Molybdenum-based polyoxometalate-haematin composite nanomaterial and preparation method and application thereof

Abstract

The application provides a molybdenum-based polyoxometalate-hemin composite nanomaterial and a preparation method and application thereof, and the method comprises the following steps: under room temperature conditions, molybdenum-based polyoxometalate and hemin are mixed by means of manual grinding, and then the obtained mixture is sequentially subjected to centrifugal separation, washing and vacuum drying, so that a solid-phase molybdenum-based polyoxometalate-hemin composite nanomaterial is prepared. 12 The nanocomposite material is synthesized by a simple manual grinding method, and is prepared through the steps of room temperature grinding, centrifugal separation, washing and vacuum drying without the need of complex equipment, high temperature and high pressure or toxic organic solvents. 12 The colorimetric sensor constructed by the nanocomposite material can rapidly and accurately quantitatively detect the AA content in fresh fruits, and has a wide application prospect in the fields of food nutrition analysis, quality control and on-site detection.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biosensing technology, and specifically relates to a molybdenum-based polyoxometalate-heme composite nanomaterial, its preparation method, and its application. Background Technology

[0002] Ascorbic acid (AA, also known as vitamin C) is an essential bioactive molecule in human metabolism, playing a crucial role in antioxidant defense, immune regulation, and collagen synthesis. As fresh fruits are a major dietary source of AA, accurately quantifying their AA content is vital for assessing nutritional value, optimizing storage conditions, and ensuring food quality.

[0003] Currently, there are numerous methods for detecting AA (acetylene oxide), among which colorimetric analysis is favored in practical food analysis due to its inherent advantages such as simple operation, rapid response, low cost, and on-site detection. However, the performance of colorimetric sensing platforms is largely constrained by the catalytic materials used: natural enzymes have limitations such as high cost, poor stability, and difficulty in recycling, which restricts their large-scale application; traditional nanozymes, although having advantages such as good stability, high cost-effectiveness, and adjustable catalytic performance, require complex processes such as hydrothermal, solvothermal, or chemical precipitation for synthesis, involving specialized equipment, harsh reaction conditions, or cumbersome post-processing steps, and are mostly in colloidal or liquid form, leading to difficulties in recycling and non-reusability, which not only increases detection costs but may also cause environmental pollution.

[0004] Polyoxometalates (POMs) and their composites have become ideal candidates for nanozymes due to their unique redox properties and structural diversity. Molybdenum-based polyoxometalates (such as phosphomolybdic acid H3PMo) 12 Nanozymes possess excellent peroxidase-mimicking activity, and heme, as a well-known catalytic center, can improve the electron transfer efficiency of composite materials. However, existing nanozymes based on heme or polyoxometalates still suffer from the aforementioned problems of complex synthesis and difficulty in recycling, which restricts their application in practical detection. Therefore, developing a nanozyme material that is easy to synthesize, recyclable, and has high catalytic efficiency is of great significance for constructing high-performance AA colorimetric sensors. Summary of the Invention

[0005] In view of this, the present invention proposes a molybdenum-based polyoxometalate-heme composite nanomaterial, its preparation method and application, to solve the problems of complex synthesis, difficult recycling and non-reusability of nanoenzyme materials for AA detection in the prior art.

[0006] In a first aspect, the present invention provides a method for preparing a molybdenum-based polyoxometalate-heme composite nanomaterial, comprising:

[0007] At room temperature, molybdenum-based polyoxometalate and heme were mixed by manual grinding. The resulting mixture was then centrifuged, washed, and vacuum dried to prepare solid-phase molybdenum-based polyoxometalate-heme composite nanomaterials.

[0008] Preferably, the molybdenum-based polyoxometalate is phosphomolybdic acid (H3PMo). 12 or H3P2Mo 18 .

[0009] Preferably, the phosphomolybdic acid H3PMo 12 The molar ratio of the mixture with heme is 27:3.

[0010] Preferably, when manually grinding the two raw materials, molybdenum-based polyoxometalate and heme, PB buffer solution at pH=5 is added, and the mixture is manually ground for 1 hour.

[0011] Preferably, the parameters for centrifugation, washing, and vacuum drying are as follows: the ground mixture is centrifuged at 8000 rpm for 5 min, and the solid product is collected; the solid product is washed with deionized water and then dried in a vacuum oven at 50 ℃ for 24 h.

[0012] Secondly, the present invention also provides a molybdenum-based polyoxometalate-heme composite nanomaterial prepared by the preparation method, wherein the composite material is a solid-phase nanomaterial, which is an aggregate of stacked sheets and irregular blocks and fragmented particles, with gaps and pores between the particles.

[0013] The material has peroxidase-mimicking activity, enabling colorimetric detection of AA, and a colorimetric detection platform for AA is constructed.

[0014] Finally, the present invention also provides an application of the aforementioned composite nanomaterial, wherein when the composite material is used as a peroxidase, the catalytic reaction conditions are as follows: composite material concentration The reaction system had a pH of 5, a TMB concentration of 1.8 mM, and an incubation time of 12 min.

[0015] This invention presents a molybdenum-based polyoxometalate-heme composite nanomaterial, its preparation method, and its applications. This material, prepared via a simple manual grinding method, exhibits excellent peroxidase-mimicking activity and, being a solid-phase material, is easily recyclable and reusable. Furthermore, its application in colorimetric detection of amino acids (AA) enables rapid, sensitive, and low-cost detection of AA in fresh fruits. The constructed colorimetric sensor can rapidly and accurately quantify the AA content in fresh fruits (such as apples), with a recovery rate of 98.4%-102.7%. The linear range of this detection method is 3-200 μM, with a detection limit as low as 1.1 μM, demonstrating high sensitivity and selectivity, and effectively resisting interference from sucrose, common metal ions, and anions. Hemin@PMo 12 It is easy to recover from the reaction system, retains more than 73% of its catalytic activity after being reused 10 times, and has good storage stability.

[0016] This invention has broad application prospects in the fields of food nutrition analysis, quality control, and on-site testing. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Provided for Example 1: (a) Hemin@PMo 12 Synthesis diagram, (b) based on Hemin@PMo 12 The colorimetric sensor is used to detect AA;

[0019] Figure 2 Hemin@PMo in Example 2 12 SEM images and EDS elemental mapping diagrams of nanocomposite materials; where (a) and (b) are SEM images at different resolutions, and (ci) are EDS mapping diagrams of C, O, N, P, Cl, Fe, and Mo, respectively.

[0020] Figure 3 Hemin@PMo in Example 2 12 XPS and FT-IR spectra of nanocomposites; where (ad) are high-resolution XPS spectra of C 1s, Cl 2p, Fe 2p, and Mo 3d, respectively; (e) is the full XPS spectrum; and (f) is the spectrum of Hemin@PMo. 12 H3PMo 12FT-IR spectra of hemin;

[0021] Figure 4 It is Hemin@PMo in Example 3 12 Figure 1 shows the peroxidase activity simulation verification and reaction condition optimization of the nanocomposite material; where (a) is the UV-vis absorption spectrum of different reaction systems and the corresponding solution photographs, and (bf) are H3PMo 12 Binding ratio with hemin, Hemin@PMo 12 The effects of concentration, buffer pH, TMB concentration, and incubation time on catalytic activity;

[0022] Figure 5 It is Hemin@PMo in Example 3 12 Steady-state kinetics analysis and catalytic mechanism investigation of nanocomposites; where (a) and (b) are the Michaelis-Menten curves and corresponding Lineweaver-Burk double reciprocal plots with TMB and H2O2 as substrates, respectively, and (c) shows the effect of different free radical scavengers on the absorbance of the reaction system.

[0023] Figure 6 These are the colorimetric detection spectra and standard curves of H2O2 and AA in Example 4; where (a) and (d) are the UV-vis absorption spectra and solution color changes of H2O2 and AA at different concentrations, respectively; (b) and (e) are the linear relationships between the concentrations of H2O2 and AA and the absorbance at 652 nm, respectively; and (c) and (f) are the standard curves of the concentrations of H2O2 and AA and the R / G ratio, respectively.

[0024] Figure 7 Hemin@PMo synthesized in Example 1 12 Selectivity and stability test results of nanocomposite materials; where (a) represents the effect of interfering substances on AA detection, (b) represents colorimetric stability, (c) represents storage stability, and (d) represents reusability. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, to avoid obscuring the invention with unnecessary details, only processing steps closely related to the solution of this invention are shown in the drawings, while other details not closely related to this invention are omitted.

[0026] like Figure 1 As shown, this invention first provides a method for preparing molybdenum-based polyoxometalate-heme composite nanomaterials, which is a simple manual grinding method. The specific steps are as follows:

[0027] (1) Weigh molybdenum-based polyoxometalate and heme in a molar ratio of 27:3.

[0028] (2) Place the two raw materials in a mortar, add PB buffer solution with pH=5, and grind manually for 1 h. Under mechanical grinding conditions, the reactant particles are continuously broken, the specific surface area increases, the lattice distortion and defects increase, the activity of the reactant is improved, and the combination of the two substances is promoted.

[0029] (3) Centrifuge the ground mixture at 8000 rpm for 5 min and collect the solid product;

[0030] (4) The solid product was washed three times with deionized water and then dried in a vacuum oven at 50 °C for 24 h to obtain the nanocomposite material.

[0031] This preparation method requires no complex equipment, high temperature and high pressure conditions, or toxic organic solvents, enabling rapid and large-scale preparation of solid-phase nanocomposites. It utilizes a simple manual grinding method, eliminating the need for complex equipment, high temperature and high pressure conditions, or toxic organic solvents. The synthesis process is rapid, low-cost, and scalable, solving the problem of complexity in traditional nanoenzyme synthesis processes.

[0032] Preferably, the molybdenum-based polyoxometalate is phosphomolybdic acid (H3PMo). 12 Following the steps above, we obtain Hemin@PMo 12 Nanocomposite material. This composite material is a solid-phase nanomaterial, exhibiting an aggregated form of stacked sheets and irregularly shaped bulk and fragmented particles, with interstitial pores between the particles; its elemental composition includes C, O, N, P, Cl, Fe, and Mo, where C, O, and N are derived from the organic part of hemin, and P and Mo are derived from H3PMo. 12 Fe originates from hemin, and the elements are evenly distributed, indicating that hemin is related to H3PMo. 12 Achieve uniform composite.

[0033] This nanocomposite material can be easily recovered from the reaction system by centrifugation or filtration. It retains high catalytic activity even after repeated use (the relative activity after 10 cycles is 73.59% of the initial activity), overcoming the inherent defects of traditional nanozymes that are difficult to separate and can only be used once. This reduces detection costs and is more environmentally friendly.

[0034] Furthermore, this embodiment also provides the application of the aforementioned composite material, which exhibits excellent peroxidase mimicry activity, and the optimal conditions for its catalytic reaction are: composite material concentration... The reaction system had a pH of 5, a TMB concentration of 1.8 mM, and an incubation time of 12 min. Under these conditions, the Michaelis constants (Km) of the composite material for TMB and H2O2 were 0.19 mM and 0.25 mM, respectively, and the maximum reaction rate (Vt) was [not specified]. max ) are respectively and It exhibits superior substrate affinity and catalytic efficiency compared to natural horseradish peroxidase (HRP).

[0035] Verified, Hemin@PMo 12 The nanocomposite material exhibits a superior affinity for both TMB and H2O2 compared to natural horseradish peroxidase (HRP).

[0036] Free radical capture experiments verified that the catalytic mechanism of this composite material is as follows: in the presence of H2O2, the composite material catalyzes the decomposition of H2O2 to generate superoxide radicals. , Further oxidation of TMB produces a blue oxide product (oxTMB), while and 1 O2 is not the main active species in this catalytic reaction.

[0037] Based on the peroxidase-mimicking activity of the above composite material, the obtained Hemin@PMo 12 The application of nanocomposite materials in AA colorimetric detection involves constructing an AA colorimetric detection platform, and the specific detection method is as follows:

[0038] (1) Preparation of the detection system: Add the following to 1.0 mL of PB buffer solution with pH=5: Hemin@PMo 12 The nanocomposite material, 1.8 mM TMB, and 50 μM H2O2 were mixed uniformly.

[0039] (2) Adding samples: Add 50 μL of AA standard solution of different concentrations or pretreated sample extract to the above detection system and incubate at 37℃ for 12 min;

[0040] (3) Signal detection: After incubation, the system was centrifuged at 8000 rpm for 5 min. The absorbance of the supernatant at 652 nm was detected by UV-vis spectrophotometer, or the system color image was captured by smartphone and the R / G value was obtained by using RGBColor Detector software.

[0041] (4) Quantitative analysis: Based on the linear relationship between absorbance or R / G value and AA concentration, the content of AA in the sample is calculated by means of calibration curve.

[0042] The sample pretreatment method is as follows: Take 5.0 g of fresh fruit pulp (such as apple), add 20 mL of 0.1 M, pH=7.4 PB buffer to homogenize, centrifuge at 8000 rpm for 15 min, take the supernatant and filter it through a 0.22 μm cellulose acetate membrane, and dilute it with PB buffer to the linear range of the calibration curve.

[0043] The AA colorimetric detection method constructed based on this material has a wide linear range (3-200 μM), a low detection limit (1.1 μM), and good selectivity, and can detect common interfering substances (sucrose, Fe). 3+ Mg 2+ Cu 2+ Na + SO4 2- Cl - The effects of various factors on the detection results are minimal. This detection method can achieve accurate quantification using a UV-Vis spectrophotometer, or semi-quantitative detection on-site using a smartphone combined with RGB analysis software. It is easy to operate, highly applicable, and has been successfully applied to the detection of AA in fresh fruits such as apples. The recovery rate is between 98.4% and 102.7% (UV detection) and 97.6% and 110.8% (smartphone detection). The results are accurate and reliable, and it has broad application prospects in food inspection, bioanalysis and other fields.

[0044] The technical solution of the present invention will be further illustrated below through specific implementation details.

[0045] Example 1

[0046] Preparation of Hemin@PMo 12 Nanocomposite materials, such as Figure 1 As shown;

[0047] (1) Reagent preparation: phosphomolybdic acid (H3PMo) 12 Both heme and phosphate buffer (PB solution) were of analytical grade. The phosphate buffer solution was prepared with NaH2PO4 and Na2HPO4 and the pH was adjusted to 5.

[0048] (2) Weighing raw materials: Weigh H3PMo at a molar ratio of 27:3 12 4.9275 g (2.7 mM) and hemin 0.1950 g (0.3 mM) were placed in an agate mortar;

[0049] (3) Grinding reaction: Add a small amount of PB solution with pH=5 to the mortar to moisten the raw material, grind manually for 1 h, and stir continuously to ensure that the raw material is fully mixed and reacted;

[0050] (4) Post-processing: The ground mixture was transferred to a centrifuge tube and centrifuged at 8000 rpm for 5 min. The bottom solid product was collected. The product was washed three times with deionized water, and centrifuged after each wash. The washed product was dried in a vacuum oven at 50℃ for 24 h to obtain a brownish-black solid powder of Hemin@PMo. 12 Nanocomposite materials.

[0051] Example 2

[0052] The Hemin@PMo obtained in Example 1 12 The nanocomposite materials were characterized as follows: Figure 2-3 As shown;

[0053] SEM-EDS characterization: The morphology of the material was observed using a scanning electron microscope (SEM, S-4800, Hitachi). The results showed that the composite material had a stacked lamellar structure with uniform particle distribution. EDS elemental mapping confirmed the uniform distribution of C, O, N, P, Cl, Fe, and Mo, indicating that hemin and H3PMo were present. 12 They got back together successfully;

[0054] XPS characterization: Elemental composition and valence state were analyzed using X-ray photoelectron spectroscopy (Thermo Scientific Axis Supra+). The results showed that Fe mainly exists as Fe²⁺. 3+ Mo exists in the form of Mo (corresponding to the 710.9 eV peak). 6+ The presence of these peaks (corresponding to peaks at 232.2 eV and 236.0 eV) confirms that the chemical structure of the material is consistent with expectations.

[0055] FT-IR characterization: Functional groups were tested using a Fourier transform infrared spectrometer (A VERTEX 70, ALPHA II), and the results showed that H3PMo 12 Characteristic peak (1060 cm⁻¹) -1 960 cm -1 873 cm -1 791 cm -1 ) and the characteristic peak of hemin (1700 cm⁻¹) -1 All of them exist, and at 503 cm -1 The presence of Fe-O bond characteristic peaks confirms the relationship between hemin and H3PMo. 12 Composite materials are formed through Fe-O bond bonding.

[0056] Example 3

[0057] Verification Hemin@PMo 12 Peroxidase-like mimicry activity of nanocomposites: such as Figure 4 and 5 As shown;

[0058] 1) Reaction system setup: Four parallel experiments were set up, namely: ① Hemin@PMo 12 +TMB+H2O2;②Hemin@PMo 12 +TMB; ③TMB+H2O2; ④TMB;

[0059] (2) Reaction conditions: In each system, Hemin@PMo 12 concentration The TMB concentration was 1.8 mM, the H2O2 concentration was 100 μM, the PB buffer pH was 5, the total volume was 1.0 mL, and the incubation was carried out at 25 °C for 12 min.

[0060] (3) Activity verification: The absorbance at 652 nm was detected using a UV-Vis spectrophotometer (K5600C, KAIAO). The results showed that only group ① had a significant absorption peak, and the solution was blue, indicating that Hemin@PMo 12 It can catalyze the oxidation of TMB by H2O2 to produce a blue oxidation product (oxTMB), exhibiting excellent peroxidase mimicry activity.

[0061] Example 4

[0062] Establish based on Hemin@PMo 12 AA colorimetric detection method, such as Figure 6 and 7 As shown;

[0063] (1) Optimization of reaction conditions: The optimal detection conditions were determined through single-factor experiments as follows: H3PMo 12 With a hemin binding molar ratio of 27:3, Hemin@PMo 12 concentration The buffer solution was at pH 5, the TMB concentration was 1.8 mM, and the incubation time was 12 min.

[0064] (2) Standard curve plotting: Prepare AA standard solutions with concentrations of 3-200 μM, react according to the optimized detection system, record the absorbance and RGB values ​​at 652 nm respectively, plot the standard curve with AA concentration as the abscissa and absorbance or R / G ratio as the ordinate, obtain the linear regression equation, and calculate the detection limit (LOD) as 1.1 μM;

[0065] (3) Selectivity test: 500 μM of interfering substances (sucrose, FeCl3, MgSO4, CuSO4, NaCl, Na2SO4, etc.) were added to the detection system and compared with the detection signal of 200 μM AA. The results showed that the interfering substances had little effect on the absorbance, indicating that the method has good selectivity.

[0066] (4) Stability test: ① Colorimetric stability: The absorbance of the reaction system was continuously monitored for 1 hour, and the fluctuation range was small; ② Storage stability: After the material was stored at room temperature for 4 weeks, the catalytic activity did not decrease significantly; ③ Reusability: After 10 cycles of use, the relative catalytic activity of the material was still 73.59%.

[0067] Example 5

[0068] Detection of AA in fresh apple samples

[0069] (1) Sample pretreatment: Take 5.0 g of Fuji apple pulp, add 20 mL of 0.1 M, pH=7.4 PB buffer, homogenize, centrifuge at 8000 rpm for 15 min, filter the supernatant through a 0.22 μm filter membrane, dilute 10 times with pH=5 PB buffer and use it as the sample to be tested.

[0070] (2) Spike recovery experiment: 50 μM, 100 μM and 150 μM AA standard solutions were added to the sample extract, and the results were measured according to the established detection method. Each concentration was measured in parallel 3 times.

[0071] (3) Results calculation: The recovery rate of the UV-Vis spectrophotometer was 98.4%-102.7%, with RSD<1.11%; the recovery rate of the smartphone combined with RGB analysis was 97.6%-110.8%, with RSD<1.91%, indicating that the method is accurate and reliable and can be used for the detection of AA in actual samples.

[0072] It should be noted that the purpose of disclosing the embodiments is to help further understand the present invention; however, those skilled in the art will understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the scope of the claims.

Claims

1. A method for preparing molybdenum-based polyoxometalate-heme composite nanomaterials, characterized in that, include: At room temperature, molybdenum-based polyoxometalate and heme were mixed by manual grinding. The resulting mixture was then centrifuged, washed, and vacuum dried to prepare solid-phase molybdenum-based polyoxometalate-heme composite nanomaterials.

2. The preparation method of the molybdenum-based polyoxometalate-heme composite nanomaterial according to claim 1, characterized in that, The molybdenum-based polyoxometalate is phosphomolybdic acid (H3PMo). 12 or H3P2Mo 18 .

3. The method for preparing molybdenum-based polyoxometalate-heme composite nanomaterials according to claim 2, characterized in that, The phosphomolybdic acid H3PMo 12 The molar ratio of the mixture with heme is 27:

3.

4. The method for preparing molybdenum-based polyoxometalate-heme composite nanomaterials according to claim 2, characterized in that, When manually grinding the two raw materials, molybdenum-based polyoxometalate and heme, PB buffer at pH=5 was added, and the mixture was manually ground for 1 hour.

5. The method for preparing molybdenum-based polyoxometalate-heme composite nanomaterials according to claim 2, characterized in that, The parameters for centrifugation, washing, and vacuum drying are as follows: the ground mixture is centrifuged at 8000 rpm for 5 min, and the solid product is collected; the solid product is washed with deionized water and then dried in a vacuum oven at 50 ℃ for 24 h.

6. The molybdenum-based polyoxometalate-heme composite nanomaterial prepared by any one of the preparation methods according to claims 1-5, characterized in that, The composite material is a solid-phase nanomaterial, which is an aggregate of stacked sheets and irregular blocks and fragmented particles, with gaps and pores between the particles.

7. The application of the molybdenum-based polyoxometalate-heme composite nanomaterial prepared by any one of the preparation methods described in claims 1-5, characterized in that, The material has peroxidase-mimicking activity, enabling colorimetric detection of AA, and a colorimetric detection platform for AA is constructed.

8. The application of the composite nanomaterial according to claim 8, characterized in that, When the composite material is used as a peroxidase, the catalytic reaction conditions are as follows: composite material concentration... The reaction system had a pH of 5, a TMB concentration of 1.8 mM, and an incubation time of 12 min.

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