TWI464267B - Cell marker and method for making the same - Google Patents

Description

Cell marker and method of producing the same

The present invention relates to a cell calibrator and a method of producing the same, and more particularly to a long-acting cell calibrator and a method of producing the same.

The formation of cancer cells comes from the abnormal proliferation of normal cells. In general, normal cells are controlled by genes in the human body, constantly undergoing processes of proliferation, differentiation, growth, and death. During the process of proliferation, some normal cells are abnormally affected by changes in internal factors such as genetic mutations or external factors such as toxic carcinogens, viral infections, etc., and the normal cell differentiation process. . Abnormal proliferation of cells can be initially recognized by the body's immune system, or eliminated, or the body's internal activation of the withering genes, resulting in cancer cells naturally die. However, when this abnormality exceeds the load of the defense mechanism, the cancer cells will be completely out of the body's control, and further unrestricted hyperplasia, and the abnormally proliferating cells will evolve into tumors. When a tumor is formed inside the body, it must be removed or treated by chemical or physical means. Before and after treatment, cancer cells that are invisible to the naked eye must use calibration to confirm the growth of cancer cells, and then use chemotherapy to completely eradicate the source of cancer cells and reduce the chance of cancer recurrence.

For a long time, organic dyes have been used to label macromolecules of organisms, interactions between various proteins, or as materials for labeling cancer cells. However, organic dyes have the following disadvantages: (1) low light stability; (2) narrow excitation spectrum; and (3) short fluorescence lifetime conditions, limiting their imaging in biomolecules, cells, and in vivo. Application. Since cancer cells need long-term tracking and labeling, it is a long-term research goal of scientists to find a material with long-acting markers applied to the interior of cells.

In summary, in view of the great potential of long-acting markers in cancer treatment, improving the shortcomings of organic dyes has naturally become an urgent need for breakthroughs in the industry. Therefore, the development of a fluorescent material with long-acting cell calibration It is necessary.

In view of this, one of the objects of the present invention is to develop a nanocomposite having good optical properties, and the nanocomposite can be applied to cell calibration.

Another object of the present invention is to develop a nanomaterial suitable for long-term tracking of cancer cells in vivo and a method of producing the same. The nano material has excellent biocompatibility and has better optical stability than the conventional organic fluorescent agent, and is more suitable for application to the human body.

To achieve the above object, the present invention provides a nanocomposite comprising: one nano-gold silver particles; and a nano-diamond-containing nano-diamond bonded to the nano-gold silver particles.

Preferably, the surface of the thiol-containing nano-diamond is obtained by sequentially modifying the nano-diamond with polyethylene glycol (PEG) and a surface modifier.

Preferably, the polyethylene glycol has a weight average molecular weight of from 300 to 3,000.

Preferably, the surface modifying agent is iminothiolane.

The invention further provides a cell calibration comprising: the nano composite material; and a target material having a surface having a thiol group, which is linked to the nano composite material.

Preferably, the target material having a thiol group on the surface is obtained by modifying a target material with a surface modifier.

Preferably, the surface modifying agent is imine cyclothiobutane.

Preferably, the target material is a transferrin or a protein comprising an RGD sequence.

Preferably, the cell calibration has an absorption range of 200 to 1100 nm.

The invention further provides a method for producing a cell calibration, comprising the steps of: providing a dispersion of a thiol-containing nano-diamond on a surface; adding a solution of nano-gold silver particles to the dispersion to obtain a mixed solution; And adding an aqueous solution of a target material having a surface having a thiol group to the mixed solution to obtain a cell calibrator.

Preferably, the method for producing the dispersion comprises the steps of: dispersing a polyethylene glycol modified nano diamond in water; and adding a surface modifying agent to react with the polyethylene glycol to obtain the dispersion. liquid.

Preferably, the surface modifying agent is imine cyclothiobutane.

Preferably, the method for producing an aqueous solution of a target material having a thiol group on the surface comprises the steps of: adding a target material to water; and adding a surface modifier to react with the target material to obtain a surface. An aqueous solution of a target material having a thiol group.

Preferably, the target material is transferrin or a protein containing an RGD sequence.

Preferably, the surface modifying agent is imine cyclothiobutane.

The nano composite material and the cell calibration material provided by the invention can be prepared in a short time and have excellent biocompatibility, and therefore are suitable for application to biomedical materials.

The present invention provides a nanocomposite and a cell calibrator excellent in biocompatibility. Specifically, the nanocomposite of the present invention comprises: one nano-gold silver particles; and a surface-containing thiol-containing nano-diamond connected to the nano-gold silver particles. The cell calibration provided by the present invention comprises the nano composite material; and a target material having a surface having a thiol group, which is linked to the nano composite material.

The term "nano-gold-silver particles" as used in the present invention refers to the disclosure of the Patent Application No. 100111493, the disclosure of which is incorporated herein by reference. In detail, the nano gold and silver particles are prepared in the following manner: (A) providing a mixed liquid containing gold ions and silver ions; (B) adding a reducing agent to the foregoing mixture; and (C) adding a chitin to obtain a growth solution, and then to stand the growth solution, and the nano gold and silver particles contain a gold component; a silver component; and chitin, and the absorption range is 600 to 1000 nm. In a preferred embodiment, the nano-gold silver particles are star-shaped nano gold and silver particles.

The surface thiol-containing nano-diamonds used in the present invention can be obtained by modifying nano-diamonds by a method known in the art, preferably by sequentially modifying the nano-diamonds with polyethylene glycol and a surface modifying agent. The surface modifying agent is not particularly limited as long as it can react with the terminal functional group of the PEG to produce a thiol group. In general, the surface modifying agent is an imine cyclothiobutane such as 2-imine cyclothiobutane. In the case of polyethylene glycol, polyethylene glycol which is modified with an amine group is generally preferred, and the amine group has a preferred reactivity to facilitate the reaction with the surface modifier. The molecular weight of the polyethylene glycol is also not particularly limited, and generally, the weight average molecular weight thereof is in the range of 300 to 3,000.

In the present invention, the surface material having a thiol group on the surface may be a target material having a thiol group on its surface, or a target material having a thiol group on the surface after being modified by a surface modifier. . For example, the target material having a thiol group on the surface can be obtained by modifying the transferrin with imine thiobutane or modifying the protein containing the RGD sequence with imine thiobutane. Specifically, the RGD-containing protein has a cancer cell target, and the amino acid contained therein is specific for the integrins alphaIIbbeta3, alphaVbeta3, and alpha5betal, but is not limited thereto.

Since the thiol group and the gold have excellent bonding ability, the thiol group-containing nano-diamond is preferably covalently bonded to the nano-gold-silver particle through the thiol group. Gold connection. Similarly, the target material having a thiol group on the surface is preferably covalently bonded to the gold in the nano-gold silver particles through the thiol group.

The invention also provides a method for producing a cell calibration, comprising the steps of: providing a dispersion of a surface thiol-containing nano-diamond; adding a solution of nano-gold silver particles to the dispersion to obtain a mixed solution; And adding an aqueous solution of a target material having a surface having a thiol group to the mixed solution to obtain a cell calibrator.

In the embodiment of the present invention, the method for producing the dispersion liquid may include the following steps: First, dispersing the polyethylene glycol modified nano diamond in water, specifically, 1 mg of polyethylene The diol-modified nano-diamond was uniformly dispersed in 20 mL of deionized water.

Then, a surface modifying agent is added and reacted with the polyethylene glycol to obtain the dispersion. Specifically, an imine cyclothiobutane (eg, 2-imine cyclothiobutane) is added, and the resulting solution is allowed to stand for 60 minutes, during which the imine cyclothiobutane reacts with the polyethylene glycol. .

Preferably, the polyethylene glycol modified nano diamond has a particle size of 1 to 500 nm after being dispersed in water.

In an embodiment of the present invention, the method for producing an aqueous solution of a target material having a thiol group on the surface comprises the steps of: adding a target material to water; and adding a surface modifier to the target material. The reaction is carried out to obtain an aqueous solution of a target material having a surface having a thiol group. In detail, after adding transferrin to water, 2-imine cyclothiobutane is added, and the resulting solution is allowed to stand for 60 minutes, during which 2-imine cyclic thiobutane reacts with the target material, and further An aqueous solution of transferrin having a thiol group on the surface is obtained.

It is to be noted that the temperature of all the reactions in the present invention is not particularly limited, but, for ease of handling, it is preferred to carry out the reaction at room temperature. In addition, if necessary, the resulting cell calibrator can be separated from the mixture by a centrifugation step. The conditions of the aforementioned centrifugation step (including the number of rotations, time and number of times, etc.) are not particularly limited as long as the purpose of separating the aforementioned cell calibrator can be achieved.

As apparent from the above, the method for producing the cell marker of the present invention is extremely simple, and therefore, it can be synthesized in a short time.

The following examples are intended to further understand the advantages of the present invention and are not intended to limit the scope of the invention.

Example 1: Preparation of Nano Composite Material of the Present Invention

The polyethylene glycol (PEG) used in the examples of the present invention is a terminally modified amine group polyethylene glycol having a weight average molecular weight of 1500 (PEG _{1500 N} ). The terminally modified amine based polyethylene glycol is commercially available from the pharmaceutical company Sigma Aldrich.

First, 43.5 mg of PEG _1500N and 625.4 mg of nano diamond were reacted at 120 ° C for three days to obtain a PEG _{1500 N} modified nano diamond.

Next, the PEG _1500N modified nano diamond was dispersed in water to obtain 0.05 mg/mL PEG _1500N modified nano diamond aqueous solution, and then 10 mg of 2-imine cyclothiobutane was added, and The mixture was uniformly mixed at room temperature and allowed to react for 60 minutes to obtain a dispersion of a thiol-containing nano-diamond.

Finally, 1 ml of a solution of nano-gold silver particles having a concentration of 20 ppm was added to the dispersion at room temperature, and uniformly mixed. Thereafter, the resulting mixture was allowed to stand for 60 minutes to obtain a mixture of nanocomposites, which was used without any treatment for the preparation of subsequent cell standards. When the mixture is centrifuged, the nanocomposite of the present embodiment can be separated. The nano-gold-silver particle solution used herein is a chitin solution containing nano-gold silver particles used in Example 1 of the Patent Application No. 100111493 of the Republic of China. The preparation method of the nano gold silver particles comprises the steps of: (A) providing a mixed liquid containing gold ions and silver ions; (B) adding a reducing agent to the mixture; and (C) adding a chitin. A growth solution was obtained, and then the growth solution was allowed to stand. .

Example 2: Preparation of cell calibrator of the invention (target material is transferrin )

1 mg of transferrin was added to 1 mL of water, and then 10 mg of 2-imine cyclothiobutane was added thereto at room temperature to react with the transferrin, and the resulting solution was allowed to stand for 60 minutes.

Next, the solution after standing was added to the mixture of the nanocomposites described in Example 1, and then the resulting solution was allowed to stand for 60 minutes to obtain a mixture of cell standards having the target properties. The mixed solution was centrifuged three times at 10,000 rpm to separate the crystalline solid therein, thereby obtaining a cell calibrator having the target properties of the present example.

Example 3: Preparation of cell calibration of the present invention (target material is a protein containing an RGD sequence)

The cell calibration of Example 3 was prepared in the same manner as in Example 2 except that the transferrin of Example 2 was changed to the protein containing the RGD sequence. The protein of the RGD sequence comprises the following three different amino acids, which are aspartic acid, glycine and arginine, respectively.

Example 4: Characterization of cell calibrators

[Exterior]

Please refer to the first A and the first B, which respectively show the PEG-modified nano-diamond modified with amino acid (ie, PEG _1500N- modified nano-diamond) and the cell calibration of the cell calibrator of Example 2. Electron micrograph. The functional group on the surface of the _{nanodiamond is} modified by PEG _1500N to have a plurality of primary amine groups distributed on the surface thereof, and the present invention utilizes the amine group to react with a surface modifier (2-imine cyclothiobutane) to make The surface functional groups of the PEG _1500N modified nanodiamonds are converted from amine groups to thiol groups. The thiol group and the gold have excellent bonding ability, and the thiol-containing nano-diamonds on the surface are reacted with the nano-gold silver particles by using this property. At the same time, the target material also has a plurality of characteristics of a single amine group, and the target material (transferrin) is modified into a surface system having a thiol group by the same method as described above, and then reacted with the nano silver gold particle bond after the reaction. To obtain a cell calibrator with targeted properties. The first A and the first panel B show that the particle size of the PEG _1500N modified nanodiamond and the cell calibrator of Example 2 is indeed a nanometer grade.

[Absorption spectrum]

The second figure is an absorption spectrum of the nano-diamond modified with PEG _1500N , nano-gold silver particles, the nano composite of Example 1, and the cell calibrator of Example 2. The figure shows that the functional groups on the surface of the _{nanodiamond are} modified by PEG _1500N to have a fluorescent property, and the absorption peak of the nano-diamond modified with PEG _1500N increases as the wavelength becomes shorter. The nano gold and silver particles have absorption peaks at 700 nm and 1000 nm, and the absorption of chitin in the nano gold and silver particles is in the ultraviolet region. The absorption of the nanocomposite of Example 1 and the cell calibrator of Example 2 combines the characteristics of the above-mentioned PEG _1500N- modified nano-diamond and nano-gold-silver particles, and some absorption peaks are slightly observed at 700 nm and 1000 nm. And the absorption peak increases as the wavelength becomes shorter.

[Fourier infrared spectroscopy]

The third panel shows the Fourier transform infrared spectrum of the PEG _1500N modified nano-diamond, the surface thiol-containing nano-diamond, and the nano-composite of the first embodiment. The functional group of the surface modification of the nanoparticle can be identified by the spectrometer. As shown in the third A diagram, 3425 cm ^-1 , 1630 cm ^-1 , 2920 cm ^-1 , and 1098 cm ^-1 have absorption peaks, which indicate that the surface of the nano-diamond modified with PEG _1500N has NH (3425 cm ^{-1 ).} ), (N) C=O (1630 cm ^-1 ), CH (2920 cm ^-1 ), CO (1098 cm ^-1 ), which means that the nano-diamond has a modified surface with PEG The functional group of the main structure.

The end of the PEG _1500N is an NH functional group which reacts with 2-imine cyclic thiobutane to carry out a ring opening reaction, and the functional group at the end thereof is converted into a thiol (SH) functional group. Although the absorption peak of the functional group of SH is not apparent on the Fourier infrared spectrum, the formation of a thiol-containing nano-diamond on the surface can be confirmed by the absorption peak of the NH functional group after the ring-opening reaction. Specifically, after the ring-opening reaction, a functional group of C=N is formed, which causes the absorption peak displacement of the original NH functional group to shift to 3137 cm ^-1 , which indirectly proves that the NH of the PEG _{1500 N} end has The 2-imine cyclothiobutane reaction further confirmed the formation of the SH functional group. From the results of the third B chart, it was found that an absorption peak was actually produced at 3137 cm ^-1 , and therefore, it was confirmed that the surface thiol-containing nano-diamond was successfully synthesized.

A nano-diamond having an S-H group on the surface can be reacted with nano-gold silver particles by the S-H functional group to form the nanocomposite of the present invention. Using a Fourier transform infrared spectroscopy, it can be found that the nano composite material has a functional signal of chitin in addition to the above functional groups, and the nano gold and silver particles used in the examples are those described in the Republic of China No. 100111493, and the nai The rice gold silver particles contain a chitin component which functions as a surface modifier and at the same time has a stable structure.

[fluorescent characteristics]

In the fourth figure, columns A to D show liver cancer cells (as a control group), liver cancer cells ingested with PEG _1500N- modified nano-diamonds, liver cancer cells ingested in the first embodiment of the nano-composite, and ingestion Example 2: Conjugate pyrogram of liver cancer cells of a cell calibration, wherein (a) is an image of a bright field; (b) an image of a cell after transfection; (c) a column derived from PEG _1500N The fluorescent signal of the modified nano diamond; and (d) the image after the merged. The fluorescence characteristics of the nanocomposite of Example 1 and the cell calibrator of Example 2 were examined by the fluorescence maps. The A column in the fourth figure is used as a control group. It can be clearly seen from the results in column A (a) of the fourth figure that the liver cancer cells (J5) have no autofluorescence but pass the fluorescent dye (such as Hochest 33342) After organic fluorescent transfection, the position of the blue nuclei in the cells can be calibrated, as shown in column A (b) of the fourth figure. Using a conjugated focal microscope to measure cell penetration spectra and nuclear location, it was determined that there was no red autofluorescence inside the cells. In the fourth figure, _column B is the conjugated fluorescein image of the cells after ingestion of PEG _1500N- modified nano-diamonds. It can be confirmed that the PEG _1500N- modified nano-diamond does have red fluorescence and undergoes cell endocytosis. The PEG _1500N modified nano diamond was swallowed inside the cell. After the nanocomposite of Example 1 or the cell calibrator of Example 2 is cultured with the cells, there is also a cell endocytosis phenomenon and the fluorescence phenomenon of the compound can be observed at the same time, as in the fourth and fourth columns.

The end-modified amine-based PEG _1500N modified nano-diamond has better optical stability than the conventional organic fluorescent dye. After the organic fluorescent dye disclosed in the literature Anal. Chem ., 2009, 81, 8687-8694 is irradiated by a conjugated focus microscope for 400 seconds, the organic fluorescent light is quenched due to structural changes of the compound. Since organic fluorescence is limited by the characteristics of photothermal instability, it cannot be used for long-term intracellular tracking, but is limited in biological experiments. However, the nanocomposite of the present invention is not exposed to light energy to cause a change in its structure, thereby affecting the intensity of its fluorescence. Please refer to the fifth figure. The fifth A is an image of the nano composite material of the present invention and organic fluorescence (calcein) irradiated 35 seconds before (left 3rd) and rear (3rd right) under a conjugated focus microscope. The two images above the six images represent the green fluorescence of the cells after organic fluorescence staining; the middle two images are the fluorescence of the nanocomposite; the next two are the two kinds of fluorescent signals and cells Pass through the map of the merged map. By comparing the image on the left with the image on the right, it can be found that after the illumination of the light source of the conjugated focal microscope, the green fluorescent light is photolyzed by illumination, whereas the fluorescence of the nano composite material does not have any obvious photolysis. At the same time, using the fifth B picture, it can be observed that the fluorescence of the two kinds of fluorescence is changed under different illumination time; the fifth C picture is the nano composite material of the invention and the organic fluorescent light By plotting the ratio of the fluorescence signal before and after the irradiation and the time, it can be found that after the organic fluorescent signal is irradiated for a short time, there is a phenomenon of photolysis, which leads to quenching of the fluorescent signal, and conversely, the nano composite of the present invention. The fluorescence of the material has not changed. The results show that the nanocomposite of the present invention is not exposed to light energy and causes a change in its structure to affect the intensity of the fluorescent light; relatively, the organic fluorescent light is irradiated, which causes quenching of the fluorescent light.

[Biocompatibility]

Since the materials used in the present invention have excellent biocompatibility, in addition to providing good light stability of the nanocomposite and cell calibrator of the present invention, excellent biocompatibility is also provided. In this test, a nano diamond (ND) modified with PEG _1500N , a nano composite material of the present invention (ND-Au Star), a cell standard of the second embodiment (ND-Au Star-Tf) or an example The cell standard (ND-Au Star-RGD) was co-cultured with liver cancer cells (J-5) or lung cancer cells (A549), and cell viability was observed to evaluate biocompatibility.

The test results are shown in Fig. 6A and Fig. BB. As the concentration increases, the cell viability of all materials co-cultured is comparable to that of the control group. The results of this experiment show that the nanocomposite and cell calibrator of the present invention have excellent biocompatibility.

[Measurement of cell endocytosis]

Figure 7A is a graph showing the amount of cell fluorescence of the flow cytometer of the nanocomposite of the present invention and the cell calibrator of Example 2. The nano composite material of the invention can be used for fluorescence measurement of nano diamond by flow cytometry, and the cell has a fluorescent signal after the cell is swallowed by the endocytosis phenomenon. Here, the number of cells with fluorescent signals can be measured by flow cytometry, and the concentration of the nano-diamond inside the cells can be confirmed. The measurement results are shown in Figure 7A. Figure 7A shows that after the nanocomposite of the present invention is linked to the modified target material (transferrin), the number of cells with fluorescence increases, indicating that the target material can increase the effect of cell endocytosis. Figure 7B is a graph showing the results of measuring the gold concentration of the nanocomposite and the cell calibrator of Example 2 by inductively coupled plasma mass spectrometry. The result of the gold concentration measurement is similar to the result shown in Fig. A, which proves that the cell calibration of the present invention has both the function of target and development.

[warm treatment]

After incubation with cancer cells modified with PEG _1500N , the composite of the present invention or the cell calibrator of Example 2, and the cancer cells for 12 hours, the results of the thermotherapy treatment by irradiation with 800 nm infrared light were shown in the eighth In the picture. Figure 8 shows the results obtained by PEG _1500N modified nano diamond, the composite material of the present invention or the cell calibrator of Example 2 and cancer cells, followed by warming treatment, wherein A is a control group, The observation results of the nano diamond modified by PEG _1500N ; B is the observation result of the composite material of the present invention; C is the observation result of the cell calibration object of the second embodiment; and the column (a) is the observation result of 1.2W; Column (b) is the observation of 1.4W; column (c) is the observation of 2.3W. In addition, the image in the lower left corner of each figure in the eighth figure is a fluorescence spectrum of the cells after the square block is irradiated. As shown in Figure 8, after using pure cancer cells and cells with only PEG _1500N- modified nano-diamonds inside the cells for low-energy (1.2 and 1.4 W) or high-energy (2.3 W), the cancer cells are not There are any changes. However, after the cancer cells with the composite material and the cell calibrator were irradiated, the dead cells were stained with trypan blue, and the cancer cells were found to have fading or death, and the cell calibrator was even Low-energy 1.2 W exposure also has a good warming effect.

In addition, please refer to the fluorescence map in the lower left corner of each figure in the eighth figure. Since the organic fluorescent light used is a heat-sensitive fluorescent light, after the temperature is changed, the fluorescent light is quenched (the black square block in the figure), which can simultaneously align the cells. The phenomenon of death. Thus, it can be demonstrated that the cell marker of the present invention is a multifunctional material having a target, a phototherapy and a thermotherapy property.

In summary, the nano composite material and the cell standard of the present invention have excellent biocompatibility and have better optical stability than the conventional organic fluorescent agent, and therefore are more suitable for application to the human body. Moreover, the cell calibration system of the present invention has the characteristics of target, phototherapy and thermotherapy, and is beneficial to the research of cancer treatment.

Other implementations

All features disclosed in this disclosure can be combined in any manner. Features disclosed in this specification can be replaced with features of the same, equivalent or similar purpose. Therefore, the features disclosed in this specification are one of a series of equivalent or similar features.

In addition, according to the disclosure of the present specification, those skilled in the art can easily make appropriate changes and modifications to different methods and situations without departing from the spirit and scope of the present invention. The implementation aspect is also included in the scope of the patent application.

The first A graph shows a tunneling electron micrograph of a nano diamond modified with PEG _1500N .

The first B panel shows a tunneling electron micrograph of the cell calibrator of Example 2.

The second figure is an absorption spectrum of the nano-diamond modified with PEG _1500N , nano-gold silver particles, the nano composite of Example 1, and the cell calibrator of Example 2.

Figure A is a Fourier infrared spectrum of nano-diamonds modified with PEG _1500N .

The third B is a Fourier infrared spectrum of a nano-diamond with a thiol group.

The third C is a Fourier infrared spectrum of the nano composite of Example 1.

The fourth picture shows liver cancer cells (as a control group), liver cancer cells ingested with PEG _1500N modified nano diamond, liver cancer cells ingested in the first embodiment of the nanocomposite, and liver cancer cells ingested in the second cell calibration sample. Conjugate focal fluorescent light.

Fig. 5A is an image of the nanocomposite of the present invention and organic fluorescent light irradiated 35 seconds before and 35 seconds after irradiation under a conjugated focus microscope.

Figure 5B is an enlarged image of the nanocomposite of the present invention and the organic fluorescence before and after irradiation under a conjugated focus microscope.

The fifth C is a quenching trend diagram of the nanocomposite of the present invention and organic fluorescence after 35 seconds of irradiation under a conjugated focus microscope.

6A is a PEG _1500N modified nano diamond, a nanocomposite of the present invention, a cell calibrator of Example 2 or a cell calibrator of Example 3, and co-cultured with liver cancer cells (J-5). Observation of cell viability.

Figure 6B is a nano-diamond modified with PEG _1500N , a nanocomposite of the present invention, a cell calibrator of Example 2 or a cell calibrator of Example 3, and co-cultured with lung cancer cells (A549), the cells survive The observation of the rate.

Figure 7A is a graph showing the amount of cell fluorescence of the flow cytometer of the nanocomposite of the present invention and the cell calibrator of Example 2, wherein ND-Au represents the nanocomposite of the present invention, ND-Au-Tf Representative of the cell calibrator of Example 2.

Figure 7B is a graph showing the gold concentration obtained by measuring the nanocomposite of the present invention and the cell calibrator of the second embodiment by an inductively coupled plasma mass spectrometer, wherein ND-Au represents the nanocomposite of the present invention, ND -Au-Tf represents the cell calibrator of Example 2.

The eighth figure is the observation obtained by PEG _1500N- modified nanodiamond, the composite material of the present invention or the cell calibrator of Example 2 and cancer cells, followed by warming treatment.

Claims (15)

A nanocomposite comprising: a star-shaped nano-gold silver particle; and a surface thiol-containing nano-diamond connected to the nano-gold silver particle. The nanocomposite according to claim 1, wherein the thiol-containing nano-diamond is obtained by sequentially modifying a nano-diamond with polyethylene glycol and a surface modifier. The nano composite material according to claim 2, wherein the polyethylene glycol has a weight average molecular weight of 300 to 3,000. The nanocomposite of claim 2, wherein the surface modifier is iminothiolane. A cell calibration comprising: a nanocomposite of claim 1; and a target material having a surface having a thiol group attached to the nanocomposite. The cell calibrator according to claim 5, wherein the target material having a thiol group on the surface is obtained by modifying a target material with a surface modifier. The cell calibrator of claim 6, wherein the surface modifying agent is imine thiobutane. The cell calibrator of claim 6, wherein the target material is transferrin or a protein comprising an RGD sequence. The cell calibrator according to claim 5, wherein the calibration range of the calibration is 200 to 1100 nm. A method for producing a cell calibrator, comprising the steps of: providing a dispersion of a thiol-containing nano-diamond on a surface; adding a solution of star-shaped nano-gold silver particles to the dispersion to obtain a mixed solution; and an aqueous solution of a target material having a surface having a thiol group added thereto to obtain a cell calibrator. The manufacturing method of claim 10, wherein the method for producing the dispersion comprises the steps of: dispersing a polyethylene glycol modified nano diamond in water; and adding a surface modifying agent to the same The polyethylene glycol was reacted to obtain the dispersion. The manufacturing method according to claim 11, wherein the surface modifying agent is imine cyclothiobutane. The manufacturing method according to claim 10, wherein the method for producing an aqueous solution having a target material having a surface of a thiol group comprises the steps of: adding a target material to water; and adding a surface modifying agent to make Reacting with the target material to obtain an aqueous solution of a target material having a surface of a thiol group. The method of manufacture of claim 13, wherein the target material is transferrin or a protein comprising an RGD sequence. The manufacturing method according to claim 13, wherein the surface modifying agent is imine cyclothiobutane.