Cerium-doped high-entropy metal-organic framework nanoenzyme and preparation method thereof, and method for detecting beta-amyloid 1-42

Abstract

This invention discloses a cerium-doped high-entropy metal-organic framework nanozyme, its preparation method, and a method for detecting β-amyloid protein 1-42, belonging to the interdisciplinary field of nanomaterial preparation and analytical chemistry. The preparation process includes: uniformly mixing N,N-dimethylformamide, anhydrous ethanol, ultrapure water, glycerol, and polyvinylpyrrolidone; adding terephthalic acid and equimolar amounts of soluble trivalent iron salt, trivalent cerium salt, divalent cobalt salt, divalent copper salt, and divalent nickel salt; adding triethylamine and stirring the reaction at pH 9.0-9.5, followed by ultrasonic treatment; then, the product is sequentially centrifuged to collect the precipitate, washed, and dried to obtain the target product. The nanozyme of this invention exhibits an ultra-wide linear detection range and good long-term stability, making it suitable for the rapid and accurate detection of the target analyte β-amyloid protein 1-42, providing a technical means for in vitro concentration monitoring and biochemical indicator evaluation of specific target proteins.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanomaterials and in vitro biochemical analysis, specifically to cerium-doped high-entropy metal-organic framework nanozymes, their preparation methods, and methods for detecting β-amyloid protein 1-42. This nanozyme is a high-entropy metal-organic framework material assembled with a five-membered transition metal as the coordination center and organic ligands. It can serve as a peroxidase-like alternative to natural biological enzymes, used to construct a highly sensitive, wide-range nano-enzyme-linked immunosorbent assay (ELISA) platform to detect β-amyloid protein 1-42 (Aβ) in samples. 1-42 The precise and quantitative determination of analytes provides technical means for in vitro concentration monitoring and biochemical indicator evaluation of specific target analytes. Background Technology

[0002] The incidence of neurodegenerative diseases such as Alzheimer's disease (AD) is rising year by year, seriously threatening the health and quality of life of the elderly population. Related biochemical mechanism studies have shown that β-amyloid protein (especially the highly toxic Aβ) is a contributing factor. 1-42 The abnormal accumulation and deposition of Aβ subtypes in the brain and the imbalance in their concentration distribution in body fluids are key fundamental biochemical events highly associated with neurodegenerative diseases. Therefore, achieving Aβ concentration analysis in complex biological samples (such as serum or cerebrospinal fluid extracts) is crucial. 1-42 Highly sensitive detection and concentration monitoring of Aβ are of significant scientific importance for related life science research, exploration of pathological mechanisms, and assessment of physiological and biochemical indicators. Currently, the determination of Aβ... 1-42 The mainstream biochemical analysis methods mainly rely on traditional enzyme-linked immunosorbent assay (ELISA). However, this technology is highly dependent on natural bioenzymes such as horseradish peroxidase (HRP) as the core catalyst for colorimetric development. Natural bioenzymes have inherent drawbacks such as extremely poor environmental tolerance, easy inactivation, high dependence on cold chain storage and transportation, and large batch-to-batch activity differences. These not only increase the cost of analysis and detection but also greatly limit their widespread application in routine, large-scale in vitro biochemical detection scenarios.

[0003] Nanozyme-based colorimetric sensing technology, with its extremely high physicochemical stability, low cost, and visual visibility, is considered an ideal choice to replace natural enzymes in constructing a new generation of highly sensitive biochemical detection platforms. In recent years, high-entropy metal-organic frameworks (HE-MOFs) have been introduced into the field of biocatalysis due to their multi-metal synergistic "cocktail effect." To achieve the detection of Aβ... 1-42 To specifically identify and measure colorless protein targets, researchers often combine nanozymes with biomolecules such as nucleic acid aptamers to construct target-responsive "nanozyme switches." This involves pre-blocking the active site of the nanozyme using an aptamer; when the target Aβ is detected... 1-42When present, it competitively binds to the aptamer, causing the aptamer to detach from the nanozyme surface, thereby releasing the catalytic site and enabling signal-activated detection. However, existing conventional transition metal-based high-entropy metal-organic frameworks (such as the FeCoNiCuMn-HE-MOF system, which is assembled with five transition metals (iron, cobalt, nickel, copper, and manganese) as the center and organic ligands coordinated, suffer from a bottleneck in electron transfer efficiency between metal centers when catalyzing the generation of free radicals from H₂O₂ due to the lack of an atom with efficient electronic control capabilities as a "central engine." This leads to limitations in their ability to handle different concentrations of target substances (such as Aβ). 1-42 During the competitive stripping of aptamers and re-release of active sites, the catalytic network experiences severe "electron transfer congestion," meaning the signal rapidly saturates in high concentration ranges, failing to maintain a linear response across a wide measurement range. This directly limits its application in the analysis of complex biological samples across measurement ranges. Furthermore, conventional HE-MOFs still face the risk of microstructure fluctuations due to environmental changes during normal storage, making it difficult to maintain high-density active site exposure over long periods, thus failing to meet the requirements for Aβ... 1-42 The need for rigorous and precise biochemical analysis. Summary of the Invention

[0004] To address at least one of the aforementioned problems, one object of the present invention is to provide a cerium-doped high-entropy metal-organic framework nanozyme, which utilizes the unique 4f orbital electronic structure of cerium (Ce) and Ce... 3+ / Ce 4+ By employing redox pairs to create abundant defect sites in HE-MOFs and combining this with a synergistic steric hindrance preparation process using glycerol-polyvinylpyrrolidone, a stacked nanosheet structure material with low crystallinity, ultra-high specific surface area, and extremely strong peroxidase-like activity was synthesized. This successfully broke through the range limitations of conventional colorimetric methods, achieving the determination of Aβ... 1-42 10 The ultra-wide detection range of 800 pg / mL provides a precise and highly reproducible biochemical analysis method for monitoring the in vitro concentration of specific target proteins and evaluating physiological and biochemical indicators.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a cerium-doped high-entropy metal-organic framework nanozyme includes the following steps: S1, according to 32 40mL:2 4mL:4 6mL: 1.5 Mix N,N-dimethylformamide, anhydrous ethanol, ultrapure water, glycerol, and polyvinylpyrrolidone thoroughly at a ratio of 2 mL:0.1 g. S2. To the mixed solution obtained in step S1, add equimolar amounts of soluble trivalent iron salt, trivalent cerium salt, divalent cobalt salt, divalent copper salt, and divalent nickel salt, then add the organic ligand terephthalic acid and mix thoroughly. The molar amount of terephthalic acid is equal to the sum of the molar amounts of all metal salts, and the ratio of the total amount of metal salts to the amount of N,N-dimethylformamide added is 1:1. 3mmol: 100mL; S3. Add triethylamine to the mixed solution obtained in step S2, wherein the volume ratio of triethylamine to N,N-dimethylformamide (DMF) is 1:1. The ratio of 5:100 should be used, and the reaction should be stirred for at least 15 minutes, while maintaining the pH of the system at 9.0. Between 9.5; In this step, the addition of triethylamine promotes the deprotonation of terephthalic acid, which rapidly initiates the coordination complexation reaction between the five metal ions and the organic ligands, and the solution gradually becomes turbid, forming a precursor colloidal suspension; S4. Transfer the suspension obtained in step S3 to a sealed container and sonicate it for more than 2 hours. Then, centrifuge the product sequentially to collect the precipitate, wash and dry it to obtain the target product. In this step, the local high temperature and high pressure generated by the ultrasonic cavitation effect further promote the formation of lattice distortion and low crystallinity amorphous structure.

[0006] In one specific embodiment of the present invention, the trivalent iron salt is ferric chloride or ferric nitrate.

[0007] In one specific embodiment of the present invention, the trivalent cerium salt is cerium nitrate or cerium chloride.

[0008] In one specific embodiment of the present invention, the divalent cobalt salt is cobalt chloride or cobalt nitrate.

[0009] In one specific embodiment of the present invention, the divalent copper salt is copper chloride or copper nitrate.

[0010] In one specific embodiment of the present invention, the divalent nickel salt is nickel chloride or nickel nitrate.

[0011] Another objective of this invention is to provide a cerium-doped high-entropy metal-organic framework nanozyme, prepared using the method described above.

[0012] Another object of the present invention is to provide a method for detecting β-amyloid protein 1-42, wherein the above-mentioned cerium-doped high-entropy metal-organic framework nanozyme is prepared by conjugating it with a specific recognition antibody via biocrosslinking technology to form a nanozyme biochemical probe, which is then used as a stable substitute for natural biological enzymes in a reaction system based on solid-phase sandwich enzyme-linked immunosorbent assay (NLISA). The absorbance (OD) value is determined by strictly timed catalytic substrate color development and acidification termination. 450This method utilizes the variation of β-amyloid protein 1-42 to specifically determine the actual distribution and abundance of β-amyloid protein 1-42 in the liquid sample being tested, thereby achieving quantitative analysis of the target analyte. The method includes the following steps: B1. Preparation of nanozyme-detection antibody probe dispersion: The cerium-doped high-entropy metal-organic framework nanozyme described in claim 7 is dispersed in a solution at pH 5.5. In a 6.0 pH buffer solution, an activator was added to activate the carboxyl groups on the surface of the nanozyme; after removing the activator, the precipitate was added to a solution with a pH of 7.2. In a 7.4 pH buffer solution, β-amyloid 1-42 detection antibody was added and incubated to allow the nanozyme to covalently bind to the detection antibody. A solution containing bovine serum albumin was then added to block non-specific binding sites. After centrifugation, the precipitate was washed and then added to a buffer solution with a pH of 7.2. In a 7.4 buffer solution, a nanozyme-detection antibody probe dispersion is formed; B2. Target-Specific Capture and Sandwich Structure Construction: Target-specific capture and sandwich structure construction of pre-coated β-amyloid protein 1-42 (Aβ) 1-42 Add the sample solution containing β-amyloid 1-42 (the target analyte) to the well plate containing the captured antibody and incubate. Then wash the plate. Next, add the pre-prepared nanozyme-detection antibody probe dispersion to the well plate and continue incubation in the dark to allow the nanozyme-detection antibody probe to specifically recognize and bind to Aβ on the well plate. 1-42 Target, construct "capture antibody-Aβ" 1-42 The solid-phase sandwich complex of "target-detection antibody-nanozyme" was washed after incubation to thoroughly remove free nanozyme probes. B3. Nanozyme Catalysis for Color Development and Signal Amplification: 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution and hydrogen peroxide (H2O2) solution were sequentially added to the washed well plates for incubation in the dark. During incubation, the cerium-doped high-entropy metal-organic framework nanozyme immobilized at the bottom of the wells exhibited strong peroxidase-like activity due to the abundant defect sites within its high-entropy framework and the efficient electron transfer mechanism of Ce3+ / Ce4+. It efficiently catalyzed the cleavage of H2O2 to generate hydroxyl radicals, rapidly oxidizing the colorless TMB to the blue oxidized state TMB (oxTMB, characteristic absorption peak at 652 nm).

[0013] B4. Acidification Termination and Absolute Quantitative Detection: After the preset incubation time, a stop solution is added to the well plate to cut off the catalytic reaction, causing the reaction system to abruptly turn into a bright yellow compound. The absorbance value at 450 nm (OD450) is measured. The amount of nanozyme retained on the well plate is related to the amount of Aβ in the sample. 1-42 The concentrations of Aβ exhibit a strict stoichiometric proportionality, combined with pre-established Aβ... 1-42A standard working curve can be used to obtain Aβ in the sample to be tested. 1-42 Precise inversion and accurate quantification of concentration.

[0014] In one specific embodiment of the present invention, in step B3, the substrate colorimetric reaction is carried out in an acetate-sodium acetate buffer solution with a pH of 4.0; in step B4, the stop solution has a concentration of 1.0. 2.0 M sulfuric acid solution.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The nanozyme of this invention has abundant Ce in its crystal lattice. 3+ / Ce 4+ Redox pairs, with their multi-electron orbital characteristics, inherently induce a large number of coordinated unsaturated metal sites. These unsaturated sites strongly couple electronically with variable-valence metal centers such as Fe, Co, Ni, and Cu, collectively constructing a highly efficient electron transfer network. In solid-phase biochemical analysis based on enzyme-linked immunosorbent assay (NLISA), even when faced with localized high-density substrate impact from high-concentration targets, this multi-metal synergistic network can still effectively promote high-speed electron flow, completely alleviating the catalytic site passivation and "electron congestion" saturation problems that easily occur in traditional single-metal nanoprobes, thus enabling them to possess an ultra-wide linear detection range.

[0016] The nanozyme of this invention benefits from the high configurational entropy brought about by the "penta-element high-entropy framework", which produces a significant "entropy stabilization effect", reducing the risk of performance degradation caused by the inactivation of a single metal site. At the same time, in combination with the redox buffering capacity given by the unique 4f orbital electronic configuration of Ce, the system can spontaneously resist and neutralize the redox shocks brought by trace amounts of oxygen or moisture in the external environment, effectively maintaining the stability of the stacked sheet-like microstructure and surface active sites inside the material during long-term exposure, giving it extremely excellent long-term storage stability. Attached Figure Description

[0017] Figure 1 This is a SEM image of the nanozyme from Example 1; Figure 2 This is a superimposed diagram of the total elemental distribution of the nanozyme in Example 1; Figure 3 This is an independent mapping distribution diagram of each metal component in the nanozyme of Example 1; Figure 4 These are the XPS peak diagrams of the five elements in the nanozyme of Example 1, and the overall XPS diagram. Figure 5 This is an XRD test result diagram of the nanozyme in Example 1; Figure 6 This is a graph showing the EPR test results of the nanozyme in Example 1. Detailed Implementation

[0018] To more clearly illustrate the present invention, specific embodiments are described below. Those skilled in the art should understand that the following description is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1 The preparation of FeCoNiCuCe-HE-MOF includes the following steps: S1. At room temperature, measure 32 mL of N,N-dimethylformamide (DMF), 2 mL of anhydrous ethanol, 4 mL of ultrapure water and 1.5 mL of glycerol into a beaker, then add 0.1 g of polyvinylpyrrolidone (PVP) and stir continuously with a magnetic stirrer until the solution is completely clear and transparent. S2. Accurately weigh and add 0.15 mmol each of ferric chloride hexahydrate, cobalt chloride hexahydrate, nickel chloride hexahydrate, copper chloride dihydrate, and cerium nitrate hexahydrate to the above solution, and continue to stir magnetically for 10 minutes to achieve atomic-level uniform dispersion of the five variable valence metal ions; weigh 0.75 mmol of the organic ligand terephthalic acid (1,4-BDC) and add it to the system, and stir for 5 minutes to dissolve it completely. S3. Using a pipette, inject 0.8 mL of triethylamine (TEA) into the above solution at a uniform rate, add sodium hydroxide, and control the pH of the system to stabilize at 9.0. The pH was between 9.5 and 9.0. At this point, the solution quickly became cloudy, forming a precursor colloid. Stirring continued for 15 minutes, during which sodium hydroxide was added to maintain the pH at 9.0. Between 9.5; S4. The reaction system was sealed and placed in an ultrasonic cleaner for 2 hours at a frequency of 50 kHz to promote lattice distortion and form an amorphous structure. After the reaction, the product was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was ultrasonically washed three times each with ultrapure water and anhydrous ethanol. Finally, the purified precipitate was placed in a vacuum freeze dryer for 12 hours and ground to obtain FeCoNiCuCe-HE-MOF nanozyme powder.

[0020] Comparative Example 1 The preparation steps, reaction conditions, and reagent amounts of FeCoNiCuMn-HE-MOF in this comparative example are the same as those in Example 1, except that in step S2, 0.15 mmol of cerium nitrate hexahydrate is replaced with 0.15 mmol of manganese chloride hexahydrate.

[0021] Comparative Example 2 The preparation steps, reaction conditions, and reagent amounts of FeCoNiCuPt-HE-MOF in this comparative example are the same as those in Example 1, except that in step S2, 0.15 mmol of cerium nitrate hexahydrate is replaced with 0.15 mmol of potassium chloroplatinate.

[0022] Comparative Example 3 The preparation steps, reaction conditions, and reagent amounts for FeCoNiCu-MOF in this comparative example are the same as in Example 1, except that no cerium salt precursor is added in step S2, and only four metal salts, Fe, Co, Ni, and Cu, are used.

[0023] Comparative Example 4 Aggregated FeCoNiCuCe-MOF (dispersion-free system group) was prepared. The metal ratio, ligand amount and reaction conditions of this comparative example were the same as those of Example 1. The difference was that in the solvent preparation of step S1, PVP, ethanol and 1.5 mL of glycerol were not added. Only a mixed solvent of 33.5 mL of LDM and 4 mL of water was used.

[0024] Test Example 1 The nanozyme of Example 1 was observed using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 1 As shown in the figure, the FeCoNiCuCeHE-MOF in this embodiment successfully suppressed the disordered aggregation of metal ions, exhibiting a clear nanosheet stacking morphology, which is beneficial for obtaining a large surface area and ensuring high-density exposure of active sites.

[0025] Elemental mapping analysis of the surface of the nanozyme in Example 1 was performed using energy-dispersive X-ray spectroscopy (EDS), and the results are shown in [Figure number missing]. Figure 2 , Figure 3 As shown in the figure, the five elements—iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and cerium (Ce)—exhibit a highly uniform atomic-level distribution within the entire nanozyme framework, with no obvious component segregation or phase separation observed. This indicates that the nanozyme of this embodiment successfully integrates five metal components with distinct properties into a single high-entropy framework, achieving a high degree of atomic-level fusion and uniform dispersion.

[0026] The surface elemental valence states of the nanozyme in Example 1 were analyzed in depth using X-ray photoelectron spectroscopy (XPS), and the results are shown in [Figure 1]. Figure 4 As shown in the figure, all five metals coexist in multiple valence states. Most importantly, Ce is clearly observed in the Ce3d spectrum. 3+ With Ce 4+The coexistence of characteristic peaks and the presence of this redox pair confirm that the system has abundant coordinated unsaturated metal sites induced by Ce doping. These unsaturated sites are strongly electronically coupled with the other transition metals, together forming an efficient electron transfer network.

[0027] The crystal structure of the nanozyme of Example 1 was analyzed in depth using X-ray diffraction (XRD), and the results are shown in the figure. Figure 5 As shown in the figure, only one broadened, low-intensity characteristic peak appeared in the diffraction pattern near 2θ≈7.08°, without any sharp diffraction planes. This broad and weak diffraction characteristic indicates that the nanozyme of this embodiment has a "low crystallinity" or "quasi-amorphous" structural characteristic. This long-range disorder and short-range order microstructure can minimize the dead volume of atoms inside the dense lattice, thereby exposing an extremely high density of coordination-unsaturated catalytic active sites on the nanosheet surface.

[0028] To verify the catalytic pathway of the nanozyme in Example 1 at the molecular level, electron paramagnetic resonance (EPR) spectroscopy was performed using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a spin trapping agent. The results are shown in [Figure number missing]. Figure 6 As shown in the figure, the addition of H₂O₂ elicits a very strong characteristic free radical signal, which exhibits a perfect 1:2:2:1 quartet splitting peak. This classic 1:2:2:1 ratio is typical. Specific characteristic maps of spin adducts. This test reveals abundant coordination unsaturation points within the high-entropy framework and Ce... 3+ / Ce 4+ Under the synergistic electron transfer mechanism of variable valence pairs, this nanozyme can efficiently catalyze the cleavage of H2O2 with extremely low activation energy, generating a large number of highly active hydroxyl radicals.

[0029] Test Example 2 The nanozyme materials from the above embodiments and comparative examples were respectively cross-linked with β-amyloid protein 1-42 (Aβ) via the carbodiimide cross-linking method (EDC / NHS method). 1-42 The detection antibody was covalently coupled to prepare a nanozyme-detection antibody probe dispersion with a uniform working concentration. Subsequently, the concentrations of each probe at different Aβ concentrations were measured. 1-42 The absorbance response limit under target capture is used to determine the quantitative detection linear range.

[0030] Test Procedure (Solid-phase Sandwich NLISA Method): B1. Preparation of nanozyme-detection antibody probe dispersion: 1.0 mg of cerium-doped high-entropy metal-organic framework nanozyme was ultrasonically dispersed in 1.0 mL of solution at pH 5.5. The nanozyme was placed in 6.0 mL of 2-(N-morpholine)ethanesulfonic acid (MES) buffer; then 2.0 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 3.0 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature in the dark for 30 minutes to fully activate the carboxyl groups on the surface of the nanozyme; the free activator was then removed by centrifugation at 8000 rpm for 5 minutes, and the precipitate was resuspended in 1.0 mL of pH 7.2 buffer. 7.4% phosphate-buffered saline (PBS) buffer; then add 50 μL of β-amyloid 1-42 (Aβ) at a concentration of 1.0 mg / mL. 1-42 Antibody detection was performed by incubating at 4°C with shaking in the dark for 8 hours. After 12 hours, the nanozyme and the detection antibody covalently bind through amide bonds. After the reaction, PBS buffer containing 1% bovine serum albumin (BSA) is added to block non-specific binding sites for 1 hour. Finally, after centrifugation and washing 3 times, the solution is resuspended in PBS buffer to prepare a nanozyme-detection antibody probe dispersion with a final working concentration of 50 μg / mL for later use.

[0031] B2. Target-Specific Capture and Sandwich Structure Construction: Pre-coated Aβ-containing... 1-42 Standard 96-well microplates containing specific capture antibodies and fully blocked with bovine serum albumin (BSA) were prepared. 100 μL of Aβ at concentrations of 0 pg / mL (blank control), 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 650 pg / mL, 700 pg / mL, 800 pg / mL, and 900 pg / mL were then precisely added to each well. 1-42 Standard solution; incubate the microplate in a 37°C constant temperature shaker for 1 hour; then wash the plate thoroughly 3 times with PBST washing buffer (phosphate buffer containing 0.05% Tween-20); then, accurately add 100 μL of the pre-prepared nanozyme-detection antibody probe dispersion to each well of the microplate, and continue to incubate at 37°C in the dark for 1 hour; after incubation, wash the plate thoroughly 5 times with PBST washing buffer to completely remove any non-specifically bound free probes from the wells.

[0032] B3. Nanozyme-catalyzed color development and signal amplification: Use a multichannel pipette to precisely add 80 μL of pH 4.0 acetate-sodium acetate buffer, 10 μL of TMB substrate solution and 10 μL of H2O2 solution to each well in sequence; incubate at room temperature (25℃) in the dark for 15.0 minutes using a timer.

[0033] B4. Acidification Termination and Absolute Quantitative Detection: Immediately after the 15.0-minute countdown ends, add 50 μL of 1.0 M sulfuric acid to each well to terminate the catalytic reaction. Then, place the microplate on a shaker and gently shake to mix. Once the system momentarily changes from blue to a stable bright yellow, immediately measure the absorbance (OD450) of the reaction solution at 450 nm using a UV-Vis microplate reader. Based on this, calculate the effect of each nanozyme on Aβ. 1-42 The quantitative linear detection range was determined, and the results are shown in Table 1.

[0034] Explanation of the mechanism for choosing 450 nm as the absolute quantitative detection wavelength: The quantitative sensing mechanism of this invention abandons the fuzzy determination of the time endpoint in traditional homogeneous reactions. Nanozymes catalyze the oxidation of TMB to generate blue oxTMB (characteristic absorption peak at 652 nm); after a strictly timed 15.0 minutes, a strong acid (H2SO4) is added as a stop solution. This strong acid environment not only instantly poisons and completely cuts off the catalytic activity of the nanozyme, achieving absolute quantification decoupled from time, but also causes the blue oxTMB generated in the system to undergo proton rearrangement, mutating into an extremely stable bright yellow compound, whose characteristic maximum absorption peak is precisely blue-shifted to 450 nm. Therefore, monitoring the absorbance (OD) at 450 nm is sufficient. 450 It fully complies with the current gold standard for biochemical analysis, ensuring the extreme rigor and high reproducibility of the test results.

[0035] Table 1. Nanozymes for Aβ in various examples and comparative examples. 1-42 Linear range performance test comparison As shown in Table 1, Example 1 exhibits extremely excellent anti-saturation ability. At 10 Its absorbance response (OD) at 450 nm is within an ultrawide concentration range of 800 pg / mL. 450 ) and Aβ 1-42 The concentration consistently maintained an excellent linear positive correlation (R0). 2 =0.998). This is thanks to the Ce present in the high-entropy framework. 3+ / Ce 4+Redox pairs intrinsically induce a large number of defect sites to maintain charge balance. These defect sites and multi-metal valence centers construct a highly efficient electron transfer network. Even under the high-density conditions of the sandwich method's densely captured probes, this network can still promote high-speed electron transfer and the cyclic regeneration of active sites, thereby greatly improving the linear response capability in the high-concentration range. In contrast, Comparative Example 1... 3. Due to the lack of efficient electronic regulation by Ce, the catalytic sites are prone to overload saturation, and their performance at 10⁻⁶ is limited. The forced fitting correlation coefficients across the entire 800 pg / mL range were all significantly lower than 0.9, indicating a significant nonlinear shift in the high concentration range. Furthermore, it should be noted that in Comparative Example 4, severe material aggregation resulted in extremely low antibody conjugation efficiency, and the antibody was easily detached during multiple washes, leading to a sharp reduction in the effective catalytic sites remaining in the wells. This resulted in an extremely weak colorimetric reaction, making it impossible to construct a statistically significant quantitative linear curve. Based on the 3σ / S method (where σ is the standard deviation of the blank control group and S is the slope of the standard curve), the theoretical limit of detection (LOD) of Example 1 of this invention reaches 2.8 pg / mL, fully meeting the requirements for highly sensitive detection of trace early biomarkers.

[0036] Test Example 3 (Long-term stability test) The nanozyme materials of each embodiment and comparative example were prepared into nanozyme-detection antibody probes (prepared according to the method in Test Example 2) and placed at room temperature (25°C) and natural humidity (RH 40%). Under normal conditions (60%), the samples were stored in a sealed container away from light. Samples were taken at regular intervals on days 1, 7, 15, 30, and 60, and probe dispersions of the same concentration were reconstituted for performance retesting. To ensure the objectivity and fairness of the evaluation and to eliminate the interference of substrate concentration differences on catalytic kinetic decay, the common intersection concentration (50 pg / mL) of the effective linear range of each nanozyme probe (except for the expired comparative example 4) was selected as the uniform Aβ value. 1-42 Standard working concentrations were used to assess long-term stability. Performance backtesting was conducted strictly according to the solid-phase sandwich method described in Test Example 2, with a strict 15.0-minute timer for color development and termination with strong acid. The OD values ​​of each nanozyme were measured on day 1. 450 Using the signal as a 100% initial activity baseline, the relative retention rate (%) of the sensor signal at each subsequent time point was calculated, and the results are shown in Table 2.

[0037] Table 2. Stability test results of nanozyme probes for each embodiment and comparative example. As shown in Table 2, the long-term storage stability of Example 1 is significantly better than that of Comparative Example 1. 3. After 60 days of storage at room temperature, its signal relative retention rate remained as high as 97.8%. This extreme stability is achieved primarily due to cerium's (Ce) unique redox self-buffering ability and the hysteretic diffusion effect of its high-entropy framework. Specifically, due to its unique 4f orbital electronic configuration, Ce readily achieves Ce... 3+ With Ce 4+ The reversible conversion between them. During long-term exposure to air and natural humidity, this low-barrier valence pair acts as a "chemical sacrificial anchor" in the crystal lattice: it can preferentially absorb redox shocks from trace amounts of oxygen or moisture in the environment, and maintain the charge balance of the entire high-entropy system through its own spontaneous adjustment of valence state, thus acting as a "shield" to protect the main catalytic active sites such as Fe, Co, Ni, and Cu from environmental passivation. In contrast, Comparative Example 1 All three exhibited significant performance degradation. Comparative Example 1, while also a high-entropy structure, lacked a Ce-like 4f ​​orbital electron self-buffering mechanism in Mn, making it unable to effectively resist environmental molecular erosion. Surface active sites slowly passivated over time, with the retention rate dropping to 81.4% after 60 days. Comparative Example 2, due to the extremely high surface energy of platinum nanoparticles, was prone to adsorbing air impurities and causing surface "poisoning" or crystal plane reconstruction during long-term storage, resulting in a retention rate drop to 74.1%. Comparative Example 3, lacking Ce, experienced a significant reduction in system configuration entropy, losing the protection of the "hysteresis diffusion effect." This led to the slow collapse (hydrolysis) of its coordination network under normal storage conditions, resulting in severe signal distortion (56.3%) on day 60. Furthermore, it should be noted that while Comparative Example 4 had a higher retention rate, the material initially experienced severe aggregation, leading to a sharp reduction in the probe's specific surface area and extremely low antibody conjugation efficiency. It also easily detached in large quantities during sandwich washing; its initial target response OD... 450 The background noise is so weak that it is close to background noise, making it completely incapable of practical biochemical analysis and detection.

[0038] Test Example 4 (Aβ in complex biological samples) 1-42 Quantitative detection of proteins) Sample pretreatment and simulated manual spiking: Serum samples from healthy volunteers, obtained through legitimate channels, were used as the background for the complex biological matrix. Considering the presence of Aβ in the serum of healthy individuals... 1-42 The basal concentration is usually extremely low. To fully verify the ability of the nanozyme of this invention to quantitatively monitor protein biomarkers at different concentration stages in real physiological fluids, this test pre-tested using a standard spiking method: accurately transfer the same batch of healthy serum stock solution, and add a quantitative amount of Aβ to each solution. 1-42 Standard solutions were artificially prepared to produce spiked serum samples with concentrations of 50.0 pg / mL, 200.0 pg / mL, and 600.0 pg / mL. Subsequently, considering the target analyte Aβ...1-42 Since these are protein biomarkers, traditional strong acid precipitation methods should be strictly avoided to prevent damage to the target conformation. To effectively reduce the matrix effect caused by high-abundance background proteins (such as serum albumin and immunoglobulins) in the complex serum matrix and the interference of non-specific adsorption on immune sandwich binding, serum samples from each group were accurately transferred and diluted 5-fold using sample dilution buffer (PBS buffer containing 1% BSA) at pH 7.4. After thorough shaking and mixing, a clear serum matrix test solution was obtained.

[0039] The detection steps of the method of this invention (solid-phase sandwich NLISA method) are as follows: C1, in the case of pre-coated Aβ 1-42 In a 96-well plate containing the captured antibody, 100 μL of the serum matrix test solution with different spiking concentrations was added sequentially and incubated at 37°C with shaking for 1 hour. The plate was then washed three times with PBST washing buffer. Next, 100 μL of the nanozyme-detection antibody probe dispersion prepared in Example 1 (preparation method as described in Test Example 2) was added to each well and incubated at 37°C in the dark for 1 hour. After incubation, the plate was thoroughly washed five times to remove the free probe.

[0040] C2. Add 80 μL of pH 4.0 acetate-sodium acetate buffer, 10 μL of TMB substrate solution (10 mM), and 10 μL of H2O2 solution (10 mM) in sequence, and incubate at room temperature in the dark for 15.0 minutes using a timer.

[0041] C3. Incubate in the dark for 15.0 minutes, then immediately add 50 μL of 1.0 M sulfuric acid stop solution to each well; the reaction solution will instantly change from blue to yellow. Immediately measure the absorbance (OD) at 450 nm using a microplate reader. 450 The measured OD 450 Substituting the values ​​into the pre-established standard working curve equation (obtained from test example 2), the Aβ values ​​in the serum sample were calculated by inversion. 1-42 The actual measured total concentration.

[0042] Classical reference method detection: Simultaneously, the "commercial human Aβ" widely used in life science research was employed. 1-42The enzyme-linked immunosorbent assay (ELISA) kit (containing natural HRP-labeled secondary antibody) was used as a reference method for parallel assays. The specific procedures were strictly followed according to the kit's official instructions: sample incubation, plate washing, addition of HRP-labeled probe for incubation, and plate washing were performed sequentially; then, the kit's accompanying single-component TMB chromogenic solution was added, and incubation was strictly timed for 15 minutes before adding the accompanying stop solution; finally, the absorbance value at 450 nm was measured using a microplate reader, and the result was substituted into the simultaneously plotted kit standard curve to calculate the Aβ concentration in the sample. 1-42 The reference concentration.

[0043] The test results of the two testing methods are shown in Table 3.

[0044] Table 3. Spike recovery test results in human serum samples in Example 1 As shown in Table 3, the Aβ levels measured by the nanozyme probe of Example 1 of this invention in complex human serum samples were [data missing]. 1-42 The total concentration (including trace background levels) showed extremely high consistency with classic commercial ELISA reference kits, and the spiked recovery rate remained stable at 98.67%. The accuracy is between 102.94%. This demonstrates that the nanozyme still exhibits excellent target recognition specificity and extremely precise absolute quantification capability when facing real complex physiological fluids.

[0045] 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 changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a cerium-doped high-entropy metal-organic framework nanozyme, characterized in that, Includes the following steps: S1, according to 32 40mL:2 4mL:4 6mL: 1.5 Mix N,N-dimethylformamide, anhydrous ethanol, ultrapure water, glycerol, and polyvinylpyrrolidone thoroughly at a ratio of 2 mL:0.1 g. S2. To the mixed solution obtained in step S1, add equimolar amounts of soluble trivalent ferric salt, trivalent cerium salt, divalent cobalt salt, divalent copper salt, and divalent nickel salt, then add terephthalic acid and mix thoroughly. The molar amount of terephthalic acid is equal to the total molar amount of all metal salts, and the ratio of the total amount of metal salts to the amount of N,N-dimethylformamide added is 1:

1. 3mmol: 100mL; S3. Add triethylamine to the mixed solution obtained in step S2, wherein the volume ratio of triethylamine to N,N-dimethylformamide is 1:

1. The ratio of 5:100 should be used, and the reaction should be stirred for at least 15 minutes, while maintaining the pH of the system at 9.

0. Between 9.5; S4. Transfer the suspension obtained in step S3 to a sealed container, sonicate for more than 2 hours, and then centrifuge the product to collect the precipitate, wash and dry it to obtain the target product.

2. The method for preparing cerium-doped high-entropy metal-organic framework nanozymes according to claim 1, characterized in that, The ferric salt is ferric chloride or ferric nitrate.

3. The method for preparing cerium-doped high-entropy metal-organic framework nanozymes according to claim 1, characterized in that, The trivalent cerium salt is cerium nitrate or cerium chloride.

4. The method for preparing cerium-doped high-entropy metal-organic framework nanozymes according to claim 1, characterized in that, The divalent cobalt salt is cobalt chloride or cobalt nitrate.

5. The method for preparing cerium-doped high-entropy metal-organic framework nanozymes according to claim 1, characterized in that, The divalent copper salt is copper chloride or copper nitrate.

6. The method for preparing cerium-doped high-entropy metal-organic framework nanozymes according to claim 1, characterized in that, The divalent nickel salt is nickel chloride or nickel nitrate.

7. A cerium-doped high-entropy metal-organic framework nanozyme, characterized in that, It was prepared using the preparation method of cerium-doped high-entropy metal-organic framework nanozymes according to any one of claims 1-6.

8. A method for detecting β-amyloid protein 1-42, characterized in that, Includes the following steps: B1. Preparation of nanozyme-detection antibody probe dispersion: The cerium-doped high-entropy metal-organic framework nanozyme described in claim 7 is dispersed in a solution at pH 5.

5. In a 6.0 pH buffer solution, an activator was added to activate the carboxyl groups on the surface of the nanozyme; after removing the activator, the precipitate was added to a solution with a pH of 7.

2. In a 7.4 pH buffer solution, β-amyloid 1-42 detection antibody was added and incubated to allow the nanozyme to covalently bind to the detection antibody. A solution containing bovine serum albumin was then added to block non-specific binding sites. After centrifugation, the precipitate was washed and then added to a buffer solution with a pH of 7.

2. In a 7.4 buffer solution, a nanozyme-detection antibody probe dispersion is formed; B2. Target-specific capture and sandwich structure construction: Add the test sample solution containing β-amyloid 1-42 to the well plate pre-coated with β-amyloid 1-42 capture antibody and incubate, then wash the plate; then add nanozyme-detection antibody probe dispersion to the well plate and continue incubation in the dark, so that the nanozyme-detection antibody probe specifically recognizes and binds to the β-amyloid 1-42 target on the well plate, and wash the plate after incubation; B3. Nanozyme-catalyzed color development and signal amplification: 3,3′,5,5′-tetramethylbenzidine substrate solution and hydrogen peroxide solution were added sequentially to the washed well plate and incubated in the dark. B4. Acidification Termination and Absolute Quantitative Detection: After incubation for the preset time, a stop solution is added to the well plate to cut off the catalytic reaction, causing the reaction system to suddenly turn into a bright yellow compound. The absorbance value at 450 nm is measured, and then the concentration of β-amyloid protein 1-42 in the sample is inverted by combining the standard working curve.

9. The method for detecting β-amyloid protein 1-42 according to claim 8, characterized in that, In step B3, the substrate colorimetric reaction is carried out in an acetate-sodium acetate buffer solution with a pH of 4.0; in step B4, the stop solution is a 1.0% solution. 2.0 M sulfuric acid solution.

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