A dual-ligand based Fe-mof nanoscale enzyme and a preparation method and application thereof

Abstract

The present application relates to the technical fields of serum creatinine detection and nanozyme preparation, and particularly relates to a double-ligand regulated Fe-MOF nanozyme and a preparation method and application thereof. The method comprises the following steps: FeCl3.6H2O, 1,10-phenanthroline and monocarboxylic acid ligand are dissolved in DMF to form solution A by stirring; terephthalic acid is dissolved in DMF to form solution B; solution B is added to solution A, ultrasonic dispersion is performed, and then the solution is transferred to a stainless steel high-pressure reaction kettle for high-temperature reaction; after the reaction is completed and cooled to room temperature, centrifugal washing and vacuum drying are performed to obtain a double-ligand regulated Fe-MOF nanozyme sample. As a ligand, n-butyric acid enhances the affinity to the substrate molecule; as a ligand, 1,10-phenanthroline increases the enzyme activity; the synergy of 1,10-phenanthroline and n-butyric acid achieves high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the fields of serum creatinine detection and nanozyme preparation technology, and in particular to a Fe-MOF nanozyme based on dual-ligand regulation, its preparation method, and its application. Background Technology

[0002] Early diagnosis of kidney injury and related diseases is crucial for preventing disease progression. Creatinine (CRE) is the final product of muscle metabolism, which is freely filtered by the glomeruli and excreted in urine. Serum creatinine concentration is closely related to the glomerular filtration rate (GFR) and is a core biochemical indicator for clinically evaluating renal function. A persistently elevated serum creatinine level often indicates impaired renal function; therefore, establishing a sensitive, stable, and visualized detection method is of significant clinical value for the early identification and progression monitoring of kidney diseases.

[0003] Traditional chemical colorimetric methods (Jaffe method) are widely used due to their simplicity, but they are significantly affected by interfering substances, leading to inaccurate results. Enzymatic methods, while offering good specificity and sensitivity, rely on a cascade reaction system of multiple natural enzymes. These enzyme preparations are expensive, prone to activity loss, and require stringent storage conditions, hindering large-scale adoption. Meanwhile, modern instrumental methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) can achieve high-precision quantification, but the equipment is expensive, the detection cycle is long, and the operation is complex, making them unsuitable for primary care and point-of-care testing (POCT) scenarios.

[0004] In recent years, nanozymes have gradually become ideal materials to replace natural enzymes due to their advantages such as high catalytic activity, chemical stability, and low cost. They can catalyze the decomposition of hydrogen peroxide or substrate oxidation reactions through surface electron transfer processes, amplifying colorimetric signals for the colorimetric quantification of target molecules. Compared with natural enzymes, nanozymes maintain good stability under varying temperatures, pH conditions, and in complex biological systems, and are tunable and feasible for large-scale synthesis. Nanozyme-based colorimetric detection systems are simple to operate, have rapid response, and provide intuitive signals, making them particularly suitable for low-cost, visual detection of serum creatinine, and they hold broad application prospects in clinical diagnosis and health monitoring.

[0005] Metal-organic frameworks (MOFs) are widely used in constructing high-performance nanozymes due to their high specific surface area, tunable pore structure, and abundant metal active sites. Composed of metal ion clusters and organic ligands, MOF materials exhibit high porosity, ease of functionalization, and excellent peroxidase mimicry capabilities, enabling catalytic signal amplification in colorimetric detection systems. However, existing MOF-based nanozymes generally suffer from insufficient affinity for substrate molecules and limited catalytic activity, severely restricting their application in biosensing. Therefore, developing structurally stable, highly affinity, and catalytically active MOF-based nanozyme sensors for highly sensitive colorimetric detection of serum creatinine is of significant research and application value. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to design and provide a technical solution based on dual-ligand-regulated Fe-MOF nanozymes, their preparation method, and applications.

[0007] The present invention is specifically implemented using the following technical solutions:

[0008] The first aspect of this invention provides a method for preparing Fe-MOF nanozymes based on dual-ligand regulation, the method comprising the following steps:

[0009] (S.1) Dissolve FeCl3·6H2O, 1,10-phenanthroline and a monocarboxylic acid ligand in DMF and stir to form solution A; disperse terephthalic acid in DMF by ultrasonication to form solution B;

[0010] (S.2) Add solution B to solution A, disperse by ultrasonication, transfer to a stainless steel high-pressure reactor, carry out high-temperature reaction, cool to room temperature after the reaction is completed, centrifuge and wash, vacuum dry to obtain the dual-ligand regulated Fe-MOF nanozyme sample.

[0011] Furthermore, in step S.1, the molar ratio of FeCl3·6H2O, 1,10-phenanthroline, and the monocarboxylic acid ligand is 1:0.5-1:2-6; the monocarboxylic acid ligand is one of acetic acid, butyric acid, hexanoic acid, and octanoic acid.

[0012] Furthermore, in step S.1, the ratio of FeCl3·6H2O to DMF is 1 mmol: 5-10 mL; the molar amount of terephthalic acid is 1.5-2.5 mmol; and the ratio of terephthalic acid to DMF is 1 mmol: 3-7 mL.

[0013] Furthermore, the centrifugal washing in step S.2 specifically involves: centrifuging and washing with DMF and ethanol respectively, then immersing the collected product in a mixture of DMF and hydrochloric acid and allowing it to stand at room temperature; and then centrifuging and washing again with DMF.

[0014] Furthermore, in step S.2, the high-temperature reaction temperature is 100-150℃; the high-temperature reaction time is 16-32h; the volume ratio of DMF (AR pure) to hydrochloric acid (12M) is 1:0.02-0.087; and the vacuum drying temperature is 90-150℃.

[0015] A second aspect of the present invention provides a dual-ligand-regulated Fe-MOF nanozyme prepared by any of the methods described above.

[0016] The third aspect of this invention provides an application of dual-ligand-regulated Fe-MOF nanozymes in the preparation of a biosensor for creatinine detection.

[0017] A fourth aspect of the present invention provides a creatinine detection biosensor containing the above-described dual-ligand regulated Fe-MOF nanozyme.

[0018] The present invention has the following beneficial effects:

[0019] (1) This invention effectively regulates the crystal growth process of MOF by introducing n-butyric acid as an auxiliary ligand during the synthesis of Fe-MOF nanozymes, and successfully constructs a nanozyme with a high specific surface area (831 m²). 2 The optimized pore structure, consisting of a large number of open mesopores (g) and a wide variety of large-sized pores, not only significantly increases the number of exposed active sites but also provides efficient diffusion channels and enrichment microenvironments for the reaction substrate, thereby greatly enhancing the affinity for substrate molecules and laying the foundation for efficient catalysis.

[0020] (2) This invention introduces 1,10-phenanthroline as a second ligand, whose nitrogen atom forms a strong coordination interaction with the Fe metal center. This interaction effectively modulates the electron cloud density and geometric configuration of the active center, optimizes the electron transfer process in the catalytic reaction, and thus significantly improves the intrinsic catalytic efficiency of the catalytic active site. This precise electronic-level regulation is a key factor in obtaining high peroxidase activity in the material.

[0021] (3) 1,10-Phenanthroline and n-butyric acid exhibit a remarkable synergistic effect, achieving a perfect combination of "electronic regulation" and "structural regulation." This effect enables the prepared nanozyme to possess both extremely high substrate affinity (Km as low as 0.0454 mM) and excellent intrinsic catalytic activity. Based on this, the creatinine biosensor constructed thus achieves ultra-high sensitivity (detection limit as low as 0.72 µM) and specificity.

[0022] (4) This method is simple to operate and low in cost, providing a high-performance solution for the accurate and rapid detection of serum creatinine in clinical practice, and has broad application prospects. Attached Figure Description

[0023] Figure 1 The image shows the X-ray powder diffraction (XRD) pattern of the sample from Example 1.

[0024] Figure 2 The images show the scanning electron microscope (SEM) image and elemental distribution (EDS) map of the sample from Example 1.

[0025] Figure 3 The graph shows the nitrogen adsorption-desorption (N2-BET) curve of the sample in Example 1;

[0026] Figure 4 This is a catalytic kinetic curve of the sample in Example 1 for H2O2;

[0027] Figure 5 The UV absorption spectra are for evaluating the peroxidase-like activity of the materials in Examples 1-3 and Comparative Examples 1-3;

[0028] Figure 6 The image shows the UV absorption spectra of the sample from Example 1 at different creatinine concentrations.

[0029] Figure 7 This is a linear relationship graph of the detection of different creatinine concentrations for the sample in Example 1;

[0030] Figure 8 This is a graph showing the activity of active peroxidases in the sample from Example 1 after 7 cycles of creatinine detection. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0032] Example 1:

[0033] I. Preparation method of Fe-MOF nanozymes based on dual-ligand regulation:

[0034] 1. Dissolve 2.5 mmol FeCl3·6H2O, 2 mmol 1,10-phenanthroline and 10 mmol n-butyric acid in 20 mL LMF by sonication and stir with a magnetic stirrer for 30 min to form solution A; at the same time, disperse 2 mmol terephthalic acid in 10 mL LMF by sonication to form solution B.

[0035] 2. Solution B was added to solution A, and after ultrasonic dispersion, it was transferred to a stainless steel high-pressure reactor and reacted at 120°C for 24 hours. After the reaction, the mixture was cooled to room temperature, washed three times each with DMF and ethanol, and then the collected product was immersed in a 20 mL mixture (DMF: hydrochloric acid (12M) (volume ratio) = 24:1) and allowed to stand at room temperature for 10 hours. Afterwards, it was washed three more times with DMF, and the solid powder was vacuum dried overnight at 120°C to obtain the Fe-MOF nanozyme-1 sample from Example 1.

[0036] II. Application and sensitivity verification of a biosensor adapted to Fe-MOF nanozymes with dual-ligand regulation in the detection of creatinine levels.

[0037] 1. Add 20 µL of 2 mg / mL Fe-MOF nanozyme suspension, 20 µL of 10 mM H2O2 and 20 µL of 5 mM TMB to 2940 µL of 0.2 M sodium acetate-acetic acid (NaAc-HAc, pH=4.5) buffer solution. After incubating at room temperature for 5 min, measure the absorbance of the reaction solution at 652 nm using a UV-Vis spectrophotometer to detect the peroxidase-like activity of Fe-MOF nanozyme.

[0038] 2. Mix 20 µL of 2 mg / mL Fe-MOF nanozyme suspension, 20 µL of 5 mM TMB, and 2940 µL of 0.2 M sodium acetate-acetic acid (NaAc-HAc, pH=4.5), and add 20 µL of H2O2 solutions of different concentrations (2.25-37.5 mM). After incubating at room temperature for 5 min, measure the absorbance of the reaction solution at 652 nm using a UV-Vis spectrophotometer to obtain the enzyme catalytic kinetic curve of the nanozyme material. Calculate the Michaelis-Menten constant Km using the Lineweaver-Burk equation.

[0039] 3. Mix 100 µL of 0.015 M phosphate-buffered saline (PBS, pH=7.5) with 20 µL of 0.2 mg / mL creatine oxidase solution, 20 µL of 0.2 mg / mL creatinine oxidase solution, 20 µL of 0.2 mg / mL creatine oxidase solution, and 20 µL of creatinine solutions of different concentrations (0-37.5 mM), and incubate at 37 °C for 50 min. Then, add 2770 µL of 0.2 M NaAc-HAc buffer (pH=4.5), 30 µL of 2 mg / mL Fe-MOF nanozyme suspension, and 20 µL of 5 mM TMB to the above solution, and incubate at room temperature for 5 min. Finally, measure the absorbance of the reaction solution at 652 nm using a UV-Vis spectrophotometer to detect the creatinine content in the solution, and then evaluate the sensitivity of the colorimetric sensing method based on this nanozyme for creatinine detection.

[0040] Example 2:

[0041] 1. Dissolve 2.5 mmol FeCl3·6H2O, 1.25 mmol 1,10-phenanthroline and 5 mmol n-butyric acid in 12.5 mL DMF by sonication and stir with a magnetic stirrer for 20 min to form solution A; at the same time, disperse 1.5 mmol terephthalic acid in 4.5 mL DMF by sonication to form solution B.

[0042] 2. Solution B was added to solution A, and after ultrasonic dispersion, it was transferred to a stainless steel high-pressure reactor and reacted at 100°C for 16 hours. After the reaction, the mixture was cooled to room temperature, washed three times each with DMF and ethanol, and then the collected product was immersed in a 20 mL mixture (DMF: hydrochloric acid (12M) (volume ratio) = 49:1) and allowed to stand at room temperature for 10 hours. Afterwards, it was washed three more times with DMF, and the solid powder was vacuum dried overnight at 90°C to obtain the Fe-MOF nanozyme-2 sample from Example 2.

[0043] 3. Same as Example 1.

[0044] 4. Same as Example 1.

[0045] 5. Same as Example 1.

[0046] Example 3:

[0047] 1. Dissolve 2.5 mmol FeCl3·6H2O, 2.5 mmol 1,10-phenanthroline and 15 mmol n-butyric acid in 25 mL DMF by sonication and stir with a magnetic stirrer for 50 min to form solution A; at the same time, disperse 2.5 mmol terephthalic acid in 17.5 mL DMF by sonication to form solution B.

[0048] 2. Solution B was added to solution A, and after ultrasonic dispersion, it was transferred to a stainless steel high-pressure reactor and reacted at 150°C for 32 hours. After the reaction, the mixture was cooled to room temperature, washed three times each with DMF and ethanol, and then the collected product was immersed in a 20 mL mixture (DMF: hydrochloric acid (12M) (volume ratio) = 23:2) and allowed to stand at room temperature for 10 hours. Afterwards, it was washed three more times with DMF, and the solid powder was vacuum dried overnight at 150°C to obtain the Fe-MOF nanozyme-3 sample of Example 3.

[0049] 3. Same as Example 1.

[0050] 4. Same as Example 1.

[0051] 5. Same as Example 1.

[0052] Comparative Example 1:

[0053] The difference between Comparative Example 1 and Example 1 is that in step (1), 1,10-phenanthroline and n-butyric acid are not added, while the rest of the methods are the same as in Example 1. The resulting sample is recorded as Comparative Example 1 sample.

[0054] Comparative Example 2:

[0055] The difference between Comparative Example 2 and Example 1 is that in step (1), no n-butyric acid is added, and the rest of the methods are the same as in Example 1. The resulting sample is recorded as Comparative Example 2 sample.

[0056] Comparative Example 3:

[0057] The difference between Comparative Example 3 and Example 1 is that 1,10-phenanthroline is not added in step (1), while the rest of the methods are the same as in Example 1. The resulting sample is recorded as Comparative Example 3 sample.

[0058] Experimental results:

[0059] The nanozymes for creatinine detection obtained in Examples 1-3 and Comparative Examples 1-3 were subjected to BET and enzyme kinetic tests. The experimental results are as follows: Figures 1-8 As shown in Table 1, the BET test results are presented in terms of specific surface area, the enzyme kinetics test results are presented in terms of Michaelis constant, and the detection limits of creatinine in the range of 5-250 µM are shown in Table 1.

[0060] Table 1. BET characterization results and creatinine detection performance of Examples 1–3 and Comparative Examples 1–3

[0061] .

[0062] Based on the Michaelis constant (Km) and limit of detection (LOD) results of Examples 1-3 in Table 1, it can be seen that Example 1 has the lowest Km, indicating a stronger affinity for the substrate and a more readily catalytic reaction, thus enabling colorimetric / detection completion in a shorter time. Simultaneously, its lowest LOD indicates a more sensitive response to changes in creatinine concentration, resulting in higher detection sensitivity. Therefore, the preparation method of Example 1 is optimal, and Comparative Examples 1-3 are set up using Example 1 as the standard.

[0063] Based on the experimental data shown in Table 1, Examples 1–3 and Comparative Examples 1–3 exhibited significant differences in substrate affinity and creatinine detection performance. The nanozymes prepared in these examples showed significantly better affinity for H2O2 (Km≤0.0798 mM) and detection limits for creatinine (LOD≤0.86 μM) than all comparative examples. This superior performance is mainly attributed to the synergistic regulatory effect of the 1,10-phenanthroline and n-butyric acid dual ligands.

[0064] By comparing the performance of Examples 1-3 with Comparative Example 1, it is evident that although the sample without any auxiliary ligands has a high specific surface area (1396 m² / g), the lack of a large-sized mesoporous structure guided by n-butyric acid results in insufficient exposure of active sites and limited substrate mass transfer. Simultaneously, the absence of the electronic regulation effect of 1,10-phenanthroline prevents effective optimization of the catalytic activity of the metal center. Therefore, Comparative Example 1 has a high Km value of 0.7841 mM for H₂O₂ and a creatinine detection limit of 12.34 μM, significantly lower than the examples. The performance comparison between Examples 1-3 and Comparative Example 2 further confirms the crucial role of n-butyric acid in structural optimization. In Comparative Example 2, without the addition of n-butyric acid, the material possesses a high specific surface area (1280 m² / g). 2 The sample (g) exhibited insufficient openness of its pore structure, severely limiting substrate diffusion kinetics and resulting in insufficient affinity for the material. Its Km value was as high as 0.5102 mM, and the detection limit was 8.59 μM, significantly lower than the examples. A comparison with Comparative Example 3 highlights the importance of 1,10-phenanthroline in electronic modulation. Although Comparative Example 3 constructed open channels using n-butyric acid, enhancing substrate adsorption and diffusion, its intrinsic catalytic activity was not fully activated due to the lack of electronic modulation of the metal center by 1,10-phenanthroline. The final Km value was 0.3681 mM, and the detection limit was 4.78 μM, still significantly lower than the dual-ligand synergistic examples. Therefore, only when 1,10-phenanthroline and n-butyric acid work together can a synergistic effect of structural optimization and electronic modulation be achieved, significantly improving the substrate affinity and catalytic performance of the nanozyme, ultimately endowing it with highly sensitive creatinine detection capabilities.

[0065] Figure 1The XRD pattern of the sample from Example 1 is shown. The results indicate that the characteristic peaks in the XRD pattern are consistent with those of the simulated standard Fe-MOF and exhibit high crystallinity, demonstrating the successful preparation of the Fe-MOF nanozyme in Example 1.

[0066] Figure 2 SEM and EDS images of the sample from Example 1 are shown. The SEM image shows that the prepared material exhibits a regular spindle-shaped morphology with a well-defined shape. The EDS elemental distribution map further indicates that N, O, and Fe are evenly distributed in the material, suggesting that 1,10-phenanthroline was successfully coordinated to the metal center during synthesis and achieved uniform doping at the molecular level. This helps optimize the distribution of active sites and thus significantly improves the material's performance in creatinine detection.

[0067] Figure 3 The BET curve and pore size distribution diagram of the sample from Example 1 are shown. A significant hysteresis loop appears in the isotherm, indicating that the material possesses a rich porous structure. The pore size distribution diagram shows that the pore size is mainly distributed in the mesoporous range, further confirming that the material is predominantly mesoporous. This high-porosity structure facilitates the diffusion and enrichment of the reaction substrate, thereby significantly improving the catalytic efficiency of the nanozyme.

[0068] Figure 4 The images show the enzyme catalytic kinetic curves of the sample from Example 1 at different H2O2 concentrations. Figure 4 As can be seen from 'a', the curve conforms to the typical Michaelis-Menten model. Figure 4 The data in 'a' were calculated using the double reciprocal plotting method. Figure 4 In the figure, b, the Michaelis constant (Km) calculated from the figure is 0.0454 mM, which is much lower than that of the comparative sample, indicating that the nanozyme prepared in this invention has a higher affinity for H2O2.

[0069] Figure 5 The peroxidase-like activities of the samples from Examples 1–3 and Comparative Examples 1–3 were compared. In the TMB-H2O2 reaction system, H2O2 generates free radicals under the catalysis of nanozymes, which then oxidize colorless TMB to produce blue oxTMB. With increasing H2O2 concentration, the absorbance at 652 nm increased, reflecting an improvement in catalytic activity. Figure 5 The order of activity detected was: Example 1 > Example 3 > Example 2 > Comparative Example 3 > Comparative Example 2 > Comparative Example 1, demonstrating that the dual-ligand synergistic strategy can significantly improve the catalytic performance of the material.

[0070] Creatinine is sequentially catalyzed by creatine oxidase, creatinine enzyme, and sarcosine oxidase to produce H₂O₂. The generated H₂O₂, under the catalysis of nanoenzyme materials, converts colorless TMB into blue oxTMB. As the creatinine concentration increases, the absorbance of oxTMB at 652 nm also increases, thus establishing a relationship between absorbance and creatinine concentration. Figure 6 The UV-Vis absorption spectra of the reaction system in the presence of different concentrations of creatinine are shown. Creatinine cascades to form H₂O₂ under the action of three enzymes, which then catalyzes the color development of TMB via nanozyme. As the creatinine concentration increases from 5 µM to 250 µM, the absorbance at 652 nm steadily increases, showing a concentration-dependent response behavior. Figure 7 The results show a linear fit between absorbance at 652 nm and creatinine concentration. A good linear relationship is observed in the range of 5–250 µM, with the fitted equation being y = 0.00416x + 0.08997 (R²). 2 = 0.997), and the detection limit is as low as 0.72 µM, indicating that the constructed sensor has excellent detection sensitivity.

[0071] Figure 8 The results show the cycle stability test results of the sample in Example 1. After seven cycles of reuse, the material still retains more than 90% of its initial catalytic activity, indicating that it has excellent structural stability and reusability, which is beneficial for practical sensing applications.

Claims

1. A method for preparing Fe-MOF nanozymes based on dual-ligand regulation, characterized in that, The method includes the following steps: (S.1) Dissolve FeCl3·6H2O, 1,10-phenanthroline and n-butyric acid ligand in DMF and stir to form solution A; disperse terephthalic acid in DMF by ultrasonication to form solution B; (S.2) Add solution B to solution A, disperse by ultrasonication, transfer to a stainless steel high-pressure reactor, carry out high-temperature reaction, cool to room temperature after the reaction is completed, centrifuge and wash, vacuum dry to obtain the dual-ligand regulated Fe-MOF nanozyme sample.

2. The preparation method of Fe-MOF nanozyme based on dual-ligand regulation as described in claim 1, characterized in that, In step S.1, the molar ratio of FeCl3·6H2O, 1,10-phenanthroline, and butyric acid ligand is 1:0.5-1:2-6.

3. The preparation method of Fe-MOF nanozyme based on dual-ligand regulation as described in claim 1, characterized in that, In step S.1, the ratio of FeCl3·6H2O to DMF is 1 mmol: 5-10 mL; the molar amount of terephthalic acid is 1.5-2.5 mmol; and the ratio of terephthalic acid to DMF is 1 mmol: 3-7 mL.

4. The preparation method of Fe-MOF nanozyme based on dual-ligand regulation as described in claim 1, characterized in that, The centrifugal washing in step S.2 specifically involves: centrifuging and washing with DMF and ethanol respectively, then immersing the collected product in a mixture of DMF and hydrochloric acid and letting it stand at room temperature; and then centrifuging and washing again with DMF.

5. The preparation method of Fe-MOF nanozyme based on dual-ligand regulation as described in claim 1, characterized in that, In step S.2, the high-temperature reaction temperature is 100-150℃; the high-temperature reaction time is 16-32h; and the vacuum drying temperature is 90-150℃.

6. The preparation method of Fe-MOF nanozyme based on dual-ligand regulation as described in claim 4, characterized in that, The DMF is AR-grade DMF, and the hydrochloric acid is 12M concentrated hydrochloric acid. The volume ratio of AR-grade DMF to 12M concentrated hydrochloric acid is 1:0.02-0.

087.

7. A dual-ligand-regulated Fe-MOF nanozyme prepared by the method described in any one of claims 1-6.

8. The application of the dual-ligand regulated Fe-MOF nanozyme as described in claim 7 in the preparation of a biosensor for creatinine detection.

9. A biosensor for detecting creatinine, characterized in that, It contains the dual-ligand regulated Fe-MOF nanozyme as described in claim 7.

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