Iron-vanadium bimetallic doped prussian blue nanoszyme, preparation method and application thereof
Patent Information
- Application Number
- CN202611179068.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-05
- Publication Date
- 2026-11-03
AI Technical Summary
Traditional colorimetric methods use natural enzymes such as horseradish peroxidase, which are unstable and easily deactivated, limiting the widespread application of tea polyphenol detection. Furthermore, existing nanoenzyme materials have limited catalytic activity and low efficiency.
A one-pot synthesis of iron-vanadium bimetallic doped Prussian blue nanozyme (FeVPBA) was performed. Under acidic conditions, it catalyzes dissolved oxygen to generate superoxide anion radicals, which oxidize 3,3',5,5'-tetramethylbenzidine (TMB). Tea polyphenols inhibit color development by scavenging free radicals through the polyphenol hydroxyl structure, thus constructing a colorimetric detection method.
It achieves highly sensitive and low-cost detection of tea polyphenols, with a detection limit as low as 0.35 μg/mL and a linear range of 0.8 μg/mL to 100 μg/mL. It is suitable for rapid detection and is ideal for primary healthcare institutions and point-of-care testing scenarios.
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Figure CN123024568A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of nanobiosensing technology and food detection technology, specifically to an iron-vanadium bimetallic doped Prussian blue nanozyme, its preparation method, and its application. Background Technology
[0002] Tea polyphenols are a collective term for various phenolic compounds in tea, including catechins, flavonoids, anthocyanins, and phenolic acids. They possess multiple functions, such as scavenging free radicals, inhibiting tumor cell proliferation, inducing apoptosis, blocking tumor angiogenesis, and enhancing the sensitivity of chemotherapy drugs, playing an important role in dietary regulation. Therefore, achieving highly sensitive and accurate quantitative detection of tea polyphenols has significant clinical guiding significance for monitoring the effects of nutritional and drug interventions and for prognostic assessment.
[0003] Currently, the main methods for detecting tea polyphenols include high-performance liquid chromatography (HPLC), fluorescence methods, and colorimetric methods. Among these, colorimetric methods have attracted much attention due to their simplicity, low cost, and the ability to observe results visually, making them particularly suitable for rapid screening in resource-limited environments such as point-of-care testing in hospitals. However, the natural enzymes used in traditional colorimetric methods (such as horseradish peroxidase) have drawbacks such as poor stability, easy inactivation, and high cost, which limits their widespread application in actual sample testing.
[0004] In recent years, nanozymes have become ideal materials to replace natural enzymes due to their advantages such as high stability, low cost, and tunable activity. Prussian blue analogues, as important members of the metal-organic framework family, possess unique three-dimensional open structures and multiple metal sites, and their catalytic performance can be optimized through multi-metal doping. Summary of the Invention
[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an iron-vanadium bimetallic doped Prussian blue nanozyme, its preparation method and application, specifically an iron-vanadium bimetallic doped Prussian blue analog nanozyme synthesized by a one-pot method and its preparation method, as well as the application of the nanozyme in the colorimetric detection of tea polyphenols and in the fields of food and pharmaceuticals.
[0006] This invention uses V6O 13Using vanadium as the source and FeCl3·6H2O as the iron source, a one-pot co-precipitation reaction with K3[Fe(CN)6] successfully synthesized a Prussian blue nanozyme (FeVPBA, an iron-vanadium bimetallic doped Prussian blue analogue). This nanozyme, under acidic conditions without the need for added hydrogen peroxide, catalyzes dissolved oxygen to generate superoxide anion radicals, which then oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue product. Tea polyphenols, due to their polyphenolic hydroxyl structure, can effectively scavenge superoxide anion radicals, thereby inhibiting the oxidation and color development of TMB. The colorimetric sensing method constructed based on this method exhibits a linear detection range of 0.8 μg / mL to 100 μg / mL for tea polyphenols, with a detection limit as low as 0.35 μg / mL, providing a technical means for the rapid detection of tea polyphenols.
[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing iron-vanadium bimetallic doped Prussian blue nanozymes, comprising the following steps: The vanadium source and the iron source are dissolved in hydrochloric acid and mixed thoroughly to obtain solution A; Dissolve K3[Fe(CN)6] in water and mix thoroughly to obtain solution B; Solution B was added dropwise to solution A under stirring conditions, and a co-precipitation reaction was carried out by stirring. After standing and aging, a precipitate was obtained. The precipitate was washed and dried to obtain iron-vanadium bimetallic doped Prussian blue nanozyme. The vanadium source includes V6O. 13 The iron source includes FeCl3·6H2O.
[0008] Preferably, the V6O 13 The preparation method includes: adding V2O5 to a mixture of ethanol and deionized water, and synthesizing V6O through a hydrothermal reaction. 13 ; The ratio of V2O5 to the mixed solution is 0.4 g~0.6 g: 30 mL~50 mL; The volume ratio of ethanol to deionized water is 1:2.5~3.5; The hydrothermal reaction temperature is 160℃~180℃, and the hydrothermal reaction time is 4 h~6 h.
[0009] Preferably, the mass ratio of the vanadium source, the iron source, and K3[Fe(CN)6] is 2~3:0.8~0.9:16~17; The concentration of the hydrochloric acid is 5.4% w / w; The temperature of the coprecipitation reaction is 20℃~30℃, and the time of the coprecipitation reaction is 15 min~25 min; The static aging time is 20 h to 28 h.
[0010] Secondly, the present invention provides an iron-vanadium bimetallic doped Prussian blue nanozyme, obtained by the aforementioned preparation method. The iron-vanadium bimetallic doped Prussian blue nanozyme contains Fe and V elements, with Fe predominating. 2+ / Fe 3+ Mixed valence state, V element with V 4+ / V 5+ Mixed valence states coexist within the Prussian blue framework.
[0011] Thirdly, the present invention provides the application of the iron-vanadium bimetallic doped Prussian blue nanozyme in the colorimetric detection of tea polyphenols.
[0012] The detection principle is as follows: The iron-vanadium bimetallic doped Prussian blue nanozyme (FeV PBA) of this invention catalyzes dissolved oxygen to generate superoxide anion free radicals under acidic conditions, which oxidize TMB to generate a blue product (ox-TMB); tea polyphenols can efficiently scavenge superoxide anion free radicals by virtue of their polyphenol hydroxyl structure, thereby inhibiting the oxidation and color development of TMB, resulting in a decrease in the absorbance of the system, thereby realizing the quantitative detection of tea polyphenols.
[0013] Fourthly, this invention provides a colorimetric detection method for tea polyphenols based on iron-vanadium bimetallic doped Prussian blue nanozymes, comprising the following steps: S1. The iron-vanadium bimetallic doped Prussian blue nanozyme is mixed with 3,3',5,5'-tetramethylbenzidine (TMB) and the sample to be tested under acidic conditions and reacted in the dark to obtain a reaction system. S2. Qualitative detection is performed based on the color change of the reaction system; Alternatively, measure the absorbance A of the reaction system at a wavelength of 652 nm. 样品 The absorbance A of the blank control group without the test sample was measured. 空白 The absorbance difference ΔA = A of the sample to be tested was calculated. 空白 - A 样品 The values were then substituted into the standard curve of absorbance difference versus tea polyphenol concentration for quantitative detection.
[0014] Preferably, S1 includes the following specific steps: The 3,3',5,5'-tetramethylbenzidine solution, HAc-NaAc buffer solution, and the sample to be tested were mixed thoroughly to obtain a mixture. The aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme was added to the mixture, vortexed and mixed, and incubated at 37°C and 200 rpm in the dark for 25 min. The volume ratio of the 3,3',5,5'-tetramethylbenzidine solution, HAc-NaAc buffer, the sample to be tested, and the aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme is 1:2:1:1. The solvent for the 3,3',5,5'-tetramethylbenzidine solution is DMSO, and the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 8 mM. The pH of the HAc-NaAc buffer solution is 2.6; The concentration of the aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme is 1 mg / mL.
[0015] Preferably, the linear detection range of the colorimetric detection method of the present invention is 0.8 μg / mL-100 μg / mL, and the detection limit is 0.35 μg / mL; the standard curve of absorbance difference versus tea polyphenol concentration is y = -0.00484x + 0.7039, R 2 =0.9979.
[0016] Fifthly, the present invention provides a kit for the detection of tea polyphenols, the kit comprising the aforementioned iron-vanadium bimetallic doped Prussian blue nanozyme with TMB and HAc-NaAc buffer.
[0017] Sixthly, the present invention provides the application of the iron-vanadium bimetallic doped Prussian blue nanozyme in the preparation of a test product for assisting in the evaluation of food and pharmaceutical products, wherein the test product is used to detect the level of tea polyphenols in a patient's body fluids.
[0018] In a seventh aspect, the present invention provides a detection product for assisting in the assessment of tumor risk and prognosis, the detection product comprising iron-vanadium bimetallic doped Prussian blue nanozyme with TMB and HAc-NaAc buffer.
[0019] The detection products include colorimetric detection kits, test strips, or microfluidic chips.
[0020] Eighthly, the present invention provides the application of the aforementioned iron-vanadium bimetallic doped Prussian blue nanozyme in the preparation of products for nutritional metabolism monitoring in the medical field, wherein the products are used to detect the level of tea polyphenols in a patient's dietary supplements.
[0021] By detecting the levels of tea polyphenols in body fluids (such as serum and urine) or dietary supplements, laboratory evidence can be provided for nutritional and pharmacological monitoring and prognostic assessment during patients' adjunctive treatment.
[0022] Ninthly, the present invention provides a product for monitoring nutritional metabolism in the field of food and pharmaceutical sciences, the product comprising iron-vanadium bimetallic doped Prussian blue nanozyme with TMB and HAc-NaAc buffer.
[0023] The products include colorimetric detection kits, test strips, or microfluidic chips.
[0024] This invention has at least one of the following beneficial effects: 1. This invention synthesizes iron-vanadium bimetallic doped Prussian blue analogues (i.e., iron-vanadium bimetallic doped Prussian blue nanozymes, abbreviated as FeV PBA) by a one-pot co-precipitation reaction, obtaining mixed valence metal centers and synergistically enhanced oxidase-like activity, thus solving the problems of single catalytic activity and low efficiency of traditional Prussian blue nanozymes.
[0025] 2. This invention constructs a colorimetric detection method for tea polyphenols. Based on the oxidase-like activity of Prussian blue nanozymes doped with iron and vanadium bimetals, it directly utilizes dissolved oxygen in the reaction system to generate superoxide anion free radicals, eliminating the need for external hydrogen peroxide. This method is simple to operate and avoids the instability and cumbersome operation caused by the addition of hydrogen peroxide. Furthermore, the detection method of this invention has good selectivity and sensitivity, with a low detection limit (0.35 μg / mL) and a wide linear range (0.8 μg / mL~100 μg / mL), which is superior to most traditional colorimetric methods. It can be applied to the detection of tea polyphenols in actual samples and related biological samples.
[0026] 3. This invention also provides the application of iron-vanadium bimetallic doped Prussian blue nanozymes in tumor medical detection products. It can be used for the quantitative detection of tea polyphenols in biological samples such as pharmaceuticals, health products, and human serum, providing a technical means for the rapid detection of tea polyphenols in medicine (such as dietary guidance for cancer chemoprevention, adjuvant therapy monitoring, and prognostic assessment). Furthermore, it is simple to operate, low in cost, and provides visualized results: the entire detection process is completed at room temperature, requiring only a UV-Vis spectrophotometer or visual observation, making it suitable for primary healthcare institutions and point-of-care testing (POCT) scenarios. The FeV PBA nanozyme of this invention, as an inorganic nanozyme, has excellent thermal and storage stability, can be stored for a long time at room temperature, and is easily commercialized into a kit, suitable for rapid screening scenarios related to food and drugs. Attached Figure Description
[0027] Figure 1 This is a schematic diagram of the preparation process of the FeV PBA nanozyme of the present invention and a schematic diagram of the principle of using FeV PBA nanozyme to detect tea polyphenols; wherein, Figure 1 In the diagram, A represents the preparation process of FeV PBA nanozymes. Figure 1 B in the diagram represents the principle of FeV PBA nanozyme for detecting tea polyphenols.
[0028] Figure 2 It is the precursor V6O in Embodiment 1 of the present invention. 13Characterization results of FeV PBA nanozymes; among which, Figure 2 In this context, A represents the precursor V6O. 13 SEM images; Figure 2 B- Figure 2 C in the text represents the precursor V6O 13 TEM images at 200nm and 100nm; Figure 2 D in the image represents the SEM image of the FeV PBA nanozyme. Figure 2 E and Figure 2 F in the image represents the TEM images of FeV PBA nanozyme at 100 nm and 50 nm, respectively. Figure 2 G in the text refers to the precursor V6O. 13 The HRTEM image, with the SAED image in the upper right inset; Figure 2 H in the image is an HRTEM image of the FeV PBA nanozyme, and the inset in the upper right corner is a SAED image. Figure 2 In this context, I represents the precursor V6O. 13 Mapping images; Figure 2 J in the image represents the mapping image of the FeV PBA nanozyme. Figure 2 K in the figure represents the EDS spectrum of the FeV PBA nanozyme.
[0029] Figure 3 These are the experimental results from Embodiment 2 of the present invention; wherein, Figure 3 A in the text is V6O 13 Feasibility analysis diagram of TMB color development catalyzed by FeV PBA, V PBA, FePBA, etc.; Figure 3 B in the image represents a real photograph of the reaction process when V PBA is doped with different metals. Figure 3 C in the figure represents a comparison of the catalytic activities of V PBA with different metal doping.
[0030] Figure 4 This is the quantitative standard curve diagram in Embodiment 3 of the present invention.
[0031] Figure 5 This is the result of the specificity test experiment of the detection method of the present invention in Embodiment 3 of the present invention. Detailed Implementation
[0032] To make the technical problems solved, the technical solutions, and the beneficial effects 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 of the invention and are not intended to limit the invention.
[0033] Example 1 This embodiment provides a method for preparing iron-vanadium bimetallic doped Prussian blue nanozymes (FeV PBA nanozymes), including the following steps: (1) Precursor V6O 13 The synthesis method is as follows: 0.5 g of V₂O₅ was added to a mixture of 10 mL of ethanol and 30 mL of deionized water. After magnetic stirring at room temperature for 1.5 h, the mixture was transferred to a reaction vessel and hydrothermally reacted at 170 °C for 5 h to obtain a dark green precipitate. The precipitate was washed with deionized water and ethanol respectively until the supernatant was clear, and then dried at 60 °C for later use to obtain V₆O₅. 13 .
[0034] (2) The synthesis method of FeV PBA nanozyme is as follows: 0.025 g V6O 13 Dissolve 0.0082 g FeCl3·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid and sonicate until clear to obtain solution A.
[0035] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0036] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain FeV PBA nanozyme.
[0037] The precursor V6O prepared in Example 1 13 The FeV PBA nanozyme was characterized, and the characterization results are shown in the figure. Figure 2 .
[0038] Figure 2 In this context, A represents the precursor V6O. 13 SEM images, Figure 2 B and Figure 2 C in the text represents the precursor V6O 13 TEM images at 200nm and 100nm, Figure 2 G in the text refers to the precursor V6O. 13 HRTEM image, Figure 2 In this context, I represents the precursor V6O. 13 The mapping image above shows that the FeV PBA nanozyme prepared in Example 1 belongs to the monoclinic crystal system, has a rod-shaped morphology, and is bonded to the layers by van der Waals forces.
[0039] Figure 2 D in the image represents the SEM image of the FeV PBA nanozyme. Figure 2 E and Figure 2 F in the image represents the TEM images of the FeV PBA nanozyme at 100 nm and 50 nm, respectively. Figure 2 H in the image represents the HRTEM image of the FeV PBA nanozyme. Figure 2 J in the image represents the mapping image of the FeVPBA nanozyme. Figure 2 K in the figure represents the EDS spectrum of the FeV PBA nanozyme. As can be seen from the figure above, the FeV PBA nanozyme prepared in Example 1 reacts with the precursor V6O. 13 Compared to the previous state, it has undergone a phase change and presents a cubic shape with relatively smooth edges, belonging to the amorphous state.
[0040] In summary, characterization results by TEM, XRD, and XPS confirmed that the FeV PBA nanozyme prepared in Example 1 has a smooth cubic morphology, and both Fe and V elements exist in multiple valence states (Fe... 2+ / Fe 3+ V 4+ / V 5+ They coexist within the Prussian blue framework.
[0041] Comparative Example 1 This comparative example provides a method for preparing V PBA nanozymes, which differs from Example 1 in that it only does not dope with Fe. The specific synthesis method is as follows: 0.025 g V6O 13 Dissolve in 4 mL of 5.4% (w / w) hydrochloric acid and sonicate until clear to obtain solution A.
[0042] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0043] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain V PBA nanozyme.
[0044] Comparative Example 2 This comparative example provides a method for preparing Fe PBA nanozymes, which differs from Example 1 in that it only does not dope with V, and the specific synthesis method is as follows: Dissolve 0.0082 g FeCl3·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid and sonicate until clear to obtain solution A.
[0045] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0046] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain Fe PBA nanozyme.
[0047] Comparative Example 3 This comparative example provides a method for preparing CuV PBA nanozymes, which differs from Example 1 in that Cu is used instead of Fe for doping. The specific synthesis method is as follows: 0.025 g V6O 13 Dissolve 0.0052 g CuCl2·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid, and sonicate until clear to obtain solution A.
[0048] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0049] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain CuV PBA nanozyme.
[0050] Comparative Example 4 This comparative example provides a method for preparing CoV PBA nanozymes, which differs from Example 1 in that Co is used instead of Fe for doping. The specific synthesis method is as follows: 0.025 g V6O 13 Dissolve 0.0040 g CoCl2·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid, and sonicate until clear to obtain solution A.
[0051] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0052] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain the CoV PBA nanozyme.
[0053] Comparative Example 5 This comparative example provides a method for preparing NiV PBA nanozymes, which differs from Example 1 in that Ni is used instead of Fe for doping. The specific synthesis method is as follows: 0.025 g V6O 13 Dissolve 0.0072 g NiCl2·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid and sonicate until clear to obtain solution A.
[0054] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0055] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain NiV PBA nanozyme.
[0056] Comparative Example 6 This comparative example provides a method for preparing MnV PBA nanozymes, which differs from Example 1 in that Mn is used instead of Fe for doping. The specific synthesis method is as follows: 0.025 g V6O 13 Dissolve 0.0030 g of MnCl2·6H2O in 4 mL of 5.4% (w / w) hydrochloric acid and sonicate until clear to obtain solution A.
[0057] Dissolve 0.1646 g K3[Fe(CN)6] in 10 mL of deionized water to obtain solution B.
[0058] Solution B was added dropwise to solution A with stirring. After stirring at room temperature for 20 min, the mixture was allowed to stand for 24 h to age. The resulting precipitate was washed with deionized water and ethanol, respectively, and dried to obtain the MnV PBA nanozyme.
[0059] Example 2 This embodiment provides a feasibility analysis method for FeV PBA nanozyme catalyzing TMB color development. Specifically, it involves combining the FeV PBA nanozyme prepared in Example 1 with the precursor V6O. 13 Furthermore, various nanozymes prepared in Comparative Examples 1 to 6 were subjected to catalytic TMB colorimetric experiments, and the specific methods are as follows: (1) Verification of oxidase-like activity of FeV PBA nanozyme (TMB colorimetric method) Step 1) includes the following operations: Add 100 μL of TMB working solution (8 mM, with DMSO as solvent), 200 μL of HAc-NaAc buffer (pH 2.6), and 100 μL of deionized water (DW) to a 1.5 mL centrifuge tube in sequence; place the centrifuge tube on a vortex mixer and vortex for 30 s to thoroughly mix the components and obtain a homogeneous TMB substrate reaction system.
[0060] Step 2) includes the following operations: Add 100 μL of pre-ultrasonically dispersed FeV PBA sample aqueous dispersion (1 mg / mL) to the TMB substrate reaction system prepared in Step 1); gently invert the centrifuge tube several times to mix, and then place the centrifuge tube in a 37 ℃ constant temperature shaker and incubate in the dark for 25 min to allow FeV PBA to exert its oxidase-like activity, catalyze dissolved oxygen to generate superoxide anion free radicals, and then oxidize TMB to produce a color reaction.
[0061] Step 3) includes the following operations: After incubation, use a pipette to take 400 μL of the mixture after reaction in step 2) and add it to a micro-quartz cuvette; place the cuvette in a UV-Vis spectrophotometer, use a blank substrate system without FeV PBA as a reference, and measure and record the absorbance value of the system at wavelengths of 500 nm to 750 nm; set up 3 parallel experiments for each sample to be tested, repeatedly measure the absorbance value, and take the average value of the 3 measurements as the final detection data.
[0062] Similarly, the precursor V6O prepared in Example 1 was processed according to the above method. 13 The oxidase-like activities of the V PBA nanozyme prepared in Comparative Example 1 and the Fe PBA nanozyme prepared in Comparative Example 2 were verified. Specifically, the FeV PBA sample aqueous dispersion in step 2) was replaced with the precursor V6O prepared in Example 1. 13 Aqueous dispersions of the samples and aqueous dispersions of nanozymes prepared in Comparative Examples 1 and 2 were used. Blank substrate systems without FeV PBA (TMB) and without TMB (FeV PBA) were used as blank controls.
[0063] Test results are shown Figure 3 A in the middle.
[0064] (2) Inhibition of colorimetric reaction and colorimetric detection of tea polyphenols by different metal-doped V PBA nanozymes Step 1) includes the following operations: Add 100 μL of TMB working solution (8 mM, with DMSO as solvent), 200 μL of HAc-NaAc buffer (pH 2.6), and 100 μL of tea polyphenol standard solutions of different concentrations to a 1.5 mL centrifuge tube in sequence; place the centrifuge tube on a vortex mixer and vortex for 30 s to mix the components thoroughly to obtain a TMB substrate reaction system containing tea polyphenols.
[0065] Step 2) includes the following operations: Add 100 μL of the pre-ultrasonically dispersed aqueous dispersion (1 mg / mL) of the V PBA nanozyme sample prepared in Comparative Example 1 to the substrate reaction system prepared in Step 1); after gently inverting the centrifuge tube several times to mix, place the centrifuge tube in a constant temperature shaker at 37 ℃ and incubate in the dark for 25 min, so that the superoxide anion free radicals generated by FeV PBA catalysis are cleared by tea polyphenols, thereby inhibiting the oxidation and color development reaction of TMB.
[0066] Step 3) includes the following operations: After incubation, use a pipette to take 400 μL of the mixture after reaction in step 2) and add it to a micro-quartz cuvette; place the cuvette in a UV-Vis spectrophotometer, use a blank substrate system without FeV PBA as a reference, measure and record the absorbance value of the system at a wavelength of 652 nm; set up 3 parallel experiments for each sample to be tested, repeatedly measure the absorbance value, calculate the absorbance difference ΔA = A blank - A sample, and substitute it into the standard curve to calculate the concentration of tea polyphenols.
[0067] Similarly, the oxidase-like activities of various nanozymes prepared in Comparative Examples 2 to 6 were verified according to the above method. Specifically, the aqueous dispersion of the V PBA nanozyme sample in step 2) was replaced with the aqueous dispersion of various nanozyme samples prepared in Comparative Examples 2 to 6.
[0068] The TMB working solution was prepared using DMSO as the solvent; the FeV PBA aqueous dispersion was prepared by ultrasonically dispersing FeV PBA samples in deionized water; the HAc-NaAc buffer (pH 2.6) was calibrated by mixing 4.625 mL of 0.3 M acetic acid and 0.375 mL of 0.2 M sodium acetate; and the tea polyphenol standard solution was prepared using deionized water as the solvent.
[0069] Test results are shown Figure 3 B in Figure 3 C in Figure 3 B in the image is a real photograph of the reaction process when V PBA is doped with different metals; Figure 3 C in the figure represents the histogram of TMB colorimetric absorption values catalyzed by V PBA with different metal doping.
[0070] Depend on Figure 3 As can be seen from A, FeV PBA has a stronger ability to catalyze the blueing of TMB under acidic conditions than FePB and V PBA alone; Figure 3 As can be seen from B, the color during the synthesis process varies depending on the metal doping; from Figure 3 As can be seen from C, compared to other metal doping, Fe doping results in the highest absorbance at 652 nm.
[0071] Therefore, FeV PBA exhibits highly efficient oxidase-like activity: under acidic conditions, it can catalyze the generation of superoxide anion radicals (O2) from dissolved oxygen without the need for external H2O2. • The superoxide radical (TSP) then oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue product (ox-TMB), which exhibits a characteristic absorption peak at 652 nm. Tea polyphenols (TP), due to their rich phenolic hydroxyl groups in their molecular structure, can effectively scavenge superoxide anion free radicals, thereby inhibiting the oxidation and color development reaction of TMB, leading to a decrease in the system's absorbance.
[0072] In summary, the results confirm that, compared with Comparative Examples 1 to 6, the FeV PBA nanozyme prepared in Example 1 can be used for colorimetric detection of tea polyphenols.
[0073] Example 3 This embodiment provides a colorimetric detection method for tea polyphenols, specifically by applying the FeV PBA nanozyme from Example 1 to the colorimetric detection of tea polyphenols. The specific method is as follows: (1) Establishment of standard curve: 100 μL of TMB solution (8 mM, dissolved in DMSO), 200 μL of HAc-NaAc buffer (0.2 M, pH 2.6), and 100 μL of tea polyphenol standard solutions of different concentrations (concentration range 0.8 μg / mL~100 μg / mL) were added sequentially to a 1.5 mL centrifuge tube and mixed thoroughly. Then, 100 μL of FeV PBA aqueous dispersion (1 mg / mL) was added and vortexed to mix. The centrifuge tube was placed in a 37 ℃ constant temperature shaker and incubated at 200 rpm in the dark for 25 min. After incubation, 400 μL of the reaction solution was transferred to a microcuvette and the absorbance at 652 nm was measured using a UV-Vis spectrophotometer. Three parallel samples were set up for each concentration.
[0074] The absorbance of the blank control group (containing no tea polyphenols) is designated as Ablank, and the absorbance of the sample group is designated as Asample. The absorbance difference ΔA = Ablank is calculated. 空白 - A 样品 Plotting tea polyphenol concentration on the x-axis, the absorbance difference ΔA (ΔA = A) is calculated. 空白 - A 样品 Plot a standard curve with y as the ordinate.
[0075] (2) Detection of the sample to be tested: Replace the tea polyphenol standard solution with the sample to be tested, measure the absorbance value at 652 nm according to the above steps, calculate ΔA, and substitute it into the standard curve equation to calculate the concentration of tea polyphenols in the sample.
[0076] The TMB solution was prepared using DMSO as the solvent; the HAc-NaAc buffer (pH=2.6) was calibrated by mixing 4.625 mL of 0.32 M acetic acid and 0.375 mL of 0.2 M sodium acetate; the FeV PBA aqueous dispersion was prepared by ultrasonically dispersing the FeV PBA sample prepared in Example 1 in deionized water; and the tea polyphenol standard solution was prepared using tea polyphenols as the solute and deionized water as the solvent.
[0077] (3) Specificity analysis: Examining various potential interfering substances (including Na) + Mg 2+ Ca 2+ K + Fe 3+ Cl - CO3 - SO4 2- PO4 3- NO2 - The concentrations of the following substances were set at 5 times the concentration of tea polyphenols: Try, L-Cys, His, Lyr, Phe, VC, GSH, UA, and DA.
[0078] (4) Actual sample testing: Commercially available L-carnitine tea polyphenol tablets were used as the actual sample for spiked recovery experiments. After grinding, ultrasonic dissolution, centrifugation, and dilution, the sample was added with three concentrations of tea polyphenol standard (10 μg / mL, 40 μg / mL, and 80 μg / mL), and analyzed according to the aforementioned colorimetric detection steps.
[0079] This embodiment also provides a kit for the detection of tea polyphenols. The kit includes FeV PBA nanozyme, TMB, and HAc-NaAc buffer. The concentration of FeV PBA nanozyme is 1 mg / mL, the concentration of TMB is 8 mM, and the concentration of HAc-NaAc buffer is 0.2 M. This kit can be used for the detection of tea polyphenols.
[0080] Figure 4 The standard solution of tea polyphenols showed a good linear relationship with the absorbance difference ΔA in the range of 0.8 μg / mL to 100 μg / mL. The linear regression equation was y = -0.00484x + 0.7039 (where y is absorbance and x is the concentration of tea polyphenols in μg / mL), with a coefficient of determination R² = 0.9979. The limit of detection (LOD, 3σ / k) was calculated to be 0.35 μg / mL.
[0081] Figure 5As a result of the specificity analysis, the test results showed that, compared with tea polyphenols, none of the interfering substances caused significant changes in absorbance, and the characteristic signal response of the target analyte, tea polyphenols, was prominent, confirming that the colorimetric detection method has excellent specificity.
[0082] The actual sample detection results are shown in Table 1. The results show that the spiked recovery rate of tea polyphenols is between 96.42% and 105.37%, and the relative standard deviation (RSD) is between 1.54% and 4.55%, indicating that the colorimetric detection method based on FeV PBA has good feasibility and reliability in actual sample detection.
[0083] Table 1 Example 4 This embodiment provides a quantitative detection method for tea polyphenols in serum. By monitoring the dynamic changes in tea polyphenol levels in patients, it provides a sensitive and reliable analytical tool for dietary guidance of cancer chemoprevention strategies, evaluation of the effectiveness of nutritional interventions during adjuvant therapy, and prognosis assessment. The specific detection method is as follows: Detection of tea polyphenols in human serum samples (spiking and recovery experiment): Step 1) Pretreatment of human serum samples: Take 1.0 mL of serum sample from a healthy person, add 3.0 mL of acetonitrile, vortex for 2 min, centrifuge at 4℃ and 12000 rpm for 15 min, collect the supernatant, and dilute it 10 times with deionized water for later use.
[0084] A serum sample was processed using the same method, but without the addition of tea polyphenols as a matrix blank.
[0085] Step 2) Preparation of spiked samples: Take 200 μL of pretreated serum dilution and add different volumes of tea polyphenol standard solution to make the final spiked concentrations 10 μg / mL, 40 μg / mL, and 80 μg / mL (covering the linear range of the standard curve), respectively. Set up 3 parallel samples for each spiked level. At the same time, set up an unspecified blank matrix control.
[0086] The absorbance of each serum spiked sample at 652 nm was determined by colorimetry according to Example 3. ΔA was calculated and substituted into the standard curve equation to calculate the concentration of tea polyphenols in each serum spiked sample.
[0087] This embodiment provides a tea polyphenol detection kit for auxiliary assessment of risk and prognosis in oncology. The kit includes FeV PBA nanozyme, TMB, and HAc-NaAc buffer. The concentration of FeV PBA nanozyme is 1 mg / mL, the concentration of TMB is 8 mM, and the pH of HAc-NaAc buffer is 2.6. This kit can be used to detect tea polyphenols for auxiliary assessment of risk and prognosis in oncology.
[0088] The results of the spiked serum samples are shown in Table 2. The results show that the recovery rate of tea polyphenols in human serum samples is between 99.2% and 102.63%, and the relative standard deviation (RSD) is between 0.15% and 5.41%, indicating that the colorimetric detection method based on FeV PBA has good feasibility and reliability in the detection of human serum samples.
[0089] Table 2 In summary, this invention successfully synthesized the iron-vanadium bimetallic doped Prussian blue analog FeV PBA via a one-pot co-precipitation reaction. This nanozyme exhibits highly efficient oxidase-like activity under acidic conditions, directly catalyzing the generation of superoxide anion radicals from dissolved oxygen, which then oxidize TMB to produce a blue product. Tea polyphenols, due to their polyphenolic hydroxyl structure, can effectively scavenge superoxide anion radicals, thereby inhibiting the oxidation and color development of TMB. Based on this principle, this invention constructs a colorimetric sensing method for the detection of tea polyphenols, applicable to the following detection fields: (1) Application of colorimetric detection of tea polyphenols The colorimetric detection method for tea polyphenols based on FeV PBA nanozymes constructed in this invention can not only accurately determine the content of tea polyphenols in commercially available health products containing tea polyphenols (such as L-carnitine tea polyphenol tablets), but can also be further extended to the field of clinical nutritional assessment and adjuvant therapy monitoring related to oncology medicine.
[0090] Tea polyphenols, as natural polyphenolic compounds, have been extensively studied and proven to possess multiple effects, including inhibiting tumor cell proliferation, inducing apoptosis, blocking tumor angiogenesis, and enhancing the sensitivity of chemotherapeutic drugs. Cancer patients experience significantly elevated oxidative stress levels due to the disease itself and the effects of radiotherapy and chemotherapy; the antioxidant properties of tea polyphenols help mitigate oxidative damage. Quantitative detection of tea polyphenols in serum or urine allows for monitoring the dynamic changes in tea polyphenol levels in patients, providing a sensitive and reliable analytical tool for dietary guidance in cancer chemoprevention strategies, evaluation of the effectiveness of nutritional interventions during adjuvant therapy, and prognostic assessment. Compared to traditional high-performance liquid chromatography (HPLC), the method of this invention eliminates the need for complex derivatization processes and expensive equipment, making it simpler, faster, and significantly reducing detection costs, thus suitable for rapid detection.
[0091] In addition, this method can be used to assess the intake compliance and metabolic level of dietary supplements (such as tea polyphenol capsules) in cancer patients, and to assist in the development of individualized nutritional intervention programs.
[0092] (2) Application of detection of tea polyphenols in human serum The colorimetric detection method for tea polyphenols based on FeV PBA nanozymes constructed by the method of the present invention can not only accurately determine tea polyphenol standards in buffer systems, but also be further extended to the detection of tea polyphenols in complex biological matrices such as human serum, providing a feasible basis for its application in clinical sample analysis.
[0093] In clinical medical applications, serum is the most commonly used biological sample type for monitoring patients' nutritional status and drug metabolism levels. However, the serum matrix contains abundant proteins, amino acids, and small molecule metabolites, which may interfere with colorimetric reactions. This method effectively eliminates the interference of the serum matrix through acetonitrile protein precipitation and dilution pretreatment steps, successfully achieving the spiked recovery detection of tea polyphenols in human serum. Spike recovery experiments show that this method maintains good accuracy and precision in complex biological matrices, and the recovery rate and relative standard deviation meet the methodological requirements for biological sample analysis, verifying the feasibility and reliability of this colorimetric sensing method in human serum samples.
[0094] The method of the present invention can be used for quantitative detection of tea polyphenols in patient serum samples. Specific application scenarios include: (1) monitoring of dietary supplement intake: for patients taking tea polyphenol health products, the bioavailability and intake compliance can be assessed by detecting serum tea polyphenol levels, providing a basis for developing individualized dietary supplement plans; (2) monitoring of adjuvant therapy drugs: the changes in serum tea polyphenol concentration can be dynamically monitored to assist in assessing treatment effects and adjusting dosing regimens; (3) prognostic assessment: by detecting the serum tea polyphenol levels of patients before and after surgery, the association between them and prognostic indicators such as progression-free survival and overall survival can be explored, providing potential new biomarkers for prognostic judgment.
[0095] Compared to traditional high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), this method eliminates the need for complex sample pretreatment and expensive equipment, making it simpler, faster, and significantly reducing detection costs. It is particularly suitable for batch screening in clinical laboratories and rapid testing in resource-limited areas. Furthermore, this method requires only a UV-Vis spectrophotometer or even visual observation, making it suitable for point-of-care testing (POCT) scenarios.
[0096] In summary, the method of this invention has advantages such as a wide detection range, low detection limit, good selectivity, convenient operation, and low cost. Furthermore, validation with human serum matrix has demonstrated its feasibility and reliability in complex biological samples. This method shows promising clinical application prospects and widespread value in multiple food and drug-related fields, including dietary nutrition assessment and prognostic evaluation.
[0097] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing iron-vanadium bimetallic doped Prussian blue nanozymes, characterized in that, Includes the following steps: The vanadium source and the iron source are dissolved in hydrochloric acid and mixed evenly to obtain solution A; Dissolve K3[Fe(CN)6] in water and mix thoroughly to obtain solution B; Solution B was added dropwise to solution A under stirring conditions, and a co-precipitation reaction was carried out by stirring. After standing and aging, a precipitate was obtained. The precipitate was washed and dried to obtain iron-vanadium bimetallic doped Prussian blue nanozyme. The vanadium source includes V6O. 13 The iron source includes FeCl3·6H2O.
2. The preparation method according to claim 1, characterized in that, The V6O 13 The preparation method includes: adding V2O5 to a mixture of ethanol and deionized water, and synthesizing V6O through a hydrothermal reaction. 13 ; The ratio of V2O5 to the mixed solution is 0.4 g~0.6 g: 30 mL~50 mL; The volume ratio of ethanol to deionized water is 1:2.5~3.5; The hydrothermal reaction temperature is 160℃~180℃, and the hydrothermal reaction time is 4 h~6 h.
3. The preparation method according to claim 1, characterized in that, The mass ratio of the vanadium source, iron source, and K3[Fe(CN)6] is 2~3:0.8~0.9:16~17; The concentration of the hydrochloric acid is 5.4% w / w; The temperature of the coprecipitation reaction is 20℃~30℃, and the time of the coprecipitation reaction is 15 min~25 min; The static aging time is 20 h to 28 h.
4. A Prussian blue nanozyme doped with iron and vanadium bimetals, characterized in that, The iron-vanadium bimetallic doped Prussian blue nanozyme, obtained by the preparation method according to any one of claims 1 to 3, contains Fe and V elements, with Fe predominating. 2+ / Fe 3+ Mixed valence state, V element with V 4+ / V 5+ Mixed valence states coexist in the Prussian blue framework.
5. The application of the iron-vanadium bimetallic doped Prussian blue nanozyme as described in claim 4 in the colorimetric detection of tea polyphenols.
6. A colorimetric detection method for tea polyphenols based on iron-vanadium bimetallic doped Prussian blue nanozymes, characterized in that, Includes the following steps: S1. The iron-vanadium bimetallic doped Prussian blue nanozyme of claim 4 is mixed with 3,3',5,5'-tetramethylbenzidine and the sample to be tested under acidic conditions, and the reaction is carried out in the dark to obtain the reaction system. S2. Qualitative detection is performed based on the color change of the reaction system; Alternatively, measure the absorbance A of the reaction system at a wavelength of 652 nm. 样品 The absorbance A of the blank control group without the test sample was measured. 空白 The absorbance difference ΔA = A of the sample to be tested was calculated. 空白 - A 样品 The values were then substituted into the standard curve of absorbance difference versus tea polyphenol concentration for quantitative detection.
7. The colorimetric detection method for tea polyphenols according to claim 6, characterized in that, S1 includes the following specific steps: The 3,3',5,5'-tetramethylbenzidine solution, HAc-NaAc buffer solution, and the sample to be tested were mixed thoroughly to obtain a mixture. The aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme was added to the mixture, vortexed and mixed, and incubated at 37 °C at 200 rpm in the dark for 25 min. The volume ratio of the 3,3',5,5'-tetramethylbenzidine solution, HAc-NaAc buffer, the sample to be tested, and the aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme is 1:2:1:
1. The solvent for the 3,3',5,5'-tetramethylbenzidine solution is DMSO, and the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 8 mM. The pH of the HAc-NaAc buffer solution is 2.6; The concentration of the aqueous dispersion of the iron-vanadium bimetallic doped Prussian blue nanozyme is 1 mg / mL.
8. A reagent kit for the detection of tea polyphenols, characterized in that, The kit comprises the iron-vanadium bimetallic doped Prussian blue nanozyme of claim 4 with TMB and HAc-NaAc buffer.
9. The application of the iron-vanadium bimetallic doped Prussian blue nanozyme according to claim 4 in the preparation of a detection product for auxiliary assessment of tumor risk and prognosis, characterized in that, The testing product is used to detect the level of tea polyphenols in a patient's body fluids.
10. The application of the iron-vanadium bimetallic doped Prussian blue nanozyme of claim 4 in the preparation of products for nutritional metabolism monitoring in the food industry, characterized in that, The product is used to detect the level of tea polyphenols in dietary supplements.