Aptamer-modified MXene nanosheet-based active targeting recognition photo-thermal probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of targeted recognition photo-thermal probes, in particular to an MXene nanosheet-based active targeted recognition photo-thermal probe modified with a nucleic acid aptamer as well as a preparation method and application of the MXene nanosheet-based active targeted recognition photo-thermal probe modified with the nucleic acid aptamer, adopted MXene is Ti3C2Tx, and the surface of the Ti3C2Tx is modified with the nucleic acid aptamer; the nucleic acid aptamer is composed of an ATP nucleic acid aptamer and a GTP nucleic acid aptamer; the photothermal conversion efficiency of the photothermal agent is improved, the laser irradiation temperature is reduced, and the problem that at present, photothermal therapy causes damage to surrounding normal tissue is solved; through dual active targeting: first targeting: active targeting of tumor cells; performing specific recognition on the FA ligand and an FR receptor highly expressed on the surface of a tumor cell membrane; the active uptake of the tumor cells to the Ti3C2Tx-ATP-GTP is increased, and the tumor cells enter the cells through endocytosis; secondary targeting: actively targeting ATP and GTP energy molecules in cells; the surface of Ti3C2Tx is modified with a nucleic acid aptamer, so that the photo-thermal agent enters tumor cells and can quickly recognize ATP / GTP molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted recognition photothermal probes, and specifically to a MXene nanosheet-based active targeted recognition photothermal probe modified with a nucleic acid aptamer, and a preparation method and application thereof. Background Art

[0002] Mild photothermal therapy is an emerging tumor treatment method with great potential in the field of targeted photothermal therapy. Mild photothermal therapy has significant advantages over traditional high-temperature photothermal therapy, which is usually a therapy with a temperature above 50°C. Although high-temperature photothermal therapy can effectively kill cancer cells, it may also cause damage to surrounding healthy tissues, leading to treatment side effects and complications. Mild photothermal therapy can avoid these side effects while regulating the photothermal conversion performance and targeting of photothermal probes to achieve precise treatment of tumor tissues within a lower temperature range, usually below 46°C. MXene is an emerging type of two-dimensional material, composed of transition metal carbides, nitrides or carbonitrides, with a unique layered structure and excellent physical and chemical properties; MXene materials have high electrical and thermal conductivity, good hydrophilicity and modifiability, high photothermal conversion efficiency, biocompatibility and degradability, and are widely used in drug delivery, bioimaging, diagnosis and treatment, etc.; By adjusting the size, morphology and surface properties of MXene materials, their photothermal conversion efficiency and targeting can be regulated, thereby achieving precise control of the photothermal therapy process; the high photothermal conversion efficiency of MXene materials means that the temperature required for treatment can be achieved at a lower light intensity, which helps to reduce damage to surrounding healthy tissues; in addition, through surface modification or combination with other molecules, MXene materials can achieve precise targeting of tumor tissues; under the irradiation of external light sources, these targeted aggregated MXene materials can generate local high temperatures, thereby effectively killing cancer cells; The nucleolus is an important structure in the cell nucleus, which is mainly responsible for the synthesis of ribosomal RNA (rRNA) and the assembly of ribosomal subunits. Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are important nucleotides in the cell, playing a key role as energy molecules and signaling molecules. In the nucleolar stress response, GTP and its related metabolism and signaling pathways may play an important role. Therefore, detecting changes in ATP and GTP content in living cells plays an important role in the diagnosis and treatment of tumor cells. Existing nanoprobes have poor specificity. Most of them focus on how to image ATP in cells. There are very few reports on simultaneous imaging of ATP and GTP in living cells. The photothermal effect of thermal killing of tumor cells needs to be improved. They have high cytotoxicity and damage to normal cells. They have low yield, high cost, and are difficult to operate. In summary, how to design a photothermal probe that can achieve live cell imaging, actively target tumor cells specifically, improve the thermal killing ability of tumor cells, and reduce damage to surrounding normal tissues during mild photothermal therapy, while also being able to specifically detect changes in the content of ATP and GTP molecules and improve the diagnostic and therapeutic effect of photothermal therapy on tumor cells, has become an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to provide a MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer and a preparation method and application thereof to solve the above problems.

[0004] In order to achieve the above objectives, the following technical solutions are provided: A MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers, wherein the MXene used is Ti 3 C 2 T x , the Ti 3 C 2 T x The surface is modified with nucleic acid aptamers; The nucleic acid aptamer is composed of ATP nucleic acid aptamer and GTP nucleic acid aptamer; the ATP nucleic acid aptamer is a DNA fragment, the 5' end of which carries a fluorescent marker peptide FAM, and the nucleotide sequence is shown in SEQ ID No: 1; the GTP nucleic acid aptamer is an RNA fragment, the 5' end of which carries a fluorescent dye anthocyanin Cy3, and the nucleotide sequence is shown in SEQ ID No: 2.

[0005] Preferably, the photothermal probe is Ti 3 C 2 T x The single-layer nanosheet is prepared, and the Ti 3 C 2 T x The thickness of a single-layer nanosheet is 1.7-1.9 nm.

[0006] The present invention also provides a method for preparing the above-mentioned MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, comprising the following steps: S1. Preparation of Ti 3 C 2 T x Single-layer nanosheets: 0.8 g of lithium fluoride powder was added to 10 mL of 9 M HCl and stirred slowly in a constant temperature water bath for 10 min. After the LiF powder was completely dissolved, 0.5 g of 200 mesh black Ti 3 AlC 2The powder was stirred at 500 rpm and the reaction lasted for 48 h at room temperature. Deionized water was used for multiple cycles of centrifugal washing at 5000 rpm. Each cycle lasted for 2 min. When the pH value reached 6, argon was introduced into the solution. Ultrasonic peeling was performed in an ice-water bath for 60 min to obtain a single layer of Ti 3 C 2 T x , centrifuged at 3500 rpm for 60 min to remove the multilayer Ti 3 C 2 T x Nanosheets, collecting Ti 3 C 2 T x The dark supernatant of the monolayer nanosheets was stored at 4°C with argon gas for later use; S2, carboxyl modified Ti 3 C 2 T x Single-layer nanosheets: Prepare 1 mg / mL of Ti-containing 3 C 2 T x Take 50 mL of the monolayer nanosheet solution, ultrasonicate in an ice bath under argon for 1 h to make the solution uniformly dispersed, add 1.25 g NaOH, stir at 60 ° C for 2 h, add 0.625 g NaOH and 3 g chloroacetic acid, continue stirring for 3 h, collect the solution, centrifuge with deionized water at 8000 rpm, single 15 min cycle centrifugation until neutral, collect the solution, and obtain Ti-containing 3 C 2 T x -COOH solution, stored at 4°C for future use; S3, modification of FA-targeted amino Ti 3 C 2 T x -PEG Nanosheets: Prepare 1 mg / mL of Ti prepared in step S2 3 C 2 T x -COOH solution, take 50 mL, add 37.5 mg EDC and 56.25 mg NHS in sequence, continue stirring for 2 h, add 50 mg FA-PEG-NH with a molecular weight of 2000 Da 2 The reaction was continued for 24 hours, and the temperature was lowered to room temperature. The solution was centrifuged at 8000 rpm for 15 minutes, the supernatant was removed, and the solution was placed in a dialyzer with a molecular weight cutoff of 3000 Da. The solution was dialyzed at room temperature for 48 hours, and the water was changed every 2 hours to obtain Ti with carboxyl-amino modification. 3 C 2 T x The solution of nanosheets is denoted as FA-Ti 3C 2 T x -PEG solution, stored at 4℃ for future use; S4. Assemble Ti 3 C 2 T x -ATP-GTP photothermal probe: First, 100 nM ATP aptamer and GTP aptamer were mixed with 1 mg / mL FA-Ti 3 C 2 T x -PEG solution, shaken and stirred at 4 ° C overnight, and centrifuged at 9500 rpm for 15 min with HEPES buffer to obtain a MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, denoted as Ti 3 C 2 T x -ATP-GTP, stored at 4℃ until use.

[0007] Preferably, the temperature of the constant temperature water bath in S1 is 40-45°C.

[0008] Preferably, all the reaction processes in S3 are carried out in an oil bath at 60-65° C. and with argon gas.

[0009] The present invention also provides the use of the above-mentioned MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers or the MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers prepared by the above-mentioned preparation method in the field of preparing photothermal agents.

[0010] The beneficial effects of the present invention are: 1. The present invention improves the photothermal conversion efficiency of the photothermal agent, reduces the laser irradiation temperature, and solves the current problem of photothermal therapy causing damage to surrounding normal tissues; through dual active targeting: the first level of targeting: active targeting of tumor cells; FA ligands and FR receptors highly expressed on the surface of tumor cell membranes are specifically recognized; increasing tumor cell Ti 3 C 2 T x -Active uptake of ATP-GTP, entering the cell through endocytosis; Second targeting: Active targeting of intracellular ATP and GTP energy molecules; 3 C 2 T x The surface is modified with nucleic acid aptamers, so that the photothermal agent can quickly recognize ATP / GTP molecules after entering the tumor cells; at the same time, the metabolic changes of ATP and GTP molecules in single living cells are monitored in real time, realizing an integrated diagnosis and treatment fluorescent nanoplatform with photothermal response.

[0011] 2. The photothermal nanoprobe constructed by the present invention utilizes the nanomaterial Ti with high photothermal conversion efficiency 3 C 2 T x Through the improved preparation method, Ti 3 C 2 T x The size is controlled at about 100nm, and the photothermal conversion efficiency is increased to 34.6% through carboxyl-amino modification, which is 13.6% higher than the 21% of the commonly used gold nanorods; 3. Ti 3 C 2 T x The size of Ti is the main reason for its high cytotoxicity. The present invention improves the preparation method of Ti 3 C 2 T x The size is controlled at about 100 nm; on the other hand, the surface chemical modification of polyethylene glycol is used to improve the biocompatibility of nanomaterials. 3 C 2 T x Modified FA-NH 2 -PEG, which not only improves the biocompatibility of nanomaterials, but also increases the endocytosis of nanomaterials by tumor cells; 4. The present invention can be applied to mild photothermal therapy, and by regulating the photothermal conversion performance and targeting of the photothermal agent, precise treatment of tumor tissue can be achieved within a relatively low temperature range, usually below 46°C; 5. Ti constructed and synthesized by the present invention 3 C 2 T x -ATP-GTP photothermal nanoprobe with simple operation, low material cost and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Ti 3 AlC 2 SEM image of ceramic block, scale bar = 1 μm; Figure 2 Ti 3 C 2 T x SEM image of multilayered lamellar structure, scale bar = 5 μm; Figure 3 Ti 3 C 2 T x SEM image of a single-layer nanosheet, scale bar = 300 nm; Figure 4 Ti 3 C 2 T xHRTEM image of a single-layer nanosheet, scale bar = 200 nm; Figure 5 Ti 3 C 2 T x HRTEM image of the lattice structure of a single-layer nanosheet, scale bar = 5 nm; Figure 6 Ti 3 C 2 T x AFM image of a single nanosheet, scale bar = 100 nm; Figure 7 Ti 3 C 2 T x Elemental mapping image of a single-layer nanosheet, scale bar = 200 nm; Figure 8 Ti 3 AlC 2 and Ti 3 C 2 T x XRD analysis diagram of Fig. 9 Ti 3 C 2 T x and Ti 3 C 2 T x -UV absorption spectrum of ATP-GTP; Fig.10 Ti 3 C 2 T x and Ti 3 C 2 T x -Surface charge change diagram of ATP-GTP; Fig.11 Ti 3 C 2 T x -Elemental mapping image of ATP-GTP photothermal probe, scale bar = 200 nm; Fig.12 Ti 3 C 2 T x Ti2p binding energy spectrum of a single-layer nanosheet; Fig.13 Ti 3 C 2 T x and Ti 3 C 2 T x -Overall spectrum of surface chemical composition and valence state analysis of ATP-GTP; Fig.14 Ti 3 C 2 T x - Fluorescence quenching performance and optimal concentration of ATP-GTP photothermal probe; Fig.15 Ti 3 C 2 T x -Response diagram of ATP-GTP photothermal probe to ATP and GTP in solution; Fig.16 Ti 3 C 2 T x -Specific response diagram of ATP-GTP photothermal probe; Fig.17 To Ti 3 C 2 T x With Ti 3 C 2 T x - ATP-GTP stability performance diagram in different solutes; Fig.18 Ti 3 C 2 T x - Live cell imaging performance of ATP-GTP photothermal probe, scale bar = 25 μm; Fig.19 Ti 3 C 2 T x -UV-visible spectra of different concentrations of ATP-GTP photothermal probe; Fig. 20 Ti 3 C 2 T x -Extinction coefficient diagram of ATP-GTP photothermal probe; Fig.21 Ti 3 C 2 T x -Photothermal conversion efficiency diagram of ATP-GTP photothermal probe; Fig. 22 Ti 3 C 2 T x -Photothermal temperature change test diagram of different concentrations of ATP-GTP photothermal probe; Fig.23 Ti 3 C 2 T x -Photothermal temperature change test diagram of ATP-GTP photothermal probe at different laser powers; Fig.24 Ti 3 C 2 T x -Photothermal temperature cycle test diagram of ATP-GTP photothermal probe; Fig.25 Ti 3 C 2 T x -UV spectrum of continuous photothermal irradiation of ATP-GTP photothermal probe; Fig.26 For different concentrations of Ti 3 C 2 T x -The effect of ATP-GTP on the cell viability of MCF-7 and MCF-10A cells after 24h incubation; Fig. 27 MCF-7 and MCF-10A cells with Ti 3 C 2 T x After 4 h of incubation with ATP-GTP, the cells were irradiated with 808 nm laser (1 W cm -2 ) of the cell viability graph; Fig.28 808nm laser irradiation (1Wcm -2 ) AM / PI images of MCF-7 and MCF-10A cells, scale bar = 100 μm; Fig.29 MCF-7 and MCF-10A cells uptake Ti 3 C 2 T x -ICP quantitative test chart of ATP-GTP; Fig.30 Ti 3 C 2 T x -ATP-GTP photothermal probe was co-incubated with MCF-7 and MCF-10A cells under 808 nm near-infrared light (1 W cm -2 ) photothermal images at different time intervals under illumination; Fig.31 Ti 3 C 2 T x -ATP-GTP photothermal probe was co-incubated with MCF-7 and MCF-10A cells under 808 nm near-infrared light (1 W cm -2 ) Fluorescence images of cells at different time intervals under illumination, scale bar = 100 μm. DETAILED DESCRIPTION

[0013] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the schematic diagrams in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] like Figure 1-31 As shown, A MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers, wherein the MXene used is Ti 3 C 2 T x , the Ti 3 C 2 T x The surface is modified with nucleic acid aptamers; The nucleic acid aptamer is composed of an ATP nucleic acid aptamer and a GTP nucleic acid aptamer; the ATP nucleic acid aptamer is a DNA fragment, the 5' end of which carries a fluorescent labeled peptide FAM, and the nucleotide sequence is shown in SEQ ID No: 1; SEQ ID No: 1: AAC CTG GGG GAG TAT TGC GGA GGA AGG T; The GTP nucleic acid aptamer is an RNA fragment, the 5' end of which carries the fluorescent dye anthocyanin Cy3, and the nucleotide sequence is shown in SEQ ID No: 2; SEQ ID No: 2: GGG ACG AAG UGG UUG GGU GUG AAA ACG UCC C; In some embodiments, the photothermal probe is Ti 3 C 2 T x The single-layer nanosheet is prepared, and the Ti 3 C 2 T x The thickness of a single nanosheet is 1.7-1.9 nm; The present invention also provides a method for preparing the above-mentioned MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, comprising the following steps: S1. Preparation of Ti 3 C 2 T x Single-layer nanosheets: 0.8 g of lithium fluoride powder was added to 10 mL of 9 M HCl and stirred slowly in a constant temperature water bath for 10 min. After the LiF powder was completely dissolved, 0.5 g of 200 mesh black Ti 3 AlC2 The powder was stirred at 500 rpm and reacted at room temperature for 48 h. Deionized water was used for multiple centrifugal washing at 5000 rpm. Each cycle lasted for 2 min until the pH value reached 6. At this time, the Ti in the solution 3 C 2 T x Automatic stratification occurred, argon was introduced into the solution, and ultrasonic stripping was performed in an ice-water bath for 60 min to obtain a single layer of Ti 3 C 2 T x , centrifuged at 3500 rpm for 60 min to remove the multilayer Ti 3 C 2 T x Nanosheets, collecting Ti 3 C 2 T x The dark supernatant of the monolayer nanosheets was stored at 4°C with argon gas for later use; S2, carboxyl modified Ti 3 C 2 T x Single-layer nanosheets: Prepare 1 mg / mL of Ti-containing 3 C 2 T x Take 50 mL of the monolayer nanosheet solution, ultrasonicate in an ice bath under argon for 1 h to make the solution uniformly dispersed, add 1.25 g NaOH, stir at 60 ° C for 2 h, add 0.625 g NaOH and 3 g chloroacetic acid, continue stirring for 3 h, collect the solution, centrifuge with deionized water at 8000 rpm, single 15 min cycle centrifugation until neutral, collect the solution, and obtain Ti-containing 3 C 2 T x -COOH solution, stored at 4℃ for later use; the purpose of this step is to make Ti 3 C 2 T x Nanosheets are carboxylated to reduce -F end groups and increase -OH end groups, and finally synthesize Ti 3 C 2 T x -COOH; S3, modification of FA-targeted amino Ti 3 C 2 T x -PEG Nanosheets: Prepare 1 mg / mL of Ti prepared in step S2 3 C 2 T x-COOH solution, take 50 mL, add 37.5 mg EDC and 56.25 mg NHS in sequence, continue stirring for 2 h, add 50 mg FA-PEG-NH with a molecular weight of 2000 Da 2 The reaction was continued for 24 hours, and the temperature was lowered to room temperature. The solution was centrifuged at 8000 rpm for 15 minutes, the supernatant was removed, and the solution was placed in a dialyzer with a molecular weight cutoff of 3000 Da. The solution was dialyzed at room temperature for 48 hours, and the water was changed every 2 hours to obtain Ti with carboxyl-amino modification. 3 C 2 T x The solution of nanosheets is denoted as FA-Ti 3 C 2 T x -PEG solution, stored at 4℃ for future use; S4. Assemble Ti 3 C 2 T x -ATP-GTP photothermal probe: First, 100 nM ATP aptamer and GTP aptamer were mixed with 1 mg / mL FA-Ti 3 C 2 T x -PEG solution, shaken and stirred at 4 ° C overnight, and centrifuged at 9500 rpm for 15 min with HEPES buffer to obtain a MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, denoted as Ti 3 C 2 T x -ATP-GTP, stored at 4℃ for future use; In some embodiments, the constant temperature water bath temperature in S1 is 40-45°C; In some embodiments, all the reaction processes in S3 need to be carried out in an oil bath at 60-65° C. and with argon gas; The present invention also provides the use of the above-mentioned MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers or the MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers prepared by the above-mentioned preparation method in the field of preparing photothermal agents; The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention; Example 1: This example provides a MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer, and the preparation method thereof is as follows: S1. Preparation of Ti 3 C 2 T x Single-layer nanosheets: 0.8 g of lithium fluoride powder was added to 10 mL of 9 M HCl and stirred slowly in a constant temperature water bath at 40 °C for 10 min. After the LiF powder was completely dissolved, 0.5 g of 200 mesh black Ti 3 AlC 2 The powder was stirred at 500 rpm and the reaction lasted for 48 h at room temperature. Deionized water was used for multiple cycles of centrifugal washing at 5000 rpm. Each cycle lasted for 2 min. When the pH value reached 6, argon was introduced into the solution. Ultrasonic peeling was performed in an ice-water bath for 60 min to obtain a single layer of Ti 3 C 2 T x , centrifuged at 3500 rpm for 60 min to remove the multilayer Ti 3 C 2 T x Nanosheets, collecting Ti 3 C 2 T x The dark supernatant of the monolayer nanosheets was stored at 4°C with argon gas for later use; S2, carboxyl modified Ti 3 C 2 T x Single-layer nanosheets: Prepare 1 mg / mL of Ti-containing 3 C 2 T x Take 50 mL of the monolayer nanosheet solution, ultrasonicate in an ice bath under argon for 1 h to make the solution uniformly dispersed, add 1.25 g NaOH, stir at 60 ° C for 2 h, add 0.625 g NaOH and 3 g chloroacetic acid, continue stirring for 3 h, collect the solution, centrifuge with deionized water at 8000 rpm, single 15 min cycle centrifugation until neutral, collect the solution, and obtain Ti-containing 3 C 2 T x -COOH solution, stored at 4°C for future use; S3, modification of FA-targeted amino Ti 3 C 2 T x -PEG Nanosheets: Prepare 1 mg / mL of Ti prepared in step S2 3 C 2 T x-COOH solution, take 50 mL, add 37.5 mg EDC and 56.25 mg NHS in sequence, continue stirring for 2 h, add 50 mg FA-PEG-NH with a molecular weight of 2000 Da 2 The reaction was continued for 24 hours, and the temperature was lowered to room temperature. The solution was centrifuged at 8000 rpm for 15 minutes, the supernatant was removed, and the solution was placed in a dialyzer with a molecular weight cutoff of 3000 Da. The solution was dialyzed at room temperature for 48 hours, and the water was changed every 2 hours to obtain Ti with carboxyl-amino modification. 3 C 2 T x The solution of nanosheets is denoted as FA-Ti 3 C 2 T x -PEG solution, stored at 4°C for future use; all reactions in this step must be carried out in an oil bath at 60°C and with argon gas; S4. Assemble Ti 3 C 2 T x -ATP-GTP photothermal probe: First, 100 nM ATP aptamer and GTP aptamer were mixed with 1 mg / mL FA-Ti 3 C 2 T x -PEG solution, shaken and stirred at 4 ° C overnight, and centrifuged at 9500 rpm for 15 min with HEPES buffer to obtain a MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, denoted as Ti 3 C 2 T x -ATP-GTP, stored at 4℃ until use.

[0015] Example 2: This example provides a MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer, and the preparation method thereof is as follows: S1. Preparation of Ti 3 C 2 T x Single-layer nanosheets: 0.8 g of lithium fluoride powder was added to 10 mL of 9 M HCl and stirred slowly in a constant temperature water bath at 45 °C for 10 min. After the LiF powder was completely dissolved, 0.5 g of 200 mesh black Ti 3 AlC 2 The powder was stirred at 500 rpm and the reaction lasted for 48 h at room temperature. Deionized water was used for multiple cycles of centrifugal washing at 5000 rpm. Each cycle lasted for 2 min. When the pH value reached 6, argon was introduced into the solution. Ultrasonic peeling was performed in an ice-water bath for 60 min to obtain a single layer of Ti 3 C2 T x , centrifuged at 3500 rpm for 60 min to remove the multilayer Ti 3 C 2 T x Nanosheets, collecting Ti 3 C 2 T x The dark supernatant of the monolayer nanosheets was stored at 4°C with argon gas for later use; S2, carboxyl modified Ti 3 C 2 T x Single-layer nanosheets: Prepare 1 mg / mL of Ti-containing 3 C 2 T x Take 50 mL of the monolayer nanosheet solution, ultrasonicate in an ice bath under argon for 1 h to make the solution uniformly dispersed, add 1.25 g NaOH, stir at 60 ° C for 2 h, add 0.625 g NaOH and 3 g chloroacetic acid, continue stirring for 3 h, collect the solution, centrifuge with deionized water at 8000 rpm, single 15 min cycle centrifugation until neutral, collect the solution, and obtain Ti-containing 3 C 2 T x -COOH solution, stored at 4°C for future use; S3, modification of FA-targeted amino Ti 3 C 2 T x -PEG Nanosheets: Prepare 1 mg / mL of Ti prepared in step S2 3 C 2 T x -COOH solution, take 50 mL, add 37.5 mg EDC and 56.25 mg NHS in sequence, continue stirring for 2 h, add 50 mg FA-PEG-NH with a molecular weight of 2000 Da 2 The reaction was continued for 24 hours, and the temperature was lowered to room temperature. The solution was centrifuged at 8000 rpm for 15 minutes, the supernatant was removed, and the solution was placed in a dialyzer with a molecular weight cutoff of 3000 Da. The solution was dialyzed at room temperature for 48 hours, and the water was changed every 2 hours to obtain Ti with carboxyl-amino modification. 3 C 2 T x The solution of nanosheets is denoted as FA-Ti 3 C 2 T x -PEG solution, stored at 4°C for future use; all reactions in this step must be carried out in an oil bath at 65°C with argon gas; S4. Assemble Ti 3 C2 T x -ATP-GTP photothermal probe: First, 100 nM ATP aptamer and GTP aptamer were mixed with 1 mg / mL FA-Ti 3 C 2 T x -PEG solution, shaken and stirred at 4 ° C overnight, and centrifuged at 9500 rpm for 15 min with HEPES buffer to obtain a MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, denoted as Ti 3 C 2 T x -ATP-GTP, stored at 4℃ until use.

[0016] Performance Testing: Ti 3 C 2 T x and Ti 3 C 2 T x -Morphological characterization performance test of ATP-GTP: like Figure 1 As shown, Ti 3 AlC 2 SEM image of the ceramic block. The image shows that the Ti 3 AlC 2 It presents a solid block structure; like Figure 2 As shown, Ti 3 C 2 T x SEM image of multilayered sheet structure; HF is generated in situ by using a mixed solution of LiF and HCl as an etchant to etch Ti 3 AlC 2 The Al layer in the 3 C 2 T x Multilayer sheet structure; This is based on Ti 3 C 2 T x The hydrogen ions in the multilayer structure exchange and intercalate with the solvated lithium ions, resulting in an increase in the interlayer distance; like Figure 3 As shown, Ti 3 C 2 T x SEM image of single-layer nanosheets; Ti was further exfoliated by improved ultrasonic liquid phase exfoliation 3 C 2 T x Further liquid phase ultrasonic peeling was performed to obtain Ti3 C 2 T x Monolayer nanosheets; the above morphological changes in the microstructure directly confirm that Ti 3 C 2 T x The single-layer nanosheet was successfully prepared, and the unpeeled Ti 3 AlC 2 The morphology of the ceramic block structure is obviously different; like Figure 4 As shown, Ti 3 C 2 T x HRTEM image of a single-layer nanosheet; the prepared Ti 3 C 2 T x The single-layer nanosheets have a typical single-layer ultrathin transparent structure with a clear surface and an average size of 85.543 ± 14.933 nm; like Figure 5 As shown, Ti 3 C 2 T x HRTEM image of the lattice structure of a single-layer nanosheet; the image clearly shows the Ti 3 C 2 T x The typical lattice fringes of a single-layer nanosheet have a lattice spacing of 0.272 nm, which directly corresponds to the Ti 3 C 2 T x The (110) plane of TiO2 shows its single crystal characteristics; the SAED image clearly shows six diffraction spots of folded symmetric structure, which further proves that Ti 3 C 2 T x The atomic arrangement has typical hexagonal structural characteristics and good crystal properties, and no obvious oxidation damage occurs; like Figure 6 As shown, Ti 3 C 2 T x AFM image of a single nanosheet; the image shows Ti 3 C 2 T x The thickness of a single nanosheet is about 1.8 nm; like Figure 7 As shown, Ti 3 C 2 T x Elemental mapping image of a single-layer nanosheet; HRTEM imaging and corresponding elemental mapping images show that Ti, C, and O elements are uniformly distributed in the Ti 3 C 2 T xOn a single nanosheet; like Figure 8 As shown, Ti 3 AlC 2 and Ti 3 C 2 T x XRD analysis; XRD results show that Ti 3 AlC 2 Structural changes before and after chemical etching: Ti 3 AlC 2 The characteristic peaks at 2θ≈9.5°(002) and 39.0°(104) are consistent with the standard card (JCPDSPDF#52-0875); after Al atoms are etched, the (002) peak representing the interlayer structure shifts from 9.5° to 7°, confirming that Ti 3 C 2 T x The formation of a multilayer structure; subsequently, after ultrasonic peeling, Ti 3 AlC 2 The strongest diffraction peak at 2θ≈39.0°(104) is greatly reduced, and most of the characteristic peaks of the (101), (103), (105) and (110) planes disappear, with only an obvious (002) peak at 6.45°, further confirming that Ti 3 C 2 T x The single-layer nanosheet was successfully prepared; Ti was calculated according to Bragg's law (2dsinθ=nλ). 3 C 2 T x The interlayer spacing of the monolayer is 13.7 Å; like Fig. 9 As shown, Ti 3 C 2 T x and Ti 3 C 2 T x -UV absorption spectrum of ATP-GTP; due to Ti 3 C 2 T x With LSPR effect, the UV-visible absorption spectrum is used to study the Ti 3 C 2 T x The optical properties of Ti 3 C 2 T x There are two obvious absorption peaks at 762nm and near-ultraviolet region; in addition, the red shift of the UV spectrum to 806nm shows that the FA-PEG-NH 2and ATP Aptamer and GTP Aptamer for Ti 3 C 2 The surface of Tx is successfully modified and is denoted as Ti from now on 3 C 2 -ATP-GTP; like Fig.10 As shown, Ti 3 C 2 T x and Ti 3 C 2 T x -Changes in the surface charge of ATP-GTP; using FA-PEG-NH 2 Modified Ti 3 C 2 T x After that, better biocompatibility was obtained, and the corresponding Zeta potential dropped to -19.93 mV, which means that Ti 3 C 2 T x The negative charges on the surface are partially removed by FA-PEG-NH 2 After further coupling with ATP Aptamer and GTP Aptamer, the zeta potential changed from -19.93 mV to -26.13 mV, indicating that GTP Aptamer was successfully loaded into Ti 3 C 2 T x ; like Fig.11 As shown, Ti 3 C 2 T x -Elemental mapping image of ATP-GTP nanoprobe. Element distribution mapping confirmed the distribution of Ti, N, and P elements in Ti 3 C 2 T x -ATP-GTP surface, indicating the successful preparation of Ti 3 C 2 T x -ATP-GTP fluorescent nanoprobe; like Fig.12 As shown, Ti 3 C 2 T x Ti2p binding energy spectrum of a single nanosheet; XPS analysis shows that 454.3 and 460.5 eV correspond to Ti-C bonds; the peaks at 455.99 and 461.53 eV are mainly attributed to Ti 2+ , while the peaks at 456.94 and 462.4 eV are attributed to Ti 3+, Ti-O (2p3 / 2) is located at 458.55 eV, and Ti–O (2p1 / 2) is located at 464.04 eV; in addition, the Ti2p binding spectrum does not show a peak at 488.8 eV, indicating that Ti 3 C 2 T x The monolayer nanosheets did not undergo oxidation; like Fig.13 As shown, Ti 3 C 2 T x and Ti 3 C 2 T x -Analysis of the surface chemical composition and valence state of ATP / GTP; 3 C 2 T x -ATP / GTP showed characteristic peaks of C1s, Ti2p, O1s, F1s, N1s and P2p in the XPS measurement range (0-800 eV), while in Ti 3 C 2 T x The XPS measurement range of does not show the characteristic peaks of N1s and P2p; with the progress of modification, the binding energy of other elements also changes slightly, Ti 3 C 2 T x -It can be observed from the XPS spectrum of ATP / GTP that as the modification proceeds, the N1s binding energy spectrum appears in the binding energy spectrum; Ti 3 C 2 T x -In vitro fluorescence performance test of ATP / GTP photothermal probe: Before verifying the fluorescence performance, Ti 3 C 2 T x - Fluorescence quenching performance and optimal concentration of ATP / GTP photothermal probe; like Fig.14 As shown, Ti 3 C 2 T x -The fluorescence quenching performance and optimal concentration of ATP / GTP photothermal probe; With the increase of Ti 3 C 2 T x -As the concentration of ATP / GTP increases, the fluorescence intensity from the FAM and Cy3 channels decreases, which is due to the Ti 3 C 2 T x FRET effect between Ti and fluorophore; 3 C 2T x -ATP / GTP concentration reaches 30 μg mL -1 When 3 C 2 T x -ATP / GTP fluorescence intensity is basically quenched; the results show that Ti 3 C 2 T x The large planar surface area of ​​the nanosheets increases the possibility of adsorbing multiple aptamers; therefore, 30 μg mL -1 Concentration of Ti 3 C 2 T x -ATP / GTP as the optimal concentration for simultaneous detection of ATP and GTP; like Fig.15 As shown, Ti 3 C 2 T x -ATP / GTP photothermal probe responds to ATP and GTP in solution; the fluorescence intensity increases with the increase of ATP and GTP concentrations, from 0 to 2.5 mM; this is due to the interaction of ATP and GTP with Ti 3 C 2 The weak binding ability of the assembly keeps the fluorophore away from the quencher surface, resulting in the fluorescence recovery of FAM or Cy3; like Fig.16 As shown, Ti 3 C 2 T x -Specific response of ATP / GTP photothermal probe; Due to the specificity of nucleic acid aptamers to ATP and GTP, nucleotide analogs including UTP, CTP and GTP / ATP were added; In the presence of ATP or GTP, it can be specifically recognized by ATP Aptamer or GTP Aptamer, while other nucleotide analogs did not show significant fluorescence intensity enhancement; The results showed that Ti 3 C 2 T x -ATP / GTP is highly specific for ATP and GTP in solution and the interaction results in the selective release of ATP Aptamer or GTP Aptamer; like Fig.17 As shown, test Ti 3 C 2 T x With Ti 3 C 2 T x - Stability of ATP / GTP in different solutes; placed in different solutes for 0-7 days; Ti 3 C2 T x It can be well dispersed in aqueous solution, showing a typical colloidal Tyndall effect, proving that Ti 3 C 2 T x It has excellent hydrophilicity and good dispersibility; Ti 3 C 2 T x -ATP / GTP has good stability in Water, DMEM, MCF-10A, and Cell specialized medium, and there is no aggregation phenomenon, which indicates that Ti 3 C 2 T x FA-PEG-NH 2 The biocompatibility was significantly improved after modification with nucleic acid aptamers; like Fig.18 As shown, Ti 3 C 2 T x -The live cell imaging performance of ATP / GTP nanoprobes; these observations successfully facilitated the detection of ATP / GTP in living cells in subsequent experiments; 3 C 2 T x -ATP / GTP and Ti 3 C 2 T x -random nanoprobes were incubated with MCF-7 cells for 4 hours; Ti 3 C 2 T x -random nanoprobes are composed of DNA randomsquence, RNA randomsquence and Ti 3 C 2 T x -PEG assembly; Ti alone 3 C 2 T x-ATP / GTP nanoprobe treated cells as the control group; the control group clearly showed the green fluorescence channel signal of FAM-labeled ATP aptamer and the red fluorescence channel signal of Cy3-labeled GTP Aptamer; when the FAM-labeled ATP Aptamer and Cy3-labeled GTP Aptamer of the nanoprobe were replaced by random sequences, no fluorescence signal was observed in living cells; in addition, after laser irradiation of the cell sample for 6 minutes, the fluorescence signals of the green fluorescence channel of FAM-labeled ATP Aptamer and the red fluorescence channel of Cy3-labeled GTP Aptamer were enhanced compared with the control group; these results indicate that Ti 3 C 2 T x -ATP-GTP nanoprobe can serve as an excellent nanosensing platform to simultaneously visualize ATP and GTP in living cells; Ti 3 C 2 T x -Photothermal performance test of ATP / GTP photothermal probe: Ti 3 C 2 T x Similar to gold nanorods, it has the localized surface plasmon resonance (LSPR) effect and good photothermal conversion ability, making it an ideal material for photothermal agents; like Fig.19 As shown, Ti 3 C 2 T x -UV-visible spectra of different concentrations of ATP-GTP nanoprobe; Ti 3 C 2 T x -ATP-GTP nanoprobe has a characteristic absorption peak around 808nm in the NIR-I window and shows good concentration dependence; like Fig. 20 As shown, Ti 3 C 2 T x -ATP-GTP nanoprobe extinction coefficient; Ti was calculated according to the Lambert-Beer law 3 C 2 T x -The extinction coefficient of ATP-GTP is 28.79Lg -1 cm -1 ; like Fig.21 As shown, Ti 3 C 2 Tx -The photothermal conversion efficiency of ATP-GTP nanoprobes; Ti was calculated by the photothermal conversion efficiency formula 3 C 2 T x -The photothermal conversion efficiency of the ATP-GTP nanoprobe is 34.6%, significantly higher than that of gold nanorods (21%); like Fig. 22 As shown, Ti 3 C 2 T x -Photothermal temperature change test of different concentrations of ATP-GTP nanoprobes; To further evaluate the photothermal performance, Ti was tested by 808nm near-infrared light irradiation. 3 C 2 T x -ATP-GTP solution temperature change; under the same conditions, 808nm near-infrared light (1Wcm -2 ) After 10 min of irradiation, Ti 3 C 2 T x -As the concentration of the ATP-GTP nanoprobe solution increased, the temperature curve showed an upward trend; when the concentration increased to 120 μg mL -1 When 3 C 2 T x -ATP-GTP nanoprobe has the highest temperature (59.1℃) after irradiation with 808nm near-infrared light, indicating that Ti 3 C 2 T x -ATP-GTP nanoprobe has concentration-dependent photothermal properties; like Fig.23 As shown, Ti 3 C 2 T x -Photothermal temperature change test of ATP-GTP nanoprobe at different laser powers; then, the Ti 3 C 2 T x -ATP-GTP nanoprobe (30 μg mL -1 ) Temperature changes of 808nm near-infrared light irradiated with different power densities; As the laser power of 808nm near-infrared light increases, Ti 3 C 2 T x -ATP-GTP nanoprobe temperature rises rapidly; under 808nm near-infrared light (1.5Wcm -2 ) under the irradiation of Ti 3 C 2 T xThe final temperature of the ATP-GTP nanoprobe was 53.5 °C, and the results showed that Ti 3 C 2 T x -ATP-GTP nanoprobe has good near-infrared light response characteristics and shows power dependence; like Fig.24 As shown, Ti 3 C 2 T x -Photothermal temperature cycle test of ATP-GTP nanoprobe; after 4 repeated heating and cooling cycles, the results showed that Ti 3 C 2 T x -ATP-GTP nanoprobe (808nm near-infrared light power = 1Wcm -2 )’s temperature distribution has no obvious change; like Fig.25 As shown, Ti 3 C 2 T x -ATP-GTP nanoprobes were irradiated with continuous photothermal irradiation. After continuous irradiation with 808nm near-infrared light for 1h, the UV-vis spectrum did not change significantly. The above results indicate that Ti 3 C 2 T x -ATP-GTP nanoprobe has good photothermal conversion efficiency and good photothermal stability; Ti 3 C 2 T x -Testing of cell biocompatibility and in vitro photothermal therapy effect of ATP-GTP nanoprobes: Ti was assessed by standard MTT assay. 3 C 2 T x - Biocompatibility and in vitro photothermal therapeutic effect of ATP-GTP nanoprobes; like Fig.26 As shown, different concentrations of Ti 3 C 2 T x -ATP-GTP affects the cell viability of MCF-7 and MCF-10A cells after 24 h of incubation; Ti 3 C 2 T x -Cytotoxicity of ATP-GTP nanoprobe; Ti 3 C 2 T x -ATP-GTP nanoprobes showed negligible cytotoxicity in MCF-7 and MCF-10A cells; this result suggests that Ti3 C 2 T x -ATP-GTP nanoprobes showed excellent biocompatibility; in order to increase cellular endocytosis, in subsequent experiments, Ti 3 C 2 T x -The concentration of ATP-GTP nanoprobe was set to 30 μg mL -1 ; like Fig. 27 As shown, MCF-7 and MCF-10A cells were treated with Ti 3 C 2 T x After 4 h of incubation with ATP-GTP, the cells were irradiated with 808 nm near-infrared light (1 W cm -2 ) of the cells; Ti 3 C 2 T x -ATP-GTP nanoprobes were incubated with MCF-7 and MCF-10A cells for 4 hours and then illuminated with 808 nm near-infrared light (1 W cm -2 ) irradiated at different time intervals; with the extension of 808nm near-infrared light irradiation time, the cell viability of cancerous MCF-7 cells decreased significantly, while normal MCF-10A cells were still able to maintain good cell viability; like Fig.28 As shown, 808nm near-infrared light irradiation (1Wcm -2 ) after MCF-7 and MCF-10A cells were imaged; then, the Ti was verified by Calcein-AM and Propidium Iodide Staining Kit (cell live / dead assay). 3 C 2 T x -The photothermal effect of ATP-GTP nanoprobe on cells; 808nm near-infrared light (1Wcm -2 ) for 6 minutes, about half of the cancerous MCF-7 cells died, and almost all of them died after 9 minutes. In contrast, 808nm near-infrared light (1Wcm -2 ) for 12 minutes, only a portion of normal MCF-10A cells died; therefore, 808 nm near-infrared light (1 W cm -2 ) laser intensity was used in subsequent experiments; these results show that Ti 3 C 2 T x -ATP-GTP nanoprobes have good photothermal properties and the ability to specifically kill tumor cells, and have good application prospects in the research field of mPTT; like Fig.29 As shown, MCF-7 and MCF-10A cells uptake Ti 3 C 2 T x -Quantitative test of ATP-GTP; in Ti 3 C 2 T x Surface modification FA-PEG-NH 2 The principle is to rely on the strong affinity of folic acid (FA) to the folate receptor (FR) on the surface of cancer cells, thereby enhancing the targeted endocytosis of the nanoprobe to tumor cells; usually, FR is highly expressed in a variety of malignant tumors, while the expression of FR in normal cells is strictly restricted, making FR a commonly used tumor biomarker; the quantitative analysis results of inductively coupled plasma resonance emission spectrometry (ICP) show that Ti 3 C 2 T x -ATP-GTP nanoprobes were treated with MCF-7 and MCF-10A cells for 4 hours, and the intracellular Ti contents of MCF-7 and MCF-10A cells were 3.84±0.25mM and 1.16±0.12mM, respectively; this result shows that in Ti 3 C 2 T x Surface modification of FA-PEG-NH 2 It is more suitable for targeted biorecognition and enhances the endocytosis of nanoprobes into tumor cells; mPTT activated ATP and GTP imaging test during nucleolar stress in living cells: like Fig.30 As shown, using Ti 3 C 2 T x -ATP-GTP nanoprobes were used for live cell imaging to monitor the levels of GTP and ATP and their changes in real time during the nucleolar stress process of live cells activated by mPTT; in order to make the temperature of the cell culture medium comparable to the mPTT treatment temperature under the same conditions, an 808 nm laser with an intensity of 1 W cm -2 , respectively directly irradiate Ti 3 C 2 T x - MCF-7 and MCF-10A cells incubated with ATP-GTP nanoprobes without PBS washing; thermal infrared images of the two cells were monitored using a Fluke thermal imager; and photothermal temperature changes of the measured cells were observed; like Fig.31As shown, the ability of nucleic acid aptamer-modified nanoprobes to display ATP and GTP in real-time fluorescence in living cells is demonstrated; for MCF-7 cells, after 3 minutes of mPTT laser irradiation, the temperature in the wells increased to 34.4°C, and the fluorescence intensity of ATP and GTP began to increase; it is obvious that ATP fluorescence is evenly distributed in the cells, while GTP fluorescence is mainly distributed in the cytoplasm; after 6 minutes of laser irradiation, the temperature in the wells increased to 39.3°C, most of the ATP fluorescence in the cells weakened, and a small part of ATP fluorescence around the nucleus increased; it is worth noting that at this time, GTP fluorescence tightly surrounded the nucleus area and was significantly enhanced, and some fluorescence even entered the nucleus; after 9 minutes of laser irradiation, the temperature of the cell culture medium increased to 43.2°C, and most of the ATP and GTP fluorescence were distributed around the nucleus; however, GTP fluorescence still partially surrounded the peripheral area of ​​the nucleus; after 12 minutes of irradiation, the temperature of the culture medium was increased to 45.5°C, and the green fluorescence of ATP and the red fluorescence of GTP were significantly weakened; this reduction in fluorescence is attributed to the consumption of energy metabolism in the process of tumor cell apoptosis induced by mPTT stimulation; For comparison with cancer cells, the same experiment was performed on normal MCF-10A cells. After 808 nm laser irradiation for 3-6 min, the temperature of MCF-10A cells (solution) increased from 33.1°C to 37.9°C, and the green and red fluorescence in the cytoplasm increased and were evenly distributed. After 9 min of irradiation, the temperature of MCF-10A cells reached 42.1°C, and the green and red fluorescence increased and were distributed around the cell nucleus, but not obviously. After 12 min of laser irradiation, the green and red fluorescence weakened. These results indicate that the content of ATP and GTP in cells increases with the extension of photothermal treatment time. However, the change trend of normal cells is not as obvious as that of tumor cells. This study confirmed the Ti for mPTT by real-time monitoring and visualization of ATP and GTP in living cells. 3 C 2 T x -ATP-GTP nanoprobes are activated upon thermal stimulation, inducing tumor cell apoptosis through a specific nuclear stress process and inhibiting the energy metabolism of GTP.

[0017] Synthesis mechanism: On the sensing platform, Ti was prepared using chloroacetic acid. 3 C 2 T x Then, under the catalysis of EDC and NHS, FA-PEG2000-NH 2 Through the condensation reaction of amino and carboxyl groups with Ti 3 C 2 T xCovalent bonding; PEG modification can effectively improve the stability of the nanoprobe, while FA as a ligand can enhance cell endocytosis and improve the therapeutic effect on cancer cells; finally, after co-incubation at 4°C, Ti was assembled through electrostatic interaction. 3 C 2 T x -PEG and fluorescently labeled aptamers, namely ATP Aptamer and GTP Aptamer; the fluorescence of the aptamer is initially 3 C 2 T x Through FRET quenching; when Ti 3 C 2 T x After the ATP-GTP photothermal nanoprobe is specifically recognized by ATP and GTP, the aptamer is separated from the nanoprobe, and the aptamer is bound to Ti 3 C 2 T x -PEG FRET disappears, fluorescence is restored, and the change in fluorescence intensity is related to the change in intracellular ATP and GTP content.

Claims

1. A MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, characterized in that: The MXene used is Ti3C2T x , the Ti3C2T x The surface is modified with nucleic acid aptamers; The nucleic acid aptamer is composed of ATP nucleic acid aptamer and GTP nucleic acid aptamer; the ATP nucleic acid aptamer is a DNA fragment, the 5' end of which carries a fluorescent marker peptide FAM, and the nucleotide sequence is shown in SEQ ID No: 1; the GTP nucleic acid aptamer is an RNA fragment, the 5' end of which carries a fluorescent dye anthocyanin Cy3, and the nucleotide sequence is shown in SEQ ID No:

2.

2. The MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers according to claim 1, characterized in that: The photothermal probe uses Ti3C2T x The single-layer nanosheets were prepared, and the Ti3C2T x The thickness of a single nanosheet is 1.7-1.9 nm.

3. A method for preparing the MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer as claimed in claim 2, characterized in that: The following steps are involved: S1. Preparation of Ti3C2T x Single-layer nanosheets: 0.8 g of lithium fluoride powder was added to 10 mL of 9 M HCl and stirred slowly in a constant temperature water bath for 10 min. After the LiF powder was completely dissolved, 0.5 g of 200 mesh black Ti3AlC2 powder was added and stirred at 500 rpm. The reaction was continued at room temperature for 48 h. Deionized water was used for multiple cycles of centrifugal washing at 5000 rpm. Each cycle lasted for 2 min. When the pH value reached 6, argon was introduced into the solution and ultrasonic peeling was performed in an ice water bath for 60 min to obtain a single layer of Ti3C2T x , centrifuged at 3500 rpm for 60 min to remove the multilayer Ti3C2T x Nanosheets, collected containing Ti3C2T x The dark supernatant of the monolayer nanosheets was stored at 4°C with argon gas for later use; S2, carboxyl modified Ti3C2T x Single-layer nanosheets: Prepare 1 mg / mL of Ti3C2T x Take 50 mL of the monolayer nanosheet solution, and perform ice bath ultrasonication for 1 h under argon to make the solution uniformly dispersed. Add 1.25 g of NaOH, stir at 60 °C for 2 h, add 0.625 g of NaOH and 3 g of chloroacetic acid, continue stirring for 3 h, collect the solution, centrifuge with deionized water at 8000 rpm, single 15 min cycle centrifugation until neutral, collect the solution, and obtain Ti3C2T x -COOH solution, stored at 4°C for future use; S3, modified FA-targeted aminated Ti3C2T x -PEG Nanosheets: Prepare 1 mg / mL of Ti3C2T in step S2 x -COOH solution, take 50mL, add 37.5mg EDC and 56.25mg NHS in turn, continue stirring for 2h, add 50mg FA-PEG-NH2 with a molecular weight of 2000Da, react for 24h, cool to room temperature, centrifuge the solution at 8000rpm for 15min, remove the supernatant, put it into a dialyzer with a molecular weight cutoff of 3000Da, dialyze at room temperature for 48h, change the water every 2h, and obtain Ti3C2T2 with carboxyl-amino modification. x The solution of nanosheets is denoted as FA-Ti3C2T x -PEG solution, stored at 4℃ for future use; S4. Assemble Ti3C2T x -ATP-GTP photothermal probe: First, 100 nM ATP aptamer and GTP aptamer were mixed with the pre-prepared 1 mg / mL FA-Ti3C2T x -PEG solution, shaken and stirred at 4 °C overnight, and centrifuged at 9500 rpm for 15 min with HEPES buffer to obtain a MXene nanosheet-based active targeting recognition photothermal probe modified with nucleic acid aptamers, denoted as Ti3C2T x -ATP-GTP, stored at 4℃ until use.

4. The method for preparing a MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer according to claim 3, characterized in that: The constant temperature water bath temperature in S1 is 40-45°C.

5. The method for preparing a MXene nanosheet-based active targeting recognition photothermal probe modified with a nucleic acid aptamer according to claim 4, characterized in that: All the reaction processes in S3 need to be carried out in an oil bath at 60-65° C. and with argon gas flowing in.

6. Use of the MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers according to any one of claims 1-2 or the MXene nanosheet-based active targeted recognition photothermal probe modified with nucleic acid aptamers prepared by the preparation method according to any one of claims 3-5 in the field of preparing photothermal agents.

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