FA / Au@COF(Fe) composite system, and preparation method and application thereof

Abstract

This invention discloses an FA / Au@COF(Fe) composite system, its preparation method, and its applications. The composite system comprises folic acid and Au@COF(Fe), wherein Au@COF(Fe) is composed of COF(Fe) and Au NP. The preparation method of the FA / Au@COF(Fe) composite system is as follows: Au solution and sodium borohydride are added to a solution of iron porphyrin COF and reacted to obtain Au@COF(Fe). Au@COF(Fe) is then dispersed in an alcohol solvent and added to a solution containing folic acid, and reacted to obtain FA / Au@COF(Fe). The preparation method of this invention is simple, inexpensive, and highly effective. This Au@COF(Fe) composite system exhibits catalase-like activity, enabling rapid colorimetric detection of H2O2 and tumor cells, demonstrating excellent detection performance and suitable for large-scale production and utilization.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, specifically relating to an FA / Au@COF(Fe) composite system, its preparation method, and its application. Background Technology

[0002] The detection of cancer biomarkers plays a crucial role in the early diagnosis and clinical research of cancer. Current detection methods include isotope internal standard methods and fluorescence time-degradation spectroscopy, but these suffer from drawbacks such as high cost, complex testing, and time consumption. Chromogenic immunoassay, as an analytical method, offers advantages such as intuitive observations, simplicity, speed, high sensitivity, and low cost, attracting significant attention in the analytical detection field and leading to the development of methods like enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and immunoarray methods. ELISA offers numerous advantages, including a simple analytical process, short processing time, high detection efficiency, and reduced sample volume and cost. However, in chromogenic immunoassays using HRP as the oxidase, an HRP-labeled secondary antibody is required to complete the oxidation of TMB; HRP is unstable and prone to denaturation, losing its peroxidase activity. Therefore, designing and synthesizing highly efficient HRP chemical mimics to simulate the function of catalase, enabling chemiluminescent immunoassay for tumor cell detection, has the potential to become a more stable and convenient immunoassay method widely used in clinical practice.

[0003] Iron porphyrin systems (heme proteins such as catalase, horseradish peroxidase, and cytochromes) are excellent hydrogen peroxide biosensors and are widely found in nature. Covalent organic frameworks (COFs) constructed from iron porphyrins are highly efficient oxidants. Through effective monomer regulation, iron porphyrin COFs materials with high catalytic oxidation activity can be designed and synthesized. Furthermore, these iron porphyrin COFs materials exhibit significant absorption peaks in the visible light region, enabling effective separation of photogenerated electrons and holes under visible light irradiation, thus improving catalytic efficiency. This makes them superior materials for redox sensors. However, the use of COFs materials as mimic enzymes in colorimetric immunoassays for the colorimetric detection of H2O2 concentration and tumor cells has not yet been reported.

[0004] Therefore, how to design and synthesize a material with high sensitivity, high stability, and simple operation method for rapid colorimetric detection of H2O2 and tumor cells is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a nanocomposite system FA / Au@COF(Fe) with catalase-like activity.

[0006] This invention provides a method for preparing a catalase-like FA / Au@COF(Fe) composite system. The prepared composite system can detect H2O2 colorimetrically, thereby enabling the colorimetric detection of tumor cells.

[0007] This invention also provides the application of the prepared FA / Au@COF(Fe) composite system in the field of biomedical detection.

[0008] Technical solution: To achieve the above objective, the present invention provides an FA / Au@COF(Fe) composite system, wherein the composite system comprises folic acid and Au@COF(Fe), and the Au@COF(Fe) is composed of COF(Fe) and Au NP.

[0009] The preparation method of the FA / Au@COF(Fe) composite system described in this invention specifically includes the following steps:

[0010] (1) First, add iron porphyrin and tetrathione monomer, then add acetic acid, 1,4-dioxane and 1,3,5-trimethylbenzene, sonicate, cool, and degas through a freeze-vacuum-thaw cycle. Heat the sample to a high temperature and keep it for several days. Wash and collect to obtain crude COF(Fe) product. Then wash with tetrahydrofuran and acetone alternately. After drying the product, obtain pure COF(Fe) material.

[0011] (2) Add HAuCl4 to a mixed solution of THF and water, stir, add NaBH4 solution, centrifuge and wash, freeze dry to obtain Au NP product;

[0012] (3) Add the Au NP prepared in step (2) to the mixed solution of DMF and water of the COF(Fe) material prepared in step (1) and stir overnight. After centrifugation and washing, vacuum drying is performed to obtain Au@COF(Fe) material, which is then dispersed in an alcohol solvent to obtain Au@COF(Fe) solution.

[0013] (4) Add the Au@COF(Fe) solution prepared in step (3) to a solution containing folic acid, centrifuge, wash, filter and dry to obtain FA / Au@COF(Fe) powder.

[0014] Further, in step (1), the molar ratio of iron porphyrin and tetrathiofulvalene monomers is 1:1, the volume ratio of acetic acid, 1,4-dioxane and 1,3,5-trimethylbenzene is 1:1-10:1-10, and the ultrasonic treatment time is 5-90 min.

[0015] Furthermore, in step (1), the sample is heated to 80℃-200℃ and the reaction is maintained for 24-96 hours.

[0016] Preferably, in step (1), the sample is heated to 120°C and reacted for 3 days.

[0017] Furthermore, in step (2), the volume ratio of HAuCl4 to NaBH4 is 1:1-10, and the volume ratio of THF to water is 1-10:1.

[0018] Furthermore, in step (3), the final concentration of the mixed solution of DMF and water of the COF(Fe) material is 1-5 mg / mL.

[0019] Furthermore, the alcohol solvent in step (4) is any one of methanol, ethanol or isopropanol, and the solvent for dissolving folic acid is acetic acid.

[0020] The application of the FA / Au@COF(Fe) composite system described in this invention in the field of biomedical detection.

[0021] The application of the FA / Au@COF(Fe) composite system described in this invention in the rapid detection of H2O2.

[0022] The application process is as follows: TMB solution and H2O2 solution are added to Au@COF(Fe) solution, and H2O2 can be detected by color development by irradiation with visible light.

[0023] Furthermore, the visible light irradiation time is 1-5 minutes.

[0024] Preferably, the visible light irradiation time is 2 minutes.

[0025] The application of the FA / Au@COF(Fe) composite system described in this invention in rapid colorimetric detection of tumor cells.

[0026] The application of the FA / Au@COF(Fe) composite system described in this invention in the preparation of reagents or materials for detecting H2O2 and tumor cells.

[0027] Preferably, the present invention adds iron porphyrin and tetrathiofulvalene monomer to a Pyrex tube, followed by the addition of acetic acid, 1,4-dioxane, and 1,3,5-trimethylbenzene, and sonicates (5-90 min); the above sample is rapidly frozen in a liquid nitrogen bath and degassed through a (1-5) freeze-vacuum-thaw cycle; the sample is heated to 80℃-200℃ and held at a constant temperature for 24-96 h, then washed and collected to obtain crude COF(Fe); the crude COF(Fe) product is extracted with 10-200 ml of tetrahydrofuran using a Soxhlet extractor for 10-80 h to obtain pure COF(Fe) material; Au NP is added to the COF(Fe) solution and reacted, followed by centrifugation and washing to obtain Au@COF(Fe) material, which is then reacted with folic acid solution, centrifuged and washed to obtain FA / Au@COF(Fe).

[0028] Mechanism of invention: This invention uses COF(Fe)-modified Au NPs to obtain Au@COF(Fe), which exhibits catalase-like activity and can catalyze the oxidation of TMB to achieve colorimetric detection of H2O2. Subsequently, folic acid is loaded onto the porous channels of Au@COF(Fe) to obtain the FA / Au@COF(Fe) composite system, which is then used for colorimetric detection of tumor cells.

[0029] This invention addresses the problem of rapid colorimetric detection of tumor cells by utilizing the excellent catalase-like activity of Au@COF(Fe) for the colorimetric detection of H2O2. Simultaneously, by utilizing the porous channels of COF to load folic acid, a FA / Au@COF(Fe) composite system is constructed. After reacting with tumor cells, this system can replace catalase, achieving targeted colorimetric detection of tumor cells with excellent detection results and significantly reducing detection costs. Furthermore, the preparation process of FA / Au@COF(Fe) is simple, rapid, and inexpensive.

[0030] This invention designs and synthesizes an FA / Au@COF(Fe) composite system, enabling targeted colorimetric detection of tumor cells with rapid detection speed. The reaction mechanism is unique; the Au@COF(Fe) component itself possesses highly efficient catalytic oxidation properties, catalyzing the generation of hydroxyl radicals from H₂O₂. Simultaneously, under visible light irradiation, Au@COF(Fe) can activate oxygen to generate hydroxyl radicals, further enhancing catalytic efficiency, resulting in high catalytic efficiency. The catalyst of this invention has multiple active sites, exhibiting single-site catalysis and high catalytic efficiency; the porous channels also promote sufficient contact between the substrate and active sites, further improving catalytic efficiency; under light irradiation, hydroxyl radicals are generated, enhancing catalytic efficiency.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] 1. The FA / Au@COF(Fe) composite system prepared by the present invention has catalase-like activity, which can realize rapid colorimetric detection of H2O2. The reaction conditions are mild, the sensitivity is high, and the stability is good.

[0033] 2. The FA / Au@COF(Fe) composite system of the present invention has a wide detection range, with the detection range of H2O2 reaching 0.461-461 μM and the lowest detection limit being 0.18 μM, which is far below the 15 μM hydrogen peroxide permissible level stipulated by the US FDA.

[0034] 3. The FA / Au@COF(Fe) material prepared by this invention is an inorganic nanozyme that can replace horseradish catalase to perform targeted colorimetric detection of tumor cells. The preparation method is simple, the cost is low, the functional effect is excellent, the biocompatibility is high, and it can be produced and utilized on a large scale. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the synthesis route of COF(Fe) of the present invention;

[0036] Figure 2 This is the powder X-ray diffraction pattern of Au@COF(Fe) of the present invention;

[0037] Figure 3 This is a comparison diagram of the three proportions of gold-supported COF(Fe) catalysts of the present invention;

[0038] Figure 4 This is a comparison chart of H2O2 detection under different conditions according to the present invention;

[0039] Figure 5 This is a standard curve for different absorbances and concentrations of H2O2 in this invention;

[0040] Figure 6 This is a colorimetric detection image of MDA-MB-231 cells using the FA / Au@COF(Fe) composite system of this invention.

[0041] Figure 7 This image shows the colorimetric detection of A549 cells using the FA / Au@COF(Fe) composite system of this invention. Detailed Implementation

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1

[0044] Synthesis of COF(Fe)

[0045] The synthesis of COF(Fe) follows Figure 1 The steps described above were performed. 5,10,15,20-tetra(p-aminophenyl)porphyrin iron (30 mg, 0.04 mmol), 2,3,6,7-tetra(4-formylphenyl)tetrathiofulvalene (24.8 mg, 0.04 mmol), 1,4-dioxane (1 mL), 1,3,5-trimethylbenzene (1 mL), and 6M acetic acid (0.4 mL) were added to a Pyrex tube. After sonication at 300 W for half an hour, the glass tube was rapidly cooled in a liquid nitrogen bath and degassed through a three-cycle freeze-vacuum-thaw cycle before sealing. The tube was then placed in an oven and reacted at 120 °C for 3 days. After cooling, the mixture was filtered and washed with 100 mL of tetrahydrofuran and acetone until a clear solution was obtained, yielding a deep red precipitate of crude COF(Fe). The precipitate was transferred to a Soxhlet extractor and washed alternately with 200 mL of tetrahydrofuran and acetone for 2 days. The product was dried to obtain 40 mg of COF(Fe) material.

[0046] Example 2

[0047] Preparation of Au NP

[0048] 10 mL of HAuCl4 (0.1% by mass) was added to a mixed solution of 40 mL of THF and 20 mL of water. After stirring at 500 rpm for one hour, 20 mL of 0.53 M NaBH4 solution was added, and the mixture was stirred overnight. After centrifugation, the product was washed three times with water and ethanol, and then freeze-dried for 24 h to obtain Au NP product.

[0049] Example 3

[0050] Preparation of Au@COF(Fe)

[0051] Take 20 mg of the COF(Fe) material synthesized in Example 1 and add it to a mixed solution of 5 mL DMF and 5 mL water. Stir at 500 rpm for 1 h, then add 0.5 mg Au NP to the above solution and stir at 500 rpm overnight. Centrifuge, wash the precipitate three times with water and ethanol, and dry it in a vacuum drying oven at 120 °C for 24 h to obtain Au@iron porphyrin COF product (Au@COF(Fe)). Disperse the product with 10 mL of ethanol to prepare a 0.1 mg / mL Au@COF(Fe) solution.

[0052] Example 4

[0053] Preparation of Au@COF(Fe)

[0054] Take 20 mg of the COF(Fe) material synthesized in Example 1 and add it to a mixed solution of 5 mL DMF and 5 mL water. After stirring at 500 rpm for 1 h, add 1 mg Au NP to the above solution and stir overnight. Centrifuge, wash the precipitate three times with water and ethanol, and dry it in a vacuum drying oven at 120 °C for 24 h to obtain the Au@iron porphyrin COF product (Au@COF(Fe)). Figure 2 As shown, the successful preparation of the sample was confirmed by powder X-ray diffraction. The product was then dispersed in 10 mL of ethanol to prepare a 0.1 mg / mL Au@COF(Fe) solution.

[0055] Example 5

[0056] Preparation of Au@COF(Fe)

[0057] Take 20 mg of the COF(Fe) material synthesized in Example 1 and add it to a mixed solution of 5 mL DMF and 5 mL water. Stir at 500 rpm for 1 h, then add 1.5 mg Au NP to the above solution and stir overnight. Centrifuge, wash the precipitate three times with water and ethanol, and dry it in a vacuum drying oven at 120 °C for 24 h to obtain Au@iron porphyrin COF product (Au@COF(Fe)). Finally, disperse the product with 10 mL ethanol to prepare a 0.1 mg / mL Au@COF(Fe) solution.

[0058] Example 6

[0059] Construction of FA / Au@COF(Fe) composite system

[0060] 60 mg of folic acid was fully dissolved in 100 mL of glacial acetic acid under magnetic stirring at 500 rpm. While stirring, the entire 10 mL of Au@COF(Fe) solution prepared in Example 4 was slowly added to the above solution, and the mixture was stirred at 500 rpm for 6 hours. Finally, after centrifugation, washing, and drying, a red powder was obtained, which is the FA / Au@COF(Fe) composite material.

[0061] Experimental Example 1

[0062] (1) Comparison of three proportions of gold-loaded Au@COF(Fe) composite systems

[0063] In a typical process, 1 mL of a simulated solution was prepared, and 100 μL of 0.1 mg / mL Au@COF(Fe) catalyst from Examples 3, 4, and 5, 100 μL of 0.08 mM TMB solution, and 120 μL of 10 mM H₂O₂ solution were added respectively. Water was added to bring the volume to 1 mL, and the reaction was allowed to proceed at room temperature for 2 minutes. The results were then measured using a UV spectrophotometer. Figure 3 As shown, the addition of the Au@COF(Fe) catalyst from Example 4 exhibits the best performance.

[0064] (1) Comparison of H2O2 detection results:

[0065] In a typical process, 1 mL of simulated solution is prepared, and 100 μL of 0.1 mg / mL Au@COF(Fe) catalyst from Example 4, 100 μL of 0.08 mM TMB solution, and 120 μL of 10 mM H2O2 solution are added. Water is added to bring the volume to 1 mL, and the reaction is carried out at room temperature for 2 minutes. By controlling the reaction conditions, the following six cases exist: 1) TMB; 2) TMB + H2O2; 3) TMB + H2O2 + Cat + Light; 4) TMB + H2O2 + Cat; 5) H2O2 + Cat; 6) TMB + Cat. Figure 4As shown, in the presence of Au@COF(Fe) catalyst (Cat), TMB, H2O2, and visible light (300W), the UV absorbance of this group is stronger than that of the other groups, indicating that the catalytic efficiency of Au@COF(Fe) is significantly improved under visible light-assisted conditions, demonstrating the best detection effect.

[0066] (2) Visible light-assisted colorimetric detection of H2O2

[0067] In a typical reaction, 1 mL of a simulated solution was prepared, and 100 μL of 0.1 mg / mL Au@COF(Fe) was added as a catalyst (prepared in Example 4). Then, 100 μL of 0.08 mM TMB solution and 120 μL of H2O2 solution (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mM) were added dropwise. After irradiation with 300 W visible light for two minutes, the final detection range of H2O2 was found to be 0.461-461 μM. Figure 5 The detection limit is 0.18 μM, which is far below the 15 μM limit for hydrogen peroxide permitted by the US FDA.

[0068] (3) Colorimetric detection of tumor cells

[0069] All cell lines were incubated at 37°C in a 5% CO2 incubator. Cells were placed in 96-well plates at a density of approximately 5000 cells per well and incubated for 24 hours. 100 μL of 0.1 mg / mL FA / Au@COF(Fe) sample obtained in Example 6 was added to the culture medium of DA-MB-231 (human breast cancer cells) and A549 (human lung cancer cells), respectively. After incubation for 4 hours, the glass slides in the culture medium were immediately washed with distilled water, and 100 μL of 0.08 mM TMB solution and 100 μL of 5 mM H2O2 solution were added. After irradiation with 300W visible light for 2 minutes, the cell state was observed using a fluorescence inverted microscope. The control group did not receive FA / Au@COF(Fe). The detection results for the two cancer cell lines are shown below. Figure 6 and Figure 7 As shown. By Figure 6 It can be seen that human breast cancer cells without FA / Au@COF(Fe) sample during culture did not show any color change, while MDA-MB-231 cells with FA / Au@COF(Fe) sample immediately turned blue. Figure 7 This also demonstrated that A549 cells with added FA / Au@COF(Fe) samples exhibited a blue color during colorimetric detection. This indicates that the FA / Au@COF(Fe)-based colorimetric immunoassay method is more convenient and faster than traditional immunoassay procedures.

[0070] The novel material prepared in this invention can be used for rapid colorimetric detection of H2O2 and tumor cells. It exhibits a unique catalytic reaction mechanism; Au@COF(Fe) itself possesses highly efficient catalytic oxidation properties, catalyzing the generation of hydroxyl radicals from H2O2. Simultaneously, under visible light irradiation, Au@COF(Fe) can activate oxygen to generate hydroxyl radicals, further enhancing catalytic efficiency. Therefore, the catalytic efficiency is high and the reaction is rapid.

[0071] Example 7

[0072] The preparation method of Example 1 was adopted, wherein the molar ratio of 5,10,15,20-tetra(p-aminophenyl)porphyrin iron and 2,3,6,7-tetra(4-formylphenyl)tetrathiofulvalene was 1:0.5, the volume ratio of acetic acid, 1,4-dioxane and 1,3,5-trimethylbenzene was 1:1:1, and the mixture was sonicated for 5 min, while other steps remained unchanged.

[0073] Example 8

[0074] The preparation method of Example 1 was adopted, wherein the molar ratio of 5,10,15,20-tetra(p-aminophenyl)porphyrin iron and 2,3,6,7-tetra(4-formylphenyl)tetrathiofulvalene was 1:2, the volume ratio of acetic acid, 1,4-dioxane and 1,3,5-trimethylbenzene was 1:10:10, and the mixture was sonicated for 90 min, while other steps remained unchanged.

[0075] Example 9

[0076] The preparation method of Example 2 was used, wherein the volume ratio of HAuCl4 to NaBH4 was 1:1, the volume ratio of THF to water was 1:1, and other steps remained unchanged.

[0077] Example 10

[0078] The preparation method of Example 2 was used, wherein the volume ratio of HAuCl4 to NaBH4 was 1:10, the volume ratio of THF to water was 10:1, and other steps remained unchanged.

Claims

1. A FA / Au@COF(Fe) composite system, characterized in that, The composite system includes folic acid and Au@COF(Fe), wherein Au@COF(Fe) is composed of COF(Fe) and Au NP; The preparation method of the FA / Au@COF(Fe) composite system specifically includes the following steps: (1) First, add iron porphyrin and tetrathione monomer, then add acetic acid, 1,4-dioxane, and 1,3,5-trimethylbenzene, sonicate, cool, degas, wash and collect to obtain crude COF(Fe) product, then wash with tetrahydrofuran and acetone alternately, and dry the product to obtain pure COF(Fe) material; (2) Add HAuCl4 to a mixed solution of THF and water, stir, add NaBH4 solution, centrifuge and wash, freeze dry to obtain Au NP product; (3) Add the Au NP prepared in step (2) to the mixed solution of DMF and water of the COF(Fe) material prepared in step (1) and stir overnight. After centrifugation and washing, vacuum drying is performed to obtain Au@COF(Fe) material, which is then dispersed in an alcohol solvent to obtain Au@COF(Fe) solution. (4) Add the Au@COF(Fe) solution prepared in step (3) to a solution containing folic acid, centrifuge, wash, filter and dry to obtain FA / Au@COF(Fe).

2. The FA / Au@COF(Fe) composite system according to claim 1, characterized in that, In step (1), the molar ratio of iron porphyrin and tetrathiofulvalene monomers is 1:0.5-2, the volume ratio of acetic acid, 1,4-dioxane and 1,3,5-trimethylbenzene is 1:1-10:1-10, and the ultrasonic treatment time is 5-90 min.

3. The FA / Au@COF(Fe) composite system according to claim 1, characterized in that, In step (2), the volume ratio of HAuCl4 to NaBH4 is 1:1-10, and the volume ratio of THF to water is 1-10:

1.

4. The FA / Au@COF(Fe) composite system according to claim 1, characterized in that, In step (3), the final concentration of COF(Fe) in the mixed solution of DMF and water of COF(Fe) material is 1-5 mg / mL.

5. The FA / Au@COF(Fe) composite system according to claim 1, characterized in that, In step (3), the alcohol solvent is any one of methanol, ethanol or isopropanol, and in step (4), the folic acid solution solvent is acetic acid.

6. The application of the FA / Au@COF(Fe) composite system according to claim 1 in the field of rapid detection of H2O2.

7. The application according to claim 6, characterized in that, The application process is as follows: add TMB solution and H2O2 solution to Au@COF(Fe) solution, and H2O2 can be detected by color development by irradiation with visible light.

8. The application according to claim 7, characterized in that, The visible light irradiation time is 1-5 minutes.

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