Star polymers covalently anchoring porphyrin indicators, methods of making and use
Patent Information
- Application Number
- CN202610624134.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-08
- Publication Date
- 2026-08-21
AI Technical Summary
[0003]由于PTTFPP分子具有强疏水性，直接注入生物体容易产生聚集并引起严重的毒副反应，现有技术中，采用两亲性嵌段共聚物（如聚乙二醇-聚己内酯，PEG-PCL）通过自组装方式，将PTTFPP分子包裹在内，形成胶束结构以改善毒副反应，然而存在热力学不稳定、指示剂分布不均、基质干扰与响应迟滞等缺陷
本发明的共价锚定卟啉指示剂的星型聚合物具有卓越的热力学稳定性、高度的检测灵敏度、低毒性、结构可调整性。
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Figure CN122608891A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fluorescent indicator technology, specifically to a star polymer of a covalently anchored porphyrin indicator, its preparation method, and its application. Background Technology
[0002] Dissolved oxygen (DO) is one of the core parameters for maintaining the life activities of organisms. Currently, optical oxygen sensors based on fluorescence or phosphorescence quenching principles have become a research hotspot for oxygen detection in living organisms. Among them, fluorescent indicators such as tetratetrafluorophenyl porphyrin platinum (PTTFPP) and Ru(dpp)3(PF6)2 are widely used as oxygen-sensitive indicators due to their high quantum yield, long phosphorescence lifetime, and excellent photochemical stability.
[0003] Because PTTFPP molecules are highly hydrophobic, direct injection into organisms can easily lead to aggregation and severe toxic side effects. Existing technologies use amphiphilic block copolymers (such as polyethylene glycol-polycaprolactone, PEG-PCL) to encapsulate PTTFPP molecules through self-assembly, forming micelle structures to mitigate toxic side effects. However, this approach suffers from drawbacks such as thermodynamic instability, uneven indicator distribution, matrix interference, and hysteresis. In summary, this invention proposes a star-shaped polymer for covalently anchored porphyrin indicators, its preparation method, and its application. By developing a structurally stable, sensitive, efficient, and low-toxicity fluorescent polymer, accurate dissolved oxygen detection can be achieved. Summary of the Invention
[0004] The purpose of this invention is to provide a star polymer for covalently anchoring porphyrin indicators, its preparation method, and its application, so as to solve the related technical problems existing in the prior art.
[0005] To achieve the above objectives, the present invention provides the following specific technical solutions: A method for preparing a star polymer covalently anchored to a porphyrin indicator includes the following steps: Step 1: Dissolve the hydroxyl-terminated hyperbranched polyester and maleic anhydride in anhydrous N,N-dimethylformamide (DMF), then add 4-dimethylaminopyridine (DMAP) for reaction. After the reaction is complete, dialyze, filter the retentate, and freeze-dry to obtain the modified hydroxyl-terminated hyperbranched polyester (H40-Mah-COOH). Step 2: H40-Mah-COOH was dissolved in DMF, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added sequentially for activation. After activation, methoxyamino-terminated polyethylene glycol (mPEG-NH2) was added for reaction. After the reaction was completed, the mixture was dialyzed, the retentate was filtered, and the mixture was freeze-dried to obtain hyperbranched polymer nanocarrier (H40-Mah-mPEG2). Step 3: Dissolve H40-Mah-mPEG2 in DMF, add triethylamine (TEA), add methyl mercaptoacetate dropwise, react, dialyze after the reaction is complete, filter the retentate, and freeze dry; Step 4: Dissolve the product obtained in the previous step in DMF, add anhydrous ethanol and hydrazine hydrate, heat the reaction, dialyze after the reaction is complete, filter the retentate, and freeze dry. Step 5: Dissolve the product obtained in the previous step in DMF, add tetratetra(pentafluorophenyl)porphyrin platinum (PTTFPP) fluorescent indicator, react, dialyze after the reaction is complete, filter, collect the filtrate, freeze dry, and obtain the finished product.
[0006] In a more optimized manner, in step one, the molar ratio of hydroxyl groups to maleic anhydride in the hydroxyl-terminated hyperbranched polyester is 1:(1~2).
[0007] In a more optimized manner, in step one, the mass ratio of DMAP to maleic anhydride is 1:(15~25).
[0008] In a more optimized manner, in step two, the molar ratio of H40-Mah-COOH to EDC is 1:(3~5), and the molar ratio of EDC to NHS is (1~3):1.
[0009] In a more optimized manner, in step three, the molar ratio of the double bond to methyl mercaptoacetate in H40-Mah-mPEG2 is 1:(1~3).
[0010] Ideally, in step three, the amount of TEA added is 1-5% of the total mass of the reaction system.
[0011] In a more optimized manner, in step four, the molar ratio of methyl ester groups to hydrazine hydrate in the reactants is 1:(9~11).
[0012] In a more optimized manner, in step four, the amount of anhydrous ethanol added is 10-20% of the total mass of the reaction system.
[0013] Ideally, DMF is used as the solvent, and the mass ratio of reactants to DMF is 1:(20~30).
[0014] In a more optimized manner, the reaction conditions in step one are: reaction at room temperature for 20-25 hours.
[0015] In a more optimized manner, in step one, the specific process of dialysis is as follows: the reaction solution is transferred to a cellulose dialysis bag and dialyzed in deionized water for 20-25 hours.
[0016] In a more optimized manner, in step two, the activation process conditions are: activation for 1-2 hours at room temperature; and the reaction process conditions are: reaction for 20-25 hours at room temperature.
[0017] In a more optimized manner, in step two, the specific process of dialysis is as follows: the reaction solution is transferred to a cellulose dialysis bag and dialyzed in deionized water for 20-25 hours.
[0018] In a more optimized manner, in step three, methyl mercaptoacetate is added dropwise at a rate of 1-2 drops / second; the reaction conditions are: at room temperature, the reaction is carried out for 20-25 hours; the specific dialysis process is: the reaction solution is transferred to a cellulose dialysis bag and dialyzed in deionized water for 20-25 hours, during which the dialysis solution is replaced 3-4 times.
[0019] In a more optimized manner, in step four, the heating reaction conditions are as follows: the reaction is carried out at a temperature of 60~65℃ for 20~25 hours; the specific dialysis process is as follows: the reaction solution is transferred to a cellulose dialysis bag, dialyzed in anhydrous ethanol for 20~25 hours, and then dialyzed in deionized water for another 20~25 hours.
[0020] In a more optimized manner, in step five, the reaction process conditions are as follows: the reaction is carried out at room temperature and in the dark for 20 to 25 hours with stirring; the specific process of dialysis is as follows: the reaction solution is transferred to a cellulose dialysis bag and placed in deionized water for dialysis for 45 to 50 hours, during which the dialysis solution is changed 6 to 7 times until there is no obvious free PTTFPP seepage outside the dialysis bag.
[0021] Ideally, the molecular weight cutoff of the cellulose dialysis bag is 3500-4000.
[0022] In a more optimized manner, the freeze-drying process conditions are: freeze-drying at a temperature of -20 to -10°C for 20 to 25 hours.
[0023] In the above technical solution, under the action of the catalyst 4-dimethylaminopyridine, carboxyl groups are introduced into the hyperbranched polyester molecular chain by reacting maleic anhydride with the hydroxyl groups in the hyperbranched polyester. Under the action of condensing agents EDC·HCl and NHS, the carboxyl groups further react with the amino groups in mPEG-NH2, incorporating PEG segments into the hyperbranched polyester molecular chain. The double bonds in the reactants from the previous step, which are derived from the maleamide structure, further undergo a thiol-Michael addition reaction with the thiol groups in methyl mercaptoacetate, introducing thioether bonds and methyl ester groups. The methyl ester groups further undergo a nucleophilic addition-elimination reaction with hydrazine hydrate to obtain a hyperbranched polymer intermediate with hydrazide active groups in the side chain. The hydrazide active groups are coupled with PTTFPP to obtain a star polymer covalently coupled with a PTTFPP probe.
[0024] The hydroxyl-terminated hyperbranched polyester, serving as the geometric center and covalently anchored backbone of the star polymer, provides mechanical support to ensure stability. Maleic anhydride ring-opening constructs a hydrophobic core, while grafted polyethylene glycol segments form a hydrophilic shell. Through chemical covalent reactions, functionalized groups are grafted into the covalently anchored backbone molecular chain, improving the thermodynamic stability of the indicator. Even at concentrations below the critical micelle concentration (CMC) during blood circulation in vivo, it will not disintegrate, preventing indicator leakage and toxic side effects. The hyperbranched structure of the fluorescent indicator in this invention provides significant steric hindrance, effectively preventing aggregation quenching (ACQ) between fluorescent indicator molecules. The PEG shell acts as a high-density hydrophilic shielding layer, effectively resisting non-specific adsorption of plasma proteins and reducing clearance by the mononuclear macrophage system, thereby extending the effective monitoring time of the fluorescent indicator in vivo. Furthermore, the star polymer probe prepared in this invention has structural tunability, allowing for changes in the molecular weight of the covalently anchored backbone hyperbranched polyester and maleic anhydride, enabling precise control of probe particle size and oxygen sensitivity range.
[0025] Application of a star polymer covalently anchored to a porphyrin indicator for detecting dissolved oxygen levels in living organisms.
[0026] Compared with the prior art, the beneficial effects of the present invention are: The star polymer of the covalently anchored porphyrin indicator of the present invention has excellent thermodynamic stability, high detection sensitivity, low toxicity, and structural adjustability. Attached Figure Description
[0027] Figure 1 The chemical reaction formula for the preparation process in Example 1 of this invention; Figure 2 This is the dynamic light scattering particle size analysis spectrum of the probe in Embodiment 1 of the present invention; Figure 3 This is a transmission electron microscope image of the probe in Embodiment 1 of the present invention; Figure 4 The responsiveness and reproducibility of the probe in Example 1 of this invention in dissolved oxygen at 0~20ppm; Figure 5 The T90 response of probe in Embodiment 1 of the present invention; Figure 6 This is the stability of Embodiment 1 of the present invention after 6 months of online use. Detailed Implementation
[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be understood that the embodiments described herein are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the protection scope of the present invention; unless otherwise specified, the related raw materials are all conventional settings.
[0029] In the following examples, the relevant raw materials are: hydroxyl-terminated hyperbranched polyester: Boltron H40; maleic anhydride, CAS: 108-31-6; mPEG-NH2, CAS: 80506-64-5, average molecular weight 5000; methyl mercaptoacetate, CAS: 2365-48-2; hydrazine hydrate, CAS: 7803-57-8; PTTFPP, CAS: 109781-47-7; the remaining raw materials are commercially available. Example 1:
[0030] A method for preparing a star polymer covalently anchored to a porphyrin indicator includes the following steps: Step 1: Dissolve the hydroxyl-terminated hyperbranched polyester and maleic anhydride in anhydrous DMF, then add 4-dimethylaminopyridine and stir the reaction at room temperature for 24 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 24 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 24 hours to obtain H40-Mah-COOH. The molar ratio of hydroxyl groups to maleic anhydride in the hydroxyl-terminated hyperbranched polyester is 1:1.5; the mass ratio of DMAP to maleic anhydride is 1:20. Step 2: Dissolve H40-Mah-COOH in DMF, add EDC·HCl and NHS sequentially, activate at room temperature for 1 hour, then add mPEG-NH2 and continue the reaction for 24 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 24 hours. After dialysis, filter the retentate and freeze-dry at -20℃ for 24 hours to obtain H40-Mah-mPEG2; the molar ratio of H40-Mah-COOH to EDC is 1:4, and the molar ratio of EDC to NHS is 2:1. Step 3: Dissolve H40-Mah-mPEG2 in DMF, add TEA, and add methyl mercaptoacetate dropwise at a rate of 1 drop / second. Stir continuously at room temperature for 24 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 24 hours, changing the dialysate 3 times during this period. After dialysis, filter the retentate and freeze-dry at -20°C for 24 hours. The molar ratio of the double bond in H40-Mah-mPEG2 to methyl mercaptoacetate is 1:2; the amount of TEA added is 3% of the total mass of the reaction system. Step 4: Dissolve the product obtained in the previous step in DMF, add anhydrous ethanol and hydrazine hydrate, and heat the mixture at 60°C for 24 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in anhydrous ethanol for 24 hours, then dialyze it in deionized water for another 24 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 24 hours. The molar ratio of methyl ester groups to hydrazine hydrate in the reactants is 1:10. Step 5: Dissolve the product obtained in the previous step in DMF, add PTTFPP fluorescent indicator, and stir the reaction for 24 hours at room temperature and in the dark. After the reaction is completed, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 48 hours, changing the dialysis solution 6 times during the process, until no obvious free PTTFPP seeps out of the dialysis bag. Finally, filter the solution, collect the filtrate, and freeze-dry it at -20℃ for 24 hours to obtain the finished product. The molar ratio of hydrazide groups to PTTFPP in the reactants is 1:1; the amount of anhydrous ethanol added is 10% of the total mass of the reaction system; in steps 1 to 5, the mass ratio of reactants to DMF is 1:20. Example 2:
[0031] A method for preparing a star polymer covalently anchored to a porphyrin indicator includes the following steps: Step 1: Dissolve the hydroxyl-terminated hyperbranched polyester and maleic anhydride in anhydrous DMF, then add 4-dimethylaminopyridine and stir the mixture at room temperature for 20 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 20 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 20 hours to obtain H40-Mah-COOH. The molar ratio of hydroxyl groups to maleic anhydride in the hydroxyl-terminated hyperbranched polyester is 1:1; the mass ratio of DMAP to maleic anhydride is 1:15. Step 2: Dissolve H40-Mah-COOH in DMF, add EDC·HCl and NHS sequentially, activate at room temperature for 1 hour, then add mPEG-NH2 and continue the reaction for 20 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 20 hours. After dialysis, filter the retentate and freeze-dry at -20℃ for 20 hours to obtain H40-Mah-mPEG2; the molar ratio of H40-Mah-COOH to EDC is 1:3, and the molar ratio of EDC to NHS is 1:1. Step 3: Dissolve H40-Mah-mPEG2 in DMF, add TEA, and add methyl mercaptoacetate dropwise at a rate of 1 drop / second. Stir continuously at room temperature for 20 hours. After the reaction, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 20 hours, changing the dialysate 3 times during this period. After dialysis, filter the retentate and freeze-dry at -20°C for 20 hours. The molar ratio of the double bond in H40-Mah-mPEG2 to methyl mercaptoacetate is 1:1; the amount of TEA added is 3% of the total mass of the reaction system. Step 4: Dissolve the product obtained in the previous step in DMF, add anhydrous ethanol and hydrazine hydrate, and heat the mixture at 60°C for 20 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in anhydrous ethanol for 20 hours, then dialyze it in deionized water for another 20 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 20 hours. The molar ratio of methyl ester groups to hydrazine hydrate in the reactants is 1:9. Step 5: Dissolve the product obtained in the previous step in DMF, add PTTFPP fluorescent indicator, and stir the reaction for 20 hours at room temperature and in the dark. After the reaction is completed, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 45 hours, changing the dialysis solution 6 times during the process, until no obvious free PTTFPP seeps out of the dialysis bag. Finally, filter the solution, collect the filtrate, and freeze-dry it at -20℃ for 20 hours to obtain the finished product. The molar ratio of hydrazide groups to PTTFPP in the reactants is 1:1; the amount of anhydrous ethanol added is 10% of the total mass of the reaction system; in steps 1 to 5, the mass ratio of reactants to DMF is 1:20. Example 3:
[0032] A method for preparing a star polymer covalently anchored to a porphyrin indicator includes the following steps: Step 1: Dissolve the hydroxyl-terminated hyperbranched polyester and maleic anhydride in anhydrous DMF, then add 4-dimethylaminopyridine and stir the mixture at room temperature for 25 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 25 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 25 hours to obtain H40-Mah-COOH. The molar ratio of hydroxyl groups to maleic anhydride in the hydroxyl-terminated hyperbranched polyester is 1:2; the mass ratio of DMAP to maleic anhydride is 1:25. Step 2: Dissolve H40-Mah-COOH in DMF, add EDC·HCl and NHS sequentially, activate at room temperature for 1 hour, then add mPEG-NH2 and continue the reaction for 25 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 25 hours. After dialysis, filter the retentate and freeze-dry at -20℃ for 25 hours to obtain H40-Mah-mPEG2; the molar ratio of H40-Mah-COOH to EDC is 1:5, and the molar ratio of EDC to NHS is 3:1. Step 3: Dissolve H40-Mah-mPEG2 in DMF, add TEA, and add methyl mercaptoacetate dropwise at a rate of 1 drop / second. Stir continuously at room temperature for 25 hours. After the reaction, transfer the reaction solution to a cellulose dialysis bag and dialyze in deionized water for 25 hours, changing the dialysate 3 times during this period. After dialysis, filter the retentate and freeze-dry at -20°C for 25 hours. The molar ratio of the double bond in H40-Mah-mPEG2 to methyl mercaptoacetate is 1:3; the amount of TEA added is 3% of the total mass of the reaction system. Step 4: Dissolve the product obtained in the previous step in DMF, add anhydrous ethanol and hydrazine hydrate, and heat the mixture at 65°C for 25 hours. After the reaction is complete, transfer the reaction solution to a cellulose dialysis bag and dialyze it in anhydrous ethanol for 25 hours, then dialyze it in deionized water for another 25 hours. After dialysis, filter the retentate and freeze-dry it at -20°C for 25 hours. The molar ratio of methyl ester groups to hydrazine hydrate in the reactants is 1:11. Step 5: Dissolve the product obtained in the previous step in DMF, add PTTFPP fluorescent indicator, and stir the reaction for 25 hours at room temperature and in the dark. After the reaction is completed, transfer the reaction solution to a cellulose dialysis bag and dialyze it in deionized water for 50 hours, changing the dialysis solution 7 times during the process, until no obvious free PTTFPP seeps out of the dialysis bag. Finally, filter the solution, collect the filtrate, and freeze-dry it at -20℃ for 25 hours to obtain the finished product. The molar ratio of hydrazide groups to PTTFPP in the reactants is 1:1; the amount of anhydrous ethanol added is 10% of the total mass of the reaction system; in steps 1 to 5, the mass ratio of reactants to DMF is 1:20.
[0033] Comparative Example 1: This comparative example provides a method for preparing a covalently anchored porphyrin indicator polymer, which uses physical encapsulation to coat PTTFPP within a hydrophilic polymer.
[0034] Comparative Example 2: This comparative example provides a method for preparing a linear polymer of covalently anchored porphyrin indicator, which is obtained by grafting PTTFPP onto a linear polymer.
[0035] Experimental test: The star-shaped polymer prepared in Example 1 above was used as a sample for performance testing; Stability testing method: After using the sample online for 6 months, test the dissolved oxygen response of the sample and record the stability of the sample before and after storage; Transmission electron microscopy (TEM) testing method: The structural and morphological characteristics of the sample are observed by scanning with a transmission electron microscope (TEM). Dissolved oxygen response test method: Test the response and reproducibility of the sample at dissolved oxygen concentrations of 0-20 ppm; Dissolved oxygen T90 response test: Test the response and reproducibility of dissolved oxygen T90 in the test samples; Cytotoxicity assay method: The MTT (thiazolyl blue) method was used to test the cytotoxicity of the samples.
[0036] The experimental data are as follows: Table 1
[0037] Table 2
[0038] Table 3
[0039] Results and Discussion: As can be seen from the data in the table above, the star-shaped polymer of this application has the characteristics of high sensitivity and good stability for dissolved oxygen detection.
[0040] Compared with Example 1, Comparative Example 1 uses a physical encapsulation process to encapsulate PTTFPP in a hydrophilic polymer, which reduces stability and easily leads to toxic side reactions. This demonstrates the technical advantage of the present invention, which uses chemical covalent grafting of PTTFPP to improve stability.
[0041] Compared with Example 1, Comparative Example 2 uses linear copolymer covalently grafted PTTFPP. Although the stability is improved compared with Comparative Example 1, it is not as stable as the star-shaped polymer fluorescent indicator prepared by the present invention, which reflects the technical advantages of the hyperbranched polymer preparation process of the present invention.
[0042] For those skilled in the art, the present invention is obviously not limited to the details disclosed in the exemplary embodiments described above. The present invention can be implemented in other specific ways without departing from its spirit and essential characteristics. Therefore, the foregoing embodiments should be considered exemplary rather than restrictive from any perspective. The scope of protection of the present invention is defined by the appended claims. Therefore, all variations falling within the meaning and scope of the equivalent elements of the claims should be attributed to the scope of the present invention.
Claims
1. A method for preparing a star polymer covalently anchored to a porphyrin indicator, characterized in that: Includes the following steps: Step 1: Dissolve the hydroxyl-terminated hyperbranched polyester and maleic anhydride in anhydrous DMF, then add DMAP to react. After the reaction is complete, dialyze, filter the retentate, freeze dry, and obtain the modified hydroxyl-terminated hyperbranched polyester. Step 2: Dissolve the modified hydroxyl-terminated hyperbranched polyester in DMF, add EDC·HCl and NHS sequentially, activate, add mPEG-NH2 to react, after the reaction is complete, dialyze, filter the retentate, freeze dry to obtain hyperbranched polymer nanocarrier; Step 3: Dissolve the hyperbranched polymer nanocarrier in DMF, add TEA, add methyl mercaptoacetate dropwise, react, dialyze after the reaction is complete, filter the retentate, and freeze-dry. Step 4: Dissolve the product obtained in the previous step in DMF, add anhydrous ethanol and hydrazine hydrate, heat the reaction, dialyze after the reaction is complete, filter the retentate, and freeze dry. Step 5: Dissolve the product obtained in the previous step in DMF, add PTTFPP fluorescent indicator, react, dialyze after the reaction is complete, filter, collect the filtrate, freeze dry, and obtain the finished product.
2. The method for preparing a star polymer covalently anchored to a porphyrin indicator according to claim 1, characterized in that: In step one, the molar ratio of hydroxyl groups to maleic anhydride in the hydroxyl-terminated hyperbranched polyester is 1:(1~2).
3. The method for preparing a star polymer covalently anchored to a porphyrin indicator according to claim 2, characterized in that: In step one, the mass ratio of DMAP to maleic anhydride is 1:(15~25).
4. The method for preparing a star polymer of a covalently anchored porphyrin indicator according to claim 1, characterized in that: In step two, the molar ratio of modified hydroxyl-terminated hyperbranched polyester to EDC is 1:(3~5), and the molar ratio of EDC to NHS is (1~3):
1.
5. The method for preparing a star polymer covalently anchored to a porphyrin indicator according to claim 1, characterized in that: In step three, the molar ratio of double bonds to methyl mercaptoacetate in the hyperbranched polymer nanocarrier is 1:(1~3).
6. The method for preparing a star polymer covalently anchored to a porphyrin indicator according to claim 1, characterized in that: In step three, the amount of TEA added is 1-5% of the total mass of the reaction system.
7. The method for preparing a star polymer of a covalently anchored porphyrin indicator according to claim 1, characterized in that: In step four, the molar ratio of methyl ester groups to hydrazine hydrate in the reactants is 1:(9~11).
8. The method for preparing a star polymer of a covalently anchored porphyrin indicator according to claim 1, characterized in that: The molecular weight cutoff of the cellulose dialysis bag is 3500~4000.
9. The method for preparing a star polymer of a covalently anchored porphyrin indicator according to claim 1, characterized in that: The freeze-drying process conditions are as follows: freeze-drying at a temperature of -20 to -10°C for 20 to 25 hours.
10. The application of a star polymer covalently anchored to a porphyrin indicator, characterized in that: It is used to detect dissolved oxygen levels in living organisms.