KR101610467B1 - Biosensor for detecting target material using graphene oxide and method for preparing the same - Google Patents

Abstract

The present invention relates to a biosensor for detecting a target material in vivo using a change in reduction potential of graphene oxide and a method for manufacturing the same. The biosensor for detecting a target substance in vivo according to the present invention is an electrochemical marker, and the sensitivity and selectivity to a target substance are excellent by using a change in the reduction potential of the graphene oxide, so that the concentration of the target substance is easy to analyze, When a target substance is detected using a sensor, it exhibits an excellent target substance detection effect by a simple and economical method.

Description

TECHNICAL FIELD The present invention relates to a biosensor for detecting a target substance using graphene oxide and a method for preparing the same,

The present invention relates to a biosensor for detecting a target material in vivo using a change in reduction potential of graphene oxide and a method for manufacturing the same.

In general, target substances present in vivo include metal ions, DNA, RNA, ATP, and proteins. Among these proteins, thrombin is a protein existing in the blood and is an important protein in the blood coagulation mechanism. However, when the concentration of thrombin normally present in the blood is deviated, a case has been reported that causes disease (J. Bichler, JA Heit, WG Owen, Thromb. Res. 84 (1996) 289-294) A method for measuring the concentration of thrombin is required. For conventional thrombin concentration measurement, immunoassay using hirudin peptide, which quantitatively reacts with thrombin in a 1: 1 ratio, was used. The thrombin-Hirudin complex formed at this time can be detected by ELISA or affinity chromatography. However, this method has a disadvantage in that, in the case of hirudin, it can not bind to thrombin covalently bound to another protein in vitro. In addition, since the immune response-based ELISA assay, which is currently used for measuring the protein concentration in a biological sample, uses a protein with low stability, special care is required for storage when applied to a sensor, The analysis equipment is very expensive.

Electrochemical biosensors have evolved into superior analytical techniques in the areas of disease diagnosis, food analysis, environmental observation and analysis due to their simplicity, sensitivity, selectivity, and economy. In addition, the biosensor can be miniaturized without deterioration in its performance as compared with other biosensors. Studies are underway to further improve the sensitivity and selectivity of electrochemical biosensors using new biosensor platforms and strategies. Among them, interest in a platform of a biosensor using graphene is increasing.

Graphene is a semimetallic nanomaterial with a thickness of one layer of carbon atoms, forming a honeycomb arrangement of sp ² bonds on a two-dimensional topology, and has a large specific surface area (2,630 m ² · g ^-1 ) Lt; / RTI > In addition, it is structurally and chemically very stable, and has excellent electrical and thermal conductivity and is known as a material that can be applied to electronic devices, sensors, super capacitors, solar cells, and heat dissipation materials.

In addition, graphene oxide (GO) and electrochemically reduced graphene oxide (ERGO) are widely used in biosensor components, cellular imaging, drug delivery, . They have a much larger surface area than conventional graphite, have high electrical conductivity and strong mechanical strength. It is also considered to be an excellent electrode surface material because it is more flexible than graphite which is brittle, has advantages in electric appliances that require flexibility, and has more uniformly distributed electrochemically active sites.

Therefore, there is a strong demand for a method for electrochemically detecting thrombin using such graphene oxide.

KR 10-2010-0046496

DISCLOSURE OF THE INVENTION In searching for a biosensor for detecting a target substance in vivo, the present inventors have confirmed that sensitivity and selectivity in detection of a target substance are excellent when a change in reduction potential of graphene oxide is used as an electrochemical marker, Thereby completing the invention.

Accordingly, the present invention provides a biosensor for detecting target substances in vivo using a change in reduction potential of graphene oxide as an electrochemical marker, and a method for producing the same.

In order to achieve the above object,

The present invention

An electrode surface-reformed with graphene oxide (GO); A target substance-specific aptamer immobilized on the electrode; A target material bound to the squib; And

And a reduction potential measuring portion of the graphene oxide.

In addition,

(1) immersing an electrode in a graphene oxide (GO) solution to produce an electrode surface-modified with graphene oxide;

(2) immobilizing a target substance-specific specific aptamer on the electrode surface-modified with the graphene oxide;

(3) adding a target material to the electrode on which the target material-specific aptamer is immobilized, thereby binding the target material to the platemaker; And

(4) electrochemically reducing the graphene oxide of the electrode in the step (3) to measure the reduction potential.

Hereinafter, the present invention will be described in detail.

The present invention relates to an electrode surface-modified with graphene oxide (GO); A target substance-specific aptamer immobilized on the electrode; A target material bound to the squib; And

And a reduction potential measuring portion of the graphene oxide.

The electrode can be used without limitation as long as it is commonly used in the field. The electrode may include gold, silver, copper, platinum carbon, indium tin oxide (ITO), and aluminum. In the present invention, a gold electrode is preferable.

Generally, "abtamer" means an abatumer capable of specifically binding to a target substance to be detected. In the present invention, an abatumer that specifically binds to a target substance is used.

The target substance-specific aptamer can be any DNA or RNA sequence that selectively binds to a target substance, and may additionally include six carbon atoms to enhance binding efficiency with the target substance. In particular, -NH ₂ may be added to the 5'-end of the target material-specific platemer for fixation on the gold electrode surface. In one example, the target substance-specific aptamer may consist of a nucleotide sequence of SEQ ID NO: 1 that specifically binds to thrombin.

The target substance can be detected in a living body, without limitation, if it is a substance that can bind to the target substance-specific tympanic membrane by physical or chemical interaction. That is, the target substance refers to a chemical or biomaterial such as metal ions, DNA, RNA, ATP, and proteins present in vivo.

Such proteins include, but are not limited to, thrombin, fibrinogen, immunoglobulin G, IgG, immunoglobulin A, IgA, IgD, hemoglobin, But are not limited to, myoglobin, albumin, casein, prolamin, actin, myosin, collagen, and keratin, and the like. Do not.

In the present invention, the measurement of the concentration of the target substance using the graphene oxide means measuring the amount of the target substance bound to the target substance-specific pressure stimulator, and the amount of the target substance bound to the pressure- Electrochemically reducing graphene oxide and measuring the reduction potential.

In order to measure the reduction current, it is preferable to use electrochemical impedance spectroscopy or cyclic voltammetry.

In the biosensor for detecting a target substance in vivo using the graphene oxide according to the present invention, the charge transfer resistance (R _ct ) of the electron transferring redox substance is further increased as the concentration of the target substance increases, And the concentration of the target substance can be accurately measured since the reduction peak potential of the GO gradually shifts in the positive direction.

The present invention also relates to a method of manufacturing a semiconductor device, comprising the steps of: (1) immersing an electrode in a graphene oxide (GO) solution to produce an electrode surface-modified with graphene oxide; (2) immobilizing a target substance-specific specific aptamer on the electrode surface-modified with the graphene oxide; (3) adding a target material to the electrode on which the target material-specific aptamer is immobilized, thereby binding the target material to the platemaker; And (4) electrochemically reducing the graphene oxide of the electrode in the step (3) to measure the reduction potential.

The method of detecting a target material may comprise continuously contacting the electrode with mercaptopropionic acid (MPA) before the step (1) to form a negatively charged layer and a positively charged layer on the electrode surface, ) And a polyethyleneimine (PEI) solution.

Further, in order to activate the carboxylic acid of the GO surface to produce an activated ester, after the step (1), the electrode is immersed in an aqueous solution of N- (3-dimethylamino-propyl) -N'-ethylcarbodiimide (EDC) and a mixture of N-hydroxy-succinimide (NHS).

The biosensor according to the present invention is advantageous in that the concentration of the target substance can be easily analyzed by using the change in the reduction potential of the graphene oxide as an electrochemical marker, and the target substance can be detected by a simple and economical method.

The biosensor for detecting a target substance in vivo according to the present invention is an electrochemical marker, and the sensitivity and selectivity to a target substance are excellent by using a change in the reduction potential of the graphene oxide, so that the concentration of the target substance is easy to analyze, When a target substance is detected using a sensor, it exhibits an excellent target substance detection effect by a simple and economical method.

FIG. 1 is a diagram showing a schematic process of a method for detecting thrombin in a target material in vivo according to the present invention.
FIG. 2 is a graph showing the Raman spectra of graphene oxide (GO) prepared in Example 1 and (b) a pure gold electrode, (c) a gold electrode modified with PEI (PEI / MPA / Au) ) Showing a SEM image of a gold electrode (GO / PEI / MPA / Au) modified by GO.
FIG. 3 is a graph showing the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of a GO-surface-modified gold electrode (GO / PEI / MPA / Au) Fig.
4 is a graph showing a measurement result of a cyclic voltammetry (CV) for a surface-modified gold electrode before and after a TBA prepared according to Example 1 is fixed.
FIG. 5 shows (a) Nyquist plot and (b) reduction peak for thrombin bound gold electrode (thrombin / TBA / GO / PEI / MPA / Au) according to the concentration of thrombin from 1 ng / Fig.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Detection of thrombin using graphene oxide

Thrombin binding aptamer (SEQ ID NO: 1), and blocking DNA (5'-TTT TTG GGT TTT T-3 ') were purchased from Genotech, All chemicals were purchased from Sigma-Aldrich.

Base sequence SEQ ID NO: 1 NH ₂ -C ₆ -TTT TTT TTT TTT TTT AGT CCG TGG TTG GTG TGG TTG GGG TGA CT

Phosphate buffered saline (PBS) and 50 mM Tris-HCl, consisting of 25 mM Tris-HCl and 300 mM and 1 M NaCl, pH 7.4 in Tris-HCl buffer, 100 mM phosphate and 100 mM NaCl Binding buffer solution consisting of mM NaCl, 1 mM MgCl ₂ , and 0.1% bovine serum albumin (BSA) was used. All electrochemical measurements including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed using CHI-A617 and Ivium Compactstat. SEM images were obtained using JEOL JSM-7001F and Raman spectra were measured using alpha 300 (WITEC).

1-1. Preparation of Graphene Oxide (GO)

A graphene oxide (GO) suspension was prepared from the graphite powder using a modified Hummers method (W. Hummers, R. Offeman, Journal of the American Chemical Society 80 (1958) 1339.). First, graphite powder (3 g) was added into an Erlenmeyer flask, and after stirring in an ice bath, NaNO ₃ (1.5 g) and concentrated H ₂ SO ₄ (69 mL) were added. Then KMnO ₄ (9 g) was added slowly and the mixture was stirred for 3 hours. Then, 120 mL of deionized water was slowly added to the mixture for 30 minutes, then 200 mL of deionized water was added, and 30% H ₂ O ₂ (3 mL) was added slowly. At this time, if the color of the suspension changed from brown to yellow, it was centrifuged twice at 4500 rpm with 5% HCl solution, and centrifuged several times at a high rate with distilled water.

1-2. Preparation of surface modified electrode by GO

Prior to surface modification, the glass electrode coated with gold was washed with a piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1), washed with water and dried with nitrogen gas. Subsequently, the cleaned gold electrode was immersed in 10 mM mercaptopropionic acid (MPA) for 12 hours to form a negatively charged layer. Subsequently, a 0.1% polyethyleneimine (PEI) solution And then immersed for 1.5 hours to form a positively charged layer. Thereafter, the negatively and positively charged gold electrode was immersed in 0.5 mg / ml graphene oxide (GO) solution prepared in Example 1-1 for 1 hour and coated with graphene oxide (GO). To prevent non-specific binding of NH ₂ -terminal TBA, blocking DNA was treated on the electrode surface-modified with GO, and the electrode surface-modified with GO was replaced with 200 mM N- (3-dimethylamino- N-ethylcarbodiimide hydrochloride (EDC) and 50 mM N-hydroxy-succinimide (NHS) were added to the solution, (1: 1) for 1 hour to activate the carboxylic acid on the GO surface.

Thereafter, the gold electrode surface-modified with GO was immersed in a thrombin binding aptamer (SEQ ID NO: 1) to immobilize thrombin-bound thrombin on the electrode. The surface was then incubated with 1% BSA solution in PBS for 1 hour to prevent nonspecific binding of thrombin to the surface.

The TBA-fixed gold electrode was exposed to various concentrations of thrombin solution and incubated for 1.5 hours. In the negative control experiment, the same procedure was performed without thrombin.

1-3. Electrochemical measurement

The surface-modified electrode was subjected to electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to determine the reduction potential.

All electrochemical measurements were performed using an electrochemical cell consisting of gold electrode modified with working electrode, Ag / AgCl as reference electrode, and Pt wire as counter electrode. The CV measurement was carried out in the voltage range of -0.4 to -1.4 V, and the EIS was measured in the presence of K ₃ [Fe (CN) ₆ ] / K ₄ [Fe (CN) ₆ ] (1 mM, It was measured between 10 kHz and 0.05 Hz at formal potential. A Randle equivalent circuit was used to fit the impedance spectrum presented by the Nyquist plot.

The process of detecting a target substance in vivo according to the present invention is schematically shown in FIG.

Experimental Example 1. Scanning electron microscope (SEM), atomic force microscope (AFM) and Raman spectrum measurement

(B) a gold electrode; (c) a gold electrode (PEI / MPA / Au) modified with PEI; and (d) a Raman spectrum of the graphen oxide (GO) prepared in Example 1, An SEM image of the gold electrode (GO / PEI / MPA / Au) is shown in FIG.

As shown in Fig. 2 (a), the Raman spectrum for GO showed typical D / G peak ratios. As shown in Figs. 2 (b) to 2 (d), the SEM image shows that graphene oxide (GO) is stably adsorbed on the gold electrode.

Also, AFM measurement results indicated that the thickness of the graphene oxide (GO) monolayer was ~ 1 nm.

Experimental Example 2. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) measurements on electrodes surface-modified with GO

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of a GO-surface-modified gold electrode (GO / PEI / MPA / Au) The measurement results are shown in Fig. CV and EIS were measured using a lumbium compactstat as a potentiostat connected to PC, respectively, and redox couple of Fe (CN) ₆ ^3- and Fe (CN) ₆ ^4- .

As shown in FIG. 3 (a), the CV measurement results for the redox pair show that the GO is modified by the insulating properties of the GO, the repulsive force between the negatively charged GO layer and the oxidation-reduction pair, and the steric effect (GO / PEI / MPA / Au) exhibited a larger peak separation and a smaller peak current than a pure gold electrode (bare Au).

As shown in Fig. 3 (b), EIS measurements are pure gold electrode (bare Au) GO surface charge transfer resistance of the modified gold electrode (GO / PEI / MPA / Au ) (R ct) is suddenly as compared to As shown in Fig. As shown in FIGS. 3 (a) and 3 (b), the gold electrode (GO / PEI / MPA / Au) Which was slightly reduced compared to the control group. This result is thought to be due to the partial loss of the GO layer during the immersion process of the surface modified electrode with GO containing TBA fixation and EDC / NHS activation.

Experimental Example 3. Electrochemical reduction measurement of graphene oxide (G0) before and after TBA fixation

FIG. 4 shows the measurement result of the cyclic voltammetry (CV) for the surface-modified gold electrode prepared before and after the TBA was fixed according to Example 1. Here, the CV measurement was performed in a voltage range of -0.4 to -1.4 V, and 20 mM NaCl was used as an electrolytic solution.

As shown in FIG. 4, the gold electrode (TBA / GO / PEI / MPA / Au) having the TBA fixed moves 22.3 mV toward the negative direction compared to the gold electrode (GO / PEI / MPA / Au) This result implies that the reduction of GO is more difficult due to the fixation of TBA on the gold electrode. The immobilized TBA has high flexibility due to the long length of poly-Thymine, and the amine groups in the base of TBA are oxygen groups present in the GO, , A carboxyl group, and a carbonyl group. This interaction can stabilize the oxygen radicals in the GO, thus leading to a shift of the reduction potential in the negative direction.

Experimental example 4. Electrochemical reduction measurement according to thrombin concentration

(Thrombin / TBA / GO / PEI / MPA / Au) with a thrombin concentration of 1 ng / ml to 1 ug / ml in order to evaluate the sensitivity to thrombin detection A) Nyquist plot and (b) reduction peak potential are shown in Fig.

As shown in FIG. 5 (a), it was shown that the charge transfer resistance (R _ct ) increased as the concentration of thrombin increased from 1 ng / ml to 1 μg / ml. However, other proteins such as avidin, mouse IgG and the like showed little change in _Rct value. This result confirms that a gold electrode with a fixed TBA (TBA / GO / PEI / MPA / Au) can be used as a platform for detecting thrombin as a target material.

As shown in Fig. 5 (b), as the concentration of thrombin increased, the reduction peak potential of GO gradually shifted in the positive direction. This result indicates that the formation of thrombin-TB complexes makes the reduction of GO easier, probably because of the collapse of the attraction between the TBA and GO oxygen groups. From the results of Fig. 5 (b), a very low detection limit of thrombin of 10.8 ng / ml was obtained. However, this trend was not observed for TBA-immobilized gold electrodes (TBA / GO / PEI / MPA / Au) without blocking-DNA treatment.

Claims (11)

(1) immersing an electrode in a graphene oxide (GO) solution to produce an electrode surface-modified with graphene oxide;
(2) immobilizing a target substance-specific specific aptamer on the electrode surface-modified with the graphene oxide;
(3) adding a target material to the electrode on which the target material-specific aptamer is immobilized, thereby binding the target material to the platemaker; And
(4) electrochemically reducing the graphene oxide of the electrode in the step (3) to measure the reduction potential,
Wherein the reduction potential of the graphene oxide is gradually shifted in the positive direction as the concentration of the target substance increases as the electrochemical marker is used to change the reduction potential of the graphene oxide.
A method for electrochemical detection of a target material. The method according to claim 1,
Wherein the electrode is any one selected from the group consisting of gold, silver, copper, platinum carbon, indium tin oxide (ITO), and aluminum. The method according to claim 1,
The target substance-specific apta electrochemical detection method of the dimmer pressure, the target material comprises a DNA or RNA sequences having a -NH ₂ in the 5'-end of Tamer. The method of claim 3,
Characterized in that the target substance-specific aptamer consists of the following SEQ ID NO: < RTI ID = 0.0 > 1 < / RTI >
[SEQ ID NO: 1]
NH ₂ -C ₆ -TTT TTT TTT TTT TTT AGT CCG TGG TTG GTG TGG TTG GGG TGA CT The method according to claim 1,
Wherein the target material binds to a target material-specific platemeral by physical or chemical interaction. The method according to claim 1,
Wherein the target substance is any one selected from the group consisting of metal ions, DNA, RNA, ATP, and proteins. The method according to claim 6,
The protein may be selected from the group consisting of thrombin, fibrinogen, immunoglobulin G, IgG, immunoglobulin M, IgA, IgD, hemoglobin, myoglobin, wherein the protein is any one selected from the group consisting of myoglobin, albumin, casein, prolamin, actin, myosin, collagen, and keratin. ≪ / RTI > wherein the electrochemical detection method comprises the steps of: The method according to claim 1,
Wherein the reduction potential of the graphene oxide is measured by a cyclic voltammetry method or an electrochemical impedance spectroscopy method. The method according to claim 1,
The method of claim 1, further comprising, before step (1), immersing the electrode sequentially in a solution of mercaptopropionic acid (MPA) and polyethyleneimine (PEI) . The method according to claim 1,
After step (1), the step of immersing the electrode in a mixture of N- (3-dimethylamino-propyl) -N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide ≪ / RTI > further comprising: an electrochemical detection of the target substance. delete

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