Preparation method and application of cascade reactor based on 2D MOF

Abstract

The invention relates to a preparation method and application of a cascade reactor based on 2D MOF. According to the method, a regular layered MOFs material is prepared by adjusting the molar ratio and concentration of different metal salts to ligands, then the material is soaked in an ethanol and isopropanol solvent, the material is activated through ultrasonic waves in an ice-water bath, layered separation is achieved through intermolecular force, a 2D MOFs material with the micron-scale size and the nano-scale thickness is obtained, finally, SOX is added, and the 2D MOFs material with the micron-scale size and the nano-scale thickness is obtained. The method comprises the following steps: carrying out a two-dimensional (2D) MOF / SOX cascade reactor to obtain a two-dimensional (2D) MOF / SOX cascade reactor capable of constructing a colorimetric sensor with TMB (3, 3 ', 5, 5'-tetramethyl benzidine). The reactor can perform 1-400 [mu] M linear quantification on sarcosine, and LOD is 0.324 [mu] M. According to the invention, high-efficiency, simple and cheap Pca capable of being rapidly detected can be synthesized without complex synthesis processes, instruments and expensive precious metals.

Description

Technical Field

[0001] The present invention relates to the field of biomedical sensors, and in particular to the preparation and application of a 2D MOF cascade reactor. Background Art

[0002] Prostate cancer is one of the most common malignant tumors of the urinary system. Prostate cancer generally develops slowly and may not show symptoms until the late stage of the disease. In the late stage of the disease, symptoms of hematuria and bladder outlet obstruction (such as straining to urinate, hesitant urination, weak or intermittent urine flow, a feeling of incomplete bladder filling, and terminal dribbling) or ureteral obstruction (such as renal colic, low back pain, and renal insufficiency) may occur. Bone metastasis (mainly to the pelvis, ribs, and vertebrae) can cause bone pain, pathological fractures, and spinal cord compression. Early diagnosis can effectively improve the patient's quality of life and prolong survival time.

[0003] PSA (prostate-specific antigen) is a classic monitoring indicator for prostate cancer screening and diagnosis. Currently, most prostate cancers are detected through screening with serum PSA levels (sometimes DRE (digital rectal examination)). Most patients with newly diagnosed prostate cancer have normal digital rectal examinations, and serum PSA measurement is not an ideal screening test at present. Although PSA is elevated in 25% to 92% of prostate cancer patients (depending on the size of the tumor), it is also moderately elevated in 30% to 50% of patients with benign prostatic hyperplasia (depending on the size of the prostate and the degree of obstruction), some smokers, and patients a few weeks after prostatitis or prostate operations (catheterization, cystoscopy, prostate biopsy). In rare cases, sexual activity or other activities such as extreme cycling can cause false increases in prostate-specific antigen. Levels ≥ 4 ng / mL are generally considered an indication for biopsy in men >50 years of age. Although very high PSA levels are meaningful (indicating extracapsular extension or metastasis of the tumor), and the likelihood of prostate cancer increases with increasing PSA levels, there is no PSA level that is considered a cutoff value for no tumor risk. At the same time, the EUA2019 guidelines pointed out that 25% of patients did not have high PSA during clinical progression, indicating that simply monitoring PSA may not meet today's needs for precise management of prostate cancer.

[0004] The level of sarcosine in urine of normal people is 1-3 μM, while the level of sarcosine in urine of prostate cancer patients increases by an order of magnitude to 10-30 μM. Sarcosine plays a key role in the early screening of prostate cancer, but the current diagnostic methods lack the sensitivity, accuracy and specificity required for clinical diagnosis. Therefore, it is necessary to find a diagnostic tool with high selectivity, sensitivity and stability.

[0005] Since the first discovery of Fe3O4 with peroxidase activity in 2007, a large number of artificial peroxidase mimics based on nanomaterials have emerged. Whether based on metal materials (Pt, Au, Ag, Bi, Zn), non-metal materials (C, B, Se), metal organic frameworks (MOFs) or their complexes, they have been prepared into a variety of nanozymes for the detection of sarcosine. The TCPP-Pt / SOX@HMUiO designed by Zhao's group can achieve linear fluorescence quantification of sarcosine in the range of 0-100 μM with a detection limit of 2.1 μM under the condition of fluorescence spectrophotometer (J. Am. Chem. Soc. 2021, 143, 15145−15151); Wang's group constructed a colorimetric sensing platform for detecting sarcosine using the granular composite material Fe3O4@SiO2@NiCo2S4 as a mimic enzyme, which can linearly quantify sarcosine 1.25-300 μM with a detection limit of 0.42 μM (Chem. Eng. J. 468 2023143612). In order to effectively solve the global problem of efficient, simple and inexpensive detection of Pca, it is of great significance to seek peroxidase activity comparable to natural peroxidase and precious metal materials and simple detection methods to construct a continuous, efficient, simple and inexpensive cascade reactor for clinical detection of Pca. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a cascade reactor based on 2D MOF and its application in order to solve the current problems. The material is prepared by adjusting the molar ratio and concentration of different metal salts and ligands to prepare a relatively regular layered MOFs material, then immersed in ethanol and isopropanol solvents, ultrasonically activated in an ice water bath, and separated by intermolecular forces to obtain a 2D MOFs material with a micrometer size and a nanometer thickness. Finally, SOX is added to obtain a 2D MOF / SOX cascade reactor that can be used to construct a colorimetric sensor with TMB (3,3',5,5'-tetramethylbenzidine). The reactor linearly quantifies sarcosine in urine in the range of 1-400μM, with a detection limit of 0.324 μM. The average sarcosine concentrations of 21.367 μM and 1.871 μM normal urine (n=5) were detected in patients and normal subjects, and the slight changes in sarcosine content between urine samples of PCa and healthy individuals were successfully identified. The present invention does not require complex synthesis processes and instruments, or expensive precious metals, and can synthesize an efficient, simple, inexpensive, and rapid Pca detection cascade reactor, which can completely replace natural enzymes (HRP) and precious metal mimetic enzymes and can greatly meet the needs of medical clinical detection of Pca.

[0007] The technical solution of the present invention is: A method for preparing a cascade reactor based on 2D MOF, the method comprising the following steps: (1) Place the metal salt and the ligand in N,N-dimethylformamide and stir to obtain a mixed solution; Wherein, the metal salt is cobalt salt and M salt; M salt is one or more of iron salt, aluminum salt, copper salt, nickel salt, and the molar ratio is M salt: Co salt = 1: (1-5); 20-60 mL of N,N-dimethylformamide is added for every 1 mmol of metal salt; The ligand is 2-aminoterephthalic acid, and the molar ratio is metal salt: ligand = 1: (1-5); (2) Add anhydrous ethanol and a deprotonating agent to the mixed solution in (1) and stir for 10-12 hours to obtain MOF; Wherein, add 0.5-1 mL of deprotonating agent to every 35 mL of N,N-dimethylformamide; add 1-3 mL of ethanol to every 1 mL of deprotonating agent; The deprotonating agent is triethylamine or sodium hydroxide; (3) Disperse the MOF obtained after the reaction in (2) into a mixed solvent, perform ultrasonic treatment in an ice-water bath for 1-6 hours, and separate by centrifugation; then continue to wash with a mixed solvent; repeat the "dispersion-ultrasound-centrifugation-washing" cycle for 4-6 cycles, and finally vacuum dry to obtain 2DMOF; The centrifuge speed is 4000-6000 r / min, the time is 5-10 minutes, and the drying conditions are all dried at 60 ℃-80 ℃ in a vacuum drying oven.

[0008] The mixed solvent is composed of anhydrous ethanol and isopropanol, and the volume ratio of the two is 1~5:1~5; (4) After dispersing 2D MOF in HEPES, add SOX, stir and react for 10-12 hours, and finally vacuum dry to obtain 2D MOF / SOX; Among them, 0.35-1 mg 2D MOF was added to every 1 mL HEPES (10 mM, pH=7.2), and 0.20-0.5 mg SOX was added to every 0.5 mg 2D MOF.

[0009] In the step (1), the cobalt salt is cobalt nitrate; the iron salt is ferric chloride; the aluminum salt is aluminum nitrate; the copper salt is copper sulfate; and the nickel salt is nickel chloride.

[0010] The amount of the metal salt used in step (1) can reach millimolar level.

[0011] In the step (4), HEPES is a hydrogen ion buffer solvent (4-hydroxyethylpiperazineethanesulfonic acid).

[0012] The cascade reactor of the 2D MOF / SOX prepared by the method is used to detect the sarcosine content.

[0013] Specifically, the method includes the following steps: mixing the 2D MOF / SOX solution, the urine to be tested, and the TMB solution, and then diluting with a HEPES (10 mM, pH=7.2) buffer to obtain a test solution; incubating at 37°C for 10 min, measuring the ultraviolet spectrum of the solution with an enzyme-labeled instrument, and obtaining the corresponding ultraviolet absorption light intensity; Then, according to the linear equation: Y (absorbance) = 0.04577X + 0.09647; the creatine content value X in the urine to be tested is obtained; When the detected sarcosine content is above 10 μM, it means that the sarcosine content exceeds the standard; Each 150 μL test solution contained 50 μL 2D MOF / SOX solution, 10 μL urine to be tested, and 50 μL TMB solution (4 mM).

[0014] The essential features of the present invention are: In response to the problem of the timeliness of current medical clinical detection of prostate cancer, 2D MOFs material was first prepared by adjusting the molar ratio and molar concentration of different metal salts and ligands, and then SOX was added and loaded on 2DMOFs through electrostatic adsorption to obtain a 2D MOF / SOX cascade reactor that can quickly detect Pca.

[0015] The present invention optimizes the synergistic state and defect structure of different metals and Co sites in 2D MOFs-(Fe, Al, Ni, Cu) / Co materials by adjusting the doping amount of different metals. The material with Fe atoms introduced exhibits extremely strong peroxidase activity compared with other materials. When detecting Pca, the Fe 2+ The strong Fenton effect causes a large number of reactive oxygen free radicals to be produced in a short period of time, significantly improving the timeliness and accuracy of Pca detection.

[0016] The beneficial effects of the present invention are: A relatively regular layered MOFs material was prepared by adjusting the molar ratio and concentration of metal ions and 2-aminoterephthalic acid ligands, and then the material was activated by ethanol / isopropanol under ultrasonic conditions to obtain 2D Fe / Co-MOF. Compared with the traditional morphology of Fe / Co-MOF, it has the following advantages: (1) a large number of active sites are exposed on the surface; (2) the electron transfer rate is fast; (3) due to the electron transfer from Co 2+ Transfer to Fe 3+ , Fe 2+The ratio of Fe / Co-MOF was significantly increased, which enhanced the Fenton effect. The 2D Fe / Co-MOF exhibited excellent peroxidase (POD) activity with a Vmax of 15.3×10 -8 MS -1 Higher than commercial natural enzymes 3.3×10 - 8 MS -1 , and even with precious metals (PtPdNDs / CNs:12.24×10 -8 MS -1 ) and high entropy alloy nanozymes (HEA(MnFeCoNiCu):13.15×10 -8 MS -1) It is comparable to the natural sarcosine oxidase (SOX), which greatly reduces the color development time (10 min) and enhances the immediacy. It forms a cascade reactor with natural sarcosine oxidase (SOX), which can linearly quantify sarcosine in the range of 1-400 μM, with a detection limit of 0.324 μM, which is more precise than the 5-40 μM detection of the precious metal Pt / MNP reactor. Compared with the prior art, the present invention prepares a 2D MOF / SOX cascade reactor by room temperature coprecipitation and electrostatic adsorption, and obtains a low-cost peroxidase with POD performance comparable to commercial natural enzymes and precious metal nanozymes. The immediacy and accuracy of detecting Pca are better than existing commercial clinical detection reagents. In the field of material application, this method comprehensively considers the distribution of active sites and catalytic mechanism, two important factors affecting material performance, and takes the realization of the optimal peroxidase activity of the material as the design principle. The atomic ratio of iron and cobalt is further studied to obtain the optimal structure and the matching efficient POD performance. Under the synergistic effect of iron and cobalt, it can also effectively promote the adsorption and desorption of substrates and the transfer of electrons in the catalytic process. In terms of biological detection, due to Co 2+ Fe 3+ The electron transfer of Fe 2+ The 2D Fe / Co-MOF / SOX has a high ratio, large specific surface area, and abundant active sites. It uses TMB (3,3', 5,5'-tetramethylbenzidine) colorimetric reagent and has a fast color development speed. It can be easily and quickly used as an early screening tool for prostate cancer patients, showing great application prospects in the field of biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the ultraviolet absorption spectrum of the MOFs material obtained in Example 1-5 obtained in the enzyme activity test.

[0018] Figure 2 This is a comparison chart of the Michaelis-Menten equation Km and Vmax obtained in the enzyme activity test of the MOFs material obtained in Examples 1-5.

[0019] Figure 3 This is the electron spin resonance (EPR) graph of the 2D Fe / Co-MOF obtained in Example 1 changing with time.

[0020] Figure 4 The ultraviolet absorption spectrum of the 2D Fe / Co-MOF / SOX cascade reactor obtained in Example 6 for detecting the sarcosine content in urine (A); and the linear graph of the relationship between the ultraviolet absorption intensity and the sarcosine content in urine (B).

[0021] Figure 5 This is a graph of the ultraviolet absorption light intensity in the cascade sensing platform of the 2D Fe / Co-MOF / SOX cascade reactor obtained in Example 6 for detecting different sarcosine concentrations.

[0022] Figure 6 This is a graph of the ultraviolet absorption intensity of various interfering substances detected by the 2D Fe / Co-MOF / SOX cascade reactor obtained in Example 6 at 652 nm.

[0023] Figure 7 The linear graph of the 2D Fe / Co-MOF / SOX cascade reactor obtained in Example 6 and the commercially available ELISA kit for detecting sarcosine, (A) 0-50 μM; (B) 0-400 μM.

[0024] Figure 8 Scanning electron microscope images of 2D Fe / Co-MOF and 2D Fe / Co-MOF / SOX obtained in Examples 1 and 6.

[0025] Fig. 9 EDS and N element content diagram of 2D Fe / Co-MOF and 2D Fe / Co-MOF / SOX obtained in Examples 1 and 6.

[0026] Fig.10 Fourier transform infrared spectra of 2D Fe / Co-MOF and 2D Fe / Co-MOF / SOX obtained in Examples 1 and 6. DETAILED DESCRIPTION

[0027] The invention is further described below in conjunction with the accompanying drawings and specific examples. The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The materials and reagents used are all commercially available.

[0028] Example 1: In order to test the effect of the ratio of metal salt to ligand on the morphology of MOFs materials, the controlled variable method was used to conduct experiments.

[0029] At room temperature, 1.262 mmol Co(NO3)2·6H2O and 0.238 mmol FeCl3·6H2O were added to 35 mL DMF (purity 99.5%) and stirred until completely dissolved. Then 1.5 mmol ligand (2-aminoterephthalic acid) was added. The molar ratio of Co(NO3)2·6H2O to FeCl3·6H2O and to 2-aminoterephthalic acid was 1:1. After stirring for 30 min, 2 mL of anhydrous ethanol and 1 mL of triethylamine were added, and the solution was stirred at room temperature for 12 h. The resulting product Fe / Co-MOF was immersed in a liquid phase exfoliator (v ethanol: v isopropanol = 3:2) and an external mechanical field energy (ultrasound in an ice water bath for 5 h) was applied to increase the intermolecular force to separate the layers. Then centrifuge at 5500 r / min for 10 min, discard the supernatant, repeat 5 cycles of "dispersion-ultrasound-centrifugation-washing" to remove unreacted impurities, and finally dry the product at 60 °C in vacuum for 12 h to obtain 2D Fe / Co-MOF, marked as No. 1.

[0030] The other steps of Example 2-5 are the same as those of Example 1, except that different metal salts are added. The metal salts added are Co(NO3)2·6H2O, Al(NO3)3·9H2O, CuSO4·5H2O, and NiCl2·6H2O, respectively. The obtained MOFs materials are dried and marked as 2, 3, 4, and 5, respectively.

[0031] Example 6 2 mg of 2D Fe / Co-MOF prepared in Example 1 was dispersed evenly in 4 mL HEPES (10 mM, pH=7.2), and then 2 mg of SOX was added and stirred for reaction for 12 hours. Finally, the product was vacuum dried to obtain 2D Fe / Co-MOF / SOX, which was marked as No. 6; Figure 1 The following are the UV absorption spectra of MOFs materials No. 1-6. The peroxidase activity test was performed in Examples 1-5. The test conditions are as follows: 1 mL of MOFs material (20 μg / mL), 100 μL of TMB (45 mM) and 1 mL of H2O2 (6 mM) were added to a quartz cuvette and the volume was fixed to 3 mL using HAc-NaAc buffer solution (0.2 M, pH = 3.6). After incubation at 37°C for 10 min, the UV spectrum of the solution was measured using a UV spectrophotometer. Figure 1 It can be seen that when different metals are doped with Co element, the ultraviolet absorbance intensity is improved to varying degrees compared with the single element Co-MOF, among which the enzyme activity of 2D Fe / Co-MOF is the most prominent.

[0032] Figure 2This is a comparison chart of the Michaelis equation Km and Vmax for MOFs materials No. 1-6. The test conditions are: 20 ug / mL nanozyme was dropped into TMB (0.25 mM~2 mM) and H2O2 (0.25 mM~2 mM) as reaction substrates, and then the volume was fixed to 150 uL with HAc-NaAc buffer solution (pH = 3.6, 0.2 M). Finally, incubate at 37°C for 10 minutes, and measure the UV spectrum of the solution using an enzyme reader. By studying the steady-state kinetics, it can be noted that the Km and Vamx of (Cu, Ni, Al) / Co-MOF are not significantly improved compared to Co-MOF, and are even slightly lower. In comparison, the Km and Vmax of 2D Fe / Co-MOF are better than the above materials and have exceeded the currently commercial natural horseradish oxidase (HRP). From the above, it can be concluded that sample No. 1 has the best enzyme activity, which means that the addition of iron ions in this material can prompt cobalt ions to form more active sites in MOFs, exerting the synergistic effect between the two metals and promoting the improvement of enzyme activity.

[0033] Figure 3 This is the electron spin resonance (EPR) graph of 2D Fe / Co-MOF changing with time, where A is superoxide anion, B is singlet oxygen, and C is hydroxyl radical. At 1 min, a large number of active oxygen radicals (•OH, O2 •－ , 1 O2), the intensity increases with time, thus accelerating the oxidation rate of TMB, indicating that the color development rate can be greatly improved, thereby meeting the needs of rapid detection of creatine in urine.

[0034] Figure 4 This is a graph of the 2D Fe / Co-MOF / SOX cascade reactor to determine the content of creatine in urine, where A is the UV absorption spectrum of creatine detection; B is a linear graph of the relationship between UV absorption intensity and the content of creatine in urine; test conditions: 50 μL 2D MOF / SOX (45 μg / mL), 10 μL of creatine in urine with different concentrations (15-6000 μM), 50 μL TMB (4mM), diluted to 150 μL with HEPES (10mM, pH=7.2) buffer, incubated at 37°C for 10 min, and the UV spectrum of the solution was measured with an enzyme marker. The reactor linearly quantified the content of creatine in urine in the range of 1-400 μM (Y(absorbance) = 0.04577X + 0.09647 (R 2 = 0.99675); the creatine content value X in the urine to be tested is obtained), and the detection limit is 0.324 μM.

[0035] Figure 5The figures are the UV absorption intensity graph and UV absorption intensity bar graph of the cascade sensing platform for detecting different sarcosine concentrations using the 2D Fe / Co-MOF / SOX cascade reactor obtained in Example 6; wherein A is the UV absorption intensity graph of the cascade sensing platform for detecting different sarcosine concentrations; and B is the UV absorption intensity bar graph of 5 PCa patients (red columns) and 5 healthy men (green columns). The test conditions of A are as follows: 50 μL 2D MOF / SOX (45 μg / mL), 10 μL urine sample containing different concentrations of sarcosine (150 μM, 300 μM, 600 μM, 1200 μM, 2100 μM, 2700 μM), and 50 μL TMB (4mM) were diluted to 150 μL with HEPES (10mM, pH=7.2) buffer, incubated at 37°C for 10 min, and the UV spectrum of the solution was measured using an enzyme marker. The test conditions of B are as follows: 50 μL 2D MOF / SOX (45 μg / mL), 10 μL urine sample diluted ten times, and 50 μL TMB (4mM) were diluted to 150 μL with HEPES (10mM, pH=7.2) buffer, incubated at 37°C for 10 min, and the UV spectrum of the solution was measured using an enzyme marker. From the data in A, it can be concluded that the accuracy is 97.27~105.8% (formula: accuracy = detected concentration / actual concentration × 100%); B reflects that the average sarcosine concentrations detected in patients and normal subjects were 21.367 μM and 1.871 μM normal urine (n=5), successfully identifying the slight changes in sarcosine content between urine samples of PCa and healthy individuals.

[0036] Figure 6 This is the ultraviolet absorption intensity diagram of various interfering substances detected by the 2D Fe / Co-MOF / SOX cascade reactor at 652 nm; A is glycine, serine, glutamic acid, tyrosine, histidine, uric acid, glucose, ascorbic acid and biological ions (Na + , Ca 2+ , Mg 2+ , K + , PO4 2- , Cl - ) samples; Test conditions: 50 μL 2D MOF / SOX (45 μg / mL), 10 μL different concentrations (0-1500 μM) of sarcosine, glycine, serine, glutamic acid, tyrosine, histidine, uric acid, glucose, ascorbic acid and biological ions (Na + , Ca 2+ , Mg 2+ , K + , PO4 2- , Cl -), 50 μL TMB (4 mM), diluted to 150 μL with HEPES (10 mM, pH = 7.2) buffer, incubated at 37 ° C for 10 min, and the UV spectrum of the solution was measured with an enzyme marker; B is glycine, serine, glutamic acid, tyrosine, histidine, uric acid, glucose, ascorbic acid and biological ions (Na + , Ca 2+ , Mg 2+ , K + , PO4 2- , Cl - ) were mixed with sarcosine; test conditions: 50 μL 2D MOF / SOX (45 μg / mL), 10 μL of various amino acids containing different concentrations (750-1500 μM) of sarcosine, 50 μL TMB (4mM), diluted to 150 μL with HEPES (10mM, pH=7.2) buffer, incubated at 37℃ for 10 min, and the UV spectrum of the solution was measured with an enzyme-labeled instrument; Figure 6 It can be seen that glycine, serine, glutamic acid, tyrosine, histidine, uric acid, glucose, ascorbic acid and biological ions (Na + , Ca 2+ , Mg 2+ , K + , PO4 2- , Cl - ) components had no effect on creatine, demonstrating that creatine levels in human urine samples can be accurately quantified.

[0037] Figure 7 The figure is a linear plot of the 2D Fe / Co-MOF / SOX cascade reactor and the commercial ELISA kit for detecting sarcosine, where A is 0-50 μM and B is 0-400 μM; the test conditions are: the commercial standard method for sarcosine is detected using a customized MesGen sarcosine kit. During the assay, sarcosine is specifically oxidized by sarcosine oxidase, and the product reacts with the colorless probe developer and displays absorbance at 570 nm. Typically, 20 uL of standard sample and the sample to be tested are added to each pre-prepared blank well. In each reaction well, 50 uL of reagent A, 50 uL of reagent B, and 2 uL of reagent C are mixed (lightly vortex before pipetting). 100 uL of working reagent is transferred to each reaction well. Tap the plate to mix. Incubate at room temperature for 10 minutes and read the optical density at 570 nm. It can be seen that there is no significant difference in the linear relationship between the cascade reactor designed by us, 2D Fe / Co-MOF / SOX, and the commercial sarcosine kit in detecting sarcosine.

[0038] Figure 8The scanning electron microscope images of 2D Fe / Co-MOF and 2D Fe / Co-MOF / SOX obtained in Examples 1 and 6, wherein A is the SEM image of the prepared sheet MOFs material, which shows clear, complete and continuous sheet MOFs material with a size of micrometers and ultra-thin thickness. B is the SEM image of loaded sarcosine oxidase (SOX), which shows that there is no effect on the morphology of the MOFs material.

[0039] Fig. 9 The EDS and N element content bar graphs of 2D Fe / Co-MOF and 2D Fe / Co-MOF / SOX clearly show the uniform distribution of Fe, Co and N in 2D Fe / Co / MOF and 2D Fe / Co-MOF / SOX. At the same time, due to the high N content of SOX, its N element content is higher than that of the original 2D MOF, proving that SOX is successfully loaded on 2D MOF.

[0040] Fig.10 This is the FTIR graph of the sheet MOFs material, which is basically consistent with the peak position of the Fe / Co-MOF material reported in the relevant literature, confirming that the prepared material is 2D Fe / Co-BDC-NH2MOFs material. When SOX is loaded on 2D MOFs by physical adsorption, the stretching vibration and bending vibration peaks of NH are red-shifted, indicating that SOX is successfully loaded on 2D MOFs.

[0041] Table 1 is a comparison of the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax). It can be noted that 2DFe / Co-MOF has significant peroxidase-like activity with H2O2 as the substrate compared with HRP and Fe3O4. More importantly, its Vmax value for H2O2 is 1.76 times that of HRP, even comparable to noble metal and high entropy nanozymes.

[0042] Table 1 is a comparison of the Michaelis constant (Km) and the maximum reaction rate (Vmax) of the 2D Fe / Co-MOF obtained in Example 1 of the present invention and the currently studied materials.

[0043]

[0044] Table 2 is a comparison of the 2D Fe / Co-MOF / SOX obtained in Example 6 of the present invention and the LOD of sarcosine detected by different materials

[0045] Table 2 is a comparison of the LOD of sarcosine detected by different samples. The detection limit (LOD: calculated using the formula 3σ / K, where σ is the deviation of the standard 11 blank samples and K is the slope of the linear equation) was calculated to be 0.324 μM, which is better than other materials.

[0046] The preparation method of the 2D MOF / SOX cascade reactor described in this embodiment is simple, safe, and low in energy consumption. There is no toxic solvent in the preparation process, which is green and environmentally friendly. The prepared 2D MOF / SOX cascade reactor has regular nanosheets, large specific surface area, many active sites, high affinity (Km), strong anti-interference ability, good immediacy, and can accurately measure the sarcosine content in urine and accurately screen prostate cancer patients. The cost and stability are far superior to commercial natural horseradish oxidase, and it has important clinical medical value.

[0047] Matters not covered by the present invention are known technologies.

Claims

1. A method for preparing a cascade reactor based on 2D MOF, characterized in that the method comprises the following steps: (1) Place the metal salt and the ligand in N,N-dimethylformamide and stir to obtain a mixed solution; in, The metal salts are cobalt salts and M salts; M salt is one or more of iron salt, aluminum salt, copper salt, nickel salt, and the molar ratio is M salt: Co salt = 1: (1-5); 20-60 mL of N,N-dimethylformamide is added for every 1 mmol of metal salt; The ligand is 2-aminoterephthalic acid, and the molar ratio is metal salt: ligand = 1: (1-5); (2) Add anhydrous ethanol and a deprotonating agent to the mixed solution in (1) and stir for 10-12 hours to obtain MOF; Wherein, add 0.5-1 mL of deprotonating agent to every 35 mL of N,N-dimethylformamide; add 1-3 mL of ethanol to every 1 mL of deprotonating agent; The deprotonating agent is triethylamine or sodium hydroxide; (3) Disperse the MOF obtained after the reaction in (2) into a mixed solvent, perform ultrasonic treatment in an ice-water bath for 1-6 hours, and centrifuge to separate. Then continue to wash with a mixed solvent. Repeat the "dispersion-ultrasound-centrifugation-washing" cycle for 4-6 cycles, and finally vacuum dry to obtain 2DMOF. The mixed solvent is composed of anhydrous ethanol and isopropanol, and the volume ratio of the two is 1~5:1~5; (4) Disperse 2D MOF in HEPES (10 mM, pH=7.2), add SOX, stir and react for 10-12 hours, and finally vacuum dry to obtain 2D MOF / SOX; Among them, 0.35-1 mg 2D MOF was added to every 1 mL HEPES (10 mM, pH=7.2), and 0.20-0.5 mg SOX was added to every 0.5 mg 2D MOF.

2. The method for preparing a cascade reactor based on 2D MOF according to claim 1, characterized in that In the step (1), the cobalt salt is cobalt nitrate; the iron salt is ferric chloride; the aluminum salt is aluminum nitrate; the copper salt is copper sulfate; and the nickel salt is nickel chloride; and in the step (4), HEPES is a hydrogen ion buffer solvent (4-hydroxyethylpiperazineethanesulfonic acid).

3. The method for preparing a 2D MOF-based cascade reactor as described in claim 1 is characterized in that the centrifuge speed is 4000-6000 r / min, the time is 5-10 minutes, and the drying conditions are drying at 60°C-80°C in a vacuum drying oven.

4. Application of the cascade reactor of 2D MOF / SOX prepared by the method of claim 1, characterized in that it is used to detect the sarcosine content.

5. The use according to claim 4, characterized in that it specifically comprises the following steps: mixing the 2D MOF / SOX solution, the urine to be tested, and the TMB solution, and then diluting with a HEPES (10 mM, pH=7.2) buffer to obtain a test solution; incubating at 37°C for 10 minutes, measuring the ultraviolet spectrum of the solution with an enzyme marker, and obtaining the corresponding ultraviolet absorption light intensity; Then, according to the linear equation: Y (absorbance) = 0.04577X + 0.09647; the creatine content value X in the urine to be tested is obtained; When the creatine content in the urine to be tested is above 10 μM, it means that the creatine content exceeds the standard; Each 150 μL test solution contains 50 μL 2D MOF / SOX solution, 10 μL urine to be tested, and 50 μL TMB solution (4 mM).

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