KR101115388B1 - Histidine specific biocensor and the manufacturing method thereof - Google Patents

Abstract

The present invention relates to histidine specific biosensors that can be used to detect histidine and histidine high content proteins, a method for preparing the same, and a method for detecting histidine using the same.

The histidine specific biosensor of the present invention is characterized in that nano-sized silver particles and lysine having a carboxyl group as the organic ligand, typically N, N-bis (carboxymethyl) -L-lysine, are combined.

The histidine-specific biosensor of the present invention is effective in detecting histidine, which is a substance to be detected, in a simple and convenient manner without using expensive equipment or complicated experimental methods. In addition, the histidine-specific biosensor of the present invention can determine whether the histidine is contained even when the concentration of histidine is very low, such as 5 micromolar or less.

Description

HISTIDINE SPECIFIC BIOCENSOR AND THE MANUFACTURING METHOD THEREOF

The present invention relates to a bioassay technique using a biosensor. More specifically, the present invention relates to histidine-specific biosensors that can be used to detect histidine and histidine-containing protein, a method for preparing the same, and a method for detecting histidine using the same.

Recently, interest in development for using metal nanoparticles modified with various organic and inorganic materials as analytical tools, that is, biosensors in the biological field is increasing. In particular, binding the organic ligand to the surface of the nanoparticles may not only impart certain functions to the nanoparticles, but also help to physically and chemically stabilize the organic ligands acting as receptors for the detection target in various solvents. It is known. According to Mirkin et al. Have developed a colorimetric assay that detects cysteines using DNA-gold nanoparticles.

In the medical field, the use of such biosensors is expected to help determine the presence of diseases. In one example, histidine and proteins containing high amounts of histidine serve as very important indicators in the human body. Abnormally high levels of protein containing high amounts of histidine in the blood or body fluids include advanced liver cirrhosis, AIDS, renal disease, asthma, pulmonary disorders, thrombotic disorders, and Diseases such as malaria can be suspected. Analytical methods for the detection of such histidine and high amounts of histidine-containing proteins have been developed in connection with immunoassays, fluorimetric detection and colorimetric detection. However, the conventionally developed histidine detection method requires expensive equipment and complicated experimental procedures, and has a problem in that a range of materials that can be effectively used is low due to low material throughput.

The present invention has been made to solve the above problems, it is an object of the present invention to provide a histidine-specific biosensor comprising nano-sized metal particles and a method of manufacturing the same. Another object of the present invention is to provide a method capable of detecting histidine and histidine-containing proteins without expensive equipment or complicated experimental procedures using the histidine-specific biosensor. In addition, an object of the present invention is to provide a method capable of effectively detecting the histidine and the histidine-containing protein in a small amount of material.

In order to achieve the above object of the present invention, the present invention provides a histidine-specific biosensor comprising a metal particle and an organic ligand bonded to the surface of the metal particle, wherein the organic ligand has a carboxyl group. .

The biosensor is characterized in that the carboxy group of the organic ligand is three. In addition, the organic ligand bound to the metal particles is characterized in that 1 to 100 moles per 1 mole of the metal particles.

In the biosensor of the present invention, the metal particles are selected from silver, gold, or platinum. The particle diameter of the metal particles is 1 nm to 30 nm, more preferably 3 nm to 10 nm.

The organic ligand is characterized in that the lysine having a carboxyl group. Specific examples of lysine having the carboxyl group are N, N-bis (carboxymethyl) -L-lysine (N, N-bis (carboxymethyl) -L-lysine).

The present invention also provides a method for detecting histidine using the histidine specific biosensor. The histidine detection method according to the present invention comprises the steps of dispersing the biosensor according to the invention in a detection buffer, mixing the aqueous solution containing histidine in the detection buffer in which the biosensor is dispersed, and the detection of the mixed histidine-containing aqueous solution Observing the color change of the buffer.

In addition, the histidine detection method, the color change of the detection buffer may further comprise the step of quantitative analysis of the content of histidine by measuring the absorbance using an ultraviolet-visible spectrophotometer.

In another aspect, the present invention is to reduce the metal oxide in the presence of a reducing agent to produce a nano-level metal colloidal particles, by mixing an aqueous solution containing an excess of lysine having a carboxyl group to the metal nano-colloids prepared above and the metal particles Binding lysine; And the lysine formed above provides a method for producing a histidine-specific biosensor comprising the step of separating the lysine-bound metal particles with an aqueous solution. In addition, the manufacturing method may further comprise the step of washing and separating the metal particles combined with the lysine produced above.

In preparing a histidine specific biosensor according to the present invention, the metal oxide may be silver nitrate (AgNO ₃ ). In addition, the lysine having a carboxyl group may be N, N-bis (carboxymethyl) -L-lysine (N, N-bis (carboxy methyl) -L-lysine).

Lysine bonded to the metal particles in the above is preferably 1 to 100 moles per 1 mole of the metal particles. The particle diameter of the metal particles formed above is preferably 1 nm to 30 nm, more preferably 3 nm to 10 nm.

Finally, the present invention provides a method for diagnosing liver cirrhosis, AIDS, kidney disease, asthma, lung disease, thrombotic disorder or malaria using the histidine specific biosensor according to the present invention. The method includes, for example, confirming that histidine is detected from a biological sample such as blood or body fluid obtained from a suspected subject using a biosensor according to the present invention. It is the same as how it is provided.

The histidine-specific biosensor of the present invention binds to histidine and causes a change in color, so that the detection result can be easily visually confirmed. Therefore, there is an effect that can selectively detect histidine and a large amount of histidine-containing protein compared to the expensive and complicated experimental procedures used in the past. As described above, the histidine-specific biosensor of the present invention is effective in detecting histidine, which is a substance to be detected, in a simple and convenient manner without using expensive equipment or complicated experimental methods. In addition, the histidine-specific biosensor of the present invention can determine whether the histidine is contained even when the concentration of histidine is very low, such as 5 micromolar or less.

In order to achieve the above object, the present invention provides a histidine-specific biosensor in which a nano-sized metal particle and an organic ligand are selectively reacted with histidine and a method for preparing the same. It also provides a method for detecting a histidine and a protein containing a large amount of histidine using the biosensor.

In the histidine-specific biosensor according to the first aspect of the present invention, the particle is a metal capable of coordinating with a non-covalent pair of electrons provided by an organic ligand, and particularly preferably a precious metal such as gold, silver or platinum.

The particles have a particle diameter of 1 to 30 nm, preferably 3 to 10 nm. When the particle size is less than 1 nm, the surface area of the particle itself is small and aggregation between particles easily occurs, so that coordination bonds with organic ligands are difficult. If the particle size exceeds 30 nm, the specific surface area is reduced, so that a sufficient amount of the organic ligand is difficult to bind.

The organic ligand serves as a receptor of histidine in the biosensor of the present invention, and has a negatively charged portion to bind to the cation of ammonium of histidine.

In the histidine specific biosensor, the organic ligand is 1 mol to 100 mol, more preferably 5 mol to 100 mol with respect to 1 mol of the metal particles. If less than 1 mole, there is a problem in that histidine cannot be effectively detected when the concentration of histidine in the biological fluid, ie, body fluid or blood, is low.

One specific embodiment of the histidine-specific biosensor, which is the first aspect of the present invention, is a nano-sized silver particle as the metal particle and a lysine having a carboxyl group as the organic ligand. Specific examples of the lysine having a carboxyl group include N, N-bis (carboxymethyl) -L-lysine. In general, biologically active molecules such as amino acids are very sensitive to environmental requirements such as external temperature or acidity of the surroundings, and thus have a large difference in activity depending on environmental requirements. Therefore, the use of such biological molecules immobilized on a solid support such as the silver particles increases the thermal stability can be maintained for a long time in a high activity state.

Method for selectively detecting histidine by the histidine-specific biosensor in which the silver particles and the N, N-bis (carboxymethyl) -L-lysine are combined is as follows.

The N, N-bis (carboxymethyl) -L-lysine is represented by the following formula (1).

As can be seen in Formula 1, the N, N-bis (carboxymethyl) -L-lysine has a carboxyl group, and thus easily loses hydrogen ions in the aqueous solution and has a positive charge. Histidine also contains three N's (nitrogen), which are negatively charged due to unshared electron pairs. Therefore, it is possible to selectively detect histidine by ionically binding the positively charged portion of the N, N-bis (carboxymethyl) -L-lysine and the negatively charged portion of histidine. 1 and 9 are schematic diagrams illustrating a process of selectively binding histidine to a histidine-specific biosensor in which N, N-bis (carboxymethyl) -L-lysine and silver particles are combined as a specific embodiment of the present invention. .

The negative charge of N, N-bis (carboxymethyl) -L-lysine (pH 9) prevents the particles from agglomerating by the repulsive force. The surface of the silver nanoparticles contains carboxylic acids that allow them to bind to cationic types of amino acids.

The N, N-bis (carboxymethyl) -L-lysine has three carboxyl groups, but the molecule is small in size, and thus may be bonded to a large amount of nanoparticles. Therefore, it is effective to secure a sufficient number of receptors capable of selectively binding histidine.

A histidine detection method using the histidine-specific biosensor according to the second aspect of the present invention includes dispersing a histidine-specific biosensor according to the first aspect of the present invention in a detection buffer; Mixing an aqueous solution containing histidine in a detection buffer in which the biosensor is dispersed; And observing the color change of the detection buffer in which the histidine-containing aqueous solution is mixed. Observing the color change may be performed by quantitative analysis of histidine content using an ultraviolet-visible spectrophotometer.

A method of manufacturing the histidine specific biosensor, which is a third aspect of the present invention, comprises: reducing a metal oxide in the presence of a reducing agent to produce a nano-level metal particle colloid; Combining the metal particles with lysine having the carboxyl group by mixing an aqueous solution containing lysine having a carboxyl group to the metal particle colloid prepared above; Separating the metal particles to which lysine having a carboxyl group formed in the step is bound.

One specific embodiment of the histidine-specific biosensor, which is a third aspect of the present invention, is prepared by reducing silver nitrate (AgNO ₃ ) to prepare a silver colloidal solution, followed by excess N, N-bis (carboxymethyl) -L By adding lysine, the silver nanoparticles of the silver colloidal solution and N, N-bis (carboxymethyl) -L-lysine may be combined. Lysine bonded to the metal particles in the above is preferably 1 to 100 moles per 1 mole of the metal particles. The particle diameter of the metal particles formed above is preferably 1 nm to 30 nm, more preferably 3 nm to 10 nm.

Hereinafter, the histidine specific biosensor of the present invention will be described in detail with reference to specific examples. The following examples are only used for the purpose of illustrating the present invention more clearly, but do not limit or limit the scope of the present invention. In addition, it will be apparent to those skilled in the art to substitute or change the minor part within the scope of the present invention.

Example Preparation of Histidine Specific Biosensors

Aqueous solution of silver nitrate (AgNO ₃ ) (5.0 mM, 1.0 mL) was stirred at room temperature, followed by sodium citrate solution (30 mM, 1.0 mL), sodium borohydride (50 mM, 1.0 mL), polyvinylpi Lollidon (polyvinylpyrrolidone) (5 mg) was added quickly. After the color of the solution was changed to pale yellow, the solution was heated under reflux for 30 minutes and cooled at room temperature to prepare a silver colloidal solution. While stirring the colloidal solution at room temperature for 24 hours, an aqueous solution containing excess N, N-bis (carboxymethyl) -L-lysine was mixed to perform a ligand exchange reaction. After the reaction was completed, the solution of silver nanoparticles to which the N, N-bis (carboxymethyl) -L-lysine was bound was centrifuged for 30 minutes, the supernatant was removed, and then redispersed in an aqueous solution. The particles were washed three more times and finally dispersed again in the detection buffer. The acidity of the solution was then adjusted to pH 9 using NaOH. Hereinafter, the biosensor according to the present invention prepared in Example 1 is abbreviated as BCML-Ag.

Test for BCML-Ag

Confirmation of binding of nanoparticle surface and organic ligand

In BCML-Ag according to Example 1, TEM (transmission electron) to confirm that N, N-bis (carboxymethyl) -L-lysine, which acts as a selective receptor for histidine, is bound to and fixed on the silver nanoparticles microscopy (transmission electron microscopy), Fourier transform Raman spectroscopy (FT-Raman spectroscopy), time of flight secondary ion mass spectroscopy (TOF-SIMS), UV-visible spectroscopy (UV-) VIS spectroscopy).

The FT-Raman spectra of the BCML-Ag showed peaks at 3263 cm ^-1 , 1684 cm ^-1 and 1284 cm ^-1 . Through this, it was confirmed that N, N-bis (carboxymethyl) -L-lysine, which is a histidine specific receptor, is bound to the surface of the silver nanoparticles. 2 shows a graph of the FT-Raman spectrum. The peak is believed to be due to N, N-bis (carboxymethyl) -L-lysine bound to the silver nanoparticle surface. FIG. 3 is a graph of the TOF-SIMS spectrum of the biosensor of the present invention, wherein the molecular mass of N, N-bis (carboxymethyl) -L-lysine is (m / z = 262.26). Is interpreted to mean a state that is completely bonded and fixed to the particle surface.

FIG. 4 is a graph showing ultraviolet-visible absorbance measurement results for BCML-Ag according to Example 1. FIG. The ultraviolet absorption peak was observed to be 400 nm, which is believed to be due to the plasmon absorption of the histidine specific biosensor surface of the present invention in the absence of amino acids. For this absorbance measurement, the spectra recorded five minutes after the start of the experiment showed the narrowest full-width-at-half-maximum (fwtn), in which the synthesized silver nanoparticles were simply dispersed (monodispersed), Means uniform.

Histidine detection confirmation experiment

In order to confirm the histidine detection capability of the histidine-specific biosensor of the present invention, the histidine aqueous solution was mixed with the detection buffer in which the BCML-Ag prepared in Example 1 was dispersed so that the final volume was 1 mL. The final concentration of BCML-Ag was 10.0 micromolar. In addition, several histidine aqueous solutions mixed in the buffer were prepared at intervals of 5 μM in the range of histidine concentration of 5.0 to 30.0 μM. In addition, in order to confirm whether the histidine-specific biosensor of the present invention selectively detects histidines, the biosensors were mixed with different aqueous amino acid solutions to observe whether the color was changed.

15 is a photograph showing a color change when histidine is added to the detection buffer. When histidine was not added, the color of the detection buffer was pale yellow, but when histidine was added, the color of the detection buffer changed to red.

FIG. 10C shows the color change state of the detection buffer after addition of various amino acid solutions (1.0 × 10 ⁻⁴ ), and FIG. 10B is its R value ( A ₅₂₀ / A ₄₀₀ ). Ten minutes after mixing the detection buffer and the aqueous amino acid solution, the histidine-containing solution changed color from yellow to red, and the absorbance was the highest at A ₅₂₀ / A ₄₀₀ . On the other hand, when other amino acids were added, no change in color or absorbance of the detection buffer was observed. Therefore, it was confirmed that the biosensor of the present invention selectively reacts with histidine.

The reason why the biosensor of the present invention binds to histidine and causes color change is because the particles of the biosensor agglomerate with each other by histidine. FIG. 11A is a TEM image of the detection buffer before and after addition of histidine and FIG. 11B is a graph showing the change in particle size of BCML-Ag before and after addition of histidine. to be. According to the TEM image, the particles of BCML-Ag were widely and uniformly dispersed in the aqueous solution before the histidine addition, and the particle diameter was observed to be about 3 to 5 nm. On the other hand, after addition of histidine, particle aggregation of BCML-Ag occurred and the particle diameter increased to 16 to 20 nm. This aggregation causes the color to change from yellow to red and is interpreted to reflect the interaction of paired plasmon excitons in the aggregated state.

In the detection of histidine, in order to determine whether the histidine specificity of the biosensor of the present invention is inhibited by a complex mixture of different amino acids, an aqueous solution of a mixture of histidine and other amino acids is added to the detection buffer to detect the detection buffer. The color change of was observed. As can be seen in FIG. 6, even when other amino acids were mixed, the color was changed to red when the biosensor of the present invention was added.

6, (1) histidine (2) alanine + histidine, (3) cytidine + histidine, (4) leucine + histidine, (5) proline + histidine in the detection buffer containing BCML-Ag according to Example 1 in FIG. , (6) valine + histidine, (7) serine + histidine, (8) tyrosine + histidine, (9) methionine + histidine, (10) phenylalanine + histidine, (11) threonine + histidine, (12) aspartic acid + Histidine is mixed. Experimental tanks (2) to (12) can also be seen that the color changed to red as in experimental tank (1) containing only histidine.

In addition, as shown in FIG. 4, even when histidine was mixed with other amino acids, there was no difference in absorbance of the aqueous solution containing histidine alone. 5 shows R ( A ₅₂₀ / A ₄₀₀ ) values of an aqueous solution containing histidine alone and an aqueous solution mixed with histidine and other amino acids. As shown in FIG. 5, even when histidine was mixed with other amino acids, the R ( A ₅₂₀ / A ₄₀₀₎ value was uniform, and it was confirmed that the histidine had a value similar to that of the mixed solution alone.

Through this, the biosensor of the present invention can selectively detect histidine, and it is confirmed that the detection ability of histidine is not affected even if other amino acids are mixed.

Confirmation of Selective Detection of Histidine-Containing Proteins

Selective color changes of the BCML-Ag were investigated using the ubiquitin protein lacking the N-terminal histidine tag and the Cdc20 protein with six histidine residues at the N terminus. The BCML-Ag particles (5.0 mM) were incubated with the protein (1.5 micrograms) for 10 minutes at room temperature, and then the absorbance of the color change of BCML-Ag was observed. 12 is a graph showing the results. In the graph, (a) is the absorbance of the detection buffer without anything, (b) is the absorbance of the detection buffer containing the ubiquitin protein lacking the histidine tag, and (c) is the absorbance result of the detection buffer containing the Cdc20 protein to be. According to this, (a) and (c) was almost no difference in absorbance, but (c) was confirmed that the change in absorbance.

The same result was obtained for the color change of the detection buffer. When the Cdc20, a histidine-labeled protein, was mixed with the detection buffer containing BCML-Ag, it was observed that the color was changed from yellow to red while increasing the absorption at A ₅₂₀ / A ₄₀₀ (c in FIG. 12B). This is because the histidine-labeled protein interacted with the BCML-Ag. On the other hand, when non-histidine-tagged protein was treated with BCML-Ag under almost the same conditions, no color change was observed (b of FIG. 12B). When the BCML-Ag was incubated with a mixture of histidine-labeled and non-histidine-labeled proteins, the color of the detection buffer containing the BCML-Ag changed from yellow to red, indicating that the histidine-labeled protein was selectively It means to bind to the surface of Ag.

In addition, in order to confirm histidine selective binding ability, analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a protein having a histidine residue and a protein without a histidine residue was performed.

The SDS-PAGE analysis was performed using 4% stacking gel and 16% resolving gel according to Bio-Rad's experimental manual. Each protein was mixed with a biosensor (10.0 μM, 1.0 mL) according to the present invention and incubated for 2 hours at room temperature by rotary mixing. The supernatant was removed by centrifugation (20 minutes) at 40,000 × g. It was. The supernatant and premixed proteins were dissolved in SDS-PAGE gels and visualized by staining with coomassie brilliant blue (CBB).

To minimize the effects of different amino acid sequence pI (isoelectric point), protein structure, and the presence of specific side chains, ubiquitin protein without the histidine label at the N-terminus and Cdc20 labeled with six histidine residues at the N-terminus Protein was also used. Figure 13 is an image of a gel containing two protein markers before adding BCML-Ag. After addition and separation of BCML-Ag (FIG. 13), non histidine labeled ubiquitin protein was still observed in the gel. On the other hand, the histidine-labeled Cdc20 protein disappeared because the complex formed between BCML-Ag and Cdc20 formed too large to enter the gel matrix. The presence of six histidine labels was confirmed by immunooblots using anti histidine antibodies. The results show that histidine labeled proteins can be effectively bound to the surface of BCML-Ag. Therefore, histidine specific biosensors according to the present invention can be usefully used to separate histidine proteins from protein mixtures.

Minimum Concentration for Histidine Detection

The experiment was prepared to find the minimum concentration of the histidine ion aqueous solution that can detect the color change. In a flask containing a mixture containing BCML-Ag according to Example 1, histidine was added in a range of 5.0 to 30.0 micromoles to 5 micromoles. The aqueous solution of each flask changed color from yellow to red. When visually confirmed, the higher the histidine concentration, the more pronounced the color change to red. In addition, it was possible to visually check the color change even when the histidine concentration was 5.0 micromolar. FIG. 7 is a graph showing the absorbance at 520 nm versus the histidine concentration in the aqueous solution containing BCML-Ag. According to the results, the absorbance results were linearly correlated with histidine concentration, and it was confirmed that the absorbance increased as the concentration of histidine increased. Therefore, by measuring the absorbance of the solution to be detected, the concentration of histidine in the solution can be calculated inversely. Linear correlation was obtained according to the concentration.

Acidity Condition of Solvent for Histidine Detection

It was confirmed that the acidity of the histidine detection buffer affects the histidine detection ability of the biosensor according to the present invention. Histidine was mixed in a detection buffer in which BCML-Ag was dispersed according to Example 1 (concentration 1.0 × 10 ⁻⁴ M), and the absorbance was measured by adjusting the acidity of the mixed solution from pH 4.0 to 12.0.

As a result, it was confirmed that the detection ability of histidine is improved under basic conditions than under acidic conditions or neutral conditions. 8 is a graph showing the results of measuring absorbance according to the acidity of the buffer. According to this, it can be seen that the absorbance is higher under basic conditions than under acidic conditions. 14 shows UV-VIS spectral results according to the acidity of the detection buffer. In the graph, (a) indicates that there is no histidine in the detection buffer, (b) indicates when histidine is added under basic conditions, and (c) indicates when histidine is added under acidic conditions. Through the experimental results, it was confirmed that similar results were obtained in the acidic condition without histidine. This is because under basic conditions, histidine tends to strongly aggregate with BCML-Ag by electrostatic interaction between the cation of ammonia of histidine and the anion of carboxylic acid of BCML-Ag.

In addition, when the histidine-labeled protein bound to BCML-Ag is incubated in a constant concentration of hydrochloric acid (HCl) solution, the protein is released from BCML-Ag to recover 95% of the UV-VIS spectral intensity, and the BCML-Ag detection buffer is It was confirmed that the color changes from red to yellow in reverse.

1 is a diagram illustrating a biosensor in which N, N-bis (carboxymethyl) -L-lysine is bound to silver nanoparticles and a state in which histidine is specifically bound to the biosensor.

2 is a graph showing peaks of the FT-Raman spectrum of the histidine specific biosensor of the present invention.

Figure 3 is a graph showing the TOF-SIMS spectral results of the histidine specific biosensor of the present invention.

4 is a graph showing the UV-VIS spectrum results of the biosensor of Example 1 and the biosensors combined with histidine and other amino acids.

Figure 5 shows the R ( A ₅₂₀ / A ₄₀₀ ) of the biosensor of the present invention selectively combined with histidine.

FIG. 6 is a color change observed after adding histidine alone and an aqueous solution mixed with histidine and other amino acids to the detection buffer including the biosensor of Example 1. FIG. 6, (1) histidine (2) alanine + histidine, (3) cystidine + histidine, (4) leucine + histidine, (5) proline + histidine, (6) valine + histidine, (7) serine + histidine , (8) tyrosine + histidine, (9) methionine + histidine, (10) phenylalanine + histidine, (11) threonine + histidine, and (12) aspartic acid + histidine.

7 is a graph showing a correction curve (520 nm) of ultraviolet absorption intensity according to the concentration of histidine in an aqueous solution.

8 is a graph showing the change in absorbance according to acidity. This confirms that the absorbance is high in the case of basic conditions.

9 illustrates a state in which the carboxyl group included in the histidine-specific biosensor of the present invention is ionically bound to the ammonia portion of histidine.

10A, 10B and 10C show that the biosensor according to the invention can selectively bind histidine.

11 shows a TEM image (A) of a histidine specific biosensor according to the present invention and a TEM image after the biosensor is combined with histidine.

12A and 12B show the detection results of the ubiquitin protein and the Cdc 20 protein by the biosensor of the present invention.

Figure 13 is a photograph showing the gel image before and after the addition of the ubiquitin protein and after the addition of the Cdc 20 protein.

14 shows UV-VIS spectral results according to the acidity of the detection buffer.

15 is a photograph showing a color changed from pale yellow to red after mixing histidine in a detection buffer including a histidine-specific biosensor according to Example 1. FIG.

Claims (16)

For histidine specific biosensors, Metal particles; And An organic ligand bonded to the metal particle surface; To include, The organic ligand is histidine specific biosensor, characterized in that the lysine having a carboxyl group. The method of claim 1, Histidine-specific biosensor, characterized in that the three carboxyl groups of the organic ligand The method according to claim 1 or 2, The organic ligand that binds to the metal particles is a histidine specific biosensor, characterized in that 1 to 100 moles per mole of the metal particles. The method according to claim 1 or 2, The metal particles are histidine specific biosensor, characterized in that selected from silver, gold, or platinum. The method according to claim 1 or 2, Particle diameter of the metal particles is histidine-specific biosensor, characterized in that 1nm to 30nm. delete The method of claim 1, Lysine having a carboxyl group is N, N-bis (carboxymethyl) -L-lysine (N, N-bis (carboxymethyl) -L-lysine) histidine specific biosensor. (a) dispersing the biosensor according to claim 1 or 2 in a detection buffer; (b) mixing the aqueous solution containing histidine in the detection buffer in which the biosensor is dispersed; And (c) observing the color change of the detection buffer in which the histidine-containing aqueous solution is mixed; Histidine detection method using a histidine-specific biosensor comprising a. The method of claim 8, The histidine detection method further comprises the step of quantitatively analyzing the content of histidine by measuring the absorbance using an ultraviolet-visible spectrophotometer of the color change detection buffer in step (c) or after step (c). Histidine detection method using a histidine specific biosensor. (a) reducing the metal oxide in the presence of a reducing agent to produce nano-level metal colloidal particles; (b) combining the metal particles with the lysine by mixing an aqueous solution containing an excess of lysine having a carboxyl group to the metal colloidal particles prepared in step (a); And (c) after the completion of step (b), separating the lysine-bound metal colloidal particles formed in step (b) with an aqueous solution; Histidine specific biosensor manufacturing method comprising a. The method of claim 10, Method for producing a histidine-specific biosensor further comprises the step of washing and separating the metal colloid particles bound to lysine, produced in step (c). The method according to claim 10 or 11, wherein The metal oxide is silver nitrate (AgNO ₃ ), characterized in that the biosensor manufacturing method. The method according to claim 10 or 11, wherein The lysine having a carboxyl group is N, N-bis (carboxymethyl) -L-lysine (N, N-bis (carboxymethyl) -L-lysine) bio-sensor manufacturing method characterized in that. The method according to claim 10 or 11, wherein Lysine binding to the metal particles in the step (b) is a biosensor manufacturing method, characterized in that 1 to 100 moles per mole of metal colloidal particles. The method according to claim 10 or 11, wherein Particle diameter of the metal colloidal particles formed in the step (a) is a biosensor manufacturing method, characterized in that 1nm to 30nm. Detecting histidine in a biological sample of an individual suspected of cirrhosis, AIDS, kidney disease, asthma, lung disease, thrombotic disorder or malaria using the histidine specific biosensor according to claim 1. Method of providing information necessary for diagnosis of disease.

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