KR101700513B1 - Method for detecting thrombin using surface-modified gold electrode - Google Patents

Abstract

The present invention relates to a method for detecting thrombin using a surface-modified gold electrode, and more particularly, to a method for detecting thrombin using surface-modified gold electrode, (Thrombin) can be precisely detected with high selectivity, high specificity and high sensitivity by optimizing the formation of the target protein (thrombin) and using ficinene labeled with ferrocene (Fc-Fib) as an electrochemically active marker And a method for electrochemical detection of thrombin using a gold electrode.

Description

METHOD FOR DETECTING THROMBIN USING SURFACE-MODIFIED GOLD ELECTRODE BACKGROUND OF THE INVENTION [0001]

The present invention relates to a method for detecting thrombin using a surface-modified gold electrode, and more particularly, to a method for detecting thrombin using surface-modified gold electrode, (Thrombin) can be precisely detected with high selectivity, high specificity and high sensitivity by optimizing the formation of the target protein (thrombin) and using ficinene labeled with ferrocene (Fc-Fib) as an electrochemically active marker And a method for electrochemical detection of thrombin using a gold electrode.

Thrombin is an essential proteolytic enzyme involved in clotting cascades, major thrombus processes and hemostasis. It is known that thrombin converts fibrinogen into fibrin to promote blood clotting and acts as a tumor marker in the diagnosis of pulmonary metastasis. In addition, abnormal levels of thrombin in the blood are known to be associated with disease, and these levels of thrombin vary greatly from low μM to low nM levels. Therefore, it is important to measure and evaluate thrombin with high sensitivity and selectivity.

Immunoassay using hirudin peptide, which quantitatively reacts with thrombin, has been used to measure the concentration of conventional thrombin. The thrombin concentration was confirmed by ELISA or affinity chromatography on the formed thrombin-hirudin complex. However, when hirudin is bound externally to other proteins, it can not bind to thrombin, which makes it difficult to measure the exact amount.

Further, a method of detecting proteins such as thrombin using an abdominal thermometer is also known. However, in this method, the platemer that binds specifically to the protein must be fixed on the surface, and in the detection method using the enzyme reaction of the bound protein, the activity of the protein is reduced due to binding with the platamer, There is a problem that detection is not easy.

In this respect, a method of electrochemically detecting thrombin has been proposed. Such electrochemical thrombin detection methods are of great interest in that they are simple, clean, efficient, economical, and easy to miniaturize.

Fibrinogen is composed of a pair of polypeptide chains linked by disulfide bonds, and each fibrinogen contains two outer D domains and one central E domain by a coiled-coil part. As the thrombin cleaves these fibrinogen chains, fibrin polymerization initiates, and charge interactions between the D and E domains generate assemblies to form the fibrin polymer.

In addition, fibrin monomers are generated from the cost-liquid state surface-bound fibrinogen by the catalytic activity of thrombin, which can interact with the fibrin as well as the original fibrinogen, which is advantageous for producing fibrin assemblies It can provide an opportunity.

What is interesting here is that the arrangement and amount of fibrin adsorbed on the surface can be changed according to different surface chemistries. Such a study of the orientation of the fibrin molecules is important in that it can affect the reactivity and accessibility of thrombin near surface-bound fibrin.

Ferrocene, on the other hand, consists of two cyclopentadiene rings and one iron atom present between these rings, and has a distinct redox peak that is electrochemically reversible. Specifically, the target molecule labeled with ferrocene can exhibit an anode peak at a low potential near 0.5 V.

Although there have been several studies to date on fibrinogen assembly in a variety of ways, there have been few reports of electrochemically detecting thrombin using ferrocene labeled fibrinogen (Fc-Fib).

Korean Patent Publication No. 10-2012-0025226

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems of the prior art, and it is an object of the present invention to provide a method of detecting thrombin electrochemically using ferrocene labeled fibrinogen (Fc-Fib) A novel type of thrombin detection method capable of optimizing the adsorption and fixation of a fibrinogen assembly associated with the enzymatic reaction of thrombin.

Specifically, as a result of intensive studies, the present inventors have found that the surface properties of gold electrode modified with alkane thiolate have an effect on the amount and orientation of surface-bound fibrinogen, and based on this, the fibrinogen assembly is efficiently Specificity and sensitivity, thereby maximizing the detection performance of thrombin. The present invention has been accomplished on the basis of this finding.

In order to achieve the above object,

(a) immersing a gold electrode in a solution of an alkanethiolate to obtain an electrode surface-modified with an alkane thiolate linker molecule;

(b) adding a fibrinogen solution to the surface-modified electrode to immobilize fibrinogen;

(c) adding thrombin to the fibrinogen-immobilized electrode to form a fibrin monomer;

(d) contacting a target material containing fibrinogen (Fc-Fib) labeled with ferrocene and a thrombin on an electrode on which fibrin monomer is formed to induce an aggregation reaction by the enzyme activity of thrombin; And

(e) electrochemically detecting thrombin by measuring an oxidation-reduction current generated by the electron transfer of the ferrocene;

(Refer to FIG. 1). FIG. 1 is a cross-sectional view illustrating a thrombin detection method according to an embodiment of the present invention. FIG.

In the step (a), an alkane thiolate layer having various functional groups as terminal linker molecules is formed on a gold electrode to modify the electrode surface. Here, the alkanethiolate serves to control the amount and orientation of the fibrinogen assembly immobilized thereon.

As the alkanethiolate (or alkanethiol), mercaptohexanol, mercaptundundecanoic acid or mercaptundecanol can be used. Specifically, the present inventors have found that when various alkanethiolates such as dodecanethiol (CH ₃ -), mercaptohexanol (MCH), mercaptundundecanoic acid (COOH-), mercaptoundecanol And the effects of the modified surface properties on the amount, arrangement and orientation of surface-bound fibrinogen were investigated. As a result, the detection performance was excellent when mercaptohexanol, mercaptoundecanoic acid and mercaptoundecanol were applied, and mercaptoundecanol (MCU) among them provided the most efficient assembly for detecting thrombin Respectively.

Further, the inventors of the present invention found that when the Fc-Fib is directly immobilized on the surface of the gold electrode modified with alkane thiolate as the first layer, and when Fc-Fib is immobilized on the fibrin monomer as the second layer, Electrochemical methods (for details, see Experimental Examples described later). Here, the latter was performed by adding a thrombin solution on the fibrinogen / alkanethiolate / electrode to form a fibrin monomer layer and then immobilizing Fc-Fib on the fibrin monomer surface. As a result, the value of the oxidation peak current measured from the Fc-Fib formed on the fibrin monomer on the surface-modified electrode was very high, about 77% of the value of the oxidation peak current measured from the Fc-Fib formed directly on the surface-modified electrode Respectively.

In this step, a gold electrode may be further rinsed with ethanol, acetone, and water before immersing in an alkanethiolate solution, followed by a pretreatment by immersing the electrode in a Piranha solution . Here, the Piranha solution is a mixed solution of sulfuric acid and hydrogen peroxide, which can be used to completely remove impurities from the electrode and prepare a precise measurement of the precise amount of thrombin.

The thrombin detection kit including the surface-modified gold electrode of the present invention may further include a bio chip or the like within a range not contradictory to the object of the present invention, and may be a film, a filter, a chip, a slide , Wafers, fiber-magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles, capillaries, and the like.

In the step (b), the fibrinogen solution is immobilized on the alkane thiolate linker layer by applying a fibrinogen solution to the surface-modified gold electrode through the step (a). Here, the characteristic of the immobilized fibrinogen assembly is determined by the specific type of alkane thiolate located below.

In the step (c), thrombin is added to the electrode on which the fibrinogen is immobilized through the step (b), and the fibrinogen is converted into the fibrin monomer by the enzymatic action of thrombin. Here, the thrombin may be applied in the form of a solution, and an incubation process may be further performed after the thrombin is applied.

In the step (d), the target material containing fibrinogen (Fc-Fib) and thrombin labeled with ferrocene is simultaneously contacted with and exposed to the surface of the fibrin monomer formed through step (c) Thereby causing flocculation by the activity. Here, the contacted thrombin catalyzes the agglutination reaction, resulting in the formation of Fc-Fibrin polymerization labeled with ferrocene on the electrode.

The ferrocene is used as an electrode active material and an electron transfer mediator, and is a molecule capable of generating an oxidation-reduction current without causing a decrease in protein activity. Ferrocene is a sandwich compound, the molecular formula is (C ₅ H ₅ ) ₂ Fe and is also called dicyclopentadienyl iron. Ferrocene is an orange crystal having stable properties such as aromatic compounds such as benzene, which can easily cause a substitution reaction, and can be used as an electrode active material and an electron transfer mediator to simultaneously realize a thrombin concentration reporter or positive charge imparting characteristic.

The target protein thrombin in the present invention performs enzymatic activity to convert fibrinogen to fibrin in the human body. Through this, blood platelets, blood cells and blood plasma around blood clots are formed, which is involved in the clotting mechanism of blood. According to the present invention, thrombin can be detected very rapidly by using a blood coagulation reaction accompanied by a reaction mechanism even in microscopic reactions in a very systematic manner in vivo.

The target substance in the mixed solution contacting the detecting electrode (kit) according to the present invention may be arbitrarily configured. When it is known that the target substance contains thrombin, the detecting electrode (kit) Lt; / RTI >

In this step, the ferrocene-labeled fibrinogen (Fc-Fib) is an active marker for obtaining an electrochemical signal generated by enzymatic aggregation reaction with thrombin. Specifically, the Fc-Fib solution in the present invention is used not only for electrochemical detection of thrombin but also for investigating an adsorption fibrinogen assembly for optimizing a sensing platform.

In one embodiment, the ferrocene-labeled fibrinogen (Fc-Fib) is dissolved by adding fibrinogen to a bicarbonate buffer of 0.01 to 1 M (e.g., 0.05 to 0.3 M) , 9.2 to 9.5), and then a ferrocene solution in which ferrocene is melted in an anhydrous solvent (for example, a polar solvent such as DMSO, DMF, THF, HMPA, or acetone) is added to the bicarbonate buffer solution to which fibrinogen is added (See Figure 1 below). ≪ tb > < TABLE >

[Figure 1]

At this time, Sephadex column chromatography may be additionally performed to separate the ferrocene-labeled fibrinogen (Fc-Fib) from the unreacted ferrocene and the unlabeled fibrinogen. G-15, G-25, G-50, and G-15 are used in the present invention in order to appropriately separate ferrocene-labeled fibrinogen (Fc-Fib) -75, G-100 and the like (preferably, G-25) can be used. In one embodiment, the Sephadex column is activated using Cleavage Buffer, and the unlabeled fibrinogen is first removed by adding the mixed solution. It is also possible to remove the ferrocene-labeled fibrinogen (Fc-Fib) in the column by eluting the column with a cleavage buffer solution after transferring the column to a clean tube.

Meanwhile, the ferrocene-labeled fibrinogen (Fc-Fib) can also be prepared by the reaction shown in FIG.

[Figure 2]

The step (e) is a step of electrochemically detecting the target protein thrombin by measuring the oxidation-reduction current generated using the ferrocene introduced as an electron transfer mediator through the step (d). Specifically, thrombin can be detected by measuring the oxidation-reduction current value by contacting a fibrinogen-labeled fibrinogen (Fc-Fib) and thrombin mixed solution in contact with a fibrin monomer layer formed on the electrode surface, Is proportional to the Fc-Fibrin moiety labeled with the ferrocene.

In this step, the measurement of the oxidation-reduction current (or the oxidation current of the ferrocene) can be performed by means of a differential pulse voltammetry (DPV) or a cyclic voltammetry (CV) The differential pulse voltage / current method (DPV) is used. The present inventors have been able to optimize the sensor performance by monitoring the DPV current signal, which is dependent on the alkane thiolate used for surface modification of the electrode. When the concentrations of thrombin contained in the various samples are different, each sample is brought into contact with an electrode on which fibrin monomers are immobilized, such as ficronin (Fc-Fib) labeled with ferrocene, so that ferrocene-labeled fibrin Fc-Fibrin) is different. Here, the intensity of each current is measured differently when measuring the intensity of the current varying by the value of the oxidation-reduction potential through the differential pulse voltage / current method (DPV).

In this step, an electrode system can be used to measure the oxidation-reduction current. The electrode may be a single electrode or two or more electrodes. For example, in the case of a three-electrode system, a working electrode, a counter electrode, and a reference electrode system may be used. As the working electrode, gold, silver, copper, platinum carbon, ITO or aluminum electrode may be used. As the counter electrode, gold, silver, copper, platinum, aluminum or platinum wire electrode may be used. , A calomel electrode or a mercury (I) sulfate electrode can be used. In one preferred embodiment, the surface-modified electrode prepared through the steps (a) to (d) is used as a working electrode, the platinum electrode as a counter electrode, and the Ag / AgCl electrode as a reference electrode.

The thrombin detection method using the surface-modified gold electrode according to the present invention can detect a wide dynamic range, for example, 1 ng / ml to 1 μg / ml of thrombin with high sensitivity.

The electrochemical detection method of thrombin using the surface-modified gold electrode according to the present invention can accurately detect a target protein (thrombin) with high selectivity, high specificity and high sensitivity.

In addition, the electrochemical detection performance of thrombin using Fc-Fib can be maximized.

In addition, it can exhibit high selectivity over a wide range of thrombin dynamic range from 1 ng / ml to 1 μg / ml.

1 is a flow chart showing a process of electrochemically detecting thrombin using a gold electrode modified with an alkane thiolate according to the present invention.
Figure 2 is the ferrocene oxidation DPVs data (0.1 M PBS) for irradiating the fibrinogen assembly against the first layer Fc-Fib and the second layer Fc-Fib, respectively, for the different alkanethiolate linkers. [(A) = for the first layer Fc-Fib formed on the alkane thiolate / gold surface, (B) = for the second layer Fc-Fib formed on the fibrin monomer / alkanethiolate / gold surface]
Figure 3 is a graph showing the dependence of DPVs on the type of alkanethiolate associated with the detection of thrombin (concentration range: 1 ng / ml to 1 [mu] g / ml) on Fc-Fibrin / alkanethiolate / gold surface. [(A) = N _{linker, (B) = CH 3 -} , (C) = COOH-, (D) = MCH, (E) = MCU and insertion view showing a bar for a peak current of ferrocene as the thrombin concentration (F) = a line graph showing the peak oxidation current for thrombin concentration for each type of linker molecule and a line graph for Fc-Fibrin / MCH / gold surface as an insertion diagram;
Figure 4 (A) is DPVs data (0.1 M PBS) showing the selectivity test for various proteins (ADH, mouse-IgG, myoglobin, trypsin and thrombin) in the case of Fc-Fibrin / Is a bar graph showing (I _protein- I _blank ) / I _blank for each protein.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

(1) Materials and conditions

Thrombin (from bovine plasma) and fibrinogen were purchased from Sigma-Aldrich, ferrocenecarboxylic acid NHS esters from Biotech, and sodium carbonate-bicarbonate buffer (SCB) packs from Pierce.

TBS (Tris buffered saline) packs were purchased from Thermo scientific and then dissolved in deionized water to provide TBS (25 mM Tris, 150 mM NaCl, pH 7.2).

FCB (Fibrinogen cleavage buffer) was prepared by adding CaCl ₂ (2.5 mM) to TBS (pH 8.4; NaOH).

A PD MiniTrap Sephadex G-25 column was obtained from GE Healthcare UK Ltd.

CHI 617c electrochemical analyzer was used for electrochemical measurements.

Gold and its modified electrode were used as a working electrode, platinum (Pt) wire as a counter electrode, and Ag / AgCl as a reference electrode.

Fc-Fib in Ko, HJ .; Park, SM .; Kim, KW. J. Electroanal . Chem . 2015 , 742 , 70. < / RTI >

(2) As alkanethiolate Surface modified  Using gold electrodes Thrombin  detection

The gold electrode was rinsed successively with ethanol, acetone and water, and the surface was immersed in a Piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1) for 5 seconds.

Subsequently, the electrode was immersed in a solution of 1 mM alkane thiolate (dodecanethiol, mercaptohexanol (MCH), mercaptundundecanoic acid, mercaptoundecanol (MCU)) dissolved in ethanol for 1 hour, The electrode surface was washed with ethanol and water and then dried with N ₂ gas.

Fibrinogen solution (4.4 mg / ml, in FCB) was added to the electrode surface on which the alkanethiolate was immobilized for 1 hour at room temperature, followed by washing with TBS and water, followed by drying with N ₂ gas.

Then, thrombin solution was spotted on the obtained surface at 37 캜 for 1 hour to form a fibrin monomer surface.

Subsequently, thrombin solution of Fc-Fib and various concentrations (1 ng / ml to 1 μg / ml) was added to the obtained surface in a dark place at 37 ° C for 1 hour and then washed with TBS and water.

On the obtained surface-modified gold electrodes, anodic scan was performed using DPV at a scan rate of 50 mVs ^-1 using 0.1 M PBS.

(3) Layer 1 Fc - Fib  And the second layer Fc - Fib  formation

In order to evaluate the amount of fibrinogen adsorption on the first layer Fc-Fib and the second layer Fc-Fib, the following procedure was performed.

In the case of the first layer, the Fc-Fib solution was spotted on the electrode surface-modified with alkanethiolate at room temperature for 1 hour, and then washed with TBS and water.

In the case of the second layer, a fibrinogen solution (4 mg / ml, in FCB) was dropped on the electrode surface-modified with alkanethiolate, exposed at room temperature for 1 hour, and then washed. Subsequently, 1 [mu] g / ml of thrombin solution containing 0.1% BSA was added to form fibrin monomers and incubated at 37 [deg.] C for 1 hour. After the fibrin monomer-modified surface was cleaned, the Fc-Fib solution was exposed on its surface and then washed with TBS and water.

DPV was performed on the modified gold electrodes at a scan rate of 50 mVs < ^-1 > using 0.1 M PBS.

Experimental Example

(1) First layer Fc - Fib  The second layer Fc - Fib  Ratio of anode current

The effect of the alkane thiolate layer was evaluated by calculating the ratio of the Fc-Fib anode current in the second layer to the Fc-Fib anode current in the first layer and measuring the oxidation current of Fc-Fib.

In each of the different alkanethiolate linkers, Figure 2 (A) shows the ferrocene oxidized DPVs for irradiating the fibrinogen assembly against the first layer Fc-Fib formed on the alkane thiolate / gold surface, Figure 2 (B) Shows the ferrocene oxidized DPVs (using 0.1 M PBS) to irradiate the fibrinogen assembly against the second layer Fc-Fib formed on the fibrin monomer / alkanethiolate / gold surface.

As shown in FIG. 2, the surface modified with COOH- and OH- showed a distinct peak of Fc-Fib near 0.45 V.

On the other hand, in the first layer of fibrinogen, the surface immobilized with CH ₃ - showed a remarkable potential shift as compared with the other alkanethiolates, as the density of the CH ₃ -alkanethiolate layer increased, It is believed that a larger overvoltage is required for electron transfer.

Also, as seen in FIG. 2 (B), no noticeable peaks were observed in the second layer on the CH ₃ - modified electrode, which resulted in the formation of a high density layer of insoluble fibrin monomers on the surface, It means to disturb delivery.

The electrochemical oxidation peak current values of the ferrocene for evaluating the amount of fibrinogen in the first layer and the second layer, and the respective calculated ratios are summarized in Table 1 for each type of alkanethiolate.

[Table 1]

The amount of adsorbed fibrinogen showed different values as the functional group was changed.

(-) charged COOH-modified surface showed the highest peak current and the highest amount of fibrinogen was adsorbed.

On the other hand, in the case of the hydrophobic surface treated with the CH ₃ -terminal linker, the peak of the ferrocene could not be secured due to the high density of the alkanethiolate linker layer and the fibrin monomer layer.

For OH- modified hydrophilic surfaces, the ferrocene current in the first layer was the lowest, but the ratio of the anode currents was the highest. This result implies that the fibrinogen assembly on the OH-modified surface has reactivity and accessibility to fibrin polymerization by thrombin.

(2) Thrombin  Detection performance Alkane thiolate  Dependency on type

Figure 3 is a graph showing the dependence of DPVs on the type of alkanethiolate associated with the detection of thrombin (concentration range: 1 ng / ml to 1 [mu] g / ml) on Fc-Fibrin / alkanethiolate / gold surface. [(A) = N _{linker, (B) = CH 3 -} , (C) = COOH-, (D) = MCH, (E) = MCU and insertion view showing a bar for a peak current of ferrocene as the thrombin concentration (F) = a line graph showing the peak oxidation current for thrombin concentration for each type of linker molecule and a line graph for Fc-Fibrin / MCH / gold surface as an insertion diagram;

As shown in FIG. 3 (B), in the case of the surface modified with CH ₃ -, the electron transport of the ferrocene was blocked by the dense layer on the surface, which is consistent with the result in FIG.

For convenience of comparison, the relationship between the oxidation peak current and the thrombin concentration is shown in Figure 3 (F) for each linker molecule. Overall, in the absence of various linkers and linkers, only MCU-modified surfaces showed a good linear relationship between peak current and thrombin concentration (dynamic range: 1 ng / ml to 1 μg / ml).

In addition, The histogram of the thrombin concentration is shown in Fig. 3 (E). As shown in Fig. 3 (E), the correlation coefficient (R ² ) value is close to 1, indicating that this method realizes excellent linearity. That is, as shown in FIG. 2, it was confirmed that the OH-terminal surface formed an efficient fibrin assembly.

Fibrinogen on the OH-terminal surface tends to have a "side-on" orientation. This orientation allows the fibrinogen to interact favorably with thrombin and soluble fibrinogen because the three domains of fibrinogen deposited in the ordered state are exposed to the solution. That is, compared to MCH, MCU-functionalized surfaces have appropriate size and functionalities.

From these results, it was confirmed that the performance of the thrombin detection depends very much on the alkanethiolate layer.

(3) Selectivity test for various proteins

In order to investigate the specificity of the protein for the catalytic polymerization of Fc-Fib, the MCU-modified surface was tested.

Various proteins including alcohol dehydrogenase (ADH), mouse-IgG, myoglobin, trypsin and thrombin were prepared at the same concentration.

4 (A) is DPVs data showing the electrochemical reaction of ferrocene against various proteins (ADH, mouse-IgG, myoglobin, trypsin and thrombin) in the case of Fc-Fib / MCU / gold surface.

As shown in FIG. 4 (B), the ferrocene current greatly changed only in the presence of thrombin, and the other cases showed very small electrochemical reaction changes.

From these results, it can be seen that only thrombin catalyzes the change of Fc-Fib and the method according to the present invention shows excellent selectivity for thrombin detection.

(4) Review the results

The present inventors have demonstrated the effect of the alkanethiolate layer on the electrochemical detection performance of thrombin using Fc-Fib.

First, the fibrinogen arrangement was examined by an electrochemical method. As a result, it was confirmed that the fibrinogen arrangement was dependent on the type of alkanethiolate.

Specifically, the immobilized surface of the MCU provided the most efficient assembly for thrombin detection, and this method showed very high selectivity for thrombin due to the specific conversion of fibrinogen to fibrin polymer.

In addition, the dynamic range of thrombin with high sensitivity was 1 ng / ml to 1 μg / ml.

Thus, it is anticipated that the present invention, in addition to providing a practicable thrombin detection technique, would be a useful step in connection with electrochemical detection of adsorption-fibrinogen assemblies.

Claims (10)

(a) immersing a gold electrode in a mercaptoundecanol solution to obtain an electrode surface-modified with a mercaptoundecanol linker molecule;
(b) adding a fibrinogen solution to the surface-modified electrode to immobilize fibrinogen;
(c) adding thrombin to the fibrinogen-immobilized electrode to form a fibrin monomer;
(d) contacting a target material containing fibrinogen (Fc-Fib) labeled with ferrocene and a thrombin on an electrode on which fibrin monomer is formed to induce an aggregation reaction by the enzyme activity of thrombin; And
(e) electrochemically detecting thrombin by measuring an oxidation-reduction current generated by the electron transfer of the ferrocene;
And a surface-modified gold electrode.
The method according to claim 1,
Wherein the value of the oxidation peak current measured from Fc-Fib formed on the fibrin monomer on the surface-modified electrode is 77% of the value of the oxidation peak current measured from Fc-Fib formed directly on the surface-modified electrode. A method for detecting thrombin using a gold electrode.
The method according to claim 1,
The ferrocene-labeled fibrinogen (Fc-Fib) in the step (d)
After adding fibrinogen to 0.01 to 1 M bicarbonate buffer to dissolve and adjust the pH to 9 to 10,
A method of detecting thrombin using a surface-modified gold electrode, wherein the ferrocene solution is prepared by adding and reacting a ferrocene solution in which ferrocene is melted in a solvent of anhydrous condition, to the bicarbonate buffer solution to which the fibrinogen is added.
The method of claim 3,
Characterized in that Sephadex column chromatography is additionally carried out to separate the ferrocene labeled fibrinogen (Fc-Fib) from unreacted ferrocene and unlabeled fibrinogen. METHOD FOR DETECTING THROMBIN USING ELECTRODE.
The method according to claim 1,
Wherein the step (e) comprises measuring a redox current using a differential pulse voltammetry (DPV) method.
6. The method of claim 5,
Wherein the surface-modified electrode prepared through steps (a) to (d) is used as a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. Detection method.
The method according to claim 1,
The gold electrode of step (a) is rinsed with ethanol, acetone and water in succession and immersed in a Piranha solution before being immersed in a mercaptoundecanol solution. A method for detecting thrombin using a modified gold electrode.
The method according to claim 1,
A method for detecting thrombin using a surface-modified gold electrode, which is capable of detecting thrombin at a concentration of 1 ng / ml to 1 μg / ml.
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