POMs type mimic enzyme and application thereof

Abstract

The application belongs to the technical field of biomolecule detection and analysis, and discloses a kind of POMs type mimic enzyme and its application.The POMs type mimic enzyme is a kind of transition metal substituted POMs, has better catalytic oxidation TMB ability, and spermine can produce enzyme activity inhibition to it for direct detection of spermine molecules.The transition metal substituted POMs is composed of 2 Keggin type three-void phosphotungstic acid polyoxy anion, 10 protons and 20 or 24 crystal water molecules.The spermine detection sensor constructed by the POMs type mimic enzyme is a new colorimetric detection biomolecule spermine strategy based on heteropoly acid, does not need to rely on complex pretreatment derivatization process and expensive instrument operation system, and can realize rapid, qualitative or quantitative determination of spermine in the system under ultraviolet detector or visual state.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomolecule detection and analysis, and particularly relates to a POMs type of artificial enzyme and application thereof. BACKGROUND

[0002] Horseradish peroxidase (HRP) is an important natural enzyme, which can be combined with antibodies to catalyze the color development of substrates for enzyme-linked immunoassay. Enzyme-linked immunoassay is simple and rapid, and is widely used in food, drug and clinical analysis. However, the preparation and storage of HRP are time-consuming and expensive, and HRP has the disadvantages of easy denaturation and inactivation. Therefore, the research on artificial peroxidase mimics has attracted widespread attention.

[0003] Nano-mimetic enzyme is a new generation of artificial synthetic enzyme with unique properties of nanomaterials and catalytic function, which is a new generation of artificial synthetic enzyme after cyclodextrin, crown ether, porphyrin and organometallic complex mimetic enzyme. Compared with natural enzymes, nano-mimetic enzyme is easy to prepare, widely available, low in price, and its activity is easy to control. It has stronger bearing capacity for extreme pH value, temperature and substrate concentration.

[0004] Polyoxometalate, referred to as polyacid, is a kind of metal-oxygen cluster compounds formed by covalent bond between transition metal ions, especially vanadium, molybdenum and tungsten, and oxygen. Polyacid has incomparable structural diversity and excellent catalytic properties, is simple to prepare, low in price and green and environmentally friendly. Referring to the published literature: Wang J, Han D, Wang X, et al. Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection [J]. Biosensors and Bioelectronics, 2012, 36(1): 18-21. Wang Xiaohong's research group first proved that polyacid has peroxidase activity, which can replace horseradish peroxidase and be used for analysis and sensing.

[0005] Polyamines (PAs) are low-molecular-weight aliphatic polycations. Due to their positively charged nature, they can readily bind to cellular components such as nucleic acids, proteins, and cell membranes, thereby protecting or regulating the physiological state of cells. Therefore, PA homeostasis plays a crucial role in the prodromal stages of many physiological and pathological processes. Spermine, as a polyamine, is present in all eukaryotic cells. In humans, spermine plays an important role in immune responses, neuronal regulation, and certain pathological events. Therefore, fluctuations in spermine levels are often associated with disease. For example, patients with stroke or chronic renal failure have low levels of spermine in their blood. Conversely, patients with liver cancer and breast cancer have detectable high concentrations of spermine in their urine and saliva. Therefore, quantitative analysis of spermine in biological fluid samples may serve as a highly effective biodiagnostic marker.

[0006] In existing technologies, the detection of spermine from biological samples typically requires complex purification and chemical derivatization procedures. Derivatization reagents include ninhydrin, o-phthalaldehyde, dansyl chloride, or benzoyl chloride. Derivatized spermine can then be quantitatively detected using methods such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), gas chromatography (GC), or capillary electrophoresis. In contrast, mass spectrometry-based detection methods can directly detect spermine without derivatization. However, these traditional spermine detection methods all require specialized knowledge to operate expensive systems, and the time-consuming procedures limit their application in clinical testing. Of course, spermine detection can also be achieved using sensors based on natural enzymes or nanomaterials. Natural enzymes, such as spermine oxidase, have high specificity for spermine detection, but they have a short shelf life. In addition to important factors such as temperature and pH, factors such as surfactants and the ionic strength of the solution also affect its activity.

[0007] Currently, the application of nanomaterials in the field of biosensing is gradually increasing. See the published literature: Jornet-Martínez N et al. Sensitive and selective plasmonic assay for spermine as biomarker in human urine.[J].Analytical chemistry,2014,86(3):1347-51. In the detection of spermine, Jornet-Martínez et al. used gold nanoparticles as colorimetric probes. Spermine was detected by inducing the aggregation of gold nanoparticles by the analyte. At higher concentrations, other polyamines such as cadaverine and spermidine can also induce the aggregation of gold nanoparticles and cause their SPR absorption peak to shift. Recent reference: AKAR, AJRB, BRKS, et al. Microwave assisted synthesis of tyrosine-protected gold nanoparticles for dual (colorimetric and fluorimetric) detection of spermine and spermidine in biological samples[J]. Biosensors and Bioelectronics, 2017, 88: 71-77. Rawat et al. reported a dual probe using tyrosine-modified gold nanoparticles for colorimetric and fluorescence excitation methods of spermine and spermidine. However, the aforementioned gold nanoparticle-based sensor system still has limitations in the specific detection of spermine in biological samples due to the strong matrix effect in complex biological samples. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies. Firstly, it provides a class of POMs-type peroxidases. Secondly, based on these peroxidase-like enzymes, it provides a colorimetric sensor for detecting the biomolecule spermine with high sensitivity, good specificity, and a low detection limit. The novel colorimetric detection strategy for spermine provided by this invention is designed based on the biomimetic enzyme activity regulation of heteropolyacids. By synthesizing heteropolyacids with biomimetic enzyme activity, and utilizing the influence of their enzyme activity on the catalytic activity of POMs-type mimic enzymes—specifically, the inhibitory effect of spermine on the catalytic activity of these enzymes—and based on the fact that the color development of the substrate TMB gradually decreases with increasing spermine concentration, a detection standard curve is plotted using ultraviolet absorption spectroscopy data to ultimately achieve the purpose of detecting spermine.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] In a first aspect, the present invention provides a class of POMs-type mimic enzymes. Specifically, the POMs-type mimic enzymes provided by the present invention are a class of transition metal-substituted polyoxometalates (hereinafter referred to as transition metal-substituted POMs), which have a better ability to catalyze the oxidation of TMB, and spermine can inhibit their enzyme activity for the direct detection of spermine molecules.

[0011] The transition metal-substituted POMs consist of two Keggin-type triple-vacant phosphotungstic acid polyoxyanions, 10 protons, and 20 or 24 molecules of water of crystallization.

[0012] Preferably, the structural formula of the transition metal substituted POMs is any one of the following structural formulas, and the following five transition metal substituted POMs are abbreviated as M4POM polyacids:

[0013] K 10 [Co4(H2O)2(PW9O 34 )2]·20H2O;K 10 [Zn4(H2O)2(PW9O 34 )2]·20H2O;

[0014] K 10 [Mn4(H2O)2(PW9O 34 )2]·20H2O;K7Na3[Cu4(H2O)2(PW9O 34 )2]·20H2O;

[0015] K6Na4[Ni4(H2O)2(PW9O 34 )2]·24H2O.

[0016] This invention also provides a method for preparing the above-mentioned POMs-type mimic enzymes, comprising the following preparation steps:

[0017] When the transition metal source is a Co, Zn, Mn, or Cu source, the preparation method of the POMs-type mimic enzyme includes: dissolving the transition metal source in ultrapure water as a first solution, and adding a completely dried powdered precursor compound Na8H[B-PW9O] to the first solution. 34The mixture was heated and stirred continuously at 60–80°C until the polyacid precursor compound was completely dissolved. The filtrate after removing the insoluble matter was used as the second solution. Excess potassium chloride powder was added to the second solution until a precipitate was formed. After the reaction cooled to room temperature, the solid was collected by vacuum filtration. The collected wet solid was completely dissolved in hot water and recrystallized at 4°C or room temperature for 12 hours. The precipitated crystals were collected by vacuum filtration and dried under vacuum at 80°C or under air at room temperature to synthesize the corresponding transition metal substituted POMs. The molar mass ratio of the transition metal source to the precursor compound ranged from (2:1) to (4:1).

[0018] When the transition metal source is a Ni source, the preparation method of the POMs-type mimic enzyme includes: dissolving Na2WO4·2H2O solid and Na2HPO4 solid in ultrapure water; adjusting the pH to 7.1 by adding glacial acetic acid dropwise, then adding an aqueous solution containing nickel acetate, resulting in a yellow solution with pH 6.9; then heating in an oil bath under reflux for 2 hours, filtering while hot and immediately adding potassium acetate; when the yellow solid begins to precipitate in the solution, filtering under reduced pressure to obtain a yellow crystalline powder mixture K6Na4[Ni4(H2O)2(PW9O)] 34 )2]·24H2O;

[0019] Among them, the precursor compound Na8HPW9O 34 The preparation process of ·24H2O includes: dissolving Na2WO4-2H2O in ultrapure water with continuous stirring; then adding 85% H3PO4 to the stirred solution, and finally adding glacial acetic acid; 1-10 seconds after adding glacial acetic acid, when the solution begins to become turbid and a white precipitate begins to form, collecting the precipitate by vacuum filtration; drying the precipitate at room temperature for 24 hours to obtain a dry powdered crude product; and continuously drying the powdered crude product at 162℃ until the acetic acid odor disappears to obtain Na8H[B-PW9O] 34 ]structure.

[0020] Secondly, this invention provides the application of the aforementioned POMs-type mimic enzyme in the fabrication of a spermine detection sensor. The spermine detection sensor comprises separately stored POMs-type mimic enzyme solution, H2O solution, chromogenic substrate TMB ethanol solution, and buffer solution. The spermine detection sensor includes the following detection steps: after obtaining the spermine sample to be tested, a reaction group and a control group are set up. In the reaction group, the H2O solution, chromogenic substrate TMB ethanol solution, and buffer solution are mixed first, then the spermine sample to be tested is added, and finally the POMs-type mimic enzyme solution is added. The reaction time is at least 5 minutes. The control group differs from the reaction group only in that the spermine sample to be tested is not added. By comparing the TMB color development degree of the control group and the reaction group, it is determined whether the spermine sample to be tested in the reaction group contains spermine. The spermine detection sensor utilizes the inhibition of the catalytic activity of the POMs-type mimic enzyme by spermine. The TMB color development degree gradually decreases as the spermine concentration increases, and rapid detection of spermine can be achieved using a simple colorimetric method.

[0021] The spermine detection sensor provided by this invention not only enables the qualitative detection of spermine, but also demonstrates accurate and quantitative detection of spermine through a colorimetric strategy designed based on the biomimetic enzyme activity regulation of heteropolyacids. Compared with existing spermine detection methods or sensors, it achieves a lower detection limit, ranging from 20 nM to 20 μM. Furthermore, it exhibits certain substrate interference resistance; for example, this invention has been verified to detect spermine in biological matrices such as artificial urine. Additionally, commercially available detection kits based on the spermine sensor described in this invention can be used as diagnostic reagents for spermine-related diseases.

[0022] In a preferred embodiment of the present invention, the application of the above-mentioned POMs-type mimic enzyme in the fabrication of a spermine detection sensor includes the following steps:

[0023] Step S1, prepare the reaction reagent solution, including the following reagent solutions:

[0024] First reagent solution: 100 μl of 1 mM M4POM solution;

[0025] Second reagent solution: 100 μl of 100 mM or 1 M H2O2 solution;

[0026] Third reagent solution: 100 μl of 10 mM TMB ethanol solution;

[0027] Fourth reagent solution: 1700 μl of sodium acetate buffer or PBS buffer;

[0028] Both the H2O2 solution and the TMB solution were prepared fresh and used immediately, and were stored in the dark before the reaction began.

[0029] Step S2, Establishing a Standard Curve: Set up a standard reaction group and a standard control group. The standard reaction group setup process includes: taking spermine samples at three standard concentrations (100 μM, 10 μM, and 1 μM) and setting up three corresponding standard reaction groups. The operation process for each standard reaction group is as follows: first, mix the second, third, and fourth reagent solutions, then add the spermine sample, and then add the first reagent solution (M4POM polyacid) and start timing. The total reaction time is 5 minutes. The standard control group setup process includes: mixing the second, third, and fourth reagent solutions, then adding the spermine sample, and finally adding the first reagent solution (M4POM polyacid) and starting timing. The solution and the fourth reagent solution were mixed, and then the first reagent solution M4POM polyacid was added to start the timing. The total reaction time was 5 minutes. After the reaction, a UV-Vis spectrophotometer was used to perform spectral scanning on each standard reaction group and control group. The spectral scanning range was 500-800 nm. The UV absorbance A of each standard reaction group and the UV absorbance A0 of the blank standard control group without spermine were obtained. A linear equation was fitted to the concentration of 1g spermine with A / A0. The detection range was 20nM-20μM. Finally, a standard curve was obtained.

[0030] Step S3, Spermine sample detection: Set up a detection reaction group and a detection control group. The detection reaction group setup process includes: mixing the second, third, and fourth reagent solutions, adding the spermine sample to be tested, then adding the first reagent solution M4POM polyacid and starting the timer. The total reaction time is 5 minutes. The detection control group setup process includes: mixing the second, third, and fourth reagent solutions, then adding the first reagent solution M4POM polyacid and starting the timer. The total reaction time is 5 minutes. After the reaction, use a UV-Vis spectrophotometer to perform spectral scanning on the detection reaction group and the detection control group respectively. The spectral scanning range is 500-800 nm. Obtain the UV absorbance A of the detection reaction group and the UV absorbance A0 of the blank detection control group without spermine. Substitute the values ​​of A and A0 into the standard curve established above to calculate the spermine concentration value in the sample.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] Compared with other POMs-type mimics, the POMs-type mimic provided by this invention has better catalytic oxidation activity of hydrogen peroxide, i.e., it possesses peroxidase-like activity. In the presence of H2O2, it can catalyze the oxidation of the chromogenic substrate TMB (3,3,5,5-dimethylaniline) to form the blue product TMB. +By using a UV-Vis spectrophotometer to scan the spectra of the reaction products, the product concentration can be calculated and differentiated by measuring the absorbance of the products. CoPOM and CuPOM also possess the ability to catalyze the oxidation of hydrogen peroxide under neutral pH conditions; and due to the varying degrees of inhibition of the catalytic activity of POMs by different concentrations of spermine, and the production of the blue product TMB... + The ultraviolet colorimetric assay shows a significant concentration-dependent change depending on the concentration, thus enabling its application in the in vitro detection of spermine.

[0033] Since spermine lacks ultraviolet absorption, both liquid chromatography and fluorescence detection require complex pretreatment derivatization processes, which is detrimental to rapid analysis. Therefore, compared with traditional spermine detection methods, the spermine detection sensor or method provided by this invention only requires simple mixing of the solution to achieve rapid detection of spermine. Furthermore, because the colorimetric intensity of TMB gradually decreases with increasing spermine concentration, the spermine detection sensor or method provided by this invention can also achieve visually visible colorimetric changes, exhibiting a high signal-to-noise ratio and great potential in rapid detection applications. Moreover, compared with other low-limit detection methods for spermine in the prior art, such as those used for biomarker detection, the spermine detection sensor of this invention, based on a novel colorimetric strategy for detecting the biomolecule spermine using heteropolyacids, can successfully detect spermine in complex biological matrices such as artificial urine, and this process is minimally affected by organic and inorganic interfering substances. Attached Figure Description

[0034] Figure 1A The Na8H[A-PW9O] prepared in Example 1 of this invention 34 Infrared spectrum;

[0035] Figure 1B The Na8H[B-PW9O] prepared in Example 1 of this invention 34 Infrared spectrum;

[0036] Figure 2 This is a comparison of the infrared spectra of five POMs prepared in Examples 1-5 of this invention;

[0037] Figure 3A The kinetic curve of CoPOM relative to the substrate H2O2 is shown.

[0038] Figure 3B The kinetic curve of CoPOM relative to the substrate TMB.

[0039] Figure 3C The kinetic curve of ZnPOM relative to the substrate H2O2 is shown.

[0040] Figure 3D The kinetic curve of ZnPOM relative to the substrate TMB is shown.

[0041] Figure 3E The kinetic curve of MnPOM relative to the substrate H2O2 is shown.

[0042] Figure 3F The kinetic curve of MnPOM relative to the substrate TMB is shown.

[0043] Figure 3G The kinetic curve of NiPOM relative to the substrate H2O2 is shown.

[0044] Figure 3H The kinetic curve of NiPOM relative to the substrate TMB is shown.

[0045] Figure 3I The kinetic curve of CuPOM relative to the substrate H2O2 is shown.

[0046] Figure 3J The kinetic curve of CuPOM relative to the substrate TMB is shown.

[0047] Figure 4 The effects of high, medium, and low concentrations of spermine on the activity of different POM-type mimic enzymes;

[0048] Figure 5 The standard curve, ultraviolet spectrum, and cuvette photograph of the spermine colorimetric detection system established in Example 6;

[0049] Figure 6 The kinetic curve of CoPOM in the presence of 1 μM spermine is shown in Test Example 3.

[0050] Figure 7A Results showing the influence of organic interfering substances on the colorimetric system;

[0051] Figure 7B The results show the influence of inorganic interfering substances on the colorimetric system;

[0052] Figure 8A This is the liquid chromatogram used for comparison in Example 7;

[0053] Figure 8B This is the derivatization-liquid chromatography detection standard curve used for comparison in Example 7;

[0054] Figure 8C This is the colorimetric detection standard curve used for comparison in Example 7;

[0055] Figure 9 A comparison table of kinetic parameters for different POMs-type enzyme mimics. Detailed Implementation

[0056] To provide a more accurate understanding of the technical features, objectives, and effects of this invention, the invention will be further described below with reference to the accompanying drawings and specific embodiments. However, this should not be construed as limiting the scope of the invention to the following embodiments. All technologies and applications implemented based on the above-described content of this invention fall within the scope of this invention.

[0057] The sources of raw materials and reagents in the following examples and test cases are:

[0058] 3,3,5,5-Bimethylaniline (TMB) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; glacial acetic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; sodium acetate was purchased from Sigma-Aldrich; potassium dihydrogen phosphate was purchased from Nanjing Experimental Testing Co., Ltd.; dipotassium hydrogen phosphate was purchased from Nanjing Experimental Testing Co., Ltd.; spermine was purchased from Mreda; horseradish peroxidase (HRP) was purchased from Yuanye Biotechnology Co., Ltd.

[0059] The artificial urine was prepared according to the method reported in the literature Kim TI, Park J, Kim YA Gold Nanoparticle-Based Fluorescence Turn-On Probe for Highly Sensitive Detection of Polyamines[J]. Chemistry–A European Journal,2011,17(43):11978-11982. The artificial urine was prepared by consisting of lactic acid (1.1 mM), citric acid (2.0 mM), sodium bicarbonate (25 mM), urea (170 mM), calcium chloride (2.5 mM), sodium chloride (90 mM), magnesium sulfate (2.0 mM), sodium sulfate (10 mM), potassium dihydrogen phosphate (7.0 mM), dipotassium hydrogen phosphate (7.0 mM), ammonium chloride (25 mM), and deionized water.

[0060] Example 1: Transition metal substituted polyacid K 10 [Co4(H2O)2(PW9O 34 Synthesis of 2]·20H2O

[0061] Synthesis and Characterization of Polyacid Precursor Compounds: The structures of five transition metal-substituted POMs were synthesized and characterized. First, Na8HPW9O was synthesized. 34To obtain the 24H2O structure, 30.0 g of Na2WO4-2H2O was dissolved in 37 mL of ultrapure water with continuous stirring. Then, 0.75 mL of 85% H3PO4 was added to the stirred solution, followed by 5.5 mL of glacial acetic acid. A few seconds after the addition of acetic acid, the solution began to become turbid, and a white precipitate began to form after approximately one minute. The precipitate was collected by vacuum filtration. The precipitate was dried at room temperature for 24 hours to obtain a dry powdered solid Na8HPW9O. 34 ·24H2O, i.e., Na8H[A-PW9O 34 The structure, its infrared characterization is as follows: Figure 1A As shown; the solid is dried continuously at 162℃ until the acetic acid odor disappears to obtain Na8H[B-PW9O]. 34 The structure, its infrared characterization is as follows: Figure 1B As shown.

[0062] Synthesis and characterization of transition metal-substituted polyacids: 0.41 g of solid CoNO3·6H2O was dissolved in 15 ml of ultrapure water. 2 g of completely dry powdered precursor compound Na8H[B-PW9O] was added to the pale pink solution. 34 The mixture was then heated to 80°C with continuous stirring for 5 to 10 minutes. When the solution turned a deep wine-red color, 4g of excess potassium chloride powder was added, resulting in a blue-purple precipitate. After the reaction cooled to room temperature, the solid was collected by vacuum filtration. The collected moist solid was completely dissolved in 5-10ml of hot water and recrystallized at 4°C for 12 hours. The precipitated crystals were collected by vacuum filtration and dried under vacuum at 80°C. K was synthesized. 10 [Co4(H2O)2(PW9O 34 )2]·20H2O.

[0063] Example 2: Transition metal substituted polyacid K 10 [Zn4(H2O)2(PW9O 34 Synthesis of 2]·20H2O

[0064] 0.19 g of solid ZnCl2 was dissolved in 15 mL of ultrapure water, and 2 g of the completely dried powdered precursor compound Na8H[B-PW9O prepared in Example 1 was added to the solution. 34The solution was heated and stirred continuously at 80°C until it became nearly homogeneous. Then, it was filtered while hot to remove insoluble matter, and the filtrate was collected. 4g of excess KCl powder was added to the collected filtrate, at which point a white precipitate formed. After the reaction cooled to room temperature, the solid was collected by vacuum filtration. The collected moist solid was completely dissolved in 5-10ml of hot water and recrystallized at 4°C for 12 hours. The precipitated crystals were collected by vacuum filtration and dried under vacuum at 80°C. KCl was thus synthesized. 10 [Zn4(H2O)2(PW9O 34 )2]·20H2O.

[0065] Example 3: Transition metal substituted polyacid K 10 [Mn4(H2O)2(PW9O 34 Synthesis of 2]·20H2O

[0066] The synthesis of MnPOM employed a method similar to that used for the synthesis of CoPOM and ZnPOM. 2 g of the precursor compound Na8H[B-PW9O] prepared in Example 1 was used. 34 Slowly add the solution to 15 ml of an aqueous solution containing 0.2366 g of MnSO4·H2O. Gently heat the orange solution and remove any insoluble substances by hot filtration. Add excess 5 g of KCl powder to the orange solution, causing an immediate precipitation of an orange solid. Collect the solid by suction filtration and recrystallize it multiple times in 20 ml of water to finally obtain KCl. 10 [Mn4(H2O)2(PW9O 34 Orange crystalline powder (2)·20H2O.

[0067] Example 4: Transition metal substituted polyacid K7Na3[Cu4(H2O)2(PW9O) 34 Synthesis of 2]·20H2O

[0068] 0.62 g of solid CuCl2·2H2O was added to 30 ml of ultrapure water and stirred until completely dissolved to form a pale blue solution. Then, 5.0 g of the precursor compound Na8H[B-PW9O] was added to this solution. 34 The mixture was stirred continuously until all solids were completely dissolved, and the solution turned light green. Then, 0.66 g of KCl powder was added, resulting in a white-green precipitate. The mixture was stirred at room temperature for ten minutes. Then, it was heated in a 60°C water bath for 2 minutes until large solid particles dissolved. The resulting slightly turbid liquid was centrifuged at 10,000 rpm for 5 minutes. The solid suspension was removed, and the supernatant was collected in a beaker and allowed to stand at room temperature for 12 hours. The solid was collected by vacuum filtration and finally dried in air. This yielded K7Na3[Cu4(H2O)2(PW9O)]34 )2]·20H2O.

[0069] Example 5: Transition metal substituted polyacid K6Na4[Ni4(H2O)2(PW9O)] 34 Synthesis of 2]·24H2O

[0070] The synthesis of NiPOM did not employ a precursor compound-based method. 33 g of solid Na₂WO₄·2H₂O and 1.57 g of solid Na₂HPO₄ were dissolved in 100 ml of ultrapure water. The pH was adjusted to 7.1 by adding glacial acetic acid, followed by the addition of 50 ml of an aqueous solution containing 5.53 g of nickel acetate. The resulting yellow solution had a pH of 6.9. The mixture was then heated under reflux in an oil bath for 2 hours, filtered while hot, and immediately supplemented with 4 g of potassium acetate. After a period of time, a yellow solid began to precipitate from the solution. Vacuum filtration under reduced pressure yielded a mixture of yellow crystalline powders, K₆Na₄[Ni₄(H₂O)₂(PW₉O)₂]. 34 )2]·24H2O.

[0071] like Figure 2 The image shows a comparison of the infrared spectra of the five POMs prepared in Examples 1-5 above.

[0072] Test Example 1: Activity Assay of M4POM, a Transition Metal Substituted Polyoxometalate Mimic Enzyme

[0073] Each polyacid was used as an experimental group. The specific reaction system for each experimental group was a 2 ml catalytic reaction system, including 100 μl of 1 mM M4POM solution, 100 μl of 100 mM or 1 M H2O2 solution, 100 μl of 10 mM TMB ethanol solution, and 1700 μl of buffer solution. For the MnPOM, NiPOM, and ZnPOM groups, sodium acetate buffer solution with pH=4 was used; for the CuPOM and CoPOM groups, PBS buffer solution with pH=6 and pH=7 was used, respectively. The H2O2 solution and TMB solution were prepared fresh and stored in the dark before the reaction started. The timing started from the addition of M4POM polyacid, and the total reaction time was 5 minutes.

[0074] Verification of whether the five synthesized polyacids catalyze peroxidase-like catalytic activity: In the presence of H2O2, they catalyze the oxidation of the chromogenic substrate TMB to form the blue product TMB. + The reaction products were spectrally scanned using a Shimadzu UV-3600Plus UV-Vis spectrophotometer with a spectral range of 500-800 nm.

[0075] Test Example 2: Determination of Steady-State Kinetic Parameters of Transition Metal Substituted Polyoxometalate Mimic Enzymes

[0076] Kinetic analyses were performed on two substrates, changing the concentration of one substrate while keeping the concentration of the other constant. For example... Figure 3A The graph shows the kinetics of CoPOM relative to the substrate H2O2. Figure 3B The graph shows the kinetics of CoPOM relative to the substrate TMB. Figure 3C The graph shows the kinetics of ZnPOM relative to the substrate H2O2. Figure 3D The graph shows the kinetics of ZnPOM relative to the substrate TMB. Figure 3E The graph shows the kinetics of MnPOM relative to the substrate H2O2. Figure 3F The graph shows the kinetics of MnPOM relative to the substrate TMB. Figure 3G The graph shows the kinetics of NiPOM relative to the substrate H2O2. Figure 3H The graph shows the kinetics of NiPOM relative to the substrate TMB. Figure 3I The graph shows the kinetics of CuPOM relative to the substrate H2O2. Figure 3J The diagram shows the kinetics of CuPOM relative to the substrate TMB. As shown above, the rate-substrate concentration curves for each polyacid with respect to hydrogen peroxide and TMB conform to the Michaelis-Menten equation kinetic model. That is, the reaction rate increases gradually with increasing concentration, and based on this, the reciprocals of both the rate and substrate concentration are taken and a linear equation is fitted. The kinetic parameters Km and Vmax are calculated using the double reciprocal curve equation.

[0077] like Figure 9 As shown, the kinetic parameters of different POM-type mimic enzymes and the natural enzyme horseradish peroxidase (HRP) for two substrates were compared. Km represents the concentration required to reach half of the maximum catalytic reaction rate; therefore, a smaller Km value indicates a higher affinity of the POM for the corresponding substrate. The comparison revealed that CuPOM's Km value for hydrogen peroxide substrate was 0.0645 mM, the smallest among the five polyacids and significantly smaller than HRP's Km value of 3.7 mM. Furthermore, CoPOM's Km value for TMB substrate, at 0.182 mM, was also superior to HRP's 0.43 mM. The Km values ​​of NiPOM, MnPOM, and ZnPOM for hydrogen peroxide are 0.5515 mM, 3.836 mM, and 0.5985 mM, respectively, and the Km values ​​for TMB are 1.042, 4.639, and 0.9747, respectively. It can be seen that MnPOM has a slightly weaker affinity for both substrates, but its overall catalytic performance is not significantly different from that of the natural enzyme horseradish peroxidase (HRP).

[0078] Comparison of the maximum catalytic rates of the Vmax-mimicking enzymes revealed that both CuPOM and CoPOM exhibited stronger catalytic rates than the natural enzyme, with CuPOM reaching a rate of 15.73 × 10⁻⁶ for catalyzing TMB substrates. -7 Ms -1It is approximately 15 times more potent than the natural enzyme horseradish peroxidase (HRP). Under high hydrogen peroxide concentrations, CoPOM also achieved a Vmax of 4.047 × 10⁻⁶. -7 Ms -1 The Vmax values ​​of the remaining POMs are not significantly different from those of the natural enzyme, except that the Vmax value of NiPOM catalyzing hydrogen peroxide is only 0.342 × 10⁻⁶. -7 Ms -1 Based on the above parameters, it can be found that the CuPOM mimic enzyme has a significantly higher affinity for both substrates than the natural enzyme, and its maximum reaction rate is also higher than that of the natural enzyme, indicating that it has extremely excellent catalytic activity.

[0079] Example 6: Application of POMs-type enzyme mimics in the fabrication of a spermine detection sensor

[0080] Three concentrations of spermine (100 μM, 10 μM, and 1 μM, representing high, medium, and low concentrations) were added to different POMs catalytic systems. The reaction group with added spermine was compared with the blank control group without added spermine. Figure 4 It is evident that, except for NiPOM, the enzyme catalytic activity of the other four POMs was significantly inhibited when 100 μM spermine was present in the system. When the spermine concentration in the system was reduced to 10 μM, the relative activity of CoPOM was still strongly inhibited, only by 32%, while the other four POMs were only inhibited by about 10% compared to the blank control group. When the system contained only 1 μM of spermine, CoPOM still experienced about 30% activity inhibition, while the effects on the other POMs were negligible. The enzyme-mimicking activity of CoPOM was most significantly affected by the spermine concentration in the system and exhibited high sensitivity to low concentrations of spermine. Therefore, this invention preferentially uses CoPOM as the basis for establishing a spermine colorimetric sensor.

[0081] like Figure 5 As shown, taking CoPOM as an example, a spermine colorimetric detection system was established according to the above process. Figure A shows the established spermine colorimetric detection standard curve; Figure B shows the ultraviolet spectrum of spermine detection; and Figure C shows the color photograph of the cuvette. As shown in Figure B, it can be observed that as the concentration of added spermine gradually increases, the ultraviolet absorption peak at 610 nm of CoPOM catalyzing TMB color development gradually decreases. Furthermore, Figure C clearly shows that from left to right, as the concentration of spermine increases, the oxidation of TMB into the blue product TMB... + The blue color gradually lightens as the concentration decreases. In Figure A, a linear equation was fitted to the concentration of lg spermine (A / A0) within the detection range of 20 nM–20 μM. Here, A0 represents the UV absorbance of the blank control group (without spermine), and A represents the UV absorbance of the experimental groups with different concentrations of spermine. The final linear equation obtained is...

[0082] A / A0=-0.2234Lg[spermine]+1.22

[0083] Where R 2 =0.995, and the detection limit (LOD) calculated with a signal-to-noise ratio of 3:1 is 10.32 nM.

[0084] Test Example 3: Experiment to Verify the Detection Mechanism of Spermine

[0085] Since spermine exists as an inhibitor in the POMs catalytic reaction system, studying the inhibition type of spermine relative to the substrate in the system is of great significance for understanding the mechanism of spermine detection. The kinetic parameters Km and Vmax of the CoPOM-mimicking enzyme catalytic reaction system in the presence of spermine were calculated using the double-reciprocal curve Michaelis-Menten equation and compared with the kinetic parameters of CoPOM in the absence of spermine in section 3.4.3. Firstly, as... Figure 6 The figures show the kinetic curves of CoPOM in the presence of 1 μM spermine. It can be seen that the rate-concentration curves for both hydrogen peroxide and TMB substrates still conform to the Michaelis-Menten equation kinetic model in the presence of spermine. Figure A shows the kinetic curve for H2O2 substrate; Figure B shows the kinetic curve for TMB substrate; Figure C shows the corresponding double reciprocal curve for Figure A; and Figure D shows the corresponding double reciprocal curve for Figure B.

[0086] Test Example 4: Substrate Interference Test for Spermine Detection

[0087] To verify the feasibility of the CoPOM spermine colorimetric detection system established in Example 6, substrate interference was tested. The organic interfering substances selected were polyamine compounds and amino acids other than spermine. The polyamine compounds included spermidine, cadaverine, putrescine, and histamine, and the amino acids included lysine, phenylalanine, tyrosine, glutamic acid, tryptophan, and proline. The spermine concentration was chosen to be 100 nM, and the interfering substance concentration was 10 times the spermine concentration, i.e., 1 μM. Figure 7A The results show that, except for spermidine, other interfering substances have a very weak effect on the enzyme activity of CoPOM; while spermidine, as a structural analog of spermine, has an effect of about 35% on the enzyme activity of CoPOM at a concentration of 10 times.

[0088] The effect of inorganic interfering substances was investigated by adding different interfering substances while keeping the concentration of spermine constant. The absorbance ratio A / A0 of spermine alone was set as 100% as a control. The selected inorganic interfering substances were calcium ions, citric acid, dipotassium hydrogen phosphate, magnesium ions, sodium sulfate, and sodium chloride. Figure 7B The results show the influence of inorganic interfering substances on the colorimetric system. The results indicate that inorganic ions have no significant effect on spermine detection. In summary, the CoPOM spermine colorimetric detection system established in this invention possesses excellent specificity.

[0089] Example 7: Detection of spermine in artificial urine

[0090] The only difference from the detection process in Example 6 is that artificial urine is used as the sample substrate solution, the blank group is artificial urine without spermine, and the reaction group with added spermine is artificial urine with the corresponding concentration of spermine added. All other operations are the same as in Example 6.

[0091] To verify the feasibility of the established method, spermine was detected in artificial urine. A pre-column derivatization-liquid chromatography method was compared with the colorimetric detection method constructed in this invention. Figure 8A The retention time of the liquid chromatography chromatogram shown is 2.879 minutes; Figure 8B and 8C Standard curves for derivatization-liquid chromatography and colorimetric detection methods are shown respectively. The linear detection range of the derivatization-liquid chromatography method in artificial urine is 5-200 μM, and the fitted standard curve R0... 2 =0.9882, while the linear detection range of colorimetric detection in artificial urine is 200 nM-10 μM, and the fitted standard curve R 2 =0.991. The experimental results clearly show that the advantage of the derivatization-liquid chromatography method lies in its wider detection range, but the minimum detectable concentration of the colorimetric method is lower. Furthermore, the derivatization reagents in the derivatization-liquid chromatography method are unstable, require stringent storage conditions, and the derivatization reaction steps are complex and time-consuming. The method constructed in this experiment is simple to operate and can achieve rapid detection of spermine. Moreover, the sensitivity and accuracy of this method are comparable to traditional methods.

[0092] Furthermore, we found that while the colorimetric detection method in artificial urine was similar to the method established in the buffer system—showing a gradual decrease in UV absorbance with increasing spermine concentration—the detection range was smaller compared to the buffer system, and the linearity was affected to some extent. This may be due to the influence of the pH and ionic strength of the artificial urine. Despite these effects, this still demonstrates the applicability of this method in the detection of real biological samples.

Claims

1. The application of a class of POMs-type enzyme mimics in the fabrication of a spermine detection sensor, characterized in that, The POMs-type mimic enzyme is a type of transition metal-substituted polyoxometalate, namely transition metal-substituted POMs. The transition metal-substituted POMs are composed of 2 Keggin-type triple-vacant phosphotungstic acid polyoxyanions, 10 protons and 20 water of crystallization molecules. The structural formula of the transition metal-substituted POMs is: K 10 [Co4(H2O)2(PW9O 34 )2]·20H2O, i.e., CoPOM; The application includes the following steps: Step S1: Prepare the reaction reagent solutions, including the following reagent solutions: First reagent solution: 100 µL of 1 mM CoPOM solution; Second reagent solution: 100 µL of 100 mM or 1 M H2O2 solution; Third reagent solution: 100 µL of 10 mM TMB ethanol solution; Fourth reagent solution: 1700 µL of sodium acetate buffer or PBS buffer; wherein, the H2O2 solution and TMB solution are freshly prepared and used immediately, and are stored in the dark before the reaction begins; Step S2, establishing a standard curve: A standard reaction group and a standard control group were set up. The standard reaction group setup process included: taking spermine samples at three standard concentrations (100 µM, 10 µM, and 1 µM) and setting up three corresponding standard reaction groups. The operation process for each standard reaction group was as follows: first, mix the second, third, and fourth reagent solutions, then add the spermine sample, and then add the first reagent solution to start timing. The total reaction time was 5 minutes. The standard control group setup process included: mixing the second, third, and fourth reagent solutions, then adding the first reagent solution to start timing. The total reaction time was 5 minutes. After the reaction, a UV-Vis spectrophotometer was used to perform spectral scanning on each standard reaction group and control group. The spectral scanning range was 500-800 nm. The UV absorbance A of each standard reaction group and the UV absorbance A0 of the blank control group (without spermine) were obtained. A linear equation was fitted to the concentration of lg spermine using A / A0, with a detection range of 20 nM-20 µM. Finally, a standard curve was obtained. Step S3, Spermine sample detection: Set up a detection reaction group and a detection control group. The detection reaction group setup process includes: mixing the second, third, and fourth reagent solutions, adding the spermine sample to be tested, then adding the first reagent solution and starting the timer. The total reaction time is 5 minutes. The detection control group setup process includes: mixing the second, third, and fourth reagent solutions, then adding the first reagent solution and starting the timer. The total reaction time is 5 minutes. After the reaction, use a UV-Vis spectrophotometer to perform spectral scanning on the detection reaction group and the detection control group respectively. The spectral scanning range is 500-800 nm. Obtain the UV absorbance A of the detection reaction group and the UV absorbance A0 of the blank detection control group without spermine. Substitute the values ​​of A and A0 into the standard curve established above to calculate the spermine concentration value in the sample.

2. The application according to claim 1, characterized in that, The linear equation for fitting the standard curve is: A / A0 = -0.2234Lg[spermine] + 1.22 Where R 2 =0.

995.

3. The application according to claim 1, characterized in that, Artificial urine was used as the sample substrate solution. The standard reaction group consisted of artificial urine with the corresponding concentration of spermine added, the blank control group and the test control group consisted of artificial urine without spermine added, and the test reaction group consisted of the spermine sample to be tested using artificial urine as the sample substrate solution.

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