Multifunctional material for colorimetric detection and photothermal killing of tumor cells, and preparation method and application thereof

Abstract

The application provides a multifunctional material for colorimetric detection and photothermal killing of tumor cells, and a preparation method and application thereof. The multifunctional material is a graphite-like carbon nitride material chelating monatomic iron, and includes a chelate formed by chelation of a polymer ligand generated by a formamide molecule through a Schiff base reaction and iron ions; wherein the chelation of the polymer ligand and the iron ions forms Fe-N active coordination, so that Fe exists in the form of an oxidized monatomic dispersion.

Description

Technical Field

[0001] This invention relates to a multifunctional material for colorimetric detection and photothermal killing of tumor cells, its preparation method and application, belonging to the field of functional materials technology. Background Technology

[0002] As the second leading cause of death worldwide, cancer is a collective term for a series of diseases characterized by abnormal cell proliferation, invasiveness, and metastasis. It can occur anywhere in the body, causing a large number of deaths annually and inflicting immense suffering and financial burden on patients and their families. Accurate diagnosis of cancer enables early detection, early treatment, and monitoring of prognosis, preventing the progression to advanced stages and recurrence, and is crucial for improving cancer cure rates. Furthermore, effective cancer treatment complements traditional methods (surgical resection, radiotherapy, and chemotherapy), enriching cancer-specific treatment modalities and reducing patient suffering and financial burden. Therefore, there is an urgent need to develop effective means for accurate diagnosis and treatment of cancer.

[0003] In tumor diagnosis, researchers and medical institutions have developed electrochemical, fluorescence, Raman, mass spectrometry, and colorimetric methods for the ultrasensitive and highly selective detection of tumor cells, thereby screening for tumors. Compared with other methods, colorimetric methods are characterized by speed, convenience, and low cost, and are widely used for tumor cell detection. However, traditional colorimetric methods rely on natural horseradish peroxidase or peroxidase-like activities from nanomaterials, and require the addition of hydrogen peroxide during the experimental process, making the operation relatively cumbersome. Currently, there are no reports on the relatively simple operation of detecting tumor cells based on peroxidase-like activities from nanomaterials. In tumor treatment, photothermal therapy is an emerging tumor treatment strategy. It utilizes exogenous photothermal conversion materials to convert light energy into heat energy, raising the temperature of the local tumor tissue above 40°C, causing protein denaturation within tumor cells, destroying the morphology and function of cancer cells, and thus killing tumor cells. It has the advantages of being controllable and non-invasive. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a multifunctional material for colorimetric detection and photothermal killing of tumor cells, its preparation method, and its application. The multifunctional material integrates colorimetric detection and photothermal killing of tumor cells into a single material system, enabling it to both detect and kill tumor cells. This provides a dual-function effect of diagnosis and treatment, helping to improve diagnostic and treatment efficiency and thereby reducing treatment costs and duration for patients.

[0005] In a first aspect, the present invention provides a multifunctional material with oxidase-like activity. The multifunctional material is a graphitized carbon nitride-like material that chelates single-atom iron, comprising a chelate formed by the chelation of a polymer ligand generated from a formamide molecule via a Schiff base reaction and an iron ion. The Fe-N active coordination formed by the chelation of the polymer ligand and the iron ion results in Fe existing in a dispersed single-atom oxidized state.

[0006] The nanomaterial exhibits excellent oxidase-like activity, catalyzing a colorimetric reaction with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). This allows for the determination of tumor cell count by detecting the absorbance at 652 nm, thus achieving colorimetric detection of tumor cells. Simultaneously, the material exhibits absorption in the near-infrared region, demonstrating good photothermal conversion properties, and can be used as a photothermal conversion material to kill tumor cells through photothermal therapy.

[0007] Preferably, the multifunctional material further includes sulfur atoms doped onto the polymer ligand in the form of CSCs. These sulfur atoms, entering the vicinity of the Fe-N active coordination sites, can modulate the electron density around the active sites, causing the charge density around the Fe-N sites to concentrate more towards Fe, thereby accelerating the electron transfer rate between TMB and dissolved oxygen.

[0008] Preferably, the multifunctional material further includes folic acid that modifies the chelate to target tumor cells. Folic acid can specifically bind to folic acid receptors highly expressed on tumor cells, thereby targeting the tumor cells.

[0009] Preferably, the N in the multifunctional material exists in the form of graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen.

[0010] Preferably, the polymer ligand has a carbon backbone formed by the self-polymerization of formamide molecules via a Schiff base reaction.

[0011] Secondly, the present invention provides a method for preparing a multifunctional material with oxidase-like activity. The preparation method includes the following steps: dispersing an iron source in formamide to form a mixed solution; reacting the mixed solution at 180–220°C for 12–24 hours; collecting and drying the product after the reaction to obtain the multifunctional material with oxidase-like activity.

[0012] Thirdly, the present invention provides a method for preparing a multifunctional material with oxidase-like activity. The preparation method includes the following steps: dispersing an iron source and thiourea in formamide to form a mixed solution; reacting the mixed solution at 180–220°C for 12–24 hours; collecting and drying the product after the reaction to obtain the multifunctional material with oxidase-like activity.

[0013] Fourthly, the present invention provides a method for preparing a multifunctional material with oxidase-like activity. The preparation method includes the following steps: dispersing an iron source and thiourea in formamide to form a mixed solution; reacting the mixed solution at 180–220°C for 12–24 hours; collecting and drying the product after the reaction; and modifying the product with folic acid via an amide reaction activated by N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain the multifunctional material with oxidase-like activity.

[0014] Preferably, the molar ratio of the iron source to formamide is 1–10 mM: 30–50 mL.

[0015] Preferably, the molar ratio of thiourea to formamide is 1–10 mM: 30–50 mL.

[0016] Preferably, the iron source is at least one of ferric acetate, ferric chloride, and ferric sulfate.

[0017] Fifthly, the present invention provides the application of the multifunctional material with oxidase-like activity described in any of the above claims in the preparation of a drug that simultaneously has colorimetric detection and photothermal killing of tumor cells. Attached Figure Description

[0018] Figure 1 XRD patterns (a) and Raman spectra (b) of Fe-SNC and Fe-NC are shown.

[0019] Figure 2 The N1s high-resolution XPS spectrum of Fe-SNC;

[0020] Figure 3 This is a schematic diagram of the structure of Fe-SNC, where the brown, blue, yellow and orange spheres represent C, N, S and Fe atoms, respectively.

[0021] Figure 4 The XRD pattern of the product obtained in Comparative Example 1 is shown.

[0022] Figure 5 The activity curves of the oxidase-like enzymes of Fe-SNC and Fe-NC are shown.

[0023] Figure 6 The UV-Vis absorption spectra of FA, Fe-SNC and Fe-SNC-FA (a) and the Zeta potentials of Fe-SNC and Fe-SNC-FA (b) are shown.

[0024] Figure 7 The peroxidase activity assay curve for Fe-SNC-FA;

[0025] Figure 8 To investigate the relationship between the absorbance of tumor cells and the number of HeLa cells using the Fe-SNC-FA colorimetric assay;

[0026] Figure 9 The relationship between the absorbance of Fe-SNC-FA for tumor cells and the number of different cell types;

[0027] Figure 10 The photothermal heating curves (a) and photothermal killing effect of Fe-SNC on tumor cells (b) are shown. In (a), the curves from bottom to top are 0 μg / mL, 60 μg / mL, 120 μg / mL, 180 μg / mL, 300 μg / mL, and 600 μg / mL. Detailed Implementation

[0028] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise specified, all percentage contents refer to mass percentage contents.

[0029] This invention provides a multifunctional material with oxidase-like activity. The multifunctional material is a graphitized carbon nitride-like material that chelates single-atom iron, comprising a chelate formed by the chelation of a polymer ligand generated from a formamide molecule via a Schiff base reaction and an iron ion. The Fe-N active coordination formed by the chelation of the polymer ligand and the iron ion results in Fe existing in a dispersed, oxidized single-atom form. Figure 1 The XRD pattern (a) shows that the 27° diffraction peak corresponds to the (002) crystal plane of graphitized carbon nitride. According to... Figure 1 The Raman spectrum (b) shows that the Raman peaks correspond to the D and G peaks of graphitic carbon. Therefore, this multifunctional material is a graphitic carbon nitride material that chelates single-atom iron. Furthermore, organic small molecule formamide, acting as both a nitrogen and carbon source, undergoes a Schiff base reaction under high temperature (sealed) and high pressure to yield a nitrogen- and carbon-rich polymer ligand. This polymer ligand chelates with iron to form Fe-N coordination sites, promoting the existence of iron in a dispersed, oxidized single-atom form. The presence of iron in a single-atom form theoretically results in 100% atomic utilization, enhancing the material's enzyme-like catalytic activity and thus improving sensitivity in colorimetric detection. Additionally, from... Figure 2 It is known that the nitrogen in the aforementioned multifunctional material exists mainly in three forms: graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen. Pyrrole nitrogen and pyridine nitrogen have strong coordination ability with metal atoms, while graphitic nitrogen has good electrical conductivity and can accelerate charge transfer during catalysis.

[0030] During the experiment, formamide was replaced with DMF, acetamide, ethanolamine, etc., but no product was obtained. This may be because the amino and carbonyl groups in the formamide molecule are highly reactive, and can obtain nitrogen-rich ligands through Schiff base reactions, which then coordinate with iron to obtain the relevant product. In contrast, the amino or carbonyl groups in DMF, acetamide, and ethanolamine molecules are less reactive, so the relevant products cannot be obtained.

[0031] In some technical solutions, the multifunctional material further includes sulfur atoms doped onto the polymer ligand in the form of CSCs. This can be seen from... Figure 3 The structural schematic diagram of Fe-SNC shown is thus demonstrated. Furthermore, Figure 1 XRD analysis showed that sulfur doping did not affect the phase composition of the multifunctional material. This invention incorporates sulfur around the Fe-N active sites, regulating the electron density around these sites and thus enhancing their oxidase-like activity. Specifically, S doping increases the adsorption capacity of Fe active sites for substrate oxygen, disrupting the symmetrical charge density around Fe, causing the charge density around the Fe-N sites to concentrate more towards Fe, accelerating the electron transfer rate between TMB and dissolved oxygen.

[0032] In some technical solutions, the multifunctional material further includes folic acid that targets tumor cells and modifies the chelate. Tumor cells (HeLa cells) highly express folic acid receptors, while normal cells do not. Therefore, only tumor cells can specifically bind to the folic acid-modified multifunctional material, subsequently catalyzing a colorimetric reaction in TMB, exhibiting absorbance at 652 nm. Normal cells, however, cannot bind to the folic acid-modified multifunctional material, are washed away during the washing process, and cannot undergo a colorimetric reaction, thus showing no absorbance at 652 nm. Therefore, the folic acid-modified multifunctional material can specifically detect tumor cells, exhibiting good selectivity for tumor cells.

[0033] The multifunctional material described in this invention catalyzes a colorimetric reaction with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) through its oxidase-like activity, thereby measuring the absorbance of the system at 652 nm. Based on this absorbance value, the presence and quantity of tumor cells can be determined, thus achieving the purpose of colorimetric detection of tumor cells. Furthermore, the sulfur-doped multifunctional material exhibits certain absorption in the near-infrared region and possesses excellent photothermal conversion properties, making it suitable as a photothermal conversion material for killing tumor cells through photothermal therapy. Therefore, the aforementioned multifunctional material can be used for tumor diagnosis and treatment.

[0034] In some technical solutions, the mass percentage of iron in the multifunctional material is 0.1% to 1%. As an example, the elemental composition of the multifunctional nanomaterial includes, by mass percentage: Fe 0.1% to 1%, N 20% to 30%, C 60% to 70%, S 0.1% to 1%, and O 5% to 15%.

[0035] The present invention also provides a method for preparing a multifunctional material with oxidase-like activity.

[0036] In some technical solutions, the preparation method includes the following steps: dispersing an iron source and thiourea in formamide to form a mixed solution, reacting the mixed solution at 180-220°C for 12-24 hours, collecting the product after the reaction and drying it to obtain the multifunctional material with oxidase-like activity.

[0037] In some technical solutions, the preparation method includes the following steps: dispersing an iron source and thiourea in formamide to form a mixed solution; reacting the mixed solution at 180–220°C for 12–24 hours; collecting and drying the product after the reaction to obtain the multifunctional material with oxidase-like activity. When the reaction temperature is below 180°C, for example, 160°C, the reaction cannot occur to form the corresponding product.

[0038] The molar ratio of the iron source to formamide is 1–10 mM: 30–50 mL. The iron source is a soluble iron salt that introduces iron ions. The iron ions introduced by the iron source can be ferrous ions (Fe2+) and / or ferric ions (Fe3+). As an example, the iron source is at least one of ferric acetate, ferric chloride, and ferric sulfate.

[0039] The molar ratio of thiourea to formamide is 1–10 mM: 30–50 mL. Thiourea has a similar molecular structure to formamide. By using thiourea, which has a similar structure to formamide, as a sulfur source, sulfur can be incorporated around the Fe-N active site during the reaction, regulating the electron density around the Fe active site and thus improving its oxidase-like activity. Trimethylthiourea, because the methyl group replaces the hydrogen atom in the amino group, cannot undergo a Schiff base reaction, thus achieving the technical objective of this invention.

[0040] The experiment found that when the concentration of iron source and thiourea relative to formamide increased to 20 mM: 30-50 mL, XRD diffraction peaks of impurities appeared in the product.

[0041] The preparation method described herein involves the self-polymerization of formamide to obtain a nitrogen-containing ligand that is coordinated with iron under high temperature and pressure. The ligand contains a carbon skeleton formed by carbon atoms, while retaining some amino groups to facilitate subsequent modification. The addition of thiourea replaces part of the self-polymerization of formamide, thus incorporating sulfur into the nitrogen-containing ligand. Sulfur doping alters the electronic structure of the Fe-N sites, improving their oxygen adsorption capacity and thereby enhancing the oxidase-like activity of the material, enabling its use for colorimetric detection of tumor cells. On the other hand, the carbon skeleton structure allows Fe-SNC to exhibit good absorption of near-infrared light, enabling the conversion of light energy into heat energy and giving it the potential for photothermal therapy.

[0042] The multifunctional material prepared by this invention not only has good dispersibility but also contains functional groups such as amino groups, making it easy to modify. In some technical solutions, the molar ratio of the iron source to thiourea is 1:(1-3), preferably 1:1.

[0043] As an example, the preparation method includes the following steps: (a) adding 1-10 mM ferric chloride and 1-10 mM thiourea to 30-50 mL of formamide; (b) stirring the solution at room temperature for 10-30 minutes to fully dissolve the ferric chloride and thiourea in the formamide; (c) transferring the solution to a stainless steel reactor, sealing the reactor, and transferring it to an oven; (d) reacting at 180-220°C for 12-24 hours; (e) after the reaction is completed, naturally cooling to room temperature, opening the reactor, transferring the solution in the reactor to a centrifuge tube, centrifuging at 10,000 rpm for 10 minutes, discarding the supernatant, repeatedly washing the centrifuged precipitate with water three times, and finally drying it at 70°C.

[0044] To specifically identify tumor cells, the preparation method further includes modifying the chelate with folic acid via an amide reaction activated by N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Through the specific binding of folic acid to tumor cells, the folic acid-modified multifunctional material can target tumor cells, subsequently catalyzing a colorimetric reaction with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) through its oxidase-like activity. The absorbance of the system is measured at 652 nm. Based on this absorbance value, the presence and number of tumor cells can be determined, thus achieving colorimetric detection of tumor cells. Simultaneously, the sulfur-doped multifunctional material exhibits absorption in the near-infrared region and possesses excellent photothermal conversion properties, making it suitable as a photothermal conversion material for killing tumor cells through photothermal therapy.

[0045] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0046] Example 1

[0047] Add 5 mM anhydrous ferric chloride to 30 mL of formamide and stir for 10 minutes at room temperature. Then transfer the mixture to a stainless steel reactor. Seal the reactor and immediately place it in a 180°C oven for 12 hours. After the reaction is complete, cool to room temperature, open the reactor, transfer the reaction mixture to centrifuge tubes, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, wash the precipitate three times with ultrapure water, and then dry it in a 70°C oven. The resulting material is designated Fe-NC.

[0048] Example 2

[0049] Add 10 mM anhydrous ferric chloride to 30 mL of formamide and stir for 15 minutes at room temperature. Then transfer the mixture to a stainless steel reactor. Seal the reactor and immediately transfer it to a 220°C oven for 12 hours. After the reaction is complete, cool to room temperature, open the reactor, transfer the reaction mixture to centrifuge tubes, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, wash the precipitate three times with ultrapure water, and then dry it in a 70°C oven.

[0050] The materials obtained in Examples 1-2 have low oxidase activity, making them difficult to use for colorimetric detection of tumor cells, or their detection performance for tumor cells is poor.

[0051] Example 3

[0052] 5 mM anhydrous ferric chloride and 5 mM thiourea were added to 30 mL of formamide and stirred at room temperature for 10 minutes, then transferred to a stainless steel reactor. The reactor was sealed and immediately placed in an oven at 180 °C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the reactor was opened, and the reaction solution was transferred to centrifuge tubes. The tubes were centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with ultrapure water, then dried in an oven at 70 °C. The resulting material was designated Fe-SNC.

[0053] Example 4

[0054] 10 mM anhydrous ferric chloride and 10 mM thiourea were added to 30 mL of formamide and stirred at room temperature for 15 minutes, then transferred to a stainless steel reactor. The reactor was sealed and immediately placed in a 220°C oven for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the reactor was opened, and the reaction solution was transferred to centrifuge tubes. The tubes were centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with ultrapure water, then dried in a 70°C oven.

[0055] Comparative Example 1

[0056] 20 mM anhydrous ferric chloride and 20 mM thiourea were added to 30 mL of formamide and stirred at room temperature for 15 minutes, then transferred to a stainless steel reactor. The reactor was sealed and immediately placed in an oven at 180 °C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the reactor was opened, and the reaction solution was transferred to centrifuge tubes. The tubes were centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with ultrapure water, then dried in an oven at 70 °C.

[0057] like Figure 4 As shown, when the concentrations of ferric chloride and thiourea increased to 20 mM, XRD diffraction peaks of impurities appeared in the obtained product.

[0058] Example 5

[0059] Evaluation of the oxidase activity of the material

[0060] The oxidase-like activity of the materials was evaluated using the TMB colorimetric method. 100 μL of 1 mg / mL Fe-SNC dispersion and 100 μL of 20 mM TMB solution were added to 800 μL of acetate-sodium acetate buffer (pH = 4, 0.1 M). After thorough mixing, the mixture was reacted at 37 °C for 30 min. Subsequently, 100 μL was taken and the full-spectrum absorbance from 450-850 nm was measured using a microplate reader. Similarly, 100 μL of 1 mg / mL Fe-SNC dispersion was replaced with 100 μL of 1 mg / mL Fe-NC dispersion. Unless otherwise specified in this invention, dispersion refers to the material (powder) dispersed in water.

[0061] Figure 5 The images show the oxidase activity test curves for materials Fe-SNC and Fe-NC. Both Fe-SNC and Fe-NC catalyze a colorimetric reaction of TMB, exhibiting absorbance at 652 nm. The absorbance value at 652 nm represents the oxidase activity of the materials. Higher absorbance values ​​indicate better oxidase activity. Figure 5The oxidase-like activity of Fe-SNC is significantly higher than that of Fe-NC. This is because S doping enhances the adsorption capacity of Fe active sites for substrate oxygen and breaks the symmetrical charge density around Fe, causing the charge density around Fe-N sites to accumulate more towards Fe, thus accelerating the electron transfer rate between TMB and dissolved oxygen.

[0062] Example 6

[0063] Preparation of Fe-SNC modified folic acid

[0064] Add 50 mg of folic acid (FA) to 10 mL of dimethyl sulfoxide and stir in the dark for 1 hour. Then add 1 mL of 0.1 M MEDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 1 mL of 0.1 M NHS (N-hydroxysuccinimide) and continue stirring in the dark for 3 hours. Then add 10 mL of 5 mg / mL Fe-SNC dispersion and continue stirring in the dark for 18 hours. Then transfer it to a centrifuge tube and centrifuge at 12,000 rpm for 10 min. Wash the centrifuged precipitate three times with water. The product obtained is denoted as Fe-SNC-FA.

[0065] The binding of Fe-SNC and FA was characterized using ultraviolet-visible absorption spectroscopy and zeta potential. Figure 6 As shown, Fe-SNC-FA exhibits an absorption peak at 280 nm similar to that of FA, while Fe-SNC itself does not show an absorption peak at this location, indicating that FA was successfully modified onto Fe-SNC. Furthermore, folic acid is negatively charged due to the ionization of its carboxyl groups; when FA is modified onto Fe-SNC, the potential of the material becomes more negative, further demonstrating the successful preparation of Fe-SNC-FA.

[0066] Example 7

[0067] Evaluation of Fe-SNC-FA oxidase activity

[0068] The oxidase-like activity of the materials was evaluated using the TMB colorimetric method. 100 μL of 0.6 mg / mL Fe-SNC-FA dispersion and 100 μL of 20 mM TMB solution were added to 800 μL of acetate-sodium acetate buffer (pH = 4, 0.1 M). After thorough mixing, the mixture was reacted at 37 °C for 30 min. Subsequently, 100 μL was taken and the full-spectrum absorbance from 450 to 850 nm was measured using a microplate reader.

[0069] Figure 7The image shows the oxidase activity assay curve for Fe-SNC-FA. Fe-SNC-FA can still catalyze the colorimetric reaction of TMB, indicating that Fe-SNC-FA still possesses good oxidase activity.

[0070] Example 8

[0071] Fe-SNC-FA colorimetric detection of tumor cells

[0072] HeLa cells (human cervical cancer cells) were seeded into 96-well plates at densities of 0, 1000, 4000, 8000, and 15000 Cell / mL and cultured in a cell culture incubator for 24 hours. Then, 0.1 mg / mL Fe-SNC-FA was added to each well, and the cells were cultured for another 2 hours. The plates were then washed three times with PBS to remove any unbound material. Next, 10 μL of TMB (20 mM) and 90 μL of sodium acetate-acetic acid buffer (pH = 4, 0.1 M) were added, and the plates were cultured for another 30 minutes. Finally, the absorbance of the solutions in the 96-well plates was measured at 652 nm using a microplate reader.

[0073] Figure 8 The relationship between the absorbance of tumor cells and the number of HeLa cells was investigated using Fe-SNC-FA colorimetric assay. As the number of HeLa cells increased, the absorbance of the solution at 652 nm increased, and a good linear relationship was observed between the absorbance and the number of HeLa cells. This indicates that the number of tumor cells can be determined by measuring absorbance, and this method can be used for the colorimetric detection of tumor cells.

[0074] Example 9

[0075] Fe-SNC-FA's selectivity for tumor cells

[0076] HeLa cells, HUVECs (human umbilical vein endothelial cells), and MC3T3-E1 cells (mouse osteoblasts) were seeded into 96-well plates at a density of 100,000 cells / mL and cultured in a cell culture incubator for 24 hours. Then, 0.1 mg / mL Fe-SNC-FA was added to the 96-well plates, and the plates were cultured for another 2 hours. The plates were then washed three times with PBS to remove any unbound material. Next, 10 μL of TMB (20 mM) and 90 μL of sodium acetate-acetic acid buffer (pH = 4, 0.1 M) were added, and the plates were cultured for another 30 minutes. Finally, the absorbance of the solutions in the 96-well plates was measured at 652 nm using a microplate reader.

[0077] Figure 9This study investigates the relationship between the absorbance of Fe-SNC-FA for tumor cells and the number of different cell types. HeLa cells exhibited high absorbance at 652 nm, while HUVECs and MC3T3-E1 cells showed no absorption peak at this temperature. This is because HeLa cells highly express folate receptors, while normal cells such as HUVECs and MC3T3-E1 cells do not. Therefore, only HeLa cells can specifically bind to Fe-SNC-FA, subsequently catalyzing a colorimetric reaction in TMB, resulting in absorbance at 652 nm. Normal cells, however, cannot bind to Fe-SNC-FA and are washed away during the washing process, failing to undergo the colorimetric reaction and thus showing no absorbance at 652 nm. This result indicates that Fe-SNC-FA can specifically detect tumor cells and exhibits good selectivity for them.

[0078] Example 10

[0079] Fe-SNC photothermal killing of tumor cells

[0080] HeLa cells were seeded into 96-well plates at a density of 100,000 cells / mL and cultured in a cell culture incubator for 24 hours. Then, 0.1 mg / mL Fe-SNC was added to each well, and the plates were irradiated with near-infrared light at 808 nm (1 W / cm²) for 10 min. 2 Then, the cells were cultured for another 12 hours, and 100 μL of Alamar Blue diluted 10 times was added. The cells were cultured for another 2 hours. Finally, the fluorescence intensity was tested using a microplate reader. The excitation wavelength was 560 nm and the emission wavelength was 590 nm.

[0081] Figure 10 Photothermal heating curve (a) and photothermal killing effect of Fe-SNC on tumor cells (b) are shown. Under near-infrared light irradiation, Fe-SNC can effectively kill tumor cells, and the survival rate of HeLa cells is almost zero.

[0082] Comparative Example 2

[0083] 5 mM anhydrous ferric chloride and 5 mM thiourea were added to 30 mL of N,N-dimethylformamide and stirred at room temperature for 10 minutes, then transferred to a stainless steel reactor. The reactor was sealed and immediately placed in an oven at 180°C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the reactor was opened, and the reaction solution was transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes. No relevant products were obtained.

Claims

1. A multifunctional material with oxidase-like activity, characterized in that, The multifunctional material is a graphitized carbon nitride material that chelates single-atom iron, comprising a chelate formed by the chelation of a polymer ligand generated by a formamide molecule through a Schiff base reaction and an iron ion; wherein, the Fe-N active coordination formed by the chelation of the polymer ligand and the iron ion results in Fe existing in a dispersed single-atom oxidized state; the multifunctional material also includes sulfur atoms doped onto the polymer ligand in the form of CSC; the multifunctional material also includes folic acid that targets tumor cells and modifies the chelate; the preparation method of the multifunctional material includes the following steps: dispersing an iron source and thiourea in formamide to form a mixed solution, reacting the mixed solution at 180~220°C for 12~24 hours, collecting the product after the reaction and drying it; and modifying the product with folic acid by an amide reaction activated by N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dimethyl sulfoxide, to obtain the multifunctional material with oxidase-like activity.

2. The multifunctional material with oxidase-like activity according to claim 1, characterized in that, The N in the multifunctional material exists in the form of graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen.

3. The multifunctional material with oxidase-like activity according to claim 1, characterized in that, The polymer ligand has a carbon backbone formed by the self-polymerization of formamide molecules through a Schiff base reaction.

4. The multifunctional material with oxidase-like activity according to claim 1, characterized in that, The molar ratio of the iron source to formamide is 1~10 mM:30~50 nL.

5. The multifunctional material with oxidase-like activity according to claim 1, characterized in that, The molar ratio of thiourea to formamide is 1~10 mM: 30~50 nL.

6. The use of the multifunctional material with oxidase-like activity as described in any one of claims 1 to 5 in the preparation of a drug that simultaneously has colorimetric detection and photothermal killing of tumor cells.

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