KR20140108937A - Method and kit for detecting protein in real-time using nano metal structure - Google Patents

Abstract

The present invention provides a method for detecting protein in real time comprising: a step for coating a positive charged polymer on a nanometal structure; a step for absorbing a DNA aptamer tagging a fluorescent material onto the coated nanometal structure; and a step for binding target protein to the nanometal structure on which the DNA aptamer tagging the fluorescent material is absorbed, and a kit for detecting the protein. The method and the kit for detecting protein in real time are capable of detecting the protein in real time, have superior sensitivity and specificity of detection, and also are easy to handle by measuring fluorescence, thereby being widely used as an optical protein sensor.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting a protein using a metal nanostructure,

The present invention relates to a real-time protein detection method and a detection kit using a metal nanostructure, and more particularly, to a real-time protein detection method for detecting fluorescence amplification of a fluorescent substance bound to a DNA aptamer using a fluorescent amplification effect of a metal nanostructure And a detection kit.

The field diagnosis and detection technology is considered to be a technical bottleneck in medical and environmental fields. The size of the global diagnostic market is growing rapidly in the fields of customized medical diagnosis, molecular diagnosis and in vitro diagnostic market. In this regard, research is focused on sample delivery, sample preparation, data analysis and detection.

Previously, there was an electrochemical sensor (Chem. Asian J. 2010, 5, pp. 293-300) in the prior art for a sensor using a gold nanostructure, but in the case of an electrochemical sensor, There was a problem of signal reliability because the disturbance signal due to the molecules having activity appeared together. In the case of a study using a quantum dot and a quencher (Biosensors and Bioelectronics 26 (2011), pp. 3870-3875), the protocol required for the construction of the initial sensor is complex and the detection of the target DNA But it does not apply to proteins. In addition, the conventional patent No. 10-2008-0084029 discloses that a DNA aptamer binding to a target protein, a single strand DNA complementary to a part of aptamer, a single strand RNA complementary to the single strand DNA, and a target protein using RNase H Detection methods and detection kits, but the use of complementary DNAs with fluorescence has been problematic in that the operation steps are complicated and real-time measurement is impossible.

Korean Patent Publication No. 10-2008-0084029

In order to solve the above problems, the present invention provides a real-time protein detection method and detection kit using a metal nanostructure that can detect proteins in real time using a fluorescence measurement method and has excellent detection sensitivity and detection specificity, The purpose is to provide.

According to an aspect of the present invention, there is provided a method of fabricating a metal nanostructure, including: coating a positive charge polymer on a metal nanostructure; Adsorbing a DNA aptamer labeled with a fluorescent substance on the coated metal nanostructure; And binding the target protein to the metal nanostructure on which the DNA aptamer labeled with the fluorescent substance is adsorbed.

In one embodiment of the present invention, the change of fluorescence intensity of the metal nanostructure may be measured in the step of binding the target protein.

In one embodiment of the present invention, the metal nanostructure may be a nanoflower or a nananol structure.

According to an embodiment of the present invention, the metal nanostructure may include a metal layer formed by depositing a seed metal having a lower reduction potential than a substituted metal on a substrate; And

And a step of reacting the metal layer with a plating solution containing a substituted metal ion.

In one embodiment of the present invention, the metal of the metal nanostructure may be at least one selected from the group consisting of gold, silver, titanium, and zinc oxide.

In one embodiment of the present invention, the positive charge polymer is selected from the group consisting of chitosan, polyethylene imine, polylysine, poly-aspartic acid, spermine, protamine, , Polyamidoamine, polypropyleneimine, polybrene, polyacrylamide, polyvinyl amine and DEAE-dextran, in the group consisting of polyvinylpyrrolidone, polyvinylpyrrolidone, polyamidoamine, polypropyleneimine, polybrene, It can be one or more selected.

In one embodiment of the present invention, the molecular weight of the positive charge polymer may be 300 to 1000.

In one embodiment of the present invention, in the step of coating the positively charged polymer on the metal nanostructure, washing may be performed by stirring at a speed of 100 to 500 rpm for 20 to 40 minutes.

In one embodiment of the present invention, the fluorescent material is one selected from the group consisting of Cy3, fluorescein, tetramethylrhodamine, Texas Red, and NADH (nicotinamide adenine dinucleotide) Or more.

In one embodiment of the present invention, the fluorescent material may have a quantum yield of 1 to 50%.

In one embodiment of the present invention, the distance between the fluorescent material and the metal nanostructure may be 20 to 50 nm.

In one embodiment of the present invention, the DNA aptamer may have a length of 10 to 30 mers.

In one embodiment of the present invention, the target protein may be thrombin.

The present invention also relates to a metal nanostructure coated with a positive charge polymer; And a DNA aptamer labeled with a fluorescent material adsorbed on the metal nanostructure coated with the positive charge polymer, wherein the target protein is bound to the DNA aptamer, to provide.

The real-time protein detection method of the present invention can be widely applied as an optical protein sensor because it can detect proteins in real time by fluorescence measurement, has excellent detection sensitivity and detection specificity, and is easy to operate.

1 is a schematic diagram showing a real-time protein detection method according to an embodiment of the present invention.
FIG. 2 is a SEM photograph showing a metal nanostructure according to a molecular weight of a chitosan and a washing method in a real-time protein detection method according to an embodiment of the present invention.
3 and 4 are graphs of fluorescence image and fluorescence intensity according to the quantum yield of the fluorescent material used in the real-time protein detection method according to an embodiment of the present invention.
5 and 6 are graphs of fluorescence intensity and fluorescence intensity according to the length of DNA aptamer used in the real-time protein detection method according to an embodiment of the present invention.
7 and 8 are graphs of fluorescence intensity and fluorescence intensity according to the target protein concentration in the real-time protein detection method according to an embodiment of the present invention.
9 and 10 are fluorescence image and fluorescence intensity graphs showing the detection specificity of the real-time protein detection method according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate the present invention by those skilled in the art.

The present invention relates to a method for manufacturing a metal nanostructure, comprising: coating a positive charge polymer on a metal nanostructure;

Adsorbing a DNA aptamer labeled with a fluorescent substance on the coated metal nanostructure; And

And binding the target protein to the metal nanostructure adsorbed by the DNA aptamer labeled with the fluorescent substance.

Hereinafter, the real-time protein detection method of the present invention will be described in more detail.

First, a positive charge polymer is coated on the metal nanostructure.

In the present invention, the metal nanostructure may be a nanoflower or a nananol structure. The nanoflower structure means that the substituted metal is in the form of a petal, and the nanorewn structure means that the substituted metal is needle-shaped. The nanoflower or nananol structure may have a needle-like structure of 200 to 250 nm. Metal nanostructures with nanoflower or nananol structures have an increased fluorescence amplification effect compared to metal nanostructures with a round structure.

Such a metal nanostructure having a nanoflower or nananol structure can be specifically manufactured by the following method. That is, the metal nanostructure may be formed by depositing a seed metal having a lower reduction potential than a substitution metal on a substrate to form a metal layer; And reacting the metal layer with a plating solution containing a substituted metal ion.

The term "electroless substitution plating" in the present invention is a plating method which is obtained when a surface containing a metal salt and a reducing agent is brought into contact with a surface to be plated, wherein electrons obtained by oxidation of the seed metal are replaced with substitution metal ions To form a metal film. Such a plating method is advantageous in forming a uniform thickness, and has an advantage that the plating process is simple.

In the present invention, a metal means a metal and metal oxide having conductivity. In the present invention, the metal may be at least one selected from the group consisting of gold, silver, titanium, and zinc oxide, and preferably may be gold. For example, when the substitution metal is gold (Au), Ni, Pt, Ag or Cu, preferably Pt, Ag or Cu, which has a lower reduction potential than gold may be a seed metal.

In the present invention, the positively charged polymer is a positively charged polymer and is not particularly limited as long as it is a polymer having various molecular weights according to the degree of polymerization. Preferably, chitosan, polyethylene imine, polylysine, poly Polypropyleneimine, polybrene, polyacrylamide, polyvinylamine (also referred to as " polyvinylamine "), polyglycolic acid, polyvinyl amine, and DEAE-dextran. More preferably, it may be chitosan. Chitosan has an amine group (-NH ₂ ) at its end, and is a polymer material having a positive charge as a whole. It plays a role of adsorbing a relatively negatively charged DNA do. Chitosan is represented by the following formula (1).

[Chemical Formula 1]

The molecular weight of the positive charge polymer may be from 300 to 1000. If the molecular weight is less than 300, the strength of the positive charge is weak and fluorescence quenching by the metal occurs. If the molecular weight is more than 1000, the effect of amplifying the fluorescence signal is weakened.

The method of coating the positive charge polymer with the metal nanostructure is not particularly limited as long as it is a coating method known in the art. For example, a method of dissolving the positive charge polymer powder in an acid solution and reacting for 3 to 5 hours can be used. After the reaction, washing is preferably carried out by soaking the polymer in distilled water and shaking the polymer at a rate of 100 to 500 rpm for 20 to 40 minutes. In the case of just dipping in distilled water without shaking, the coating becomes too thick to retain the metal nanostructure prior to coating, creating a surface that has no effect on nanostructures. On the contrary, when the polymer is immersed in distilled water and shaking, the nanoflower or nananol structure of the metal nanostructure is maintained, and the shaking is performed at a rate of 100 to 500 rpm for 20 to 40 minutes shaking), the structure is best maintained.

When the positively charged polymer is coated on the metal nanostructure, the fluorescence amplification effect is increased. When the distance between the metal nanostructure and the fluorescent material is too close to each other, the energy of the fluorescent molecule is completely transferred to the metal, so that the fluorescence signal intensity is reduced due to the quenching phenomenon in which no fluorescence appears. To enhance the fluorescence amplification effect by the metal nanostructure. The positive charge polymer is preferable because it can be used for maintaining an appropriate distance between the metal and the fluorescent material because the thickness of the coating layer can be easily controlled by varying the molecular weight. In the present invention, the thickness of the coating layer coated with the positive charge polymer is preferably 20 to 50 nm.

Accordingly, the distance between the fluorescent material and the nanostructure is preferably 20 to 50 nm. If it is less than 20 nm, the fluorescence intensity decreases due to the generation of chromatic effect, while if it exceeds 50 nm, the signal amplification effect due to the nanostructure decreases.

Then, a DNA aptamer labeled with a fluorescent substance is adsorbed on the coated metal nanostructure.

In the present invention, the DNA aptamer is a fluorescent substance labeled with DNA. Because DNA aptamers are electronegative, adsorption occurs by electrostatic attraction to metal nanostructures coated with positively charged polymers. The adsorption reaction time is about 1 hour, and DNA solution reaction can be performed.

In the present invention, the fluorescent material may be one or more selected from the group consisting of Cy3, fluorescein, tetramethylrhodamine, Texas Red and NADH (nicotinamide adenine dinucleotide). Preferably Cy3.

In addition, the fluorescent material may have a quantum yield of 1 to 50%. Preferably 1 to 10%. When the quantum yield is within the above range, the fluorescence amplification effect is further increased. For example, it can be seen that the quantum yield of Cy3 is 10% and the fluorescence amplification effect is 10 to 15 times larger.

In the present invention, the DNA aptamer may have a length of 10 to 30 mers. If the DNA aptamer length is less than 10 mer, immobilization due to negative charge is impossible. If the DNA aptamer is more than 30 mer, the DNA aptamer can not be separated from the surface when the protein is bound to the DNA aptamer. Real time detection of the protein is possible when the DNA aptamer has a length in the above range.

Finally, the fluorescent substance binds the target protein to the metal nanostructure on which the labeled DNA aptamer is adsorbed.

The injected target protein reacts with the DNA aptamer, where charge shielding occurs, resulting in the separation of the fluorescent DNA-labeled aptamer. Therefore, the concentration of the protein can be measured by measuring the change in fluorescence intensity of the metal nanostructure before and after the injection of the target protein. FIG. 1 is a schematic view showing a process of separating a fluorescent substance by binding a DNA aptamer having a fluorescent substance to a protein.

In the present invention, the target protein is not particularly limited as long as it is a protein that selectively binds to an aptamer. Preferably thrombin. The reason why the target protein can be selectively detected in the present invention is that it selectively binds to a fluorescently labeled aptamer and induces it to be peeled off from the surface, thereby reducing fluorescence intensity.

The present invention also relates to a metal nanostructure coated with a positive charge polymer; And a DNA aptamer labeled with a fluorescent substance adsorbed on the metal nanostructure coated with the positive charge polymer. The protein detection kit measures a change in fluorescence intensity caused by binding of a target protein to a DNA aptamer.

In the measurement of fluorescence intensity change by the real-time protein detection kit according to the present invention, the concentration of the target protein showed a decrease of fluorescence signal of 30% or more to the level of 1 nM, indicating that the fluorescence amplification effect was sufficient and the detection sensitivity was excellent.

The present invention will be described in more detail through the following examples. However, the examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Example 1] A DNA aptamer labeled with a fluorescent substance adsorbed on a metal nanostructure coated with a positive charge polymer

Crystalline (100) p type Si wafers were cleaned by the standard RCA method and then 100 Å of Ti was deposited on the Si surface. Cu was then deposited on each sample to a thickness of 1000 Angstroms. Both the Ti layer (100 Å) and the metal layer (1000 Å) were deposited using an e-beam evaporator (Ultech) at a rate of 3 Å / s. The evaporation pressure was 2 × 10 ^-6 Torr. After metal deposition, the substrate was immersed in an electroless plating solution containing a diluted Au metal salt [KAu (CN) _{2 aqua} ]. The Au metal salt present in this solution displaced the metal layer with an Au metal layer. The progress temperature of this solution was maintained at 80 ° C using a double boiler. The substitution time was 2 minutes. The solution was stirred with a magnetic stirrer. After a reaction time of 2 minutes, the gold particles were distributed as seeds on the metal substrate. The substrate contained in the gold plating solution remained undisturbed. The reaction vessel containing the gold plating solution was removed from the hot plate. The gold plating solution containing the substrate was cooled at room temperature. Thus, the growth of nanoflowers at room temperature was achieved in the metal layer.

Then, the chitosan powder was dissolved in acetic acid (220 mg / 1.5 ml), and the obtained test piece was immersed in the acetic acid solution and reacted for 4 hours to conduct chitosan coating. The molecular weight of chitosan used was 1000. Then, in order to wash the chitosan, it was immersed in distilled water with shaking at 280 rpm for 30 minutes.

After coating with chitosan, aptamer DNA labeled with a fluorescent substance was adsorbed on the gold nanostructure. At this time, since the aptamer DNA is electrically negative, the chitosan adsorbed by the electrostatic attraction. The fluorescent material used was Cy3, which was a fluorescent molecule with a quantum yield of 10%. The adsorption reaction time of the aptamer DNA was 1 hour, and 10 mM of DNA solution was reacted.

[Test Example 1] Gold nanostructure according to molecular weight and washing method of chitosan

The DNA aptamer labeled with the fluorescent material adsorbed on the gold nanostructure coated with the chitosan (molecular weight 1000) prepared in Example 1 was observed in the molecular weight of chitosan. The DNA aptamers of Comparative Examples 1 and 2 were the same as those of Example 1, except that chitosan having a molecular weight of 1000 to 3000 and chitosan having a molecular weight of 5000 were used.

The chitosan on the surface was removed by spraying distilled water with each of Example 1 and Comparative Examples 1 and 2, washing with immersion for 30 minutes in distilled water, and distilled water were sprayed to remove the chitosan on the surface and immersed in distilled water for 15 minutes and 30 minutes Washing was performed with shaking at a speed of 280 rpm. The structure formed on the surface after washing was confirmed by SEM. 2 is a SEM measurement photograph for each condition.

As can be seen in FIG. 2, it was difficult to find gold nanostructures before chitosan coating when immersed in distilled water for 30 minutes without shaking, the first of two Washing methods. This means that the chitosan coating becomes thicker and the effect on the nanostructure can not be seen. The shaking washing method was shaken at 280 rpm while immersed in distilled water for 15 minutes and 30 minutes. This method was confirmed to maintain the original structure before the coating, unlike the washing method which was only immersed in distilled water. In particular, the structure was observed more clearly when shaking for 30 minutes. However, the original structure could not be confirmed at molecular weights of 1000 to 3000, and two molecular weights of 1000 and 5000 showed almost the same structure as the original structure. Therefore, the chitosan coating to be used as a fluorescence amplification sensor is preferably shaken at 280 rpm in distilled water for 30 minutes, and the molecular weight of chitosan is in the range of 1,000 to 3,000.

Test Example 2 Fluorescence intensity according to quantum yield of fluorescent substance

The fluorescence intensities of the fluorescent material (Cy3) labeled with the gold nanostructure coated with the chitosan (molecular weight 1000) prepared in Example 1 were measured according to the quantum yield of the fluorescent material. The fluorescent molecule of Example 1 uses Cy3 with a quantum yield of 10%. For comparison, the same conditions as in Example 1 were used in Comparative Example 3, except that FAM fluorescent molecules having a quantum yield of 90% were used, and in Example 1 and Comparative Example 2 except that the molecular weight of chitosan was 5000 3 were designated as Comparative Examples 4 and 5, respectively.

In order to compare the fluorescence amplification effect, DNAs with fluorescent molecules were adsorbed and confirmed on a gold surface having a general gold surface and a nanostructure. Chitosan having a molecular weight of 1000 and 5000 was used for the chitosan coating used.

Thereafter, DNA was treated with two different fluorescent molecules on each surface, and adsorption reaction was carried out for 1 hour each. And fluorescence image measurement was performed through a Scan Array Lite device. FIG. 3 is a fluorescence image of Cy3 fluorescent molecule and FAM fluorescent molecule, and FIG. 4 is a graph showing fluorescence intensity. The fluorescence intensity is examined according to fluorescent molecule, chitosan coating, and nanostructure. The fluorescence intensities of the prepared gold nanostructure and general gold were calculated for fluorescent molecules and chitosan coating, and the results are shown in Table 1 below.

Molecular weight of chitosan Cy3 staining
(Quantum yield 10%) FAM staining
(Quantum yield 90%) 1000 (Example 1)
Inano / Ibare: 15 (Comparative Example 3)
Inano / Ibare: 3.69 5000 (Comparative Example 4)
Inano / Ibare: 3.0 (Comparative Example 5)
Inano / Ibare: 3.05

In the case of chitosan coating, fluorescence signal of about 1.5 times higher than that of nanostructured substrate. After the chitosan coating, the nanostructured effect was found to be about three times as large as that of the coating. However, it was confirmed that the effect on the nanostructure was only 5 to 10 times as high as that on the surface using the Cy3 fluorescent molecule among the chitosan having a molecular weight of 1,000. These results show that the surface with gold nanostructures is advantageous while using Cy3 fluorescent molecules with low quantum yield as the surface for optical sensors.

[Test Example 3] Fluorescence intensity according to DNA aptamer length

The fluorescence intensity of the chitosan-coated gold nanostructure prepared in Example 1 was measured with a DNA aptamer having a fluorescent substance adsorbed on the gold nanostructure. The DNA aptamer of Example 1 had a length of 15 mers and the DNA aptamer of 21 mers length for comparison was used as Comparative Example 6. In the DNA aptamer, the region that reacts with thrombin is 15 mer long, so we want to check the change of fluorescence intensity using 15mer and longer 21mer. The purpose of this change is to find an aptamer DNA of suitable length as an optical sensor using a fluorescent amplified signal.

After the injection of thrombin, experiments on the presence or absence of a solution were carried out by real-time detection. The concentration of thrombin used was relatively high, 10 μM, for clarity, and the change in fluorescence intensity before and after the reaction was measured for 15 min after thrombin injection. The results are shown in FIGS. 5 and 6. FIG.

As can be seen in FIGS. 5 and 6, the fluorescence intensity change with the length of the DNA aptamer showed different results with respect to the presence or absence of the solution. In other words, when there is no solution on the surface, almost similar fluorescence intensity reduction was observed. However, when the solution was present, only the short-length 15-mer showed that the fluorescence intensity varied with or without the solution. This refers to a surface capable of real-time sensing of thrombin infusion at the surface where the solution is present. As can be seen in Figures 5 and 6, a more suitable length for the sensor was found to be the shorter length of the aptamer being the adsorbed surface. This is because the electrically absorbed DNA aptamer is separated from the surface with a specific reaction to thrombin, and the relatively short DNA aptamers are easily separated from the surface.

[Test Example 4] Detection sensitivity test: Fluorescence intensity according to thrombin concentration

The detection sensitivity (sensitivity) test, which is an important characteristic of the sensor, was carried out using the DNA aptamer labeled with the fluorescent material adsorbed on the gold nanostructure coated with the chitosan prepared in Example 1 above. Fluorescence intensities before and after thrombin injection were compared using various concentrations of thrombin for sensitivity experiments. The concentrations of thrombin used were 100 pM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM. Three experiments were performed for each concentration. Each reaction was measured with a reaction time of 10 minutes after the injection of thrombin. The results are shown in Figs.

8 is a graph showing the amount of decrease in fluorescence intensity before and after thrombin injection. As can be seen from FIG. 8, it was confirmed that the detection was possible up to a concentration of about 1 nM.

[Test Example 5] Detection specificity experiment

A DNA aptamer labeled with a chitosan-coated gold nanostructure-adsorbed fluorescent substance prepared in Example 1 was evaluated for the specific reaction of a material important in a sensor. In this experiment, HSA (Human serum albumin, ZLB Behring, USA) was injected as a comparison with thrombin for a specific response of aptamer-thrombin. DNA was used. The results are shown in FIGS. 9 and 10. FIG.

As can be seen from FIG. 10, when HAS is injected and when thrombin is ref. When DNA was reacted, the fluorescence reduction rate was within 4%. This is in contrast to the reaction between the thrombin-aptamer according to the present invention showing a fluorescence reduction rate of 30% or more. As a sensor of the present invention, a DNA aptamer having a fluorescent substance adsorbed on a gold nanostructure coated with chitosan, Detection was possible.

Claims (22)

Coating a positive charge polymer on the metal nanostructure;
Adsorbing a DNA aptamer labeled with a fluorescent substance on the coated metal nanostructure; And
And binding the target protein to the metal nanostructure on which the DNA aptamer labeled with the fluorescent substance is adsorbed. The method according to claim 1,
Wherein a change in fluorescence intensity of the metal nanostructure is measured in the step of binding the target protein. The method according to claim 1,
Wherein the metal nanostructure is a nanoflower or nananol structure. The method according to claim 1,
Depositing a seed metal having a lower reduction potential than the substituted metal on the substrate to form a metal layer; And
And reacting the metal layer with a plating solution containing a substituted metal ion. The method according to claim 1,
Wherein the metal of the metal nanostructure is at least one selected from the group consisting of gold, silver, titanium, and zinc oxide. The method according to claim 1,
The positive charge polymer may be selected from the group consisting of chitosan, polyethylene imine, polylysine, poly-aspartic acid, spermine, protamine, polyamidoamine, poly Wherein the at least one protein is at least one selected from the group consisting of polypropyleneimine, polybrene, polyacrylamide, polyvinyl amine, and DEAE-dextran. The method according to claim 1,
Wherein the positive charge polymer has a molecular weight of 300 to 1,000. The method according to claim 1,
Wherein the step of coating the positively charged polymer with the metal nanostructure is performed by stirring at a rate of 100 to 500 rpm for 20 to 40 minutes. The method according to claim 1,
Wherein the fluorescent material is at least one selected from the group consisting of Cy3, fluorescein, tetramethylrhodamine, Texas Red, and nicotinamide adenine dinucleotide (NADH). The method according to claim 1,
Wherein the fluorescent substance has a quantum yield of 1 to 50%. The method according to claim 1,
Wherein the distance between the fluorescent substance and the metal nanostructure is 20 to 50 nm. The method according to claim 1,
Wherein the DNA aptamer has a length of 10 to 30 mers. The method according to claim 1,
Wherein the target protein is thrombin. Positively charged polymer coated metal nanostructures; And
And a DNA aptamer in which a fluorescent substance adsorbed on the metal nanostructure coated with the positive charge polymer is labeled,
Wherein the target protein binds to the DNA aptamer. 15. The method of claim 14,
The positive charge polymer may be selected from the group consisting of chitosan, polyethylene imine, polylysine, poly-aspartic acid, spermine, protamine, polyamidoamine, poly Wherein the at least one protein is selected from the group consisting of polypropyleneimine, polybrene, polyacrylamide, polyvinyl amine, and DEAE-dextran. 15. The method of claim 14,
Wherein the positive charge polymer has a molecular weight of 300 to 1000. 15. The method of claim 14,
Wherein the metal nanostructure is a nanoflower or nananol structure. 15. The method of claim 14,
Wherein the fluorescent material is at least one selected from the group consisting of Cy3, fluorescein, tetramethylrhodamine, Texas Red, and nicotinamide adenine dinucleotide (NADH). 15. The method of claim 14,
Wherein the fluorescent substance has a quantum yield of 1 to 50%. 15. The method of claim 14,
Wherein the distance between the fluorescent substance and the metal nanostructure is 20 to 50 nm. 15. The method of claim 14,
Wherein the DNA aptamer has a length of 10 to 30 mers. 15. The method of claim 14,
Wherein the target protein is thrombin.

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