Material for real-time detection of stem cell differentiation, preparation method and real-time detection and analysis method of stem cell differentiation

Abstract

The invention provides a material for real-time detection of stem cell differentiation, 2DMXene is taken as a matrix material, the matrix material of 2DMXene is Nb2CTx, chiral amino acid, nanogold and methylene blue are loaded on the matrix material of 2DMXene, the matrix material is recorded as chiral RH6G-Au / Nb2CTx, RH6G represents methylene blue, Au represents nanogold, chirality represents that Au / Nb2CTx reacts with the chiral amino acid to form a chiral material, the matrix material of 2DMXene is Nb2CTx, and the matrix material of 2DMXene is Nb2CTx and is recorded as chiral RH6G-Au / Nb2CTx is recorded as chiral RH6G-Au / Nb2CTx. The thickness of the material for real-time detection of stem cell differentiation is 2.5-3.5 nm, and the ratio of the longest diameter to the shortest diameter of a two-dimensional surface, perpendicular to the thickness direction, of the material for real-time detection of stem cell differentiation is 1.0-1.5; in the material for real-time detection of stem cell differentiation, the weight ratio of the nanogold material to the Nb2CTx material is x: (100-x), and x is more than or equal to 10 and less than or equal to 30. The material provided by the invention has high resolution on the functional neuron marker dopamine, clear strip and higher sensitivity, and realizes high-sensitivity and high-selectivity detection on the functional neuron marker dopamine differentiated by stem cells.

Description

Technical Field

[0001] The present invention relates to the field of stem cell secretion detection, and in particular to a material and a preparation method for real-time detection of stem cell differentiation and a real-time detection and analysis method for stem cell differentiation. Background Art

[0002] Due to their pluripotent properties, neural stem cells are expected to replace damaged cells and rebuild neural networks after differentiation, and are widely used in the treatment of neurodegenerative diseases. In order to improve the survival rate of transplanted cells, avoid fluctuations in transplantation treatment effects and safety risks caused by cell heterogeneity, it is necessary to detect and monitor the differentiation of neural stem cells. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a material and preparation method for real-time detection of stem cell differentiation and a real-time detection and analysis method for stem cell differentiation.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a material for real-time detection of stem cell differentiation, wherein the material for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as a matrix material, the 2DMXene matrix material is Nb2CTx, and the 2DMXene matrix material is loaded with chiral amino acids, nanogold, and methylene blue, which is recorded as chiral RH6G-Au / Nb2CTx, wherein RH6G represents methylene blue, Au represents nanogold, and chirality represents that Au / Nb2CTx reacts with chiral amino acids to form a chiral material;

[0005] The material for real-time detection of stem cell differentiation is a nanomaterial, which is a 2D material as a whole. The thickness of the material for real-time detection of stem cell differentiation is 2.5-3.5 nm. The ratio of the longest diameter to the shortest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation perpendicular to the thickness direction is (1.0-1.5);

[0006] In the material for real-time detection of stem cell differentiation, the weight ratio of the nano-gold material to the Nb2CTx material is x:(100-x), wherein 10≤x≤30.

[0007] Dopamine, a functional neuron marker, is a neural stem cell marker. The material used for real-time detection of stem cell differentiation in the present invention is a chiral material. Raman spectroscopy has a sensitive response to molecular structure, symmetry, electronic environment and bonding relationship. The above material can identify dopamine, a functional neuron marker. The material used for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as a matrix material. The 2DMXene matrix material is Nb2CTx. The 2DMXene matrix material is loaded with chiral amino acids, nanogold and methylene blue. The combination of Nb2CTx material and chiral amino acids, nanogold and methylene blue has high resolution and clear bands for dopamine, a functional neuron marker, and has higher sensitivity, thereby realizing highly sensitive and highly selective detection of dopamine, a functional neuron marker. The material used for real-time detection of stem cell differentiation in the present invention is a chiral material combined with Raman spectroscopy signals. Real-time functional stem cell detection can be realized through the detection of dopamine.

[0008] Preferably, the chiral amino acid is L-alanine, L-cysteine, L-methionine, L-glutamic acid or L-isoleucine.

[0009] Preferably, the weight ratio of the chiral amino acid to the Nb2CTx material is 1:(0.8-1.2).

[0010] Preferably, the weight ratio of the methylene blue to the Nb2CTx material is: (3-5):10.

[0011] Preferably, the longest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation, which is perpendicular to the thickness direction, ranges from 500 to 800 nm.

[0012] Preferably, the method for preparing the material for real-time detection of stem cell differentiation comprises the following steps:

[0013] (1) Nb2AlC powder is heated to 58-65°C in a 45%-50% HF solution and reacted for 12-18 hours to obtain Nb2CT x Dispersion liquid;

[0014] (2) Add tetramethylammonium hydroxide to the terminal product of step (1), stir and react for 12 to 24 hours, then centrifuge and collect the solid, wash it alternately with ethanol and water, and then dry it to obtain Nb2CT x Nanomaterials;

[0015] (3) Nb2CT x The nanomaterials were ultrasonically treated in an ice-water bath for 15 to 20 minutes, and the supernatant was collected after centrifugation to obtain Nb2CT x Nanosheet suspension;

[0016] (4) In Nb2CT xAdd chloroauric acid to the nanosheet suspension, stir and mix, then add sodium borohydride ice water solution, stir, reduce chloroauric acid to obtain nano-gold loaded on the Nb2CT x On the nanosheet, Au / Nb2CT was obtained x Nanodispersions,

[0017] (5) adding chiral amino acid to the Au / Nb2CTx nano-dispersion, and ultrasonicating for 60 to 120 minutes under the protection of inert gas to obtain a chiral Au / Nb2CTx nano-material dispersion;

[0018] (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and methylene blue to the chiral Au / Nb2CTx nanomaterial dispersion and shake or stir at 20-30°C for 4-8 hours, collect the supernatant by centrifugation, and freeze-dry the supernatant to obtain chiral RH6G-Au / Nb2CTx nanomaterial dispersion. x .

[0019] Preferably, in step (5), Au / Nb2CT x The concentration of the nano-dispersion is 0.3-0.8 mg / L; Au / Nb2CT x Au / Nb2CT in Nanodispersion x The weight ratio of the nano material to the chiral amino acid is (0.8-1.2):1.

[0020] Preferably, the amount of methylene blue used in step (6) is about the same as that of Au / Nb2CT x Au / Nb2CT in Nanodispersion x The ratio of nanomaterials is (3-5):10.

[0021] The present invention also provides a method for preparing a material for real-time detection of stem cell differentiation, the method comprising the following steps:

[0022] (1) heating Nb2AlC powder in 45% to 50% HF solution to 58 to 65° C. and reacting for 12 to 18 hours to obtain a Nb2CTx dispersion;

[0023] (2) adding tetramethylammonium hydroxide to the terminal product of step (1), stirring and reacting for 12 to 24 hours, and then centrifuging and collecting the solid, washing it alternately with ethanol and water, and then drying it to obtain a Nb2CTx nanomaterial;

[0024] (3) treating the Nb2CTx nanomaterials by ultrasonic treatment in an ice water bath for 15 to 20 minutes, and collecting the supernatant after centrifugation to obtain a Nb2CTx nanosheet suspension;

[0025] (4) adding chloroauric acid to the Nb2CTx nanosheet suspension, stirring and mixing, then adding an ice-water solution of sodium borohydride, stirring, reducing the chloroauric acid to obtain nano-gold loaded on the Nb2CTx nanosheet, and obtaining an Au / Nb2CTx nano-dispersion.

[0026] (5) adding chiral amino acid to the Au / Nb2CTx nano-dispersion, and ultrasonicating for 60 to 120 minutes under the protection of inert gas to obtain a chiral Au / Nb2CTx nano-material dispersion;

[0027] (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and methylene blue to the chiral Au / Nb2CTx nanomaterial dispersion, shake or stir at 20-30°C for 4-8 hours, collect the supernatant by centrifugation, and lyophilize the supernatant to obtain chiral RH6G-Au / Nb2CTx.

[0028] Preferably, in step (1), the ratio of Nb2AlC powder to 45% to 50% HF solution is 0.08 g Nb2AlC powder per ml of 45% to 50% HF solution.

[0029] Preferably, in step (4), the weight ratio of chloroauric acid to sodium borohydride is 1:(1.1-1.5).

[0030] Preferably, the power of the ultrasound in step (4) is 100W to 150W.

[0031] Preferably, the power of the ultrasound in step (5) is 100W to 150W.

[0032] The present invention also provides a method for real-time detection of stem cell differentiation, which comprises the following steps: (a) mixing any of the above-mentioned materials for real-time detection of stem cell differentiation with neural stem cells during the differentiation of neural stem cells; (b) acquiring Raman spectral signals; (c) establishing a mapping relationship between a known dopamine quantity parameter and the intensity of the Raman spectral signal; and (d) performing a function operation on the Raman spectral signal in step (b) according to the mapping relationship in step (c).

[0033] The beneficial effects of the present invention are as follows: the present invention provides a material for real-time detection of stem cell differentiation, a preparation method and a real-time detection and analysis method of stem cell differentiation. Dopamine, a functional neuron marker, is a neural stem cell marker. The material for real-time detection of stem cell differentiation of the present invention is a chiral material. Raman spectroscopy has a sensitive response to molecular structure, symmetry, electronic environment and bonding relationship. The material can identify dopamine, a functional neuron marker. The material for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as a matrix material. The 2DMXene matrix material is Nb2CTx. The 2DMXene matrix material is loaded with chiral amino acids, nanogold and methylene blue. The combination of Nb2CTx material and chiral amino acids, nanogold and methylene blue has high resolution, clear bands and higher sensitivity, and realizes highly sensitive and highly selective detection of dopamine, a functional neuron marker. The material for real-time detection of stem cell differentiation of the present invention is a chiral material combined with Raman spectroscopy signals. Real-time functional stem cell detection can be realized by detecting dopamine. DETAILED DESCRIPTION

[0034] Figure 1 This is an atomic force microscope image of a material for real-time detection of stem cell differentiation according to an embodiment of the present invention.

[0035] Figure 2 This is an atomic force microscope test dimension diagram of the material for real-time detection of stem cell differentiation according to an embodiment of the present invention.

[0036] Figure 3 This is a Raman spectrum signal diagram of dopamine, a functional marker of stem cell differentiation, detected using the material for real-time detection of stem cell differentiation according to an embodiment of the present invention.

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] Example 1

[0040] As a material for real-time detection of stem cell differentiation in an embodiment of the present invention, the material for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as a matrix material, and the 2DMXene matrix material is Nb2CT x , chiral amino acids, nano-gold, and methylene blue are loaded on the 2DMXene matrix material, recorded as chiral RH6G-Au / Nb2CT x , where RH6G represents methylene blue, Au represents nanogold, and chirality represents Au / Nb2CT x react with chiral amino acids to form chiral materials;

[0041] The material for real-time detection of stem cell differentiation is a nanomaterial, which is a 2D material as a whole. The thickness of the material for real-time detection of stem cell differentiation is 2.5-3.5 nm. The ratio of the longest diameter to the shortest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation perpendicular to the thickness direction is (1.0-1.5);

[0042] In the material for real-time detection of stem cell differentiation, the nano-gold material and Nb2CT x The weight ratio of the materials is x:(100-x), where the theoretical calculation result of x is 12.6;

[0043] The chiral amino acid is L-alanine, L-cysteine, L-methionine, L-glutamic acid or L-isoleucine;

[0044] Nb2C x The weight of the material is obtained by separately preparing Nb2CT from Nb2AlC powder in step (1) of Example 1. x After dispersion, freeze-drying was performed to obtain Nb2CT x Materials, from Nb2AlC powder to Nb2CT by theoretical calculation x In the preparation method of the material for real-time detection of stem cell differentiation, the yield of the chiral amino acid and Nb2CT is theoretically calculated by the raw material Nb2AlC powder and the raw material amounts of gold, methylene blue, and chiral amino acid. x The weight ratio of the materials is 1:1.03; the methylene blue and Nb2CT x The weight ratio of the materials is 4.16:10;

[0045] The longest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation perpendicular to the thickness direction is in the range of 500 to 800 nm;

[0046] As a method for preparing a material for real-time detection of stem cell differentiation according to an embodiment of the present invention, the method for preparing a material for real-time detection of stem cell differentiation comprises the following steps:

[0047] (1) Nb2AlC powder was heated to 60°C in 45% HF solution and reacted for 15 hours to obtain Nb2CT x Dispersion; the ratio of Nb2AlC powder to 45% HF solution is 0.08g Nb2AlC powder / ml 45% HF solution;

[0048] (2) Add tetramethylammonium hydroxide to the terminal product of step (1), stir and react for 15 hours, then centrifuge and collect the solid, wash it alternately with ethanol and water, and then dry it to obtain Nb2CT x Nanomaterials;

[0049] (3) Nb2CT x The nanomaterials were ultrasonically treated in an ice-water bath for 20 min, and the supernatant was collected after centrifugation to obtain Nb2CT x Nanosheet suspension;

[0050] (4) In Nb2CT x Add chloroauric acid to the nanosheet suspension, stir and mix, then add sodium borohydride ice water solution, stir, reduce chloroauric acid to obtain nano-gold loaded on the Nb2CT x On the nanosheet, Au / Nb2CT was obtained x The weight ratio of chloroauric acid to sodium borohydride in the nano-dispersion liquid is 1:1.1; the power of ultrasound in step (4) is 120W;

[0051] (5) To Au / Nb2CT x Chiral amino acids were added to the nanodispersion and ultrasonicated for 90 minutes under inert gas protection to obtain chiral Au / Nb2CT x Nanomaterial dispersion; the power of ultrasound in step (5) is 120W

[0052] (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), methylene blue and chiral Au / Nb2CT x The nanomaterial dispersion was shaken or stirred at 25±1°C for 5 h, the supernatant was collected by centrifugation, and the supernatant was freeze-dried to obtain chiral RH6G-Au / Nb2CT x .

[0053] The chiral RH6G-Au / Nb2CT in the dispersion of step (6) was detected by atomic force microscope (AFM). x The size thickness, such as Figure 1 , Figure 2 As shown, chiral RH6G-Au / Nb2CT in the dispersion x The thickness is about 2.8nm, and the radial size is between 425 and 625nm.

[0054] Example 2

[0055] As a material for real-time detection of stem cell differentiation according to an embodiment of the present invention, the difference between this embodiment and Example 1 is that the amounts of chloroauric acid and sodium borohydride in step (4) are adjusted, the weight ratio remains unchanged, and the amount of chloroauric acid is 1.5 times the amount in Example 1.

[0056] The theoretical calculation result of x is 25.2.

[0057] Comparative Example 1

[0058] As a comparative example of the present invention, a material for real-time detection of stem cell differentiation is different from Example 1 in that step (4) is omitted and the preparation is performed in sequence. That is, the prepared material does not contain nano-gold (Au).

[0059] Experimental methods

[0060] 1. Material Characterization

[0061] (1) 1 mL of 10 mg / mL materials of Example 1, Example 2, and Comparative Example 1 were respectively dispersed by ultrasonic, and 0.3 mL of a 0.01 μg / L dopamine aqueous solution was added. The mixture was ultrasonically treated for 15 minutes for sufficient adsorption, and the Raman spectrum of the sample solution was characterized.

[0062] (2) 1 mL of 10 mg / mL materials of Example 1, Example 2, and Comparative Example 1 were respectively ultrasonically dispersed, 0.3 mL of a 0.01 μg / L dopamine aqueous solution was added, and the mixture was ultrasonically treated for 15 minutes. The Raman spectra of the materials of Example 1, Example 2, and Comparative Example 1 were characterized at an excitation wavelength of 780 nm.

[0063] The experimental results show that compared with the comparative example, the embodiment has a greater response intensity. And the response intensity of Example 2 is greater. Under the action of 780nm excitation wavelength, the response intensity of the material increases, and the degree of increase in Example 2 is much higher than that in Comparative Example 1, indicating that the material used for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as the matrix material, and the 2DMXene matrix material is Nb2CTx. The 2DMXene matrix material is loaded with chiral amino acids, nanogold, and methylene blue. The combination of Nb2CTx material and chiral amino acids, nanogold, and methylene blue has high resolution, clear bands, and higher sensitivity, achieving highly sensitive and highly selective detection of dopamine, a functional neuron marker. The material used for real-time detection of stem cell differentiation of the present invention is a chiral material combined with Raman spectroscopy signals. Through the detection of dopamine, real-time functional stem cell detection can be achieved.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A material for real-time detection of stem cell differentiation, characterized in that: The material used for real-time detection of stem cell differentiation is a nanomaterial with 2DMXene as the matrix material, and the 2DMXene matrix material is Nb2CT x , chiral amino acids, nano-gold, and methylene blue are loaded on the 2DMXene matrix material, recorded as chiral RH6G-Au / Nb2CT x , where RH6G represents methylene blue, Au represents nanogold, and chirality represents Au / Nb2CT x Reacts with chiral amino acids to form chiral materials; The material for real-time detection of stem cell differentiation is a nanomaterial, which is a 2D material as a whole. The thickness of the material for real-time detection of stem cell differentiation is 2.5-3.5 nm. The ratio of the longest diameter to the shortest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation perpendicular to the thickness direction is (1.0-1.5); In the material for real-time detection of stem cell differentiation, the nano-gold material and Nb2CT x The weight ratio of the materials is x:(100-x), where 10≤x≤30.

2. The material for real-time detection of stem cell differentiation according to claim 1, characterized in that: The chiral amino acid is L-alanine, L-cysteine, L-methionine, L-glutamic acid or L-isoleucine.

3. The material for real-time detection of stem cell differentiation according to claim 1, characterized in that: The chiral amino acid and Nb2CT x The weight ratio of the materials is 1:(0.8~1.2).

4. The material for real-time detection of stem cell differentiation according to claim 1, characterized in that: The methylene blue and Nb2CT x The weight ratio of the materials is (3-5):

10.

5. The material for real-time detection of stem cell differentiation according to claim 1, characterized in that: The longest diameter of the two-dimensional surface of the material for real-time detection of stem cell differentiation that is perpendicular to the thickness direction ranges from 500 to 800 nm.

6. The material for real-time detection of stem cell differentiation according to claim 1, characterized in that: The method for preparing the material for real-time detection of stem cell differentiation comprises the following steps: (1) Nb2AlC powder is heated to 58-65°C in a 45%-50% HF solution and reacted for 12-18 hours to obtain Nb2CT x Dispersion liquid; (2) Add tetramethylammonium hydroxide to the terminal product of step (1), stir and react for 12 to 24 hours, then centrifuge and collect the solid, wash it alternately with ethanol and water, and then dry it to obtain Nb2CT x Nanomaterials; (3) Nb2CT x The nanomaterials were ultrasonically treated in an ice-water bath for 15 to 20 minutes, and the supernatant was collected after centrifugation to obtain Nb2CT x Nanosheet suspension; (4) In Nb2CT x Add chloroauric acid to the nanosheet suspension, stir and mix, then add sodium borohydride ice water solution, stir, reduce chloroauric acid to obtain nano-gold loaded on the Nb2CT x On the nanosheet, Au / Nb2CT was obtained x Nanodispersions, (5) To Au / Nb2CT x Chiral amino acids were added to the nano-dispersion and ultrasonicated for 60 to 120 minutes under inert gas protection to obtain chiral Au / Nb2CT x Nanomaterial dispersions; (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), methylene blue and chiral Au / Nb2CT x The nanomaterial dispersion was shaken or stirred at 20-30°C for 4-8 hours, the supernatant was collected by centrifugation, and the supernatant was freeze-dried to obtain chiral RH6G-Au / Nb2CT x .

7. The material for real-time detection of stem cell differentiation according to claim 6, characterized in that: In step (5), Au / Nb2CT x The concentration of the nano-dispersion is 0.3-0.8 mg / L; Au / Nb2CT x Au / Nb2CT in Nanodispersion x The weight ratio of the nanomaterial to the chiral amino acid is (0.8-1.2):1; The amount of methylene blue used in step (6) is related to the Au / Nb2CT x Au / Nb2CT in Nanodispersion x The ratio of nanomaterials is (3-5):

10.

8. A method for preparing a material for real-time detection of stem cell differentiation according to any one of claims 1 to 7, characterized in that: The method for preparing the material for real-time detection of stem cell differentiation comprises the following steps: (1) Nb2AlC powder is heated to 58-65°C in a 45%-50% HF solution and reacted for 12-18 hours to obtain Nb2CT x Dispersion liquid; (2) Add tetramethylammonium hydroxide to the terminal product of step (1), stir and react for 12 to 24 hours, then centrifuge and collect the solid, wash it alternately with ethanol and water, and then dry it to obtain Nb2CT x Nanomaterials; (3) Nb2CT x The nanomaterials were ultrasonically treated in an ice-water bath for 15 to 20 minutes, and the supernatant was collected after centrifugation to obtain Nb2CT x Nanosheet suspension; (4) In Nb2CT x Add chloroauric acid to the nanosheet suspension, stir and mix, then add sodium borohydride ice water solution, stir, reduce chloroauric acid to obtain nano-gold loaded on the Nb2CT x On the nanosheet, Au / Nb2CT was obtained x Nanodispersions, (5) To Au / Nb2CT x Chiral amino acids were added to the nano-dispersion and ultrasonicated for 60 to 120 minutes under inert gas protection to obtain chiral Au / Nb2CT x Nanomaterial dispersions; (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), methylene blue and chiral Au / Nb2CT x The nanomaterial dispersion was shaken or stirred at 20-30°C for 4-8 hours, the supernatant was collected by centrifugation, and the supernatant was freeze-dried to obtain chiral RH6G-Au / Nb2CT x .

9. The method for preparing a material for real-time detection of stem cell differentiation according to claim 8, characterized in that: In step (1), the ratio of Nb2AlC powder to 45%-50% HF solution is 0.08 g Nb2AlC powder / ml 45%-50% HF solution; in step (4), the weight ratio of chloroauric acid to sodium borohydride is 1:(1.1-1.5); in step (4), the power of ultrasound is 100W-150W; in step (5), the power of ultrasound is 100W-150W.

10. A method for real-time detection of stem cell differentiation, characterized in that: The detection method comprises the following steps: (a) during the differentiation process of neural stem cells, mixing the material for real-time detection of stem cell differentiation as claimed in any one of claims 1 to 7 with neural stem cells; (b) acquiring Raman spectral signals; (c) establishing a mapping relationship between known dopamine quantity parameters and Raman spectral signal intensity; (d) performing a function operation on the Raman spectral signal in step (b) according to the mapping relationship in step (c).

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