Patent Application
20130015062
1. Field of the Invention
The present invention relates to an electrode for measuring glycoprotein, and more particularly, to an electrode for measuring glycoprotein, the surface of which can bind an increased amount of boronic acid capable of binding to glycoprotein and which can more accurately measure the amount of glycoprotein, and to a preparation method thereof.
2. Description of the Prior Art
Blood glucose and glycated hemoglobin (HbAlc) are most frequently used as important indexes that reflect the current status of diabetes. Particularly, in the case of glycated hemoglobin, a test error caused by an empty stomach and a drug is low and it is possible to predict long-term changes in the blood glucose levels of diabetic patients before blood collection. Thus, the measurement of glycated hemoglobin is useful for assessment of the long-term regulation of plasma glucose. In addition, glycated hemoglobin has good specificity, and thus is considered as a good index for diabetes and related complications.
In the prior art, in order to electrochemically measure glycated hemoglobin, the electrode surface was modified with boronic acid (BA) capable of binding specifically to glycated hemoglobin. Boronic acid is frequently used in sensors for measuring glycoprotein or glucose, because it has the property of binding specifically to sugars and glycoprotein by interaction with the cis-diol. Specifically, a boronic acid group is exposed to the electrode surface to which glycated hemoglobin is then immobilized, and the concentration of the glycated hemoglobin is measured in the resulting change in electrochemical signals.
For example, Korean Patent Laid-Open Publication No. 2011-0002293 discloses preparing an electrode by binding a dendrimer to the electrode surface and then binding 4-formyl-phenylboronic acid thereto, and also discloses reacting glycated hemoglobin with the 4-formyl-phenylboronic acid and then electrochemically measuring the amount of the glycated hemoglobin. However, even when the 4-formyl-phenylboronic acid is used, there are shortcomings in that the reaction of glycated hemoglobin is insufficient and the density of 4-formyl-phenylboronic acid that is bound to the electrode surface by the dendrimer is low.
Accordingly, the present invention has been made in view of the above-described problems occurring in the prior art, and it is an object of the present invention to increase the amount of boronic acid that is bound to the electrode surface and to more accurately measure the amount of glycoprotein.
Another object of the present invention is to prepare an electrode having a boronic acid bound thereto in a simple and easy manner.
To achieve the above objects, the present invention provides an electrode for measuring glycoprotein, comprising: an electrode; a cystamine (Cys) bound to the surface of the electrode; and a boronic acid (BA) conjugated to the cystamine.
Herein, the boronic acid is preferably formylphenyl boronic acid (FPBA), more preferably 3-formylphenyl boronic acid (3-FPBA), and most preferably a 3-formylphenyl boronic acid (3-FPBA) represented by the following formula 1:
Moreover, the boronic acid may consists of two 3-formylphenyl boronic acids which are conjugated to one cystamine.
Furthermore, the cystamine may be bound to the electrode surface by a disulfide group.
In addition, the electrode surface may be made of gold (Au).
In one embodiment, the present invention provides an electrode for measuring glycoprotein, comprising: an electrode; and a cystamine-boronic acid conjugate bound to the surface of the electrode.
Herein, the boronic acid is preferably formylphenyl boronic acid (FPBA), and more preferably 3-formylphenyl boronic acid (3-FPBA).
Moreover, the conjugate may be Cys-FPBA2 represented by the following formula 2:
Furthermore, the conjugate may be bound to the electrode surface by the disulfide group of cystamine.
In addition, the electrode surface may be made of gold (Au).
In another aspect, the present invention provides a method for preparing an electrode for measuring glycoprotein, the method including the steps of: preparing a cystamine-boronic acid conjugate by conjugating cystamine to boronic acid; and binding the prepared conjugate to the surface of an electrode.
Herein, the step of preparing the conjugate may include a step of reacting the cystamine with the boronic acid for 3-5 hours, preferably 3-4 hours.
Furthermore, the step of preparing the conjugate may also include a step of reacting the cystamine with the boronic acid in the presence of triethylamine (TEA).
In addition, the step of preparing the conjugate preferably includes a step of reacting the cystamine with the boronic acid at a temperature of 40˜60° C., more preferably 45˜55° C.
Details of other embodiments of the present invention are included in the following detailed description and the accompanying drawings.
Although the present invention can be modified variously and have several embodiments, exemplary embodiments are illustrated in the accompanying drawings and will be described in detail in the detailed description. However, the present invention is not limited to the specific embodiments and should be construed as including all the changes, equivalents and substitutions included in the spirit and scope of the present invention. In the following description, the detailed description of related known technology will be omitted when it may obscure the subject matter of the present invention.
Terms used in this specification are used only to describe a specific embodiment and are not intended to limit the scope of the present invention. Singular expressions include plural expressions unless specified otherwise in the context thereof. In this specification, the terms “comprise”, “have”, etc., are intended to denote the existence of mentioned characteristics, numbers, steps, operations, components, parts, or combinations thereof, but do not exclude the probability of existence or addition of one or more other characteristics, numbers, steps, operations, components, parts, or combinations thereof.
The present invention is directed to an electrode for measuring glycoprotein, and more particularly to an electrode capable of detecting the presence or measuring the amount of a glycoprotein having the cis-diol group of the carbohydrate chain, for example, glycated hemoglobin (HbAlc) or glycated albumin.
The electrode that is used in the present invention may be a conductive electrode, such as a gold (Au) electrode or a platinum (Pt) electrode, or a semiconductor electrode such as a carbon electrode. Preferably, the gold electrode is used.
In order for the inventive electrode to bind to a glycated protein having the cis-diol group of the carbohydrate chain, the inventive electrode preferably has on its surface a boronic acid (BA) capable of binding the cis-diol group by interaction with the cis-diol. In order to bind the boronic acid to the electrode surface, a cystamine (Cys) is present between the electrode and the boronic acid. Herein, the boronic acid is not specifically limited and may be any boronic acid which is widely known in the art. For example, the boronic acid may be formylphenyl boronic acid (FPBA), preferably 3-formylphenyl boronic acid (3-FPBA), and more preferably a 3-formylphenyl boronic acid (3-FPBA) represented by formula 1.
Specifically, in the inventive electrode for measuring glycoprotein, the cystamine is bound to the electrode surface, and the boronic acid is conjugated to the cystamine. The cystamine includes a disulfide group which can bind to the electrode surface made of, for example, a metal, and it has an amine group at both ends, and thus can also bind to a boronic acid having an aldehyde group. Thus, one cystamine can be conjugated to two 3-formylphenyl boronic acids by amino groups at both ends.
In addition, in order to more effectively bind the cystamine, the electrode of the present invention may further comprise a surface modification layer which apparent to those skilled in the art.
In the prior art, in order to bind boronic acid to the electrode surface, 3,3′-dithio-bis-propionic acid N-hydroxysuccinimide ester (DTSP) having a functional group capable of reacting with an amine was bound to the electrode surface, and a dendrimer such as poly(amidoamine) or poly(alkyleneimine) was bound thereto, and then boronic acid was bound to the dendrimer. In other words, a structure such as BA/dendrimer/DTSP/electrode was prepared.
However, according to the present invention, cystamine is bound to the electrode surface, and boronic acid is bound directly thereto. Thus, a structure of BA/cystamine/electrode which is simpler than the prior art structure is provided, and the preparation method is simpler and easier than the prior art.
This electrode of the present invention may be prepared by binding a cystamine to the electrode surface and conjugating a boronic acid to the cystamine. Specifically, this electrode structure is preferably prepared by a conjugation method in which a cystamine is conjugated with a boronic acid to prepare a Cys-FPBA2 conjugate which is then bound to the electrode surface by dipping the electrode once in a solution containing the conjugate. Alternatively, the electrode structure may also be prepared by a bottom-up method in which a cystamine is bound to the electrode surface and then a boronic acid is bound to the cystamine.
The inventive electrode structure consisting of boronic acid/cystamine/electrode surface could not be conceived of from the prior art. The reason is as follows. First, the disulfide group of cystamine is not located at the end of the cystamine, but is located at the middle of the cystamine. Second, the electrode having the boronic acid bound thereto is preferably prepared by the conjugation method compared to the bottom-up method which is a general method.
Thus, in one embodiment, the present invention provides an electrode for measuring glycoprotein, in which a cystamine-boronic acid conjugate is bound to the electrode surface. Herein, the conjugate may be a Cys-FPBA2 synthesized by reacting cystamine with 3-formylphenyl boronic acid (3-FPBA) according to the following reaction scheme:
Moreover, the conjugate is preferably bound to the electrode surface such that the diol group of the boronic acid faces outward so as to bind to glycoprotein. Thus, the conjugate is preferably bound to the electrode surface by the disulfide of the cystamine.
As described above, the present invention is characterized in that cystamine is bound to the electrode surface and boronic acid is conjugated to the cystamine. Thus, according to the present invention, an increased amount of boronic acid can be bound to the electrode surface, so that the amount of glycoprotein can be more accurately measured.
Another aspect of the present invention is directed to a method for preparing an electrode for measuring glycoprotein, the method comprising the steps of: preparing a cystamine-boronic acid conjugate by conjugating a cystamine with a boronic acid; and binding the prepared conjugate to an electrode surface. Specifically, the electrode for measuring glycoprotein is prepared in a simple and easy manner by a conjugation method in which cystamine and boronic acid are conjugated with each other to prepare a Cys-FPBA2 conjugate, and then an electrode is then dipped once in a solution containing the conjugate. As described below, the electrode prepared by this method has significantly excellent effects compared to an electrode prepared by a conventional bottom-up method.
The method of preparing the conjugate is not specifically limited. For example, the cystamine can be prepared by reacting cystamine with 3-FPBA in a solvent at room temperature for several hours. Herein, the solvent is preferably DMSO (dimethyl sulfoxide).
In addition, the method of binding the conjugate is also not specifically limited. For example, the binding can be performed by washing a gold-deposited electrode surface and then dipping the electrode in a solution of Cys-FPBA2 in DMSO for several hours. Preferably, before the dipping process, the electrode is washed with Piranha solution to remove contaminants, and after the dipping process, the electrode is washed with DMSO and ethanol.
As described in the following examples of the present invention, the present inventors carried out tests under various conditions in order to find optimal conditions for synthesizing Cys-FPBA2 with high yield. As a result, it was found that the reaction for preparing the conjugate is preferably carried out for 3-5 hours, and more preferably 3-4. This is because if the reaction time is shorter than 3 hours, the synthesis of Cys-FPBA2 will be insufficient so that the signal of glucose oxidase (GOX) will be weak, and if the reaction time is longer than 5, the reaction signal decreases, rather than increases. Furthermore, the reaction is carried out in the presence of triethylamine (TEA) as a catalyst. This is because the use of triethylamine as the reaction catalyst has excellent effects compared to HCl which is frequently used as a catalyst in the general Schiff base synthesis reaction. In addition, the reaction temperature is preferably 40˜60° C., and more preferably 45˜55° C. This is because, if the reaction temperature is lower than 40° C., the synthesis of Cys-FPBA2 will be insufficient (the signal of glucose oxidase (GOX) will be weak), and if the reaction temperature is higher than 60° C., the conjugate can be decomposed.
As shown therein, when the electrode surface is modified with boronic acid and then various concentrations of HbAlc and a constant concentration of glucose oxidase (GOX) are simultaneously allowed to react with the electrode surface, relatively high concentrations of HbAlc interfere with the immobilization of GOX onto the electrode surface, thus reducing the electrochemical signal of GOX. Herein, GOX that is a glycoprotein can react with boronic acid even when it is not biochemically treated. When GOX is immobilized onto the electrode surface by competition with HbAlc, the amplified electrochemical signal thereof can be detected due to a biological catalytic reaction.
The present invention can be better understood by the following examples, which are merely for illustrative, not for limiting the scope of the present invention.
To form a boronic acid self-assembled monolayer (BA SAM), the following two methods were used: a conjugation method in which a self-assembled monolayer (SAM) is formed using a synthesized cystamine/boronic acid (BA) conjugate; and a bottom-up method in which cystamine and BA are sequentially reacted with the electrode surface.
Then, in order to find optimal conditions for synthesizing Cys-FPBA2 with high yield, tests were carried out under various conditions.
First, 99.999% pure gold was resistively evaporated on a titanium-treated silicon wafer (20 nm Ti) to a thickness of 200 nm, thereby preparing an electrode.
Cystamine (5 mM) and 3-FPBA (20 mM) were allowed to react with each other in a DMSO (dimethyl sulfoxide) solvent at room temperature for 4 hours, thereby synthesizing a Cys-FPBA2 conjugate. Herein, the amino group of the cystamine was mixed and bound to the aldehyde group of FPBA by the Schiff's base. As a catalyst, triethylamine (TEA) was added to the mixed solution before a basic atmosphere was formed.
Then, the deposited gold surface was immersed in Piranha solution for 5 minutes and washed with distilled water. Then, in order to form a boronate-modified self-assembled monolayer (SAM), the electrode was dipped in a solution of the Cys-FPBA2 in DMSO for 2 hours. Then, the electrode was washed with DMSO and ethanol, followed by rinse with distilled water (DDW). Finally, the electrode was stored in phosphate buffered saline (PBS).
An electrode was prepared in the same manner as Example, and 5 mM cystamine, 20 mM 3-FPBA and a DMSO solvent were used as in Example 1.
Specifically, cystamine was reacted with the gold electrode such that the amino group was exposed to the electrode surface. Then, 3-FPBA having an aldehyde group capable of reacting with the amino group was reacted with the electrode surface so as to be bound to the electrode surface by a Schiff's base.
In order to confirm whether the electrodes of Examples 1 and 2 were modified with the BA SAM, 25 μg/ml of glucose oxidase (GOX) was reacted with each of the BA-SAM/Au/Si electrodes, and the intensity of the amplified electrochemical signal of GOX, which appeared by the interaction between the glucose chain of GOX and the cis-diol of BA, was measured. In the measurement, 0.1 mM ferrocene (Fc) solution containing 10 mM glucose as a substrate for GOX was used as an electron transmitter. Also, the electrochemical measurement was carried out using cyclic voltammetry at a scan rate of 5 mv/s.
Thus, it could be seen that the conjugation method of forming a BA self-assembled monolayer (SAM) using a synthesized Cys-FPBA2 conjugate is significantly effective compared to the bottom-up method. Thus, all subsequent tests were carried out using BA-SAM/Au/Si electrodes obtained by the conjugation method.
In order to determine the optimal reaction time for the synthesis of Cys-FPBA2, the reaction for synthesis of Cys-FPBA2 was carried out for various times between 2 hours and 8 hours, and the degree of synthesis of Cys-FPBA2 was analyzed as a function of reaction time. As described in Example 1, the synthesis of Cys-FPBA2 was performed by reacting 5 mM cystamine with 20 mM 3FPBA in DMSO. Also, the degree of modification with BA SAM as a function of the synthesis yield and reaction time was examined using a GOX immobilization test in the same manner as Example 1 (BA SAM modification test). The results of the electrochemical test as a function of synthesis time are shown in
As can be seen in
In order to make sensitive HbAlc measurement possible, the yield of Cys-FPBA2 that is synthesized should be maximized. In order to more efficiently synthesize Cys-FPBA2 with high yield, catalysts were used. Based on the results of Example 1, HCl and triethylamine (TEA) were selected as catalysts, and in a control, no catalyst was used. With reference to catalysts previously used in Schiff base synthesis reactions by the present inventors, 100 μl of 0.35% HCl was added to Cys-FPBA2 conjugates, and 1.1 μl of TEA was added to other Cys-FPBA2 conjugates.
Meanwhile, in order to maximize the synthesis yield of Cys-FPBA2, the synthesis of Cys-FPBA2 was carried out at 25° C. (room temperature) and 50° C. based on the results of Example 1, and the results obtained using the Cys-FPBA2 conjugates synthesized at various temperatures were compared.
Under the optimal conditions for synthesizing Cys-FPBA2 as determined in Examples 1 to 5 and Test Example 1, 5 mM cystamine and 20 mM 3-formylphenyl boronic acid were reacted with each other in a DMSO solvent at 50° C. for 4 hours in the presence of 1.1 μl of TEA as a catalyst.
In order to confirm whether the surface of Cys-FPBA2 SAM/Au thus synthesized was modified as desired, a comparative test with cystamine having no BA conjugated thereto was carried out. In the same manner as in the tests in which modification with BA was confirmed, 25 μg/ml of GOX was reacted with each of cystamine having no BA conjugated thereto and a gold electrode having Cys-FPBA2 immobilized thereon, and the patterns of immobilization with GOX were compared.
It was expected that, in the case of the electrode modified with BA, GOX would be immobilized on the electrode surface by the interaction between BA and cis-diol so that the electrochemical signal of GOX would be detected, and in the case of the cystamine which was not modified with BA, GOX would not be immobilized, because a functional group to which GOX can be immobilized does not exist.
In fact, as can be seen in
Using Cys-FPBA2 synthesized under the optimal conditions confirmed in the above examples and test examples, a BA SAM was formed on the electrode surface, and electrochemical measurement was carried out at various concentrations of HbAlc. For electrochemical measurement of HbAlc, native GOX and competition assays were performed.
The competition assay was performed using each of a buffer sample consisting of HbAlc alone (Test Example 3-1), an HbAlc containing BSA (Test Example 3-2), and a reference sample (Test Example 3-3).
In order to determine whether the competition assay of the present invention is suitable for measurement of HbAlc %, a electrochemical signal was detected as a function of the concentration of HbAlc. Specifically, 20 μg/ml of an HbAlc sample (corresponding to a 7500-fold dilution of 150 mg/ml which is the average total hemoglobin concentration of normal persons) was diluted to a concentration ranging from 0% to 15% (0.5 μg/ml to 3 μg/ml), and a signal was measured as function of the concentration of HbAlc.
In order to verify reproducibility, three dependent repeated tests were carried out. As a result, a low HbAlc concentration of 2.5% (0.5 μg/ml), a middle HbAlc concentration of 7% (1.4 μg/ml) and a high HbAlc concentration of 15% (3 μg/ml) were selected, and COV (coefficient of variation) was calculated. The COV of the standard sample solution consisting of HbAlc alone was 9% in an HbAlc concentration of 2.5% (0.5 μg/ml), 17% in a HbAlc concentration of 7% (1.4 μg/ml), and 29% in a HbAlc concentration of 15% (3 μg/ml), suggesting that COV increases as the concentration of HbAlc increases. This is believed to be because the signal (current) of GOX decreases as the concentration of HbAlc increases, and thus the COV value increases. This test example could demonstrate that the BA SAM-based competition assay is useful for measurement of HbAlc.
Because the HbAlc samples in Test Example 3-1 consist of HbAlc alone, the total concentration proteins of the samples which are allowed to react with the electrode modified with GOX differ from each other. The measurement of HbAlc % is carried out to measure HbAlc in a total Hb sample which comprises HbAlc (glycated hemoglobin (Hb)) and HbA0 (non-glycated hemoglobin).
Before a test was carried out in a total Hb sample comprising HbA0 and HbAlc, the signal of HbAlc % comprising HbAlc and BSA in place of HbA0 was measured. Similarly, 20 μg/ml of total protein (assumed as 100% Hb) was diluted to a concentration ranging from 2.5% to 15% to prepare HbAlc samples, and a competition assay of each sample with GOX was carried out.
In this test example, it could be seen that the competition assay can be applied in a test based on total Hb and that the ratio for the Hb sample and the GOX sample for the competition assay is suitable. The ratio of concentration between the two samples is very important in carrying out the competition assay. This is because when the ratio of GOX is too high or low, the electrochemical signal is too large or small to accurately detect the signal of HbAlc.
In order to measure HbAlc % in blood, a test was carried out using HbAlc reference samples (Lyphochek Hemoglobin Linearity set) purchased from Bio-Rad, Inc. The samples used were reference samples close to whole blood, which were lyophilized according to their HbAlc levels. Because no total hemoglobin level was indicated on the four samples included in the linearity set, the total hemoglobin and glucose levels of each sample were using Reflotron (Roche), before the competition assay was carried out. As a result, it could be seen that the amount of total Hb was different between the samples, and each of the samples contained a significant amount of glucose. Because glucose in blood can react with BA, like HbAlc, it interferes with the measurement of the concentration of HbAlc %. For accurate measurement of HbAlc, glucose was removed from each sample, and then the total Hb of each sample was adjusted to 20 μg/ml and used in the test.
As shown in
As described above, according to the present invention, cystamine is bound to the electrode surface, and boronic acid is conjugated to the cystamine. Thus, an increased amount of boronic acid can be bound to the electrode surface, so that the amount of glycoprotein can be more accurately measured using the electrode.
In addition, according to the present invention, the electrode having boronic acid bound thereto can be prepared in a simple and easy manner by preparing a cystamine-boronic acid conjugate and dipping an electrode once in a solution containing the conjugate.
Although the preferred embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2011-0070646 | Jul 2011 | KR | national |
