Ferroporphyrin conjugated acetylene polymer peroxidase material as well as preparation method and application thereof

Abstract

The invention discloses a ferriporphyrin conjugated acetylene polymer peroxidase-like material as well as a preparation method and application thereof, and belongs to the field of functional polymer materials, a ferriporphyrin structural unit is connected with 1, 4-diacetylene benzene through a Sonogashira coupling reaction to construct a polymer skeleton with an extended conjugated structure, the ferriporphyrin unit is used as a peroxidase-like active center, and the polymer skeleton with the extended conjugated structure is used as a peroxidase-like active center. The material has a porous structure (the specific surface area is 100-800 m / g) and excellent catalytic activity (the specific surface area is 1.0-2.5 mM), hydrogen peroxide can be detected with high sensitivity, the detection limit reaches 0.5 mu M, and the detection range is 0.5-500 mu M. The material is simple and convenient in preparation method, good in chemical stability and thermal stability and recyclable, is an ideal substitute of natural peroxidase, and has wide application prospects. The method has wide application prospects in the fields of biological analysis, clinical diagnosis, food safety, environment monitoring and the like.

Description

Technical Field

[0001] This invention belongs to the field of functional polymer materials, specifically relating to iron porphyrin conjugated acetylene polymer peroxidase materials, their preparation methods and applications, and particularly to the application of this material in the colorimetric detection of hydrogen peroxide. Background Technology

[0002] hydrogen peroxide ( As an important reactive oxygen species, it participates in various physiological processes in organisms, such as cell signal transduction and immune defense, and is also a substrate or product of many enzymatic reactions. Abnormal changes in concentration are closely related to various diseases such as oxidative stress, aging, and cancer. Therefore, establishing a sensitive and selective [system / mechanism] is crucial. Detection methods are of great significance for biomedical research and clinical diagnosis. Furthermore, It is also widely used in the food industry, environmental monitoring, chemical production and other fields. The accurate determination of its concentration is crucial for product quality control and environmental safety.

[0003] Natural peroxidases such as horseradish peroxidase (HRP) can catalyze The color change produced by the oxidation of the chromogenic substrate is a classic method. Natural enzymes are the core components of colorimetric detection methods. However, natural enzymes have inherent drawbacks such as high preparation costs, poor stability, easy inactivation, and harsh storage conditions, which severely limit their widespread application in practice. To overcome these problems, in recent years, researchers have focused on developing nanomaterials with enzyme-like activity (nanozymes) in order to replace natural enzymes in the fields of bioanalysis and catalysis.

[0004] Various nanoenzyme materials have been reported in the prior art, such as metal oxide nanoparticles ( , Examples of active materials include noble metal nanoparticles, carbon-based nanomaterials, and metal-organic frameworks. The active center of natural heme peroxidases is an iron porphyrin structure, where the conjugated structure of the porphyrin macrocycle facilitates electron transfer, and the central iron ion acts as the catalytic active site. The decomposition of enzymes and the oxidation of substrates are significant challenges. Therefore, incorporating iron porphyrin structures into polymer frameworks is an important strategy for constructing highly active peroxidase-like materials. Conjugated microporous polymers (CMPs) are a new class of porous organic polymer materials whose framework is composed of conjugated units linked by covalent bonds. They possess advantages such as high specific surface area, abundant pore structure, good chemical stability, and tunable electronic structure. Alkyne bonding is one of the important ways to construct conjugated microporous polymers. The Sonogashira coupling reaction can achieve efficient linkage between aromatic halogenated compounds and terminal alkynes to form a rigid conjugated polymer framework.

[0005] However, there are currently no examples of constructing conjugated acetylene polymers by linking iron porphyrin structural units through alkyne bonds, and applying them to peroxidase-like catalysis and... Research reports on colorimetric detection. Therefore, there is an urgent need to develop a novel iron porphyrin-based peroxidase material with high catalytic activity, good stability, and ease of preparation to meet the needs. The practical need for high-sensitivity and high-selectivity detection. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an iron porphyrin conjugated acetylene polymer-based peroxidase material, its preparation method, and its applications. This material connects iron porphyrin structural units to 1,4-diacetylenebenzene via a Sonogashira coupling reaction, constructing a polymer backbone with an extended conjugated structure. The iron porphyrin unit serves as the active center for the peroxidase-like enzyme. The porous structure of the polymer facilitates mass transfer of substrate molecules and sufficient exposure of active sites, thereby enabling peroxidase activity. High-sensitivity colorimetric detection.

[0007] To achieve the above objectives, the first aspect of the present invention provides an iron porphyrin conjugated acetylene polymer peroxidase material, the material comprising an iron porphyrin structural unit and a benzene ring structural unit linked by an alkyne bond; wherein the iron porphyrin structural unit is 5,10,15,20-tetra(4-substituted phenyl)iron porphyrin, and the substituent is selected from at least one of bromo, iodo, and acetylene groups; the benzene ring structural unit is 1,4-diacetylenene; the iron porphyrin structural unit and the benzene ring structural unit are linked by an alkyne bond formed by a Sonogashira coupling reaction to form a conjugated acetylene polymer backbone; the number average molecular weight of the polymer is 5000-50000 Da.

[0008] Preferably, the iron porphyrin structural unit is 5,10,15,20-tetra(4-bromophenyl)iron porphyrin or 5,10,15,20-tetra(4-iodophenyl)iron porphyrin.

[0009] Preferably, the molar ratio of iron porphyrin structural units to 1,4-diacetylenebenzene structural units in the polymer is 1:(2-20), more preferably 1:(5-15), and even more preferably 1:10.

[0010] Preferably, the material has a porous structure and a specific surface area of ​​100-800 m². / g, with a pore size distribution range of 1-50nm.

[0011] Preferably, the material has an apparent Michaelis constant K for 3,3',5,5'-tetramethylbenzidine (TMB). The concentration is 0.5-1.5 mM. The apparent Michaelis constant K It ranges from 1.0 to 2.5 mM.

[0012] A second aspect of the present invention provides a method for preparing the iron porphyrin conjugated acetylene polymer peroxidase material, comprising the following steps:

[0013] (1) Monomer preparation: 5,10,15,20-tetra(4-bromophenyl)porphyrin or 5,10,15,20-tetra(4-iodophenyl)porphyrin is reacted with iron salt in a solvent to obtain 5,10,15,20-tetra(4-bromophenyl)ironporphyrin or 5,10,15,20-tetra(4-iodophenyl)ironporphyrin;

[0014] (2) Sonogashira coupling polymerization: The iron porphyrin monomer obtained in step (1) and 1,4-diacetylenebenzene were coupled and polymerized in an organic solvent in the presence of a palladium catalyst, a copper co-catalyst, and a base. The reaction temperature was 60-100℃ and the reaction time was 12-72 h.

[0015] (3) Product processing: After the reaction is completed, the solid product is filtered and washed with methanol, acetone and tetrahydrofuran in sequence, and dried under vacuum to obtain iron porphyrin conjugated acetylene polymer peroxidase material.

[0016] Preferably, the iron salt mentioned in step (1) is , , or One or more of the following: the solvent is N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or pyridine; the reaction temperature is 120-160℃, and the reaction time is 4-12 h.

[0017] Preferably, the palladium catalyst in step (2) is , or One of them; the copper co-catalyst is , or The base is triethylamine ( The organic solvent is piperidine or diisopropylethylamine (DIPEA); the organic solvent is DMF, tetrahydrofuran (THF) or toluene; the molar ratio of the iron porphyrin monomer to 1,4-diacetylenebenzene is 1:(2-20), the amount of palladium catalyst is 5-15% of the molar amount of iron porphyrin monomer, and the amount of copper co-catalyst is 10-30% of the molar amount of iron porphyrin monomer.

[0018] A third aspect of the present invention provides the application of the iron porphyrin conjugated acetylene polymer peroxidase material in the preparation of a colorimetric reagent for hydrogen peroxide detection.

[0019] Preferably, the hydrogen peroxide colorimetric detection method includes the following steps: mixing a dispersion of iron porphyrin conjugated acetylene polymer peroxidase material, a 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the sample to be tested; reacting at 25-45℃ for 10-60 min; measuring the absorbance at 652 nm using a UV-Vis spectrophotometer; and calculating based on the absorbance value. Concentration; the The detection range is 0.5-500 μM, and the detection limit is 0.5 μM.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) Innovative polymer backbone design: This invention constructs an iron porphyrin conjugated acetylene polymer via the Sonogashira coupling reaction. The rigid conjugated backbone formed by the alkyne bonds promotes efficient electron transport along the polymer chain, enhances the electronic interaction between the iron porphyrin active center and the polymer backbone, and significantly improves catalytic activity. Compared with existing technologies using non-conjugated linkage methods such as the Williams ether reaction, the conjugated structure of this invention improves catalytic efficiency by approximately 2-3 times.

[0022] (2) Excellent peroxidase-like activity: The material of this invention exhibits an apparent Michaelis constant K for TMB. The concentration is 0.5-1.5 mM. The apparent Michaelis constant K The concentrations ranged from 1.0 to 2.5 mM, all lower than the K+ of natural horseradish peroxidase (HRP) for TMB. Approximately 0.434 mM, for K The concentration (approximately 3.7 mM) indicates that the material of this invention has higher affinity for the substrate and higher catalytic efficiency.

[0023] (3) High sensitivity detection performance: The material of this invention is used for Colorimetric detection achieves a detection limit of 0.5 μM, significantly lower than the 1.37 μM reported in existing technologies, and improves detection sensitivity by approximately 2.7 times. The detection range covers 0.5-500 μM, meeting the detection needs of various application scenarios.

[0024] (4) Good structural stability: The material of this invention constructs a polymer framework through covalent bonds, and the iron porphyrin units are firmly anchored in the polymer structure, effectively preventing the shedding and deactivation of the active components. The rigid polymer framework endows the material with good chemical and thermal stability, and it can maintain high catalytic activity in a wide pH range (3-9) and temperature range (25-60℃).

[0025] (5) Advantages of porous structure: The material of the present invention has a porous structure with a specific surface area of ​​100-800 m². / g, the abundant pore structure is conducive to the rapid mass transfer and diffusion of substrate molecules, so that the active sites are fully exposed and utilized, and the catalytic efficiency is further improved.

[0026] (6) Simple preparation method: This invention uses the classic Sonogashira coupling polymerization reaction, which has mild reaction conditions, is easy to operate, and uses readily available raw materials, making it suitable for large-scale preparation. The polymerization product only requires simple filtration and washing to obtain a high-purity product, without the need for complicated post-processing steps.

[0027] (7) Broad application prospects: The material of this invention can be used as an artificial enzyme in multiple fields such as bioanalysis and detection, clinical diagnosis, food safety, and environmental monitoring. It is expected to replace expensive and unstable natural enzymes, and has important scientific value and broad application prospects. Attached Figure Description

[0028] Figure 1 Schematic diagram of the structure of :P(DEB-FeTBrPP) polymer.

[0029] Figure 2 UV-Vis DRS of P(DEB-FeTBrPP) and FeTBrPP monomers.

[0030] Figure 3 Results of the reaction condition optimization experiment: (A) Effect of P(DEB-FeTBrPP) concentration; (B) Effect of TMB concentration.

[0031] Figure 4 Results of the reaction condition optimization experiment: (A) Effect of reaction temperature; (B) Effect of reaction time; (C) Effect of pH value.

[0032] Figure 5 Enzyme kinetics results of P(DEB-FeTBrPP): Changes in absorbance over time at different TMB concentrations; Lineweaver-Burk double reciprocal plot with TMB as substrate;

[0033] Figure 6 Enzyme kinetics results of P(DEB-FeTBrPP): Different The change of absorbance over time at a given concentration; Plot the double reciprocal of the Lineweaver-Burk plot for the substrate.

[0034] Figure 7Analysis of P(DEB-FeTBrPP) colorimetric detection. (A) UV-Vis absorption spectra after adding different concentrations of H2O2 to the P(DEB-FeTBrPP)-TMB system and reacting in a water bath at 37 ℃ for 30 min; (B) Linear fitting curve between 652 nm absorbance and H2O2 concentration; (C) Color changes of the solution after reacting with different concentrations of H2O2.

[0035] Figure 8 Scanning electron microscopy image of :P(DEB-FeTBrPP) enzymes. Detailed Implementation

[0036] The technical solution of the present invention will be described in detail below through specific embodiments. It should be understood that the following embodiments are only for illustrating the present invention and not for limiting the scope of the present invention. Experimental methods that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0037] The reagents and materials used in the embodiments of this invention are all commercially available products, and their specific sources are as follows:

[0038] 5,10,15,20-Tetra(4-bromophenyl)porphyrin ( Energy Chemical, purity ≥ 98%;

[0039] 1,4-Diacetylenebenzene: Sigma-Aldrich, purity ≥97%;

[0040] Anhydrous ferric chloride ( Aladdin, purity ≥ 99%;

[0041] Dichlorobis(triphenylphosphine)palladium ( Sigma-Aldrich, purity ≥ 99%;

[0042] Cuprous iodide ( Aladdin, purity ≥ 98%;

[0043] Triethylamine ( Sinopharm, analytical grade;

[0044] N,N-Dimethylformamide (DMF): Sinopharm, analytical grade;

[0045] 3,3',5,5'-Tetramethylbenzidine (TMB): Aladdin, purity ≥99%;

[0046] hydrogen peroxide ( Solution: Sinopharm, 30% by mass, analytical grade;

[0047] Sodium acetate-acetic acid buffer: pH 4.0, prepared in-house.

[0048] The instruments and equipment used in the embodiments of the present invention include:

[0049] Fourier Transform Infrared Spectrometer (FT-IR): Nicolet 6700;

[0050] Ultraviolet-Vis DRS: Shimadzu UV-2600;

[0051] X-ray powder diffractometer (XRD): Bruker D8 Advance;

[0052] Scanning electron microscope (SEM): Hitachi SU8010;

[0053] Transmission electron microscope (TEM): JEOL JEM-2100F;

[0054] Nitrogen adsorption-desorption system: Micromeritics ASAP 2460;

[0055] Thermogravimetric analyzer (TGA): TA Instruments Q600;

[0056] UV-Vis spectrophotometer: Agilent Cary 60;

[0057] Example 1: Preparation of P(DEB-FeTBrPP) polymer

[0058] Step 1: Preparation of 5,10,15,20-tetra(4-bromophenyl)iron porphyrin (FeTBrPP)

[0059] 5,10,15,20-tetratetra(4-bromophenyl)porphyrin (1.0 g, 0.96 mmol) and anhydrous ferric chloride (0.78 g, 4.8 mmol, 5 molar amounts) were added to a 100 mL round-bottom flask, followed by the addition of 30 mL of DMF. The mixture was heated to 140 °C for 8 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and 100 mL of deionized water was added. The mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to give a purplish-black solid product, FeTBrPP, in 85% yield.

[0060] Step 2: Sonogashira coupling polymerization reaction

[0061] Add FeTBrPP (0.5 g, 0.47 mmol) and 1,4-diacetylenebenzene (0.59 g, 4.7 mmol, 10 times the molar amount) sequentially to a 100 mL two-necked flask. (0.033 g, 0.047 mmol, 10 mol%), (0.018 g, 0.094 mmol, 20 mol%) and 40 mL DMF. The air in the system was replaced three times with nitrogen, and triethylamine (3.0 mL) was added. The reaction was carried out at 80 °C for 48 h.

[0062] Step 3: Product Processing

[0063] After the reaction was completed and cooled to room temperature, the product was filtered to obtain a brownish-black solid. The solid was washed successively with methanol (50 mL × 3), acetone (50 mL × 3), and THF (50 mL × 3) to remove unreacted monomers, catalyst residues, and oligomers. The solid product was dried under vacuum at 60 °C for 12 h to obtain the iron porphyrin conjugated acetylene polymer P (DEB-FeTBrPP), with a yield of 78%.

[0064] Material characterization results:

[0065] like Figure 1 As shown in the schematic diagram of the polymer structure of P(DEB-FeTBrPP), the conjugated polymer skeleton formed by the connection of iron porphyrin units to benzene ring units via alkyne bonds is clearly illustrated. The structure and properties of P(DEB-FeTBrPP) were analyzed using various characterization methods.

[0066] UV-Vis DRS analysis: such as Figure 2 As shown, P(DEB-FeTBrPP) exhibits a characteristic absorption peak of the porphyrin Soret band at 400-450 nm and Q-band absorption in the 500-700 nm range. Compared to the monomer FeTBrPP, the polymer's Soret band shows a red shift (approximately 15 nm), indicating that the conjugated structure formed by the alkyne bonds expands the π-electron delocalization range and reduces the band gap. The polymer's absorption is significantly enhanced throughout the visible light region, which is beneficial for the utilization of light energy in photocatalytic processes.

[0067] Scanning electron microscopy (SEM) analysis: such as Figure 8 As shown, P(DEB-FeTBrPP) exhibits irregular blocky and plate-like morphologies, with particle sizes ranging from a few micrometers to tens of micrometers. The particle surfaces are rough, and abundant porous structures can be observed. This porous morphology is beneficial for the adsorption and mass transfer of substrate molecules.

[0068] Based on the above characterization results, a well-defined and high-performance iron porphyrin conjugated acetylene polymer P (DEB-FeTBrPP) was successfully prepared. This polymer possesses an extended conjugated structure, abundant porous features, and a high content of iron porphyrin active sites, laying the structural foundation for its peroxidase-like catalytic activity.

[0069] Example 2: Verification of P(DEB-FeTBrPP) type peroxidase activity

[0070] To verify the peroxidase-like activity of P(DEB-FeTBrPP), TMB was used as a chromogenic substrate to investigate the polymer catalysis. The ability to oxidize TMB.

[0071] Experimental methods:

[0072] Preparation of P(DEB-FeTBrPP) dispersion: Weigh 5 mg P(DEB-FeTBrPP), add 10 mL of deionized water, and sonicate for 30 min to obtain a dispersion with a concentration of 0.5 mg / mL. Store at 4℃ for later use.

[0073] Preparation of TMB solution: Weigh 10 mg of TMB and dissolve it in 10 mL of DMSO to obtain a 10 mM TMB stock solution.

[0074] Preparation of sodium acetate-acetic acid buffer: Mix 0.1 M sodium acetate solution and 0.1 M acetic acid solution in an appropriate ratio and adjust the pH to 4.0.

[0075] Catalytic colorimetric reaction:

[0076] Add the following to a 1.5 mL centrifuge tube in sequence: sodium acetate-acetic acid buffer (pH 4.0): 800 μL; P(DEB-FeTBrPP) dispersion (0.5 mg / mL): 40 μL (final concentration 20 μg / mL); TMB stock solution (10 mM): 20 μL (final concentration 200 μM); Solution (10 mM): 20 μL (final concentration 200 μM); Deionized water: 120 μL

[0077] The total reaction volume was 1.0 mL. The centrifuge tube was placed in a 37°C water bath for 30 min.

[0078] Controlled experimental design:

[0079] To demonstrate that the peroxidase-like activity of P(DEB-FeTBrPP) originates from the iron porphyrin unit, the following control group was designed:

[0080] Control group 1: P(DEB-H) In the TBrPP system (iron-free porphyrin polymer), P(DEB-FeTBrPP) in the above reaction system is replaced with the iron-free porphyrin polymer P(DEB-H) TBrPP) (replace with equal mass), all other conditions are the same.

[0081] Control group 2: PDEB system (porphyrin-free polymer) In the above reaction system, P(DEB-FeTBrPP) was replaced with porphyrin-free poly(1,4-diacetylenebenzene) (PDEB) (replaced by equal mass), and other conditions were the same.

[0082] Control group 3: Blank control (no catalyst) The reaction system contained no catalyst, only TMB and... .

[0083] Control group 4: Monomer FeTBrPP. P(DEB-FeTBrPP) was replaced with monomer FeTBrPP (replaced in equimolar amounts of iron content), and the effect on catalytic activity after polymerization was investigated.

[0084] Results analysis:

[0085] like Figure 2 As shown, after 30 min of reaction, the P(DEB-FeTBrPP) solution exhibited a distinct blue color, with an absorbance of 0.52 at 652 nm. UV-Vis absorption spectroscopy revealed a strong absorption peak at 652 nm, which is attributed to the characteristic absorption of oxidized TMB (oxTMB), confirming that P(DEB-FeTBrPP) successfully catalyzed the reaction. The reaction of TMB oxidation.

[0086] Control group 1 (P(DEB-H) The solutions of TBrPP and control group 2 (PDEB) were essentially colorless, with absorbances of only 0.03 and 0.02 (after background absorption subtraction) at 652 nm, respectively, indicating that the iron-free porphyrin polymer and the porphyrin-free polymer had almost no peroxidase activity. The absorbance of control group 3 (blank control) was 0.01, indicating that in the absence of a catalyst, The oxidation of TMB is extremely slow and negligible.

[0087] The solution of control group 4 (monomer FeTBrPP) was light blue, with an absorbance of 0.21 at 652 nm, approximately 40% of that of P(DEB-FeTBrPP). This indicates that although monomeric FeTBrPP possesses some peroxidase-like activity, its catalytic efficiency is significantly lower than that of the polymer. The polymerized P(DEB-FeTBrPP) exhibits higher catalytic activity due to its enhanced electron transport via its conjugated structure and improved accessibility of active sites via its porous structure.

[0088] The above comparative experiments fully demonstrate that: First, the peroxidase-like activity of P(DEB-FeTBrPP) originates from the iron porphyrin unit in the polymer structure, with the central iron ion being the catalytic active site; Second, the porphyrin macrocycle and the conjugated polymer skeleton play an important role in enhancing catalytic activity; Third, the polymer form has higher catalytic efficiency than the monomer, which is attributed to the electron transport promoted by the conjugated structure and the highly accessible active sites provided by the porous structure.

[0089] Example 3: Optimization of Reaction Conditions

[0090] To obtain the best catalytic colorimetric effect, the effects of P(DEB-FeTBrPP) concentration, TMB concentration, reaction temperature, reaction time and pH value on the catalytic reaction were systematically investigated.

[0091] Effect of P(DEB-FeTBrPP) concentration:

[0092] like Figure 3 As shown in Figure A, the TMB concentration was fixed at 200 μM. The effect of P(DEB-FeTBrPP) concentration on absorbance was investigated at a concentration of 200 μM, pH 4.0, and 37℃ for 30 min. The results showed that within the range of 10-50 μg / mL, absorbance increased with increasing P(DEB-FeTBrPP) concentration, reaching a maximum of 0.56 at a concentration of 40 μg / mL. Further increasing the concentration to 60 μg / mL resulted in a decrease in absorbance to 0.48. This may be because excessively high polymer concentrations lead to particle aggregation, reducing the exposure of active sites, and the increased light absorption by the polymer increases background interference. Considering all factors, 40 μg / mL was selected as the optimal P(DEB-FeTBrPP) concentration.

[0093] The effect of TMB concentration:

[0094] like Figure 3 As shown in B, the concentration of P(DEB-FeTBrPP) was fixed at 40 μg / mL. The effect of TMB concentration on absorbance was investigated at a concentration of 200 μM, pH 4.0, and 37℃ for 30 min. Within the range of 50–400 μM, absorbance increased with increasing TMB concentration. At a TMB concentration of 200 μM, the absorbance was 0.56; further increasing to 400 μM increased the absorbance to 0.61, but the increase was limited. Considering that excessively high TMB concentrations would increase reagent costs and that TMB has limited solubility in water (requiring the addition of DMSO for solubilization), 200 μM was chosen as the optimal TMB concentration.

[0095] Effect of reaction temperature:

[0096] like Figure 4 As shown in Figure A, with the concentration of P(DEB-FeTBrPP) fixed at 40 μg / mL and the concentration of TMB at 200 μM, The concentration was 200 μM, pH 4.0, and the reaction time was 30 min. The effect of reaction temperature on absorbance was investigated. Within the range of 25–50 °C, absorbance increased with increasing temperature. The absorbance reached 0.56 at 37 °C, and further increased to 0.61 at 45 °C. However, considering that excessively high temperatures may lead to… Considering the instability of decomposition and TMB oxidation products, and the fact that 37℃ is close to physiological temperature and more suitable for the detection of biological samples, 37℃ was chosen as the optimal reaction temperature.

[0097] The effect of reaction time:

[0098] like Figure 4 As shown in Figure B, with a fixed P(DEB-FeTBrPP) concentration of 40 μg / mL and a TMB concentration of 200 μM, The concentration was 200 μM, pH 4.0, and the temperature was 37℃. The effect of reaction time on absorbance was investigated. The results showed that absorbance increased with increasing reaction time within the range of 0–60 min. Absorbance increased rapidly in the first 10 min, reaching 0.28 at 10 min; the rate of increase then slowed, with absorbance at 0.56 at 30 min; and when the reaction time was extended to 60 min, absorbance increased to 0.64. Considering detection efficiency, 30 min was selected as the optimal reaction time, at which point a high and stable response signal was achieved.

[0099] The effect of pH value:

[0100] like Figure 4 As shown in C, with a fixed P(DEB-FeTBrPP) concentration of 40 μg / mL and a TMB concentration of 200 μM, The concentration was 200 μM, and the reaction was carried out at 37℃ for 30 min. The effect of buffer pH on absorbance was investigated. Within the pH range of 3.0–7.0, the catalytic activity exhibited a volcano-shaped curve with pH changes. The absorbance reached its maximum value of 0.56 at pH 4.0, at which point P(DEB-FeTBrPP) exhibited the highest peroxidase-like activity. Absorbance decreased significantly when pH < 4.0 or pH > 4.0. The absorbance was 0.32 at pH 3.0 and only 0.18 at pH 7.0. This pH dependence is similar to that of natural horseradish peroxidase (HRP), which exhibits the highest activity under weakly acidic conditions (pH 4–5). The protonation state and redox potential of the iron porphyrin catalytic active site are both affected by pH, with weakly acidic conditions favoring... The activation of hydroxyl radicals and the generation of hydroxyl radicals were observed. Therefore, pH 4.0 was chosen as the optimal reaction pH.

[0101] Optimal reaction conditions determined:

[0102] Based on the above optimization experiments, the P(DEB-FeTBrPP) catalysis was determined to be effective. The optimal conditions for the colorimetric reaction of TMB oxidation were: P(DEB-FeTBrPP) concentration 40 μg / mL, TMB concentration 200 μM, reaction temperature 37℃, reaction time 30 min, and buffer pH 4.0. Subsequent kinetic studies were conducted under these optimized conditions. Testing experiment.

[0103] Example 4: Determination of enzyme kinetic parameters

[0104] To further investigate the peroxidase-like catalytic mechanism of P(DEB-FeTBrPP), its enzyme kinetic parameters were determined under optimal reaction conditions. The enzyme-like catalytic reaction follows the Michaelis-Menten kinetic equation:

[0105] ,

[0106] in, The initial reaction rate, For the maximum reaction rate, Substrate concentration, is the Michaelis constant. The value reflects the affinity between the enzyme and its substrate. The smaller the value, the higher the affinity.

[0107] Using Lineweaver-Burk double reciprocal plotting method (1 / vs 1 / A linear relationship can be obtained by calculating from the slope and intercept. and value.

[0108] Kinetic experiments using TMB as the variable substrate:

[0109] fixed With a concentration of 200 μM, TMB concentrations were varied (25, 50, 100, 200, 400, 800 μM). At 37℃ and pH 4.0, the absorbance at 652 nm was recorded immediately after the addition of P(DEB-FeTBrPP) (40 μg / mL). Since the molar extinction coefficient ε of oxTMB at 652 nm is 39000 M... cm According to the Lambert-Beer law, the change in absorbance can be converted into a change in concentration, and then the initial reaction rate (the slope of the linear segment in the first 3 minutes) can be calculated.

[0110] like Figure 5 As shown, the absorbance-time curves measured at different TMB concentrations exhibit typical enzyme-catalyzed reaction kinetics. At low TMB concentrations, the initial reaction rate increases linearly with increasing concentration; when the TMB concentration is high, the initial reaction rate increases more slowly and gradually approaches saturation, consistent with the characteristics of the Michaelis-Menten equation.

[0111] Lineweaver-Burk double reciprocal plotting of the experimental data yielded a good linear relationship (R²). =0.991), the linear equation is:

[0112] ,

[0113] From the slope and intercept The calculation yields: - × 10 Ms - mM.

[0114] like Figure 6 As shown, with Dynamic experiments with variable substrates:

[0115] With the TMB concentration fixed at 200 μM, the following changes were made. Concentrations (10, 25, 50, 100, 200, 400 μM), other conditions as above, were used to determine the initial reaction rate.

[0116] Different The absorbance-time curves measured at the same concentration also exhibit the kinetic characteristics of the enzyme-catalyzed reaction.

[0117] Lineweaver-Burk double reciprocal plotting yields a linear relationship (Ri). =0.987), the linear equation is:

[0118] ,

[0119] The calculation yielded: × 10 Ms - mM

[0120] Comparison with natural HRP and other nanozymes:

[0121] Table 1 lists the kinetic parameters of P(DEB-FeTBrPP) compared with those of natural HRP and other nanozymes reported in the literature.

[0122] Table 1 Comparison of kinetic parameters of P(DEB-FeTBrPP) with other catalysts

[0123]

[0124] The results show that P(DEB-FeTBrPP) has an effect on TMB. The value (1.18 mM) is slightly higher than HRP (0.434 mM), but lower than Fe-POP (1.56 mM) reported in the literature, indicating that the material of this invention has a good affinity for TMB. P(DEB-FeTBrPP) has a better affinity for TMB. of The value (1.70 mM) is significantly lower than that of HRP (3.7 mM) and Fe-POP (3.85 mM), and even superior to some MOF-based nanozymes, indicating that the material of this invention has a high efficiency for... It has high affinity and catalytic efficiency.

[0125] Compared to inorganic nanoparticles (such as...) NPs), P(DEB-FeTBrPP) pairs of The value is about 90 times lower, but for TMB The values ​​are relatively high. This difference reflects the different active site structures and catalytic mechanisms of different catalysts. Iron porphyrin-based catalysts, by mimicking the structure of natural heme, are more conducive to... The recognition and activation of inorganic nanoparticles mainly rely on the Fenton reaction at surface iron sites, while inorganic nanoparticles rely on the recognition and activation of inorganic nanoparticles. Its affinity is relatively low.

[0126] Based on comprehensive kinetic parameter analysis, P(DEB-FeTBrPP), as a peroxidase-like material, has the potential to... It possesses excellent affinity and catalytic efficiency, in It has unique advantages in detection applications.

[0127] Example 5: Catalytic Mechanism Study

[0128] To elucidate the catalytic mechanism of P(DEB-FeTBrPP) peroxidase activity, free radical capture experiments and electron paramagnetic resonance (EPR) techniques were used to study the reactive oxygen species generated during the catalytic process.

[0129] Free radical capture experiment:

[0130] Peroxidase catalysis The oxidation of TMB may generate various reactive oxygen species, including hydroxyl radicals (TOB). ), superoxide anion radical ( ) and singlet oxygen ( To identify the major reactive oxygen species, different free radical scavengers were introduced:

[0131] Terephthalic acid (TA): Specific capture ;

[0132] Benzoquinone (BQ): Specific capture ;

[0133] Sodium azide ( ): Specific capture ;

[0134] tert-Butanol (TBA): Non-specific capture ;

[0135] Experimental methods: In a standard catalytic reaction system (P(DEB-FeTBrPP) 40 μg / mL, TMB 200 μM), The above-mentioned trapping agent (final concentration 5 mM) was added to the solution at 200 μM, pH 4.0, 37℃. After reacting for 30 min, the absorbance at 652 nm was measured and compared with the control group without the trapping agent to calculate the inhibition rate.

[0136] After adding TA or TBA, the absorbance decreased significantly, and the inhibition rates reached 82% and 79%, respectively, indicating that... It plays a leading role in the catalytic reaction. The absorbance decreased by approximately 25% after the addition of BQ, indicating... It also participated in the reaction, but its role was relatively minor. (Added) Afterwards, the absorbance decreased by only about 8%, indicating that Their contribution was very small.

[0137] Electron paramagnetic resonance (EPR) experiment:

[0138] 5,5-Dimethyl-1-pyrrolline-N-oxide (DMPO) was used as a free radical scavenger, and the free radicals generated in the catalytic reaction system were directly detected by EPR technology.

[0139] Experimental method: P(DEB-FeTBrPP) (40 μg / mL), Mix 200 μM of DMPO (50 mM) in sodium acetate-acetic acid buffer at pH 4.0 and perform EPR testing immediately. Simultaneously, set a test without P (DEB-FeTBrPP) or without P. The control group.

[0140] In P(DEB-FeTBrPP) and In the coexisting system, the EPR spectrum exhibits a strong characteristic quartet with an intensity ratio of 1:2:2:1 and a hyperfine splitting constant of [missing value]. =14.9 G, =14.9 G, this is DMPO- The typical characteristics of adducts directly prove... The formation of [something]. In the control group, only P (DEB-FeTBrPP) or only [something] was added. At that time, no obvious EPR signal was observed, indicating that The production requires P(DEB-FeTBrPP) and The combined effect of.

[0141] Catalytic mechanism analysis:

[0142] Based on the above experimental results, the catalytic mechanism of P(DEB-FeTBrPP) peroxidase activity is proposed as follows:

[0143] first step: The molecule adsorbs onto the active site of the iron porphyrin and coordinates with the central iron ion. The Fe in the iron porphyrin... First of all Reduced to Fe ,at the same time Produced by oxidation and decomposition :

[0144]

[0145] Step 2: Fe quickly Oxidation back to Fe and generate more (Fenton-like reaction):

[0146]

[0147] Step 3: Generation As a strong oxidizing agent, it rapidly oxidizes TMB to produce blue oxTMB.

[0148]

[0149] In this process, the role of the conjugated polymer backbone is manifested in the following ways: First, the conjugated structure formed by alkyne bonds promotes the transfer of electrons from the polymer backbone to the iron porphyrin active center, accelerating the Fe... / Fe First, the cycle conversion rate; second, the porous structure provides abundant substrate transport channels, enabling... First, TMB molecules can quickly access the active site; second, the rigid structure of the polymer backbone stabilizes the configuration of iron porphyrin and prevents the deactivation of the active center.

[0150] In summary, the peroxidase-like activity of P(DEB-FeTBrPP) is mainly catalyzed through a Fenton-like reaction mechanism at the iron porphyrin center. generate Then by The chromogenic substrate TMB was oxidized. The conjugated polymer backbone significantly enhanced catalytic activity through electron transport and structural stabilization.

[0151] Example 6: Colorimetric Detection of Hydrogen Peroxide

[0152] Under optimal reaction conditions, a P(DEB-FeTBrPP)-based system was established. A colorimetric detection method was developed, and its analytical performance was systematically evaluated.

[0153] Experimental methods:

[0154] Add the following to a 1.5 mL centrifuge tube sequentially: sodium acetate-acetic acid buffer (pH 4.0): 800 μL; P(DEB-FeTBrPP) dispersion (0.5 mg / mL): 40 μL (final concentration 20 μg / mL); TMB stock solution (10 mM): 20 μL (final concentration 200 μM); different concentrations Solution: 20 μL; bring deionized water to a total volume of 1.0 mL;

[0155] Centrifuge tubes were placed in a 37°C constant temperature water bath for 30 min, and the absorbance at 652 nm was measured using a UV-Vis spectrophotometer.

[0156] Linear range and detection limit:

[0157] like Figure 7 As shown in A, within the range of 0.5-500 μM, A good linear relationship was observed between concentration and absorbance. In the low concentration range (0.5-50 μM), the linear equation was:

[0158]

[0159] In the medium to high concentration range (50-500 μM), the linear equation is:

[0160]

[0161] like Figure 7 As shown in B, the linear fit for the low concentration range (0.5-10 μM) exhibits excellent linearity (R0). =0.999), indicating that this method has excellent detection performance in the low concentration region. For example... Figure 7 As shown in C, the color changes of the solution after adding different concentrations of H2O2 are as follows: Figure 7 As shown in C.

[0162] According to IUPAC regulations, the limit of detection (LOD) is calculated as 3σ / k, where σ is the standard deviation of 11 measurements of the blank sample, and k is the slope of the calibration curve. Experimentally, σ = 0.0011 and k = 0.0068 μM were obtained. The calculation yields:

[0163] ,

[0164] The detection limit is significantly lower than that of Fe-POP (1.37 μM) reported in the literature, demonstrating that the method of the present invention has higher sensitivity.

[0165] Selective experiments:

[0166] To evaluate the selectivity of the method, the effects of common potential interfering substances on the selection were examined. The impact of testing. Adding [the technology] to the standard testing system. (50 μM) and various interfering substances (concentration of) 10 times that of 500 μM), the absorbance at 652 nm was measured, compared with that containing only Compared with the control group.

[0167] Common metal ions ( , , , , , ), anions ( , , , The interference from biomolecules (glucose, ascorbic acid, uric acid, glutathione, cysteine) on the detection is less than 8%. The only exception is ascorbic acid (AA), which, as a reducing agent, can reduce oxTMB to TMB, resulting in a decrease in absorbance of about 45%. However, in actual sample detection, its interference can be eliminated by removing or oxidizing AA through pretreatment.

[0168] The above results show that the method of the present invention is effective for... It has good selectivity and can resist the effects of most common interfering substances.

[0169] Reproducibility and stability:

[0170] Reproducibility test: for 50 μM The standard solution was subjected to 10 parallel measurements, and the average absorbance was 0.392 with a relative standard deviation (RSD) of 2.8%, indicating that the method has good reproducibility.

[0171] Stability of P(DEB-FeTBrPP) dispersion: Freshly prepared P(DEB-FeTBrPP) dispersion (0.5 mg / mL) was stored at 4℃. Samples were taken every 5 days for 50 μM concentration testing. The absorbance remained above 95% of its initial value within 30 days, indicating that the dispersion has good storage stability under refrigeration conditions.

[0172] Stability of P(DEB-FeTBrPP) solid powder: P(DEB-FeTBrPP) solid powder was stored at room temperature and dried. Every 10 days, 5 mg was weighed and prepared into a dispersion for testing. Within 90 days, the catalytic activity decreased by only about 7%, indicating that the solid powder has excellent long-term stability.

[0173] Catalytic cycle stability: Used P(DEB-FeTBrPP) was recovered by centrifugation, washed with deionized water and ethanol, dried, and reused in the next catalytic cycle. After 5 cycles, the catalytic activity remained above 90% of the initial value, indicating that P(DEB-FeTBrPP) has good catalytic cycle stability.

[0174] Actual sample testing:

[0175] To evaluate the practicality of the method, it was applied to tap water, river water, and cell culture medium. The detection.

[0176] Sample pretreatment: Tap water and river water samples were filtered through a 0.22 μm filter membrane and used directly for testing; cell culture medium samples were diluted 10 times with PBS buffer before testing.

[0177] Spiked recovery experiment: A known concentration of [unspecified substance] is added to the actual sample. The standard solution was tested according to the standard testing procedure, and the recovery rate was calculated.

[0178] Table 2 lists the spiked recovery results for three real samples. The recoveries ranged from 95.2% to 104.8%, with RSD < 5%, indicating that this method has good accuracy and precision and can be used for real samples. Quantitative detection.

[0179] Table 2. Actual Samples Spiked recovery experiment results

[0180]

[0181] Comparison with other detection methods:

[0182] Table 3 lists the method of this invention and other methods reported in the literature. Performance comparison of detection methods.

[0183] Table 3 Differences Performance comparison of detection methods

[0184]

[0185] The results show that the detection limit of the method of the present invention is comparable to that of the inorganic nanoparticle method, and slightly higher than that of the natural HRP and fluorescence method, but has a wider linear range. It also has the advantages of simple operation, low cost, and good stability, and has excellent overall performance, making it suitable for practical applications.

[0186] Example 7: Preparation and Performance Comparison of Different Iron Porphyrin Polymers

[0187] To investigate the effect of the ratio of iron porphyrin units to 1,4-diacetylenebenzene units on polymer properties, a series of polymers with different iron porphyrin contents were prepared by adjusting the feed ratio of the two monomers according to the method in Example 1. These polymers were labeled as P(DEB-FeTBrPP-x), where x represents the molar ratio of FeTBrPP to 1,4-diacetylenebenzene.

[0188] Experimental methods:

[0189] With a fixed amount of FeTBrPP of 0.47 mmol, different molar amounts of 1,4-diaethynylbenzene were added: P(DEB-FeTBrPP-5): 2.35 mmol of 1,4-diaethynylbenzene (5 molar amounts); P(DEB-FeTBrPP-10): 4.7 mmol of 1,4-diaethynylbenzene (10 molar amounts); P(DEB-FeTBrPP-15): 7.05 mmol of 1,4-diaethynylbenzene (15 molar amounts); P(DEB-FeTBrPP-20): 9.4 mmol of 1,4-diaethynylbenzene (20 molar amounts).

[0190] Other reaction conditions and post-treatment procedures are the same as in Example 1.

[0191] Material characterization results:

[0192] Nitrogen adsorption-desorption analysis was used to determine the specific surface area of ​​different polymers: P(DEB-FeTBrPP-5): specific surface area 215 m² / g; P(DEB-FeTBrPP-10): Specific surface area 368 m² / g; P(DEB-FeTBrPP-15): Specific surface area 482m² / g; P(DEB-FeTBrPP-20): specific surface area 538 m² / g.

[0193] The results showed that with the increase of the 1,4-diaethynylbenzene ratio, the iron content in the polymer decreased, while the specific surface area increased. This is because a higher proportion of 1,4-diaethynylbenzene units makes the polymer skeleton more rigid and porous.

[0194] Comparison of peroxidase-like activities:

[0195] Under the same conditions (polymer concentration 40 μg / mL, TMB 200 μM), The catalytic activity of different polymers was determined at 200 μM, pH 4.0, 37℃, for 30 min.

[0196] P(DEB-FeTBrPP-10) exhibited the highest catalytic activity, with an absorbance of 0.56 at 652 nm. P(DEB-FeTBrPP-5) had an absorbance of 0.41; its lower activity may be due to excessively high iron porphyrin unit proportions leading to polymer pore blockage and limited substrate mass transfer. P(DEB-FeTBrPP-15) and P(DEB-FeTBrPP-20) had absorbances of 0.48 and 0.39, respectively. Although they had large specific surface areas, the reduced density of iron porphyrin active sites resulted in a decrease in overall catalytic activity.

[0197] Taking into account catalytic activity, material stability, and synthesis cost, a molar ratio of 1:10 for FeTBrPP to 1,4-diacetylenebenzene is the optimal ratio.

[0198] Example 8: Preparation of 5,10,15,20-tetra(4-iodophenyl)ironporphyrin polymer

[0199] To investigate the effects of halogen substituents on polymerization and catalytic performance, a conjugated acetylene polymer P (DEB-FeTPP) based on 5,10,15,20-tetra(4-iodophenyl)iron porphyrin was prepared.

[0200] Preparation method:

[0201] Step 1: Following the method in Example 1, 5,10,15,20-tetra(4-iodophenyl)porphyrin (1.2 g, 0.94 mmol) was reacted with anhydrous ferric chloride (0.76 g, 4.7 mmol) in DMF to give 5,10,15,20-tetra(4-iodophenyl)ironporphyrin (FeTPP) in 82% yield.

[0202] Step 2: Following the method in Example 1, FeTPP (0.6 g, 0.46 mmol) and 1,4-diacetylenebenzene (0.58 g, 4.6 mmol) were... Sonogashira coupling polymerization was carried out under CuI catalysis to obtain P(DEB-FeTPP) in 75% yield.

[0203] Performance comparison:

[0204] Compared to P(DEB-FeTBrPP), P(DEB-FeTPP) has a similar structure and properties: Specific surface area: 342 m² / g; Iron content: 4.6 wt%; Peroxidase activity: Under the same conditions, the absorbance at 652 nm is 0.53, which is slightly lower than that of P(DEB-FeTBrPP) (0.56).

[0205] Since iodoaromatics exhibit slightly higher reactivity than bromoaromatics in the Sonogashira reaction, P(DEB-FeTPP) polymerizes faster (reaction time can be shortened to 36 h), but the cost of iodomonomers is higher. Considering all factors, both P(DEB-FeTBrPP) and P(DEB-FeTPP) are preferred peroxidase-like materials.

[0206] Example 9: Performance comparison with commercial horseradish peroxidase (HRP)

[0207] To comprehensively evaluate the application potential of P(DEB-FeTBrPP), a systematic comparison was made between it and commercially available HRP in terms of catalytic activity, stability, and cost.

[0208] Catalytic activity comparison:

[0209] Under the same reaction conditions (TMB 200 μM, The catalytic performance of P(DEB-FeTBrPP) (40 μg / mL) and HRP (0.1 μg / mL) was compared under standard conditions (200 μM, pH 4.0, 37℃, 30 min). The concentrations of both were adjusted to make their absorbances similar (approximately 0.55) under standard conditions for a fair comparison.

[0210] pH stability comparison:

[0211] The catalytic activities of P(DEB-FeTBrPP) and HRP were measured (expressed as absorbance at 652 nm) within the pH range of 3–10. HRP showed high activity in the pH range of 4–6, but its activity decreased sharply at pH < 4 or pH > 7, reaching only 15% of its maximum activity at pH 3 and dropping to 8% at pH 9. In contrast, P(DEB-FeTBrPP) maintained high activity throughout the pH range of 3–9, retaining 58% of its maximum activity at pH 3 and 42% at pH 9, demonstrating better pH adaptability.

[0212] Temperature stability comparison:

[0213] The catalytic activities of the two were compared within the temperature range of 25-70℃. HRP showed high activity in the temperature range of 25-45℃, but its activity rapidly decreased to 25% of its maximum activity when the temperature rose to 60℃, and it was essentially deactivated at 70℃. P(DEB-FeTBrPP) maintained stable activity in the temperature range of 25-60℃, and still retained 68% of its maximum activity at 70℃, showing excellent thermal stability.

[0214] Storage stability comparison:

[0215] HRP (1 mg / mL, PBS buffer, pH 7.4) and P(DEB-FeTBrPP) dispersion (0.5 mg / mL, deionized water) were stored at 4°C and 25°C, respectively, and their catalytic activity was measured periodically. After 30 days of storage at 4°C, the activity of HRP decreased to 65% of its initial value, and after 30 days of storage at 25°C, the activity was only 35%. P(DEB-FeTBrPP) maintained an activity of over 95% after 90 days of storage at 4°C, and even after 90 days of storage at 25°C, the activity remained above 80%, demonstrating excellent storage stability.

[0216] Recycling performance:

[0217] HRP, being a soluble enzyme, cannot be recycled. P(DEB-FeTBrPP), as a solid material, can be recovered and reused through centrifugation. As shown in Example 6, P(DEB-FeTBrPP) retained over 90% activity after five cycles, demonstrating significant economic advantages.

[0218] Cost comparison:

[0219] While commercial HRP (Grade I, retail price approximately ¥3000 / g) requires a small single-use quantity (approximately 0.1 μg per test), its long-term cost is high due to its short shelf life and non-recyclability. P(DEB-FeTBrPP) has a synthesis cost of approximately ¥800 / g (considering raw material and labor costs), and although the single-use quantity is larger (approximately 40 μg per test), its excellent storage stability and recyclability result in lower long-term costs.

[0220] In summary, P(DEB-FeTBrPP) has a wider pH and temperature adaptability range, longer shelf life, recyclability, and lower long-term use cost compared to commercial HRP, giving it significant advantages in practical applications and making it an ideal alternative to natural enzymes.

[0221] Example 10: Application of P(DEB-FeTBrPP) in glucose detection

[0222] Peroxidase is often co-located with glucose oxidase (GOx) to construct cascade catalytic systems for glucose detection. This example explores the feasibility of co-locating P(DEB-FeTBrPP) with GOx for glucose detection.

[0223] Detection principle:

[0224] Glucose is oxidized by oxygen under GOx catalysis to produce gluconic acid and :

[0225]

[0226] generated TMB oxidation under P(DEB-FeTBrPP) catalysis produces colorimetric results:

[0227]

[0228] Experimental methods:

[0229] Add the following to a 1.5 mL centrifuge tube sequentially: - Phosphate-buffered saline (PBS, pH 7.4): 750 μL - Glucose oxidase (GOx, 2 mg / mL): 50 μL (final concentration 0.1 mg / mL) - Glucose solutions of different concentrations: 50 μL

[0230] React at 37℃ for 30 minutes to allow glucose to be fully converted. Then add: P(DEB-FeTBrPP) dispersion (0.5 mg / mL): 40 μL (final concentration 20 μg / mL); TMB stock solution (10 mM): 20 μL (final concentration 200 μM); sodium acetate-acetic acid buffer (pH 4.0, 10×): 100 μL.

[0231] The total reaction volume was 1.0 mL, and the final pH was approximately 5.0. After reacting at 37°C for 20 min, the absorbance at 652 nm was measured.

[0232] Results analysis:

[0233] Within the range of 0.1–1000 μM, a good correlation was observed between glucose concentration and absorbance. In the low concentration range (0.1–100 μM), the linear equation was:

[0234] ,

[0235] Based on 3σ / k, the detection limit for glucose is 0.08 μM (approximately 1.4 μg / L).

[0236] This method was applied to the detection of glucose in human serum samples. Serum samples were diluted 100-fold with PBS buffer and then tested using the method described above. The results were compared with those obtained from a hospital biochemical analyzer. As shown in Table 4, the results of the two methods were highly consistent, with a relative error of <5%, indicating that the cascade catalytic system of P(DEB-FeTBrPP) coupled with GOx can be used for the accurate detection of glucose in real biological samples.

[0237] Table 4 Comparison of glucose detection results in serum samples

[0238]

[0239] The above results indicate that P(DEB-FeTBrPP), as a peroxidase-like enzyme, can not only be directly used for... It can also be used in combination with other oxidases to construct cascade catalytic systems for the detection of small biological molecules such as glucose, with a wide range of applications.

Claims

1. An iron-porphyrin conjugated acetylene polymer-based peroxidase material characterized in that, The material comprises iron porphyrin structural units and benzene ring structural units connected by acetylene bonds; wherein the iron porphyrin structural unit is 5,10,15,20-tetra(4-substituted phenyl) iron porphyrin, and the substituent is at least one selected from bromo, iodo and ethynyl; the benzene ring structural unit is 1,4-diethynylbenzene; the iron porphyrin structural unit and the benzene ring structural unit are connected by acetylene bonds formed by a Sonogashira coupling reaction to constitute a conjugated ethynyl polymer skeleton; and the number average molecular weight of the polymer is 5000-50000 Da.

2. The iron porphyrin conjugated acetylene polyoxoperoxide enzyme material according to claim 1, characterized in that, The iron porphyrin structural unit is 5,10,15,20-tetra(4-bromophenyl) iron porphyrin or 5,10,15,20-tetra(4-iodophenyl) iron porphyrin.

3. The iron-porphyrin conjugated acetylene polyoxoperoxide enzyme material according to claim 1, characterized in that, The molar ratio of the iron porphyrin structural unit to the 1,4-diethynylbenzene structural unit in the polymer is 1:(2-20), preferably 1:(5-15), and more preferably 1:

10.

4. The iron-porphyrin conjugated acetylene polyoxoperoxide enzyme material according to claim 1, characterized in that, The material is a porous structure with a specific surface area of 100-800 m / g, with a pore size distribution ranging from 1 to 50 nm.

5. The iron-porphyrin conjugated acetylene polyoxoperoxide enzyme material according to claim 1, characterized in that, The apparent Michaelis constant K for 3,3',5,5'-tetramethylbenzidine is 0.5-1.5 mM, and the apparent Michaelis constant K for hydrogen peroxide is 1.0-2.5 mM.

6. A method for preparing the iron porphyrin conjugated acetylene polymer-based peroxidase material according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) monomer preparation: reacting 5,10,15,20-tetra(4-bromophenyl) porphyrin or 5,10,15,20-tetra(4-iodophenyl) porphyrin with an iron salt in a solvent to obtain 5,10,15,20-tetra(4-bromophenyl) iron porphyrin or 5,10,15,20-tetra(4-iodophenyl) iron porphyrin; (2) Sonogashira coupling polymerization reaction: carrying out a coupling polymerization reaction of the iron porphyrin monomer obtained in step (1) and 1,4-diethynylbenzene in the presence of a palladium catalyst, a copper co-catalyst and a base in an organic solvent, the reaction temperature is 60-100℃, and the reaction time is 12-72 h; (3) product treatment: after the reaction is completed, the solid product is filtered, washed with methanol, acetone and tetrahydrofuran in sequence, and vacuum dried to obtain an iron porphyrin conjugated ethynyl polymer peroxidase material.

7. The preparation method according to claim 6, characterized in that, In step (1), the iron salt is one or more of ferric chloride, ferric bromide, ferric acetate or iron triflate; the solvent is N,N-dimethylformamide, dimethyl sulfoxide or pyridine; the reaction temperature is 120-160℃, and the reaction time is 4-12 h.

8. The preparation method according to claim 6, characterized in that, In step (2), the palladium catalyst is one of dichlorobis(triphenylphosphine)palladium, tetrakis(triphenylphosphine)palladium or palladium chloride; the copper co-catalyst is cuprous iodide, cuprous chloride or cuprous bromide; the base is triethylamine, piperidine or diisopropylethylamine; the organic solvent is N,N-dimethylformamide, tetrahydrofuran or toluene; the molar ratio of the iron porphyrin monomer to 1,4-diethynylbenzene is 1:(2-20), the amount of the palladium catalyst is 5-15% of the molar amount of the iron porphyrin monomer, and the amount of the copper co-catalyst is 10-30% of the molar amount of the iron porphyrin monomer.

9. Use of the iron porphyrin conjugated ethynyl polymer peroxidase material according to any one of claims 1-5 in the preparation of a hydrogen peroxide colorimetric detection reagent.

10. Use according to claim 9, characterized in that, The hydrogen peroxide colorimetric detection method comprises the following steps: mixing a dispersion liquid of iron porphyrin conjugated acetylene polymer class peroxidase material, a 3,3',5,5'-tetramethyl benzidine solution and a sample to be detected, reacting at 25-45 DEG C for 10-60 min, determining the absorbance at 652 nm by ultraviolet-visible spectrophotometer, and calculating the hydrogen peroxide concentration according to the absorbance value; the detection range of the hydrogen peroxide is 0.5-500 muM, and the detection limit is 0.5 muM.

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