A labeling probe for stem cell detection and preparation method and kit thereof

Abstract

The present invention provides a labeling probe for stem cell detection, a preparation method thereof and a kit, comprising the following steps: preparing a Nb2CTx nanosheet suspension; adding a silver nitrate solution to the Nb2CTx nanosheet suspension to disperse uniformly, heating to 90-100°C and adding a sodium citrate solution under stirring or ultrasonic conditions, reacting for 40-80 minutes, reducing silver ions to obtain nanosilver loaded on the Nb2CTx nanosheets, and obtaining an Ag / Nb2CTx nanodispersion; adding a small molecule compound containing a carboxyl group to the Ag / Nb2CTx nanodispersion to react at 70-120°C for 30-50 minutes; collecting the solid phase particles in the obtained dispersion, drying them, mixing them with 5-aminofluorescein in an organic solvent, adding an active phosphate N-acylated coupling reagent, stirring and reacting for 25-30 minutes, and collecting the solid particles. The labeling probe for stem cell detection of the present invention has good biocompatibility and antibacterial properties, and has strong fluorescence specificity and high response signal.

Description

Technical Field

[0001] The present invention relates to the field of stem cell detection, and in particular to a labeling probe for stem cell detection, a preparation method thereof and a kit. Background Art

[0002] Stem cell-mediated regenerative medicine activates endogenous stem cells and promotes the renewal of damaged neuronal tissue by directly replacing damaged cells in relevant tissues or by stimulating paracrine signaling networks at the damaged site. In order to fully realize its therapeutic potential, the homing, survival, and differentiation characteristics of transplanted stem cells must be clearly understood. In recent years, based on their unique physicochemical properties, nanomaterials have been applied to medical enhanced imaging after stem cell labeling, to identify transplanted stem cells in recipient tissues, and to track their survival and development status in vivo. Therefore, it is necessary to develop new stem cell tracing materials and methods. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a labeled probe for stem cell detection and a preparation method and a kit thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a labeled probe for stem cell detection, the preparation method comprising the following steps:

[0005] (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;

[0006] (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;

[0007] (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;

[0008] (4) In Nb2CT x Silver nitrate solution is added to the nanosheet suspension to disperse it evenly, heated to 90-100°C and sodium citrate solution is added under stirring or ultrasonic conditions, reacted for 40-80 minutes, and silver ions are reduced to obtain nanosilver loaded on Nb2CT x On the nanosheet, Ag / Nb2CTx nanodispersion was obtained;

[0009] (5) Towards Ag / Nb2CT xA small molecule compound containing a carboxyl group is added to the nano-dispersion liquid and reacted at 70 to 120° C. for 30 to 50 minutes;

[0010] (6) After the solid particles in the dispersion obtained in step (5) are collected and dried, they are mixed with 5-aminofluorescein in an organic solvent, an active phosphate N-acylated coupling reagent is added, and the mixture is stirred for 25 to 30 minutes. The solid particles are then collected and washed alternately with ethanol and deionized water to obtain a labeled probe for stem cell detection.

[0011] Preparation of Ag / Nb2CT labeled probe for stem cell detection x Nanoparticles with excellent antibacterial properties, Ag / Nb2CT x After the nanoparticles are coupled to a small molecule compound containing a carboxyl group, the 5-aminofluorescein fluorescent group is connected through the reaction of the carboxyl group and the amino group to obtain a labeled probe for stem cell detection. The labeled probe for stem cell detection has good biocompatibility and antibacterial properties, and has strong fluorescence specificity and a high response signal.

[0012] Preferably, in the Ag / Nb2CTx nano-dispersion liquid of step (4), nanosilver and Nb2CT x The mass ratio of the nanosheets is (15-30):100.

[0013] Nanosilver and Nb2CT x The mass ratio of the nanosheets was determined by the silver nitrate solution and Nb2CT x Nb2CT in nanosheet suspension x Scaling control of nanosheets.

[0014] Preferably, in step (5), Ag / Nb2CT x Solid content in nanodispersion Ag / Nb2CT x The weight ratio of the small molecule compound containing a carboxyl group is 10:(2.5-5).

[0015] Preferably, the amount of 5-aminofluorescein used is such that the mass ratio of 5-aminofluorescein to the small molecule compound containing a carboxyl group is (2.5-4):1.

[0016] Preferably, the active phosphate N-acylated coupling reagent is benzotriazolyl diethyl phosphate or bis(4-nitrophenyl)-phenyl-phosphite.

[0017] Preferably, the small molecule compound containing a carboxyl group is aspartic acid, glutamic acid, or succinic acid, and the organic solvent in step (6) is dichloromethane or tetrahydrofuran.

[0018] Preferably, in step (4), the concentration of silver nitrate in the mixed system is 0.15 to 0.25 g / L.

[0019] The present invention also provides a labeled probe for stem cell detection prepared by any of the above-mentioned methods for preparing a labeled probe for stem cell detection.

[0020] The present invention also provides a labeling probe kit for stem cell detection, wherein the labeling probe kit comprises the labeling probe for stem cell detection described above.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a labeling probe for stem cell detection, a preparation method thereof and a kit; the labeling probe for stem cell detection of the present invention is used to prepare Ag / Nb2CTx nanoparticles, which have excellent antibacterial properties; after the Ag / Nb2CTx nanoparticles are coupled to a small molecule compound containing a carboxyl group, the 5-aminofluorescein fluorescent group is connected through the reaction of the carboxyl group and the amino group to obtain a labeling probe for stem cell detection; the labeling probe for stem cell detection has good biocompatibility and antibacterial properties, and has strong fluorescence specificity and a high response signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the experimental results of the labeled probe used for stem cell detection according to an embodiment of the present invention. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] As a labeled probe for stem cell detection according to an embodiment of the present invention, the preparation method of the labeled probe for stem cell detection comprises the following steps:

[0026] (1) 1 g Nb2AlC powder was heated to 60 °C in 10 mL 48% HF solution for 12 hours to obtain Nb2CT x Dispersion liquid;

[0027] (2) The terminal product of step (1) was added with 70 mL of tetramethylammonium hydroxide and stirred for 15 hours, and then the solid was collected by centrifugation and washed alternately with ethanol and water and then dried to obtain Nb2CT x Nanomaterials 0.832 g;

[0028] (3) Under the protection of nitrogen, 0.832gNb2CT x The nanomaterials were resuspended in deionized water, ultrasonically treated in an ice water bath for 15 min at an ultrasonic power of 80 W, and centrifuged at 3500 rpm for 30 min to collect the supernatant to obtain Nb2CT. x Nanosheet suspension, according to Nb2CTx Nanomaterial, resuspended deionized water volume, dry weight of precipitate after centrifugation, calculated Nb2CT x Nb2CT in nanosheet suspension x The concentration of nanosheets was 0.804 g / L;

[0029] (4) Nb2CT x The nanosheet suspension and silver nitrate solution were evenly dispersed in equal volumes, and the concentration of silver nitrate in the mixed system was 0.1566 g / L. The mixture was heated to 95 °C and sodium citrate solution was added under stirring or ultrasonic conditions. The reaction lasted for 50 minutes to reduce silver ions and obtain nanosilver loaded on Nb2CT. x On the nanosheets, Ag / Nb2CTx nanodispersion was obtained; the theoretical mass ratio of nanosilver to Nb2CTx nanosheets was 25:100;

[0030] (5) Towards Ag / Nb2CT x A small molecule compound containing a carboxyl group is added to the nano-dispersion and reacted at 80° C. for 40 minutes; the weight ratio of the solid content of Ag / Nb2CTx in the Ag / Nb2CTx nano-dispersion to the small molecule compound containing a carboxyl group is 10:3; the small molecule compound containing a carboxyl group is succinic acid;

[0031] (6) After the solid particles in the dispersion obtained in step (5) are collected and dried, they are mixed with 5-aminofluorescein in a dichloromethane organic solvent, an active phosphate N-acylated coupling reagent is added, and the mixture is stirred for reaction for 25 to 30 minutes. The solid particles are collected and washed alternately with ethanol and deionized water to obtain a labeled probe for stem cell detection. The amount of 5-aminofluorescein used is such that the mass ratio of 5-aminofluorescein to the small molecule compound containing a carboxyl group is 3:1, and the active phosphate N-acylated coupling reagent is benzotriazolyl diethyl phosphate or bis(4-nitrophenyl)-phenyl-phosphite.

[0032] Example 2

[0033] As a labeling probe for stem cell detection in an embodiment of the present invention, the only difference between this embodiment and embodiment 1 is that the silver nitrate solution and Nb2CT x Nb2CT in nanosheet suspension x The ratio of nanosheets is adjusted, and the theoretical mass ratio of nanosilver to Nb2CTx nanosheets is 15:100.

[0034] Comparative Example 1

[0035] As a labeling probe for stem cell detection in a comparative example of the present invention, the only difference between this comparative example and Example 1 is that in step (5), no small molecule compound containing a carboxyl group is added, and deionized water is used instead.

[0036] Comparative Example 2

[0037] As a labeling probe for stem cell detection in a comparative example of the present invention, the only difference between this comparative example and Example 1 is that in step (4), the silver nitrate solution is not added and is replaced by deionized water.

[0038] Experimental methods

[0039] 1. Fluorescence characterization

[0040] 0.5 g of the labeled probes for stem cell detection of Examples 1 to 2, Comparative Examples 1 and 2 were ultrasonically dispersed in 10 mL of water, and the fluorescence spectrum was tested by full wavelength scanning at 400 to 580 nm. The fluorescence spectrum was as follows: Figure 1 As shown, the fluorescent tracer material used for stem cell tracing has an excitation wavelength of 450-550 nm, and the highest excitation light peak is around 503 nm.

[0041] No response was detected in the sample of comparative example 1, and the fluorescence signal intensity of embodiments 1 to 2 was higher than that of comparative example 2, indicating that Ag / Nb2CT x After the nanoparticles are coupled with a small molecule compound containing a carboxyl group, the 5-aminofluorescein fluorescent group is connected through the reaction of the carboxyl group and the amino group, thereby enhancing the fluorescence intensity.

[0042] 2. Fluorescence biocompatibility experiment

[0043] The cultured human umbilical cord mesenchymal stem cells were diluted with culture medium to a concentration of 1×10 5 , take 2 mL of cell solution into a centrifuge tube, accurately weigh 50 mg of the labeled probes for stem cell detection of Examples 1-2 and Comparative Example 2, respectively, and culture them in a cell culture incubator at 37° C. and 5% CO 2 for 5 hours.

[0044] The cells were collected by centrifugation, rinsed with PBS, and then suspended in 5 mL of PBS solution. The fluorescence intensity at 503 nm was measured and recorded as C1.

[0045] 50 mg of the labeled probes for stem cell detection in Examples 1-2 and Comparative Example 2 were accurately weighed and dispersed in 5 mL of PBS solution, and the fluorescence intensity at 503 nm was measured and recorded as C0.

[0046] Biocompatibility coefficient (%) = C1 / C0×100%.

[0047] The experimental results are shown in Table 1.

[0048] sample Biocompatibility coefficient (%) Example 1 87.4 Example 2 86.3 Comparative Example 2 83.4

[0049] As shown in Table 1, the preparation of Ag / Nb2CT marker probes for stem cell detection according to the present invention xNanoparticles with excellent antibacterial properties, Ag / Nb2CT x After the nanoparticles are coupled to a small molecule compound containing a carboxyl group, the 5-aminofluorescein fluorescent group is connected through the reaction of the carboxyl group and the amino group to obtain a labeled probe for stem cell detection. The labeled probe for stem cell detection has good biocompatibility and antibacterial properties, and has strong fluorescence specificity and a high response signal.

[0050] 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 method for preparing a labeled probe for stem cell detection, characterized in that: The preparation method comprises the following steps: (1) Heat Nb2AlC powder in 45%~50% HF solution to 58~65℃ and react 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 resuspended in deionized water and sonicated in an ice-water bath for 15–20 min; (4) In Nb2CT x Silver nitrate solution is added to the nanosheet suspension to disperse it evenly, heated to 90-100°C and sodium citrate solution is added under stirring or ultrasonic conditions, reacted for 40-80 minutes, and silver ions are reduced to obtain nanosilver loaded on Nb2CT x Nanosheets are formed to obtain Ag / Nb2CTx nanodispersion; in the Ag / Nb2CTx nanodispersion, nanosilver and Nb2CT x The mass ratio of nanosheets is (15~30):100; (5) Towards Ag / Nb2CT x A small molecule compound containing a carboxyl group is added to the nano-dispersion and reacted at 70-120°C for 30-50 minutes; in step (5), Ag / Nb2CT x Solid content in nanodispersion Ag / Nb2CT x The weight ratio of the small molecule compound containing carboxyl group is 10: (2.5~5); (6) After the solid particles in the dispersion obtained in step (5) are collected and dried, they are mixed with 5-aminofluorescein in an organic solvent, an active phosphate N-acylated coupling reagent is added, and the mixture is stirred for 25 to 30 minutes. The amount of 5-aminofluorescein used is such that the mass ratio of 5-aminofluorescein to the small molecule compound containing a carboxyl group is (2.5 to 4):

1. The solid particles are collected and washed alternately with ethanol and deionized water to obtain a labeled probe for stem cell detection.

2. The method for preparing a labeled probe for stem cell detection according to claim 1, characterized in that: In the Ag / Nb2CTx nano-dispersion liquid of step (4), nanosilver and Nb2CT x The mass ratio of nanosheets is (15~25):

100.

3. The method for preparing a labeled probe for stem cell detection according to claim 1, characterized in that: Step (5) Ag / Nb2CT x Solid content in nanodispersion Ag / Nb2CT x The weight ratio of the small molecule compound containing carboxyl group is 10: (3~5).

4. The method for preparing a labeled probe for stem cell detection according to claim 1, characterized in that: The dosage of 5-aminofluorescein is such that the mass ratio of 5-aminofluorescein to the small molecule compound containing a carboxyl group is (3~4):

1.

5. The method for preparing a labeled probe for stem cell detection according to claim 1, characterized in that: The active phosphate N-acylation coupling reagent is benzotriazolyl diethyl phosphate or bis(4-nitrophenyl)-phenyl-phosphite.

6. The method for preparing a labeled probe for stem cell detection according to claim 1, characterized in that: The small molecule compound containing a carboxyl group is aspartic acid, glutamic acid, or succinic acid, and the organic solvent in step (6) is dichloromethane or tetrahydrofuran.

7. A labeled probe for stem cell detection prepared by the method for preparing a labeled probe for stem cell detection as claimed in any one of claims 1 to 6.

8. A labeling probe kit for stem cell detection, characterized in that: The labeling probe kit comprises the labeling probe for stem cell detection as claimed in claim 7.

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