticle modified with a glycosylated albumin binding substance,
The kit is a glycated albumin measurement kit, characterized in that colorimetric detection and electrochemical detection are possible.
10. The method of claim 9,
The beads are made of agarose, cellulose, sepharose, polystyrene, polymethyl methacrylate, polyvinyltoluene, latex beads, and glass beads. A glycated albumin measurement kit, characterized in that at least one selected from the group. 10. The method of claim 9,
The glycosylated albumin binding material is a glycosylated albumin measurement kit, characterized in that at least one selected from the group consisting of boronic acid, concanavalin A, and an antibody, respectively. 10. The method of claim 9,
A glycosylated albumin measurement kit, characterized in that the glycated albumin binding material bound to the bead is boronic acid, and the binding substance modified to the nanoparticles is an antibody.
10. The method of claim 9,
The sea urchin-shaped platinum nanoparticles are 1% citric acid and 1.25% L-ascorbic acid in a mixed solution of the platinum seed and 0.4M chloroplatinic acid after synthesizing a platinum seed of 5 nm or less. It is prepared by adding (L-ascorbic acid) dropwise every 3 seconds, and the surface is coated with polyvinylpyrrolidone (PVP) except for the surface on which the glycated albumin binding material is modified, characterized in that, A glycated albumin measurement kit.

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