A preparation method of a pyrophosphate strong anchoring platinum-based alloy nanoprobe, and products and applications thereof
By controlling the interfacial structure of nanoprobes, platinum-based alloy nanoprobes were prepared, solving the problem of insufficient recognition of PPi by traditional nanozymes. This enabled the detection of PPi with high affinity and selectivity, which can be applied to biomedical research and clinical diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HANGZHOU NORMAL UNIVERSITY
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-29
AI Technical Summary
Traditional nanozymes lack high affinity and specificity for recognizing pyrophosphate (PPi), making it difficult to achieve accurate detection in complex biological samples.
By precisely controlling the interfacial atomic structure of the nanoprobe, a platinum-based alloy nanoprobe with strong pyrophosphate anchoring was prepared. The coordination effect of polyvinylpyrrolidone and transition metal precursors was utilized to form alloy nanoclusters through a solvothermal reaction, exposing abundant interfacial binding sites and enhancing the specific binding with PPi.
It achieves high affinity and selectivity for PPi detection, enabling specific responses in complex physiological environments, and can be applied to biomedical research and clinical diagnosis.
Smart Images
Figure CN122109066A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of nanomaterial synthesis and biosensing technology, specifically to a method for preparing a platinum-based alloy nanoprobe that achieves strong pyrophosphate anchoring, as well as its products and applications, namely, a method for synthesizing a platinum-based alloy nanoprobe that achieves strong pyrophosphate anchoring and its oxidase activity response characteristics inhibited by pyrophosphate. Background Technology
[0002] Pyrophosphate (PPi) is an important byproduct of many key metabolic processes in organisms (such as ATP hydrolysis and DNA synthesis), and its concentration level is closely related to various diseases (such as tumor calcification and arthritis). Therefore, developing highly sensitive and selective PPi detection methods is of great significance for disease diagnosis and physiological process research. Traditional detection methods, such as chromatography and enzyme-linked immunosorbent assays (ELISA), have limitations such as high cost, complex operation, and long processing time. Nanozymes, especially nanomaterials with oxidase (OXD) activity, have shown great potential in the field of biosensing due to their good stability, low cost, and high catalytic efficiency. However, conventional nanozymes often lack high affinity and specificity for PPi recognition, making it difficult to achieve accurate detection of PPi in complex biological samples. Precisely controlling the interfacial atomic structure of nanomaterials to increase their specific binding sites with PPi is key to improving PPi detection performance.
[0003] Previous studies have primarily optimized the catalytic performance of nanoprobes through elemental doping or morphology modulation, but most have neglected the interfacial interactions between the probe and substrate: insufficient anchoring leads to weak binding affinity and low catalytic efficiency. Therefore, designing a platinum-based alloy nanoprobe with strong PPi anchoring effect can overcome the limitations of traditional nanozyme detection. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing probes by providing a method for precisely controlling the atomic structure of the nanoprobe interface to achieve strong anchoring of substrates (PPi), which can produce a specific response to PPi concentration and can be applied to the monitoring of complex physiological diseases.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:
[0006] In a first aspect, the present invention provides a method for preparing a platinum-based alloy nanoprobe that achieves strong anchoring of pyrophosphate, the method comprising the following steps:
[0007] Step (1): Dissolve polyvinylpyrrolidone in N,N-dimethylformamide to obtain a polyvinylpyrrolidone solution;
[0008] Step (2): Platinum acetylacetonate and transition metal precursors are added to the polyvinylpyrrolidone solution. The coordination between polyvinylpyrrolidone and transition metal precursors achieves dispersion and preliminary regulation.
[0009] Step (3): Dissolve the reducing agent and morphology modifier in ultrapure water, and then add them to the solution treated in step (2) under continuous stirring. Stir at room temperature to mix them thoroughly.
[0010] Step (4): The mixture from step (3) is transferred to a high-pressure reactor and subjected to a solvothermal reaction at high temperature. During this process, transition metal atoms are reduced, nucleated, and grown, and self-assembled into platinum-based alloy nanoclusters with rich interface structures under the regulation of polyvinylpyrrolidone and N,N-dimethylformamide.
[0011] Step (5): After the reaction is complete, the precipitate is collected by centrifugation and washed alternately with ethanol and acetone solutions to remove residual reagents and byproducts, thus obtaining the nanoprobe PtM NPs.
[0012] Preferably, the transition metal precursor in step (2) is one or more of iron acetylacetonate and cobalt acetylacetonate.
[0013] Preferably, the reducing agent in step (3) is L-ascorbic acid, and the morphology modifier is NaBr.
[0014] Preferably, in step (3), the reducing agent and morphology modifier are added to the solution after treatment in step (2) and stirred at room temperature for 30 min.
[0015] Preferably, the solvothermal reaction temperature in step (4) is 140-160°C and the reaction time is 6-8 hours.
[0016] Preferably, in step (1), the ratio of polyvinylpyrrolidone to N,N-dimethylformamide is (100-120) mg: 6 mL; in step (2), the mass ratio of platinum acetylacetonate to the transition metal precursor is (55-60): (12-18).
[0017] Secondly, the present invention provides a platinum-based alloy nanoprobe that achieves strong anchoring of pyrophosphate, which is prepared by the above method, and the platinum-based alloy nanoprobe exhibits a specific response to the concentration of pyrophosphate.
[0018] Preferably, the platinum-based alloy nanoprobe is a nanocluster self-assembled from alloy seeds of platinum atoms and transition metal atoms, and the exposed interfacial atomic sites on the surface provide binding sites for the subsequent strong anchoring of the substrate pyrophosphate.
[0019] The platinum-based alloy nanoprobe has oxidase activity and can catalyze the generation of reactive oxygen species from O2. After adsorbing and anchoring pyrophosphate, the oxidase activity is significantly inhibited.
[0020] Thirdly, the present invention provides the application of the platinum-based alloy nanoprobe in the detection of pyrophosphate or biomarkers related to pyrophosphate metabolism.
[0021] Preferably, the application specifically involves: adding an aqueous solution of platinum-based alloy nanoprobe to the test solution, then adding a tetramethylbenzidine solution, and vortexing to mix evenly; if the test solution does not contain pyrophosphate, the platinum-based alloy nanoprobe can catalyze the oxidation of tetramethylbenzidine to generate a blue product; if the test solution contains pyrophosphate, the ability of the platinum-based alloy nanoprobe to catalyze the oxidation of tetramethylbenzidine to generate a blue product is inhibited.
[0022] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:
[0023] Advantages of the synthesis process: This invention adopts a one-step solvothermal method, which is simple, has controllable parameters, good reproducibility, and is easy to prepare on a large scale;
[0024] Unique structure: The prepared PtM NPs are alloy nanoclusters with a high density of interface atoms, providing abundant anchoring sites for PPi;
[0025] High affinity and selectivity: Based on the strong adsorption between the interface atoms and PPi, it exhibits high affinity and good selectivity for PPi;
[0026] It has a wide range of applications: it can be used directly for PPi detection, or indirectly for detecting biomarkers related to PPi metabolism (such as acid phosphatase activity), and has broad application prospects in biomedical research and clinical diagnosis. Attached Figure Description
[0027] Figure 1 This is a TEM image of the PtFe NPs solution prepared in Example 1.
[0028] Figure 2 This is a TEM image of the PtCo NPs solution prepared in Example 2.
[0029] Figure 3 The oxidation properties of PtFe NPs after anchoring PPi are shown in Application Example 1.
[0030] Figure 4 The oxidation properties of PtCo NPs after anchoring PPi are shown in Application Example 2. Detailed Implementation
[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0032] At least one embodiment provides a method for preparing platinum-based alloy nanoprobes (PtM NPs) that achieve strong pyrophosphate anchoring, synthesized via a solvothermal method, the method comprising the following steps:
[0033] Step (1): Dissolve polyvinylpyrrolidone (PVP) in N,N-dimethylformamide (DMF) to obtain a polyvinylpyrrolidone solution;
[0034] In one embodiment, in step (1), the ratio of polyvinylpyrrolidone to N,N-dimethylformamide is (100-120) mg:6 mL;
[0035] In one embodiment, in step (1), the molecular weight Mw of polyvinylpyrrolidone is 1,300,000.
[0036] Step (2): Platinum acetylacetonate and transition metal precursors are added to the polyvinylpyrrolidone solution. The coordination between polyvinylpyrrolidone and transition metal precursors achieves dispersion and preliminary regulation.
[0037] In one embodiment, the transition metal precursor in step (2) is one or more of iron acetylacetone Fe(acac)3 and cobalt acetylacetone Co(acac)3.
[0038] In one embodiment, in step (2), the mass ratio of platinum acetylacetonate to the transition metal precursor is (55-60):(12-18).
[0039] Step (3): Dissolve the reducing agent and morphology modifier in ultrapure water, and then add them to the solution treated in step (2) under continuous stirring. Stir at room temperature to mix them thoroughly.
[0040] In one embodiment, in step (3), the reducing agent and morphology modifier are added to the solution after treatment in step (2) and stirred at room temperature for 30 min.
[0041] In one embodiment, the reducing agent in step (3) is L-ascorbic acid (AA), and the morphology regulating agent is NaBr. L-ascorbic acid is used to reduce metal ions, and NaBr helps to regulate the nucleation and growth of nanocrystals. Preferably, the ratio of L-ascorbic acid, NaBr, and ultrapure water is 55-65 mg: 450-550 mg: 0.5 mL.
[0042] Step (4): The mixture from step (3) is transferred to a high-pressure reactor and subjected to a solvothermal reaction at high temperature. During this process, transition metal atoms are reduced, nucleated, and grown, and self-assembled into platinum-based alloy nanoclusters with rich interface structures under the regulation of polyvinylpyrrolidone and N,N-dimethylformamide.
[0043] In one embodiment, the solvothermal reaction temperature in step (4) is 140-160°C and the reaction time is 6-8 hours, preferably 160°C for 6 hours.
[0044] Step (5): After the reaction is complete, the precipitate is collected by centrifugation and washed alternately with ethanol and acetone solutions to remove residual reagents and byproducts, and nanoprobes PtM NPs are obtained, where M represents a transition metal.
[0045] Step (6): Take the dispersion of PtM NPs in water and place it in a 0.5 mL centrifuge tube. Add PPi solution and acetate buffer solution (pH=4.5) respectively, then add TMB solution (concentration 10 mM). Vortex for 5 s to mix thoroughly. Measure the absorbance of each reaction solution at 652 nm using a UV spectrophotometer and record the absorbance values and trends.
[0046] In one embodiment, the concentration of the dispersion of nanoprobe PtM NPs in step (6) is 0.5-1 mg / mL.
[0047] In one embodiment, the preferred solution volume ratio of PtM NPs, PPi, acetate buffer, and TMB in step (6) is 1:1:7:1.
[0048] At least one embodiment also provides a platinum-based alloy nanoprobe that facilitates strong anchoring of pyrophosphate, prepared by the above method, wherein the platinum-based alloy nanoprobe exhibits a specific response to pyrophosphate concentration.
[0049] Furthermore, the platinum-based alloy nanoprobe is a nanocluster self-assembled from alloy seeds of platinum atoms and transition metal atoms, and the exposed interfacial atomic sites on the surface provide binding sites for the subsequent strong anchoring of the substrate pyrophosphate.
[0050] The platinum-based alloy nanoprobe has oxidase activity and can catalyze the generation of reactive oxygen species from O2. After adsorbing and anchoring pyrophosphate, the oxidase activity is significantly inhibited.
[0051] The oxidase activity of the nanoprobes exhibits a concentration-dependent response to PPi: as the concentration of PPi increases, the OXD activity of the nanoprobes gradually decreases; by controlling the surface atoms, PtM nanoprobes with different adsorption capacities for PPi can be obtained, wherein the stronger the adsorption of PPi, the more obvious the OXD inhibition effect.
[0052] At least one embodiment also provides the application of platinum-based alloy nanoprobes in the detection of pyrophosphate or biomarkers related to pyrophosphate metabolism.
[0053] Furthermore, the platinum-based alloy nanoprobe aqueous solution is added to the test solution, followed by the addition of tetramethylbenzidine solution, and the mixture is vortexed and oscillated to achieve uniform mixing. If the test solution does not contain pyrophosphate, the platinum-based alloy nanoprobe can catalyze the oxidation of tetramethylbenzidine to produce a blue product. If the test solution contains pyrophosphate, the ability of the platinum-based alloy nanoprobe to catalyze the oxidation of tetramethylbenzidine to produce a blue product is inhibited.
[0054] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products. The present invention will be further described below with reference to specific embodiments and accompanying drawings.
[0055] Example 1
[0056] A method for preparing a PPi-anchored platinum-based alloy nanoprobe includes the following steps:
[0057] (1) Dissolve 100 mg of PVP in 6 mL of DMF to form a clear, colorless solution.
[0058] (2) Dissolve 57 mg of Pt(acac)2 and 17 mg of Fe(acac)3 in the solution obtained in step (1).
[0059] (3) Dissolve 60 mg AA and 500 mg NaBr in 0.5 mL of ultrapure water and add them rapidly to the solution obtained in step (2) with continuous stirring. Stir for 30 min at room temperature.
[0060] (4) Put the solution into a stainless steel autoclave and heat it at 160°C for 6 hours.
[0061] (5) After cooling to room temperature, the precipitate was collected by centrifugation. The obtained product was washed three times alternately with ethanol and acetone solutions to remove excess PVP and inorganic ions, yielding the nanoprobe PtFe NPs. TEM images are attached. Figure 1 As shown.
[0062] Example 2
[0063] A method for preparing a PPi-anchored platinum-based alloy nanoprobe includes the following steps:
[0064] (1) Dissolve 100 mg of PVP in 6 mL of DMF to form a clear, colorless solution.
[0065] (2) Dissolve 57 mg of Pt(acac)2 and 17 mg of Co(acac)3 in the solution obtained in step (1).
[0066] (3) Dissolve 60 mg AA and 500 mg NaBr in 0.5 mL of ultrapure water and add them rapidly to the solution obtained in step (2) with continuous stirring. Stir for 30 min at room temperature.
[0067] (4) Put the solution into a stainless steel autoclave and heat it at 160°C for 6 hours.
[0068] (5) After cooling to room temperature, the precipitate was collected by centrifugation. The obtained product was washed three times alternately with ethanol and acetone solutions to remove excess PVP and inorganic ions, yielding the nanoprobe PtCo NPs. TEM images are attached. Figure 2 As shown.
[0069] Application Example 1
[0070] Take 20 μL of the PtFe NPs (0.5 mg / mL) nanoprobe dispersion from Example 1 and place it in a 0.5 mL centrifuge tube. Add 20 μL of PPi solutions of different concentrations (0, 2, 4, 6 mM), 140 μL of acetate buffer solution, and then add 20 μL of LMB solution (10 mM). Vortex for 5 s to mix thoroughly. Measure the absorbance of each reaction solution at 652 nm using a UV spectrophotometer and record the absorbance values and trends. The results are shown in the attached figure. Figure 3 As shown.
[0071] Application Example 2
[0072] Take 20 μL of the PtCo NPs (0.5 mg / mL) nanoprobe dispersion from Example 2 and place it in a 0.5 mL centrifuge tube. Add 20 μL of PPi solutions of different concentrations (0, 2, 4, 6 mM), 140 μL of acetate buffer solution, and then add 20 μL of TMB solution (10 mM). Vortex for 5 s to mix thoroughly. Measure the absorbance of each reaction solution at 652 nm using a UV spectrophotometer and record the absorbance values and trends. The results are shown in the attached figure. Figure 4 As shown.
[0073] The embodiments and application examples described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing a platinum-based alloy nanoprobe that achieves strong anchoring of pyrophosphate, characterized in that, The method includes the following steps: Step (1): Dissolve polyvinylpyrrolidone in N,N-dimethylformamide to obtain a polyvinylpyrrolidone solution; Step (2): Platinum acetylacetonate and transition metal precursors are added to the polyvinylpyrrolidone solution. The coordination between polyvinylpyrrolidone and transition metal precursors achieves dispersion and preliminary regulation. Step (3): Dissolve the reducing agent and morphology modifier in ultrapure water, and then add them to the solution treated in step (2) under continuous stirring. Stir at room temperature to mix them thoroughly. Step (4): The mixture from step (3) is transferred to a high-pressure reactor and subjected to a solvothermal reaction at high temperature. During this process, transition metal atoms are reduced, nucleated, and grown, and self-assembled into platinum-based alloy nanoclusters with rich interface structures under the regulation of polyvinylpyrrolidone and N,N-dimethylformamide. Step (5): After the reaction is complete, the precipitate is collected by centrifugation and washed alternately with ethanol and acetone solutions to remove residual reagents and byproducts, thus obtaining the nanoprobe PtM NPs.
2. The method according to claim 1, characterized in that, The transition metal precursor mentioned in step (2) is one or more of iron acetylacetone and cobalt acetylacetone.
3. The method according to claim 1, characterized in that, The reducing agent in step (3) is L-ascorbic acid, and the morphology modifier is NaBr.
4. The method according to claim 1, characterized in that, In step (3), the reducing agent and morphology modifier are added to the solution after treatment in step (2) and stirred at room temperature for 30 min.
5. The method according to claim 1, characterized in that, In step (4), the solvothermal reaction temperature is 140-160°C and the reaction time is 6-8 hours.
6. The method according to claim 1, characterized in that, In step (1), the ratio of polyvinylpyrrolidone to N,N-dimethylformamide is (100-120) mg:6 mL; in step (2), the mass ratio of platinum acetylacetonate to transition metal precursor is (55-60):(12-18).
7. A platinum-based alloy nanoprobe for strong pyrophosphate anchoring, prepared by the method according to any one of claims 1-6, characterized in that, The platinum-based alloy nanoprobe exhibits a specific response to pyrophosphate concentration.
8. The platinum-based alloy nanoprobe according to claim 7, characterized in that, The platinum-based alloy nanoprobe is a nanocluster self-assembled from alloy seeds of platinum atoms and transition metal atoms. The exposed interfacial atomic sites on the surface provide binding sites for the subsequent strong anchoring of the substrate pyrophosphate. The platinum-based alloy nanoprobe has oxidase activity and can catalyze the generation of reactive oxygen species from O2. After adsorbing and anchoring pyrophosphate, the oxidase activity is significantly inhibited.
9. The application of the platinum-based alloy nanoprobe according to any one of claims 7-8 in the detection of pyrophosphate or the detection of biomarkers affecting pyrophosphate concentration.
10. The application according to claim 9, characterized in that, Specifically, the platinum-based alloy nanoprobe aqueous solution of any one of claims 7-8 is added to the test solution, and then a tetramethylbenzidine solution is added and vortexed to mix evenly; if the test solution does not contain pyrophosphate, the platinum-based alloy nanoprobe can catalyze the oxidation of tetramethylbenzidine to generate a blue product; If the test solution contains pyrophosphate, the ability of the platinum-based alloy nanoprobe to catalyze the oxidation of tetramethylbenzidine to produce a blue product is suppressed.