Method for detecting hydrogen peroxide content and pH value using nanoparticles and surface enhanced Raman scattering

Abstract

The present disclosure provides a nanoparticle, wherein the nanoparticle includes a hollow mesoporous silica microsphere, the outer wall of the hollow mesoporous silica microsphere is coated with a bimolecular lipid membrane layer, the inner wall of the hollow mesoporous silica microsphere is loaded with nanometal particles, and the nanometal particles are combined with 3-mercaptophenylboric acid and 4-mercaptobenzoic acid. The present disclosure also provides a method for preparing the above-mentioned nanoparticles, the nanoparticles prepared by the method, the use of the nanoparticles, and a method for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering. The nanoparticles disclosed in the present disclosure can realize surface enhanced Raman scattering to simultaneously detect the intracellular hydrogen peroxide content and pH value of cells, and have higher detection sensitivity.

Description

Technical Field

[0001] The present disclosure relates to the field of measurement, and in particular, to a nanoparticle, a method for preparing the nanoparticle, the nanoparticle prepared by the method, the use of the nanoparticle, and a method for simultaneously detecting the intracellular hydrogen peroxide content and pH value of a cell by surface enhanced Raman scattering. Background Art

[0002] Increased hydrogen peroxide content in cells can directly or indirectly induce malignant transformation of cells. Moreover, the malignant phenotype of tumor cells can be reversed by reducing the intracellular hydrogen peroxide content. If the hydrogen peroxide content in tumor cells is increased, it may also induce apoptosis of tumor cells. Therefore, the determination of intracellular hydrogen peroxide content is very important for the study of cell biology and the development of tumor drugs.

[0003] Changes in intracellular pH are also typical features of many common diseases, such as cancer. The intracellular pH of cancer cells is also abnormally acidic. Therefore, the detection of intracellular pH is also very important for the study of cell biology and the development of tumor drugs.

[0004] CN111474162A discloses a SERS-labeled nanoprobe of gold-hexanethiol-mesoporous silicon structure, which uses hexanethiol (HT) as a molecular separator to ensure the stability of the signal molecule, and mesoporous silica encapsulates gold nanoparticles to integrate nanoprobes to monitor the pH inside tumor cells. However, experiments have shown that the nanoprobe still cannot distinguish between intracellular pH and extracellular pH, and cannot detect the hydrogen peroxide content inside cells. Summary of the invention

[0005] The purpose of the present disclosure is to realize surface enhanced Raman scattering to simultaneously detect the intracellular hydrogen peroxide content and pH value of cells.

[0006] In order to achieve the above-mentioned purpose, the present disclosure provides a method for preparing nanoparticles, which comprises: mixing hollow mesoporous silica microspheres whose inner walls are loaded with nano metal particles with an ethanol solution containing 3-mercaptophenylboric acid and 4-mercaptobenzoic acid, and then washing them by centrifugation with water to obtain washed microspheres; mixing the washed microspheres with water and liposomes to obtain microspheres coated with a bimolecular lipid membrane layer.

[0007] Optionally, the particle size of the nano metal particles is 15-20 nm; the particle size of the hollow mesoporous silica microspheres is 300-700 nm, and the wall thickness is 50-200 nm.

[0008] Optionally, the method further includes preparing the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall by the following operations: mixing amino-functionalized polyethylene nanoparticles with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles bound on the particle surface; mixing the polyethylene nanoparticles with the nano-metal particles bound on the particle surface with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles wrapped with a mesoporous silica shell layer; washing the polyethylene nanoparticles wrapped with the mesoporous silica shell layer with N,N-dimethylformamide to dissolve and remove the polyethylene nanoparticles to obtain hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall.

[0009] Optionally, the particle size of the polyethylene nanoparticles is 100-650nm.

[0010] Optionally, the method further includes: dispersing the polyethylene nanoparticles in ethanol, and then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles, relative to 100 mg of the polyethylene nanoparticles, the amount of ethanol used is 10-30 mL, and the amount of 3-aminopropyltriethoxysilane used is 50-400 μL.

[0011] Optionally, the nano-metal particles are nano-gold, and the method further comprises preparing the nano-gold by the following operation: heating the chloroauric acid aqueous solution to boiling, and then adding the trisodium citrate aqueous solution so that the liquid turns purple-red to obtain the nano-gold; the concentration of the chloroauric acid aqueous solution is 0.1-10mM, the concentration of the trisodium citrate aqueous solution is 0.5-2wt%, and the amount of trisodium citrate is 1-2g relative to 1mmol of chloroauric acid. The time for mixing the amino-functionalized polyethylene nanoparticles with the nano-metal particles can be 10-40 hours, and the temperature can be 10-40°C.

[0012] Optionally, relative to 100 mg of the polyethylene nanoparticles, the amount of the nanogold is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL. By adjusting the amount of tetraethoxysilane, the wall thickness of the hollow mesoporous silica microspheres can be appropriately adjusted.

[0013] Optionally, relative to 100 mg of the hollow mesoporous silica microspheres whose inner walls are loaded with nano-metal particles, the amount of 3-mercaptophenylboric acid is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid is 0.1-0.5 mmol, the amount of ethanol is 10-30 mL, and the amount of liposomes is 10-100 mg.

[0014] Optionally, the liposome contains phospholipids and cholesterol.

[0015] The present disclosure also provides nanoparticles prepared by the method described above.

[0016] The present disclosure also provides a nanoparticle, wherein the nanoparticle comprises a hollow mesoporous silica microsphere, the outer wall of the hollow mesoporous silica microsphere is coated with a bimolecular lipid membrane layer, the inner wall of the hollow mesoporous silica microsphere is loaded with nanometal particles, and the nanometal particles are combined with 3-mercaptophenylboric acid and 4-mercaptobenzoic acid.

[0017] Optionally, the particle size of the nano metal particles is 15-20 nm; the particle size of the hollow mesoporous silica microspheres is 300-700 nm, and the wall thickness is 50-200 nm.

[0018] Optionally, the molar ratio of the metal element in the nano-metal particles to the silicon element in the hollow mesoporous silica microspheres is 1:100-10000.

[0019] Optionally, the nano metal particles are nano gold, and the bilayer lipid membrane contains phospholipids and cholesterol.

[0020] The present disclosure also provides use of the nanoparticles as described above in preparing a kit for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering.

[0021] The present disclosure also provides a method for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering, the method comprising: mixing the nanoparticles as described above with the cells to be tested, then irradiating with laser and collecting Raman spectral information, and using the Raman spectral information to quantify the intracellular hydrogen peroxide content and pH value.

[0022] Optionally, using I 998 / I 882 The peak intensity ratio of I 1580 / I 1356 The intracellular pH was determined by the peak intensity ratio of

[0023] Through the above technical scheme, the nanoparticles disclosed in the present invention can allow the hollow mesoporous silica microspheres loaded with nano-metal particles on the inner wall to enter the cells through the bimolecular lipid membrane layer, and the hydrogen peroxide and hydrogen ions in the cells enter the inner cavity of the hollow mesoporous silica microspheres through the mesopores of the hollow mesoporous silica microspheres, and the 3-mercaptophenylboric acid bound to the nano-metal particles loaded on the inner wall of the hollow mesoporous silica microspheres reacts with hydrogen peroxide to be oxidized to 3-hydroxythiophenol, and the surface enhanced Raman scattering (SERS) signal of the 3-hydroxythiophenol bound to the nano-metal particles can be used to quantify the concentration of hydrogen peroxide, and the -COO group contained in the 4-mercaptobenzoic acid bound to the nano-metal particles loaded on the inner wall of the hollow mesoporous silica microspheres has pH sensitivity, and it has different SERS peak intensities under different pH conditions, so it can be used to determine the pH value. However, outside the cell, due to the coating of the bimolecular lipid membrane layer, hydrogen peroxide and hydrogen ions are isolated outside the hollow mesoporous silica microspheres loaded with nano-metal particles. Therefore, the present disclosure realizes the simultaneous detection of intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering.

[0024] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0026] Figure 1 It is a schematic diagram of the structure of nanoparticles provided by an exemplary embodiment of the present disclosure. Specific implementation methods

[0027] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0028] The present disclosure provides a method for preparing nanoparticles, the method comprising: mixing hollow mesoporous silica microspheres whose inner walls are loaded with nano metal particles with an ethanol solution containing 3-mercaptophenylboric acid and 4-mercaptobenzoic acid, and then washing them by centrifugation with water to obtain washed microspheres; mixing the washed microspheres with water and liposomes to obtain microspheres coated with a bimolecular lipid membrane layer.

[0029] Optionally, the particle size of the nano metal particles is 15-20 nm; the particle size of the hollow mesoporous silica microspheres is 300-700 nm, and the wall thickness is 50-200 nm.

[0030] Optionally, the method further includes preparing the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall by the following operations: mixing amino-functionalized polyethylene nanoparticles with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles bound on the particle surface; mixing the polyethylene nanoparticles with the nano-metal particles bound on the particle surface with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles wrapped with a mesoporous silica shell layer; washing the polyethylene nanoparticles wrapped with the mesoporous silica shell layer with N,N-dimethylformamide to dissolve and remove the polyethylene nanoparticles to obtain hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall.

[0031] Optionally, the particle size of the polyethylene nanoparticles is 100-650nm.

[0032] Optionally, the method further includes: dispersing the polyethylene nanoparticles in ethanol, and then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles, relative to 100 mg of the polyethylene nanoparticles, the amount of ethanol used is 10-30 mL, and the amount of 3-aminopropyltriethoxysilane used is 50-400 μL.

[0033] Optionally, wherein the nanometal particles are nanogold, the method further comprises preparing the nanogold by the following operation: heating an aqueous solution of chloroauric acid to boiling, and then adding an aqueous solution of trisodium citrate to make the liquid turn purple-red to obtain nanogold; the concentration of the aqueous solution of chloroauric acid is 0.1-10 mM, and the concentration of the aqueous solution of trisodium citrate is 0.5-2 wt %, and the amount of trisodium citrate used is 1-2 g relative to 1 mmol of chloroauric acid.

[0034] Optionally, relative to 100 mg of the polyethylene nanoparticles, the amount of the nanogold is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL.

[0035] Optionally, relative to 100 mg of the hollow mesoporous silica microspheres whose inner walls are loaded with nano-metal particles, the amount of 3-mercaptophenylboric acid is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid is 0.1-0.5 mmol, the amount of ethanol is 10-30 mL, and the amount of liposomes is 10-100 mg.

[0036] Optionally, the liposome contains phospholipids and cholesterol.

[0037] The present disclosure also provides nanoparticles prepared by the method described above.

[0038] The present disclosure also provides a nanoparticle, wherein the nanoparticle comprises a hollow mesoporous silica microsphere, the outer wall of the hollow mesoporous silica microsphere is coated with a bimolecular lipid membrane layer, the inner wall of the hollow mesoporous silica microsphere is loaded with nanometal particles, and the nanometal particles are combined with 3-mercaptophenylboric acid and 4-mercaptobenzoic acid.

[0039] Optionally, the particle size of the nano metal particles is 15-20 nm; the particle size of the hollow mesoporous silica microspheres is 300-700 nm, and the wall thickness is 50-200 nm.

[0040] Optionally, the molar ratio of the metal element in the nano-metal particles to the silicon element in the hollow mesoporous silica microspheres is 1:100-10000.

[0041] Optionally, the nano metal particles are nano gold, and the bilayer lipid membrane contains phospholipids and cholesterol.

[0042] The present disclosure also provides use of the nanoparticles as described above in preparing a kit for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering.

[0043] The present disclosure also provides a method for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering, the method comprising: mixing the nanoparticles as described above with the cells to be tested, then irradiating with laser and collecting Raman spectral information, and using the Raman spectral information to quantify the intracellular hydrogen peroxide content and pH value.

[0044] Optionally, using I 998 / I 882 The peak intensity ratio of I 1580 / I 1356 The intracellular pH was determined by the peak intensity ratio of

[0045] The present invention is further described in detail below by way of examples.

[0046] Example 1

[0047] 150 μL of HAuCl4 (100 mM) was added to 30 mL of ultrapure water, and the mixture was heated to 100°C to boil. Trisodium citrate dihydrate solution (1.5 mL) was slowly added to the above solution via a syringe. The color of the mixture changed from brown to purple, and gold particles with a particle size of about 15-20 nm were obtained.

[0048] 50 mg of polyethylene (PS) particles (particle size of 150 nm) were uniformly dispersed in 10 mL of ethanol solution, 75 μL of APTES was added, and stirred at room temperature for 12 hours to obtain amino-functionalized PS particles (PS-NH2), and the product was washed 3-5 times by centrifugation with ethanol. The obtained PS-NH2 nanoparticles were added to the above-synthesized gold particles and stirred at room temperature for 24 hours. The nanoparticles obtained by centrifugation were washed 3 times with ethanol and dispersed in 20 mL of water to obtain polyethylene nanoparticles with the nanometal particles bound to the particle surface.

[0049] 30 mg of triethanolamine (TEOA) and 50 mg of hexadecyltrimethylammonium bromide (CTAB) were added to the polyethylene nanoparticles with the nanometal particles bound on the particle surface. The mixed solution was heated to 80 ° C in an oil bath. 100 μL of tetraethoxysilane (TEOS) was slowly added dropwise. After 2 hours of reaction, a layer of mesoporous silica will grow on the surface of the particles to obtain polyethylene nanoparticles wrapped with a mesoporous silica shell. After the reaction is completed, wash 5 times with N, N-dimethylformamide (DMF) to dissolve the polyethylene nanoparticles to obtain hollow mesoporous silica microspheres with nanometal particles loaded on the inner wall.

[0050] Use electronic balance to accurately weigh 3-mercaptophenylboric acid and 4-mercaptobenzoic acid, and add ethanol to be mixed with 3-MPBA ethanol solution and 4-MBA ethanol solution (100mM). The hollow mesoporous silica microspheres loaded with nano metal particles on the inner wall are mixed with the ethanol solution containing 3-mercaptophenylboric acid and 4-mercaptobenzoic acid, and then centrifuged with water to obtain the washed microspheres; the washed microspheres are mixed with water and liposomes to obtain the microspheres coated with bimolecular lipid membrane layers, i.e. the nanoparticles of the present embodiment. Relative to 100mg of the hollow mesoporous silica microspheres loaded with nano metal particles on the inner wall, the amount of 3-mercaptophenylboric acid is 0.3mmol, the amount of 4-mercaptobenzoic acid is 0.3mmol, the amount of ethanol is 20mL, and the amount of liposome is 50mg. The liposomes contain 90% by weight of lecithin and 10% by weight of cholesterol.

[0051] Comparative Example 1

[0052] Nanoparticles were prepared according to the method of Example 1, except that liposomes were not used. The nanoparticles prepared in this comparative example were not coated with a bimolecular lipid membrane layer.

[0053] Comparative Example 2

[0054] Nanoparticles were prepared according to the method of Example 1, except that the polyethylene nanoparticles were not dissolved and removed. The nanoparticles prepared in this comparative example did not have a hollow structure.

[0055] Test Example 1

[0056] This test example uses MHCC97 cells as an example of tumor cells and L-02 cells as an example of normal cells. The intracellular hydrogen peroxide content of MHCC97 cells is higher than that of L-02 cells, and the intracellular pH value of MHCC97 cells is lower than that of L-02 cells.

[0057] MHCC97 cells and L-02 cells were cultured in DMEM medium containing 10% fetal bovine serum. The culture conditions included: temperature 37°C, 5% CO2, and seeding density of approximately 5×10 6 / mL, adherently cultured until the confluence reached about 70%, discarded the culture medium, and washed the cells with phosphate buffered saline (PBS) with a pH value of 7.4 for later use.

[0058] Equal weights of the nanoparticles of Example 1 and Comparative Examples 1-2 were suspended in phosphate buffered saline (PBS) with a pH value of 7.4 (the particle concentration was about 1 mg / mL), and added to MHCC97 cells and L-02 cells as SERS labeling agents, respectively, and allowed to stand for 30 minutes before starting Raman spectroscopy detection.

[0059] The detection conditions of Raman spectroscopy include: the excitation light is 633nm, and each Raman spectrum is recorded in 400-1600cm -1 The signal value in the range of 20 seconds is 520cm -1 The signal value of the peak at is used for calibration. 998 / I 882 The peak intensity ratio of I 1580 / I 1356 The intracellular pH value was determined by the peak intensity ratio of . The results are shown in Table 1.

[0060] Table 1

[0061]

[0062] According to the data in Table 1, it can be seen that the SERS labeling reagent of Example 1 can significantly distinguish the hydrogen peroxide concentration in MHCC97 cells and L-02 cells, while the SERS labeling reagent of Comparative Example 1 is difficult to enter the cells and can only measure the hydrogen peroxide concentration outside the cells. Since the hydrogen peroxide concentration in the extracellular environment of MHCC97 cells and L-02 cells is not much different, it is impossible to distinguish MHCC97 cells from L-02 cells; the SERS labeling reagent of Comparative Example 2 lacks a hollow structure and is not as good as the SERS labeling reagent of Example 1 in terms of detection sensitivity. This shows that the nanoparticles disclosed in the present invention can realize surface enhanced Raman scattering to simultaneously detect the intracellular hydrogen peroxide content and pH value of cells, and have higher detection sensitivity.

[0063] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0064] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0065] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A method for preparing nanoparticles, characterized in that: The method comprises: The hollow mesoporous silica microspheres loaded with nano-metal particles on the inner wall are mixed with an ethanol solution containing 3-mercaptophenylboric acid and 4-mercaptobenzoic acid, and then washed by centrifugation with water to obtain washed microspheres; the particle size of the nano-metal particles is 15-20nm; the particle size of the hollow mesoporous silica microspheres is 300-700nm, and the wall thickness is 50-200nm; The washed microspheres are mixed with water and liposomes to obtain microspheres coated with a bimolecular lipid membrane layer; relative to 100 mg of the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall, the amount of 3-mercaptophenylboric acid is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid is 0.1-0.5 mmol, the amount of ethanol is 10-30 mL, and the amount of liposomes is 10-100 mg; The liposomes contain phospholipids and cholesterol.

2. The method according to claim 1, wherein: The method further comprises preparing the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall by the following operation: Mixing amino-functionalized polyethylene nanoparticles with nanometal particles to obtain polyethylene nanoparticles with the nanometal particles bound to the particle surface; The polyethylene nanoparticles having the nano-metal particles bound to the surface of the particles are mixed with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles coated with a mesoporous silica shell layer; The polyethylene nanoparticles wrapped with the mesoporous silica shell layer are washed with N,N-dimethylformamide to dissolve and remove the polyethylene nanoparticles, thereby obtaining the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall.

3. The method according to claim 2, wherein: The particle size of the polyethylene nanoparticles is 100-650nm; The method also includes: dispersing polyethylene nanoparticles in ethanol, then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles, wherein the amount of ethanol used is 10-30mL and the amount of 3-aminopropyltriethoxysilane used is 50-400μL relative to 100mg of the polyethylene nanoparticles.

4. The method according to claim 2, wherein: The nano metal particles are nano gold, and the method further comprises preparing the nano gold by the following operations: The chloroauric acid aqueous solution is heated to boiling, and then a trisodium citrate aqueous solution is added to make the liquid turn purple-red to obtain nanogold; the concentration of the chloroauric acid aqueous solution is 0.1-10mM, and the concentration of the trisodium citrate aqueous solution is 0.5-2wt%. Relative to 1mmol of chloroauric acid, the amount of trisodium citrate used is 1-2g.

5. The method according to claim 2, wherein: Relative to 100 mg of the polyethylene nanoparticles, the amount of the nanogold is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL.

6. A nanoparticle prepared by the method according to any one of claims 1 to 5, characterized in that: The nanoparticles include hollow mesoporous silica microspheres, the outer walls of which are coated with a bimolecular lipid membrane layer, the inner walls of which are loaded with nanometal particles, and the nanometal particles are combined with 3-mercaptophenylboric acid and 4-mercaptobenzoic acid.

7. The nanoparticle according to claim 6, wherein The particle size of the nano-metal particles is 15-20 nm; the particle size of the hollow mesoporous silica microspheres is 300-700 nm, and the wall thickness is 50-200 nm; The molar ratio of the metal element in the nano-metal particles to the silicon element in the hollow mesoporous silica microspheres is 1:100-10000; The nano metal particles are nano gold, and the bimolecular lipid membrane layer contains phospholipids and cholesterol.

8. Use of the nanoparticles according to any one of claims 6 to 7 in preparing a kit for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering.

9. A method for simultaneously detecting the intracellular hydrogen peroxide content and pH value of cells by surface enhanced Raman scattering, characterized in that: The method includes: The nanoparticles according to any one of claims 6 to 7 are mixed with cells to be tested, then irradiated with laser light and Raman spectroscopy information is collected, and the Raman spectroscopy information is used to quantify the intracellular hydrogen peroxide content and pH value.

10. The method according to claim 9, wherein: Use I 998 / I 882 The peak intensity ratio of I 1580 / I 1356 The intracellular pH was determined by the peak intensity ratio.

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