KR20110071336A - Gold nano particles and manufacturing method of the same - Google Patents

Abstract

PURPOSE: A nanoparticle cluster and a manufacturing method thereof are provided to control a size and optical features of the gold nanoparticle cluster by controlling amounts of a cross-linking agent and a receptor. CONSTITUTION: The nanoparticle cluster is composed of a gold nanoparticle, a cross-linking agent combining the gold nanoparticles to each other; and a receptor. One end side of the receptor is combined in the surface of the gold nanoparticle. A recognition part of target materials is exposed to the outside of the nanoparticle cluster. The cross-linking agent contains a chain type organic molecule selected among diaminoalkane, dithiol and dicarboxylate which have an amine functional group(-NH2), a thiol(-SH) and a carboxyl functional group(-COO-) in the respective chain both ends. The cross-linking agent is included to the receptor at a rate of 10-40 mole per the receptor Gram formula weight. The manufacturing method of the gold nanoparticle cluster includes following steps.(a) The gold nanoparticle is manufactured.(b) The gold nanoparticle, the receptor and the cross-linking agent are mixed.(c) An aqueous solution of sodium chloride is added.

Description

GOLD NANO PARTICLES AND MANUFACTURING METHOD OF THE SAME

The present invention relates to a cluster of gold nanoparticles that can be usefully used as a biosensor and a method of manufacturing the same, and more particularly, to gold nanoparticles, a crosslinking agent for bonding the gold nanoparticles to each other, and one end of the gold nanoparticles. The gold nanoparticle cluster coupled to the surface, and the target material recognition site is composed of a receptor exposed to the outside of the gold nanoparticle cluster, and a method of manufacturing the same.

The present invention also relates to a biosensor using the gold nanoparticle cluster.

Research on a method of detecting a biomaterial (deoxyribose nucleic acid (DNA), protein, etc.) using metal nanoparticles has been progressing for about 10 years, and a biosensor based on a new platform technology is being developed. Gold nanoparticles exhibit physical, chemical and optical properties due to specific surface plasmon resonance, which is mainly used for signal detection of biomolecules.

Surface plasmon resonance is a surface plasmon wave generated by the collective vibration of electrons on the surface of a metal thin film, which is excited by surface electromagnetic waves traveling along the interface of the dielectric material adjacent to the metal. It is a phenomenon. It is possible to precisely measure the physical change of the dielectric constant occurring in the metal surface, that is, the change in concentration and thickness refractive index, so that the quantitative analysis of the material is a part of the signal conversion technique for analyzing nanoscale interface phenomena. Various sensors have been devised.

In particular, when the surface plasmon wave occurs in the metal nanoparticles, it appears as a local surface plasmon wave on the surface of the nanoparticles, and because of this phenomenon, the nanoparticle solution exhibits a specific color under visible light. It is known to be used as a biosensor or chemical sensor without labeling because it can be easily detected by change.

The method using gold nanoparticles can provide faster and easier analysis with higher sensitivity and show high reproducibility compared to technologies for attaching fluorescent materials to form an array. In addition, gold nanoparticles can form stable bonds with various organic molecules on the surface, and can maintain stable bonds even at high physiological salt concentrations in which biological materials (eg, oligonucleotides and proteins) can maintain their original structure. It has several advantages.

Therefore, when the biosensor using the gold nanoparticles uses oligonucleotides (fragments of DNA) or proteins as biological materials as the receptors, the oligonucleotides may form strong hydrogen bonds with target DNA having complementary sequences. Proteins can form strong bonds with target proteins through antigen-antibody reactions, allowing detection of specific targets.

However, the conventional biosensor using gold nanoparticles has a problem in that it is difficult to control the size of the gold nanoparticles to be formed and thus function as a biosensor. Therefore, the development of a method for controlling the size of the gold nanoparticles in various ways and the development of a biosensor capable of various colors using the same has been required.

In order to solve the above problems, an object of the present invention is to provide a gold nanoparticle cluster, a manufacturing method thereof, and a biosensor using the same.

One embodiment of the present invention for solving the above problems is a gold nanoparticles, a crosslinking agent for binding the gold nanoparticles, and one end is bonded to the surface of the gold nanoparticles, the target material recognition site of the gold nanoparticle cluster It provides a cluster of gold nanoparticles consisting of receptors exposed to the outside.

In addition, another embodiment of the present invention comprises the steps of preparing a gold nanoparticles, the gold nanoparticles, the step of mixing a receptor and a crosslinking agent comprising a target material recognition site that can bind or react with the target material, and aqueous sodium chloride solution It provides a method for producing a gold nanoparticle cluster comprising the step of adding.

In addition, an embodiment of the present invention provides a biosensor for detecting a target material that binds to or reacts with a target material recognition site of the receptor using the gold nanoparticle cluster.

The above objects, features, and advantages will become more apparent from the following description taken in conjunction with the accompanying drawings. Hereinafter, the present invention will be described in detail.

Gold nanoparticle cluster according to an embodiment of the present invention is a gold nanoparticle, a crosslinking agent for bonding the gold nanoparticles, and one end is bonded to the surface of the gold nanoparticles, the target material recognition site is the gold nanoparticle cluster It characterized in that it comprises a receptor exposed to the outside.

Gold nanoparticles have strong affinity with many organic molecules, which can form stable bonds with organic molecules, even at high physiological salt concentrations where biomolecules such as DNA or proteins can maintain their original structure. Have stability. Therefore, in the present invention, in the gold nanoparticle cluster formed by bonding gold nanoparticles to each other with a crosslinking agent and bonding a receptor to the surface thereof, the size of the gold nanoparticle cluster is controlled by controlling the amount of the crosslinking agent and the receptor. It is possible to apply to a biosensor that can exhibit various optical properties.

In one embodiment of the present invention, the size of the gold nanoparticles is preferably 1 nm to 1000 nm, more preferably 1 nm to 500 nm, but is not limited thereto.

In addition, gold nanoparticles forming a gold nanoparticle cluster according to an embodiment of the present invention may be bonded to each other by a crosslinking agent. The crosslinking agent may include one type of chained organic molecule selected from diaminoalkane, dithiol, and dicarboxylate in which an amine functional group (-NH2), thiol (-SH), and a carboxyl functional group (-COO-) are positioned across the chain, respectively. Of these, dithiol containing thiol groups at both ends of the chain is preferable. Dithiothretol is most preferred as dithiol.

In one embodiment of the invention, the receptor comprises a target material recognition site capable of binding or reacting with the target material.

In one embodiment of the present invention, the binding or reaction of the receptor and the target material may be performed by covalent bonds, hydrogen bonds, antigen-antibody reactions or electrostatic attraction, but is not limited thereto.

Non-limiting examples of such receptors can be one or more selected from the group consisting of enzyme substrates, ligands, amino acids, peptides, proteins, antibodies, nucleic acids, oligonucleotides, lipids, cofactors and carbohydrates.

In one embodiment of the present invention, one end of the receptor is coupled to the surface of the gold nanoparticle cluster, the target material recognition site is exposed to the outside of the gold nanoparticle cluster.

Gold nanoparticle cluster according to an embodiment of the present invention may include a deoxyribonucleotide (DNA) as a receptor.

As described above, in one embodiment of the present invention, the gold nanoparticles are basically bonded to each other by a crosslinking agent to form a cluster, and the target material recognition site of the receptor whose one end is bonded to the surface of the gold nanoparticle cluster is gold. Exposed to the outside of the nanoparticle cluster.

According to the present invention, when the gold nanoparticles are bonded to each other by a crosslinking agent to form clusters, signal amplification characteristics using various optical properties can be efficiently utilized by controlling the size of the clusters formed according to the ratio of the crosslinking agent and the receptor. . In the present invention, it is preferable to include a crosslinking agent per mole of the receptor in a 10 to 40 molar ratio to exhibit the size control and color development effect of the gold nanoparticle cluster. Therefore, the present invention can be applied to detect various biological materials with very high sensitivity, and has an advantage of obtaining more quantitative detection results.

In addition, in one embodiment of the present invention, the receptor may further comprise a functional group that mediates the bond with the gold nanoparticles. Examples of the functional group may be at least one selected from the group consisting of an amine group, a carboxyl group, a thiol group, and a phosphoric acid group.

On the other hand, the method for producing a gold nanoparticle cluster according to an embodiment of the present invention comprises the steps of preparing a gold nanoparticles, the gold nanoparticles, a receptor and a crosslinking agent comprising a target material recognition site capable of binding or reacting with the target material It characterized in that it comprises the step of mixing, and adding an aqueous sodium chloride solution.

In addition, in the method for producing a gold nanoparticle cluster according to an embodiment of the present invention, the crosslinking agent per mole of the receptor comprises a 10 to 40 molar ratio, the gold nanoparticle cluster according to the molar ratio of the receptor and the crosslinking agent The size of the is characterized in that it is adjusted.

On the other hand, in the method for producing a gold nanoparticle cluster according to an embodiment of the present invention, the sodium chloride aqueous solution is characterized in that it is added in a 0.1 to 0.2 molar concentration.

In one embodiment of the present invention, the gold nanoparticles may be used in a state in which the surface stabilizer is added or not added. In addition, the gold nanoparticles can be used in a state dispersed in an organic solvent or an aqueous solution, it is preferable to use in a state dispersed in an aqueous solution.

In one embodiment of the present invention, gold nanoparticles are known to have a strong affinity with various organic molecules can form a stable bond with the organic molecules. For example, one end of the receptor including the target material recognition site capable of binding or reacting with the target material may be bound to the surface of the gold nanoparticles by covalent bonding or electrostatic attraction.

On the other hand, an embodiment of the present invention relates to a biosensor for detecting a target material that binds or reacts with a target material recognition site of a receptor by using the gold nanoparticle cluster according to the present invention described above.

The biosensor according to the present invention has excellent optical properties and specific binding properties to target materials by using gold nanoparticle clusters, and thus, multiple detections of target materials such as various biological materials are possible with high efficiency and sensitivity.

On the other hand, an embodiment of the present invention relates to a method for detecting a target material that binds to or reacts with a target material recognition site of a receptor of the biosensor, characterized in that it uses the biosensor according to the present invention described above.

The detection may be performed using any one or more from the group consisting of colorimetric analysis, UV spectroscopy, Raman spectroscopy, optical microscopy, electric sensing method and scanometric method.

In one embodiment of the invention, the target material is a material that can react with or bind to the target material recognition site of the receptor of the biosensor, preferably a biomaterial. Even more preferred are enzymes, proteins, nucleic acids, oligonucleotides, oligosaccharides, peptides, amino acids, carbohydrates, lipids, cells, cancer cells, cancer stem cells, antigens, aptamers or other bioderived substances.

In one embodiment of the present invention, when using a DNA or oligonucleotide as a receptor, the binding of the target material and the target material recognition site of the receptor may be made by complementary hydrogen bonding. Further, in another embodiment of the present invention, when using a protein as a receptor, the binding of the target material and the target material recognition site of the receptor may be made through an antigen-antibody reaction.

In one embodiment of the present invention, when DNA is used as a receptor and DNA is used as a target material, the receptor DNA and the target DNA are complementary to each other, but when the temperature is heated above Tm, the complementary binding is dissociated. Reversible reaction to restore

As such, using the biosensor using the gold nanoparticle cluster according to the present invention, it is possible to selectively and specifically detect a specific target material of interest.

The nanoparticle cluster according to the present invention may be made of any metal as long as it is a metal nanoparticle generating surface plasmon waves. Specifically, in addition to gold according to the present invention, the nanoparticle cluster may be used as a nanoparticle cluster. Can be made.

Gold nanoparticle cluster according to the present invention can control the size of the gold nanoparticle cluster formed by adjusting the amount of the cross-linking agent and the receptor added in the manufacturing process, thereby can exhibit excellent optical properties.

The biosensor using the gold nanoparticle cluster according to the present invention can efficiently perform detection according to a target material, in particular, a reversible DNA complementary hydrogen bond with a target biomaterial, and the fields of medicine and pharmacy where detection of a biomaterial is important. It can be used in various ways.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

The gold nanoparticles used in this example were synthesized by citrate reduction of geum chloride (HAuCl ₄ ), and deoxyribonucleotides (DNA) bound to thiol groups were purchased from Genotech to deprotect the thiol groups. Used.

Example 1 DNA-Gold Nanoparticle Cluster Preparation

Example 1-1 Preparation of Gold Nanoparticles

To prepare 150 ml of 15 nm spherical gold nanoparticle solution, the precursors of hydrogen tetrachloroaurate (III) trihydrate (HAuCl ₄ , 3H 2 O, Aldrich) and reducing agent trisodium citrate (Na ₃ C ₆ H ₅ O ₇ , 2H 2 O, Mallinckrodt) were prepared. The stock solution was prepared using ultrapure water as a solvent at concentrations of 0.254 mM and 38.8 mM, respectively.

50 mL of the prepared stock solution of HAuCl ₄ was vigorously stirred while heating to 150 ° C. in a flask connected to a reflux condenser. 38.8 ml of the prepared trisodium citrate was added when the aqueous solution started to boil. The color of the solution was changed from yellow to dark red. After the color was changed, heating was continued for 5 minutes and then slowly cooled to room temperature. TEM image of the prepared gold nanoparticles is shown in Figure 1, the diameter was 15 nm.

Example 1-2 Preparation of Monothiol DNA

Sigma Aldrich, a 5'-alkylthiol modified deoxynucleotide (5'-HS-A10-ATTATCACT-3 ') purchased from Genotech having thiol groups at the 5' and 3 'ends, respectively, capable of binding gold nanoparticles The deprotection reaction was carried out by adding dithiothretol (0.1 M) purchased from and left at room temperature for 2 hours.

The deprotected solution was purified by passing through a NAP-5 Sephadex column (deoxy ribose nucleic acid (DNA) grade) to prepare oligonucleotide 1A: (5'-HS-A ₁₀ -ATTATCACT-3 '). Purified to 20 μM.

Example 1-3 Preparation of DNA-Gold Nanoparticle Clusters

Example of using 225 μL of 20 μM deoxyriboso nucleic acid to which the deprotected thiol group was bonded through the procedure of Example 1-2 and Dithiothretol as a crosslinking agent, Dithiothretol ) Was not used, and in each case was added to 1 ml of a 15 nm diameter gold nanoparticle solution prepared in Example 1-1 and stirred at room temperature for at least 12 hours.

The composition of the solution was then adjusted so that the concentration of phosphate was 10 mM and the concentration of sodium dodecylsulfonic acid salt was about 0.01%. After further stirring for 30 minutes, the final salt concentration was adjusted to 0.15 M NaCl concentration. 2 shows a schematic diagram of a process of preparing the gold nanoparticle cluster.

After further left for 2-3 hours or more, the solution is centrifuged to discard the supernatant and buffered (0.15 M NaCl, 0.01% Tween 20, and 10 mM phosphate (pH 7.4)). The process of diluting by adding 1 ml of the solution was repeated twice, and the colors indicated by the solutions in the Comparative Examples and Examples are shown in Table 1 and FIG. 3A, respectively.

Dithiothretol concentration (μM) usage
(μL) Color Comparative Example 1 0 10 Red Example 1-3-1 300 10 Purple Example 1-3-2 500 10 Violet Example 1-3-3 700 10 Blue

The absorbance of the solution was measured using a UV-visible spectrometer for the Comparative Examples and Examples, and FIG. 3B shows the change in absorbance according to the ratio of the crosslinking agent and deoxyribose nucleic acid in each Comparative Example and Example. It was.

As shown in FIG. 3B, the comparative example without the addition of Dithiothretol shows the maximum absorbance at 520 nm, but the wavelength showing the maximum absorbance with the addition amount of Dithiothretol increases to 520. It can be seen that the wavelength becomes wider while moving from nm to 700 nm. As the amount of dithiothretol is increased, the aggregation of gold nanoparticles occurs a lot and the dielectric constant of the surface of the nanoparticles is changed, so that the color of the nanoparticle solution itself changes as the surface plasmon absorption condition is changed. It is judged that the result is consistent with the color indicated by each solution shown in FIG. 2A.

In addition, the shape and size of the gold nanoparticle clusters prepared by the dynamic light scattering method and the TEM photograph were confirmed as shown in FIGS. 4A and 4B, respectively.

As shown in FIG. 4A, in the comparative example without adding dithiothretol, the diameter of the gold nanoparticles was measured to be 35 nm by dynamic light scattering, and added dithiothretol. As the amount of is increased, as the agglomeration of gold nanoparticles increases, the diameter increases to 42 nm, 49 nm, and 115 nm, and according to the present invention, the amount of dithiothretol added is increased according to the present invention. It can be seen that the size of the gold nanoparticle cluster is increased.

Example 2 Reversible Binding of DNA-Gold Nanoparticle Clusters, Dissociation Ability, and Temperature Safety Experiments

Example 2-1 Preparation of Monothiol DNA

Deoxynucleotide 2A: (5'-HS-A10-ATTATCACT-3 ') was prepared by the same method as Example 1-1, and deoxynucleotide 2B having a binding complementary thereto: (5'-HS-A10). -AGTGATAAT-3 ') was prepared.

Example 2-2 Preparation of Gold Nanoparticle Cluster

Gold nanoparticle clusters of three sizes were prepared by varying the amount of dithiothretol added in the same manner as in Example 1-2 for the deoxynucleotides 2A and 2B produced in Example 2-1. Prepared.

Example 2-3 Reversible Complementary Coupling and Dissociation Ability Between DNA-Gold Nanoparticle Clusters

When the DNA-gold nanoparticle cluster is mixed with other DNA-gold nanoparticle clusters having complementary bonds, the gold nanoparticle cluster is formed by complementary hydrogen bonding between the DNA of the DNA-gold nanoparticle cluster at a temperature lower than the melting temperature (Tm). It exists as an assembly, and at a temperature higher than the melting temperature (Tm), the aggregated DNA-gold nanoparticle clusters are dissociated.

Using DNA-gold nanoparticles prepared in Example 2-2, it was confirmed whether the binding and dissociation of DNA-AuNP cluster is a reversible reaction according to temperature change. Clusters of DNA-gold nanoparticles with complementary DNA sequences were mixed with each other to observe the color change, and the color change was observed as each solution was heated.

The process of binding gold nanoparticle clusters comprising deoxynucleotides 2A and 2B having complementary sequences is schematically illustrated in FIG. 5A, and the change in color developed by mixing and heating is shown in FIGS. 5B and 2. . In FIG. 5B, U represents the state of the gold nanoparticles containing each deoxynucleotide 2A, 2B before hybridization, H is the hybridized state, M is not hybridized again when the complementary hydrogen bond is broken by heating to a temperature above Tm. Re-dehybridized.

Dithiothretol
(Dithiothretol) Color density
(μM) usage
(μL) unhybridized
(U) hybridized
(H) re-dehybridized (M) Comparative Example 2 0 10 Red purple Red Example 2-3-1 300 10 Purple pale violet Purple Example 2-3-2 500 10 Violet pale blue Violet Example 2-3-3 700 10 Blue pale indigo Blue

In the case of FIG. 5B (1), in Comparative Example 2, without adding dithiothretol, the deoxynucleotide 2A: (5'-HS-A10-ATTATCACT-3 ') and complementary binding thereto Deoxynucleotide 2B having: (5'-HS-A10-AGTGATAAT-3 ') is a solution obtained by mixing gold nanoparticles, respectively. The solution was red (unhybridized (U)) and the mixed solution was purple (hybridized (H)) when they hybridized at a temperature lower than Tm. When it is heated again to a temperature higher than Tm, the aggregates of the DNA-gold nanoparticle clusters are dissociated to exist as individual DNA-gold nanoparticle clusters, and the mixed solution turns red again (re-dehybridized). (M)). This reversible change with temperature is very sensitive to the peak width.

In case of FIG. 5B (2), 300 μM of dithiothretol was added to Example 2-3-1 in Table 2, and the deoxynucleotide 2A: (5′-HS-A10-ATTATCACT-3 ') And a deoxynucleotide 2B having complementary binding to it: (5'-HS-A10-AGTGATAAT-3'), respectively, are mixed with gold nanoparticles.

The solution was purple (unhybridized (U)), and the solution hybridized at a temperature lower than Tm showed pale violet (hybridized (H)). When it is heated to a temperature higher than Tm, the aggregates of the DNA-gold nanoparticle clusters are dissociated to exist as DNA-gold nanoparticle clusters, and the mixed solution turns purple again (re-dehybridized (M )).

In the case of FIG. 5B (3), 500 μM of dithiothretol was added to Example 2-3-2 in Table 2, and the deoxynucleotide 2A: (5′-HS-A10-ATTATCACT-3 ') And a deoxynucleotide 2B having complementary binding to it: (5'-HS-A10-AGTGATAAT-3'), respectively, are mixed with gold nanoparticles. The solution showed violet color (unhybridized (U)), and the solution hybridized at a temperature condition lower than Tm showed pale blue (hybridized (H)). When it is heated to a temperature higher than Tm, the aggregates of the DNA-gold nanoparticle clusters are dissociated to exist as respective DNA-gold nanoparticle clusters, and the mixed solution is changed to violet color again (re-dehybridized). (M)).

5B (4) was added 700 μM of dithiothretol as Example 2-3-3 in Table 2, and the deoxynucleotide 2A: (5′-HS-A10-ATTATCACT-3 ') And a deoxynucleotide 2B having complementary binding to it: (5'-HS-A10-AGTGATAAT-3'), respectively, are mixed with gold nanoparticles. The mixed solution was blue (unhybridized (U)), and the solution hybridized at a temperature lower than Tm showed pale indigo (hybridized (H)). When it is heated again to a temperature higher than Tm, the aggregates of the DNA-gold nanoparticle clusters dissociate to exist as DNA-gold nanoparticle clusters, and the mixed solution turns blue again (re-dehybridized (M )).

 The melting point change of the hybridized gold nanoparticle clusters was monitored by measuring the change in surface plasmon peak (change in extinction) at 260 nm, heating each solution from 25 ° C to 70 ° C at a rate of 1 ° C / min. It was measured and shown in FIG. 5B.

 When heated to a temperature above the melting point, the solution originally changed to the color it was originally created in. This means that the gold nanoparticle clusters made according to the present invention reversibly change to the original gold nanoparticle clusters when heated even after complementary bonding with the target material, and the FWHM is about 2-3 ° C. Complementary binding of clusters is produced and broken in a short time, i.e., the detection performance of the target material is excellent.

In addition, it will be shown that the nanoparticle cluster of the present invention shows a safety that does not change even with temperature changes.

Example 2-4 Temperature Stability Experiments of DNA-Gold Nanoparticle Clusters

The gold nanoparticle clusters representing the violet color produced in Example 2-2 were mixed with each other, and hybridization and heating were repeated to measure and monitor the change in surface plasmon peak (change in extinction) at 260 nm. This is shown in FIG. 6.

As shown in FIG. 6, the change of the peak during the first heating process and the change of the peak during the tenth heating process are almost identical, which indicates that the gold nanoparticle cluster of the present invention exhibits safety according to temperature change. Will show.

Example 3 Colorimetric Detection Experiment

Example 3-1 Preparation of Monothiol DNA

Deoxynucleotide 3A in the same manner as in Example 1-1: (5'-HS-A10-ATCCTTATCAATATT-3 ') and deoxynucleotide 3B: (3'-HS-A10-CTCCCTAATAACAAT-5'), and 3C: 3'-TAGGAATAGTTATAA-ATTGTTATTAGGGAG-5 'which complementarily binds 3A and 3B was prepared.

Deoxynucleotides 3A and 3B do not bind complementarily to each other, but complementarily bind to deoxynucleotide 3C. The binding relationship between them is shown in Fig. 7A.

Example 3-2 Preparation of Gold Nanoparticle Cluster

For the oligonucleotides 3A and 3B prepared in Example 2-1, 500 μM of dithiothretol was added as a binder to prepare 450 μL of gold nanoparticle clusters showing violet color.

Example 3-3 Binding to Target DNA Strands

After preparing the target DNA strand prepared in Example 3-1 at a concentration of 0.5, 1, 2, 5, 10, 20, 50 nM, and reacted with the gold nanoparticle cluster prepared in Example 3-2, respectively The change in color was observed and shown in FIG. 7B.

The absorbance of the solution was measured every 15 minutes for 2 hours with a UV-Visible Spectrometer. As shown in FIG. 7C, the maximum peak wavelength red shifted during binding to the target DNA strand. This indicates that as the nanoparticle clusters aggregate as they bind to the target DNA strand, the nanoparticle clusters grow in size, causing changes in the dielectric constant of the nanoparticle surface and changing local surface plasmon absorption conditions.

After the reaction between the target DNA strand and the DNA-gold nanoparticle cluster, the melting point change according to the concentration of the target DNA strand was observed at 260 nm wavelength, and is shown in FIG. 7D. The melting point change according to the concentration of the target DNA strand was shown. Shown in 7E. It can be seen that the higher the concentration of the target DNA strand, the higher the melting point, which causes a change in the permittivity of the surface of the gold nanoparticles as the concentration of the target DNA strand increases with the binding of the gold nanoparticle cluster of the present invention. It can be seen that the surface plasmon absorption conditions are changed.

1 is a TEM photograph of gold nanoparticles prepared in Example 1-1.

Figure 2 is a schematic diagram briefly showing the manufacturing process of the DNA-gold nanoparticle cluster according to the present invention.

3 is a view showing extinction according to the color development degree and wavelength of the DNA-gold nanoparticle cluster according to the amount of dithiothretol added according to an embodiment of the present invention. 3A and 3B, Red shows Comparative Example 1, Purple shows Example 1-3-1, violet shows Example 1-3-2, and Blue shows Examples 1-3-3.

FIG. 4 is a diagram showing dynamic light scattering spectra and TEM images of DNA-gold nanoparticle clusters according to the amount of dithiothretol added according to an embodiment of the present invention. In FIG. 4A, Red shows Comparative Example 1, Purple shows Examples 1-3-1, violet shows Examples 1-3-2, Blue shows Examples 1-3-3, and FIG. 4B (1) shows Comparative Examples. 1, FIG. 4B (2) shows Example 1-3-1, FIG. 4B (3) shows Example 1-3-2, and FIG. 4B (4) shows Example 1-3-3.

Figure 5A is a schematic diagram showing the complementary binding and dissociation process between DNA-gold nanoparticle cluster according to another embodiment of the present invention, Figure 5B is a view showing the change in color, melting point and extinction accordingly . In FIG. 5B, Red represents Comparative Example 2, Purple represents Example 2-3-1, violet represents Example 2-3-2, and Blue represents Example 2-3-3.

FIG. 6 is a diagram illustrating a melting point, extinction and extinction when the heating process is performed once and the heating process is performed 10 times.

FIG. 7A is a schematic diagram showing the colorimetric analysis of the gold nanoparticle clusters according to another embodiment of the present invention. FIG. 7B is a hybridization of the DNA-gold nanoparticle cluster representing violet (U) before hybridization. (H), and the figure showing the color shown after heating to a temperature above the melting point (M), Figure 7C is a diagram showing the change in extinction in the process of binding to the target DNA strand, Figure 7D and Figure 7E It is a graph showing the change of melting point according to the concentration of DNA strand.

Claims (15)

Gold nanoparticle cluster consisting of a gold nanoparticles, a crosslinking agent for bonding the gold nanoparticles, and one end is bonded to the surface of the gold nanoparticles, the target material recognition site is exposed to the outside of the gold nanoparticle cluster. The method of claim 1, The crosslinking agent is a gold nano that is a kind of chain organic molecules selected from diaminoalkane, dithiol, and dicarboxylate each of the amine functional group (-NH2), thiol (-SH), and carboxyl functional group (-COO-) is located at the both ends of the chain Particle Clusters. The method of claim 2, The crosslinking agent is dithiothretol (Dithiothretol) gold nanoparticle cluster. The method of claim 1, The receptor is gold nanoparticle cluster is one or more selected from the group consisting of enzyme substrate, ligand, amino acid, peptide, protein, antibody, nucleic acid, oligonucleotide, lipid, cofactor and carbohydrate. The method of claim 1, The receptor is gold nanoparticle cluster that is deoxyribonucleotide. The method of claim 1, Gold nanoparticle cluster, characterized in that the crosslinking agent per mole of the receptor comprises 10 to 40 molar ratio. Preparing gold nanoparticles; Mixing a receptor and a crosslinking agent comprising a gold nanoparticle, a target material recognition site capable of binding to or reacting with a target material; And Method of producing a gold nanoparticle cluster comprising the step of adding an aqueous sodium chloride solution. The method of claim 7, wherein The crosslinking agent includes one type of chained organic molecule selected from diaminoalkane, dithiol, and dicarboxylate in which amine functional group (-NH2), thiol (-SH), and carboxyl functional group (-COO-) are positioned at both ends of the chain, respectively. Method for preparing gold nanoparticle clusters. The method of claim 7, wherein The crosslinking agent is dithiothretol (Dithiothretol) is a method for producing a cluster of gold nanoparticles. The method of claim 7, wherein The receptor is one or more selected from the group consisting of enzyme substrates, ligands, amino acids, peptides, proteins, antibodies, nucleic acids, oligonucleotides, lipids, cofactors and carbohydrates. The method of claim 7, wherein Wherein said receptor is a deoxyribonucleotide. The method of claim 7, wherein The aqueous sodium chloride solution is added to the 0.1 to 0.2 molar concentration method of producing a gold nanoparticle cluster. The method of claim 7, wherein The crosslinking agent per mole of the receptor comprises a 10 to 40 molar ratio, the method of producing a gold nanoparticle cluster, characterized in that the size of the gold nanoparticle cluster is adjusted according to the molar ratio of the receptor and the crosslinker. A biosensor for detecting a target substance that binds to or reacts with a target substance recognition site of a receptor by using the gold nanoparticle cluster according to any one of claims 1 to 6. The method of claim 14, When DNA is used as the receptor and DNA is used as the target material, the receptor DNA and the target DNA are complementary to each other, and when the temperature is heated to more than Tm, the complementary binding is dissociated to restore the original state. Biosensor, characterized in that.

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