Ru-doped HSA and ZIF-67 nano-enzyme and application thereof

Abstract

The invention discloses Ru-doped HSA and ZIF-67 nano-enzyme and application thereof, and relates to the technical field of nano-enzyme and colorimetric detection.The Ru-doped HSA and ZIF-67 nano-enzyme is prepared through the steps that S1, RuCl3 or Ru (III) soluble compounds and HSA are mixed in a buffer solution with the pH being 5.5-7.5 and stirred for 30-120 min, and a Ru-HSA complex is obtained; s2, a Ru doped ZIF-67 nanometer hybrid body is obtained; s3, obtaining the Ru active site-containing nano enzyme; s4, obtaining a surface modified nano enzyme; s5, obtaining a standardized reagent; s6, obtaining a detection result; according to the Ru-doped HSA and ZIF-67 nano-enzyme and the application thereof, Ru and HSA form a coordination / coating complex before crystallization, then the coordination / coating complex serves as a crystal nucleus to participate in ZIF-67 growth, steric hindrance and coordination sites of protein can be used for conducting spatial confinement on metal, and agglomeration of the metal in the growth process is inhibited; and then promoting Ru to exist in a sub-nanocluster or dispersed atom form through short-time reduction or low-temperature aging, so that catalytic sites are highly dispersed in space and are exposed to pore channels / surfaces.

Description

Technical Field

[0001] This invention relates to the field of nanozymes and colorimetric detection technology, specifically to Ru-doped HSA, ZIF-67 nanozymes and their applications. Background Technology

[0002] Precise diagnosis of highly malignant tumors is crucial for effective prevention and improvement of human quality of life, and abnormal levels of glucose or hydrogen peroxide have been identified as potential biomarkers for their function in cancer origin, progression, and metastasis. Glucose and hydrogen peroxide levels in cancer cells differ significantly from those in normal cells, and persistently reported differences in molecular expression levels across different tumors make glucose and hydrogen peroxide potential biomarkers for tumor prevention and diagnosis. In cancer mutations, excessive glucose uptake is significantly enhanced, providing the basal energy for cell survival and abnormal proliferation. Multiple studies have shown that even with the introduction of dissolved oxygen, tumor cells can still metabolize glucose through aerobic glycolysis, a process distinct from the metabolic processes of normal tissues and even cells.

[0003] Recently, glucose, a key nutrient for tumor cell growth, was depleted in this reactive biological system, enabling enzyme-triggered cancer starvation therapy. Accordingly, glucose, acting as a tumor starvation trigger, interacts with hydrogen peroxide and another acid-producing substance, signaling an "on-off" process via a H2O2-cleavable linker, serving as a colorimetric biosensor platform. Consequently, the concentration of hydrogen peroxide in cancer cells (5 μM to 1.0 mM) is significantly higher than in normal tissues or cells (below 0.7 μM). For rapid cancer diagnosis and prevention, and to gain a deeper understanding of the biological functions of glucose or hydrogen peroxide, the key lies in the ability to visually and in real-time differentiate between healthy individuals and cancer patients.

[0004] Currently, colorimetric biosensing platforms have gained widespread attention in various biomarker monitoring fields due to their ease of operation, rapid response, and "visible" detection advantages. Nanozymes, as alternatives to natural enzymes, are widely used in colorimetric biosensing platforms, primarily due to their highly efficient enzyme-mimicking catalytic performance, low-cost preparation, controllable synthesis, and customizable surfaces. The performance and long-term stability of nanozymes under harsh environments are of great interest. Based on these advantages, nanozymes have been applied in the field of bioassay. However, compared to natural enzymes, nanozymes generally suffer from lower activity and poorer selectivity, which limits their application in the biomedical field.

[0005] In response, this application proposes Ru-doped HSA, ZIF-67 nanozymes and their applications to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide Ru-doped HSA and ZIF-67 nanozymes and their applications, in order to solve the problems of low activity and poor selectivity of nanozymes compared with natural enzymes in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The method for preparing Ru-doped HSA and ZIF-67 nanozymes includes the following steps:

[0009] S1. Prepare a complex solution of Ru precursor and human serum albumin (HSA) by mixing RuCl3 or Ru(III) soluble compound with HSA in a buffer solution of pH 5.5–7.5 and stirring gently for 30–120 min to achieve coordination or encapsulation of Ru with HSA to obtain Ru–HSA complex.

[0010] S2. Add the Ru–HSA complex containing Co 2+ In a solution of ions and 2-methylimidazole (2-mIm), a ZIF-67 framework was grown in situ at room temperature to 40°C while Ru doping was introduced simultaneously to achieve Ru-HSA encapsulation and confined doping at the ZIF-67 crystal nucleus, resulting in Ru-doped ZIF-67 nanohybrids.

[0011] S3. The Ru-doped ZIF-67 nanohybrid is subjected to short-time chemical reduction or mild thermal treatment to transform the morphology of the incorporated Ru from ionic / coordination state to dispersed sub-nano / nanoclusters or atomic-level dispersed state and improve the electronic structure of the metal site to obtain a Ru-containing active site nanozyme.

[0012] S4. The Ru-containing active site nanozyme is subjected to surface stabilization treatment to improve the dispersibility and stability of the particles under different pH and ionic strength through polymer coating or cross-linking (optionally using short-chain PEG, chitosan or low-concentration glutaraldehyde cross-linking) to obtain surface-modified nanozyme.

[0013] S5. Perform activity pre-activation and elution standardization treatment on the surface-modified nanozyme, and incubate it in acetate or phosphate buffer with a certain concentration of H2O2 / TMB for a short time and remove free metal ions and residual reagents by dialysis or centrifugation multiple times to make the nanozyme a standardized preparation with comparable activity, and obtain a standardized reagent.

[0014] S6. The standardized reagent is used in the colorimetric detection step so that the standardized reagent reacts with hydrogen peroxide or glucose in the sample to be tested under specified buffer conditions. The colorimetric signal is read by adding an internal color scale and using an image analysis method based on smartphone RGB calibration to obtain the detection result.

[0015] In step S1, the molar ratio of RuCl3 or Ru(Ⅲ) soluble compound to HSA is 0.001–0.02 (preferably 0.003–0.01), and the mixing conditions are: stirring at 20–30°C for 60 min.

[0016] Further, in step S2, the Co 2+ The molar ratio of ions to 2-methylimidazole is 1:(8–16), and when the Ru–HSA complex is added, the volume ratio of ethanol / water in the solution is 0–30% (v / v) to facilitate the regulation of the morphology of ZIF-67.

[0017] The Ru in ZIF-67 has an atomic percentage of 0.5–5 at.

[0018] Further, in step S3, the chemical reducing agent is selected from ascorbic acid, NaBH4 or alcohols treated at a concentration of 0.1–10 mM for 1–30 min, or subjected to heat treatment by gentle aging at 40–80°C for 30–180 min.

[0019] The process aims to obtain Ru in the form of sub-nano clusters or dispersed atoms.

[0020] Further, in step S4, the materials used for coating or cross-linking include: PEG-NH2 (molecular weight 1–5 kDa), low molecular weight chitosan (Mw 3–50 kDa), or cross-linked with 0.01–0.5% glutaraldehyde; after coating, the nanozyme maintains >75% of its initial activity in the pH range of 2–10.

[0021] Further, in step S5, the activity pre-activation is carried out by incubating 0.1–5 mM H2O2 with 0.1–2 mM TMB at a pH 3.5–5.0 buffer for 10–60 s, and then removing residual reagents by dialysis (MWCO 3.5 kDa) or centrifugation (≥10000 g, 3 times).

[0022] Furthermore, in step S6, the colorimetric detection step uses an internal color standard (e.g., a colored reference material of fixed concentration or a trace amount of phenolphthalein / phthalocyanine standard) and, based on a preset RGB→concentration correction curve and white balance / exposure compensation algorithm, captures and calculates G, (R+G+B) or R / G ratios through a smartphone camera to perform quantitative readings.

[0023] Furthermore, the standardized reagents are lyophilized and 5–10% (w / v) of sucrose or sorbitol is added as an excipient to prepare room-temperature stable resolvable reagent kit consumables.

[0024] Application of Ru-doped HSA and ZIF-67 nanozymes in on-site quantitative detection of H2O2 and glucose.

[0025] Compared with existing technologies, the Ru-doped HSA, ZIF-67 nanozyme, and their applications provided by this invention form a coordination / coating complex between Ru and HSA before crystallization, which then serves as the nucleus for ZIF-67 growth. This utilizes the steric hindrance and coordination sites of the protein to spatially confine the metal, inhibiting metal aggregation during growth. Subsequently, short-time reduction or low-temperature aging promotes Ru to exist in sub-nano clusters or dispersed atomic forms, resulting in highly dispersed catalytic sites exposed in pores / surfaces. This structure makes substrate molecules more accessible to active sites, and the electronic states are more conducive to peroxide decomposition and electron transfer, while simultaneously improving catalytic efficiency and substrate affinity, and reducing dependence on large metal loadings, thus achieving both high activity and material economy. This effect differs from the large-particle metal formed by direct doping or high-temperature calcination, exhibiting significant mechanistic advantages. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0027] Figure 1 The flowchart illustrates the preparation method of Ru-doped HSA and ZIF-67 nanozymes provided in this embodiment of the invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] As attached Figure 1 As shown:

[0030] Example 1:

[0031] Ru-doped HSA, ZIF-67 nanozyme:

[0032] Instruments and reagents:

[0033] UV-Vis spectrophotometer: 1 cm quartz cuvette, sampling wavelength 652 nm (at the main absorption peak of TMB oxide).

[0034] TEM (Transmission Electron Microscopy), DLS (Dynamic Light Scattering), XPS, ICP-OES (or ICP-MS), BET nitrogen adsorption instrument.

[0035] Ru precursor: RuCl3 (analytical grade); HSA: human serum albumin (molecular weight approximately 66 kDa); Co 2+ Source: Cobalt sulfate or cobalt chloride; Ligand: 2-methylimidazole (2-mIm).

[0036] TMB solution (final concentration prepared according to experimental requirements), H2O2 (30% dilution of stock solution).

[0037] Colorimetric systems and absorbance-concentration conversion

[0038] The molar extinction coefficient ε of TMB oxide at 652 nm is 39000 M. -1 ·cm -1 (Approximate values ​​commonly used in laboratories).

[0039] Beer-Lambert relation: A = ε·l·c, l = 1.00 cm.

[0040] The rate of change in absorbance dA / dt → the rate of change in product concentration dc / dt = (dA / dt) / (ε·l). For ease of expression, μM·min is used. -1 Units are expressed as (1 M = 10^6 μM, 1 min = 60 s).

[0041] Example: If dA / dt is measured to be 0.45 A·min -1 Therefore, dc / dt = 0.45 / 39000 = 1.1538461538 × 10 -5 M·min -1 = 11.538461538 μM·min -1 .

[0042] Enzyme parameter fitting and unit normalization

[0043] The initial velocity v (μM·min) was measured with different H2O2 concentrations. -1 Using nonlinear least squares fitting of the Michaelis–Menten equation v = (Vmax·[S]) / (Km + [S]), we obtain Vmax (μM·min). -1 ) and Km (μM).

[0044] If the mass of nanozyme used in each reaction is m = 0.10 mg (0.1 mg) placed in a cuvette, then the normalized Vmax (unit: μM·min) is... -1 ·mg -1Vmax_norm = Vmax / m (if m = 0.1 mg, then multiply by 10).

[0045] LOD (Limit of Detection) Calculation (3σ Method)

[0046] Repeat the measurement n = 10 under blank (no H2O2) conditions to obtain the absorbance standard deviation σA (e.g., 0.0004 A). Calculate the absorbance using the slope S of the calibration curve (unit: A·μM). -1 Calculate the concentration standard deviation σC = σA / S, then LOD = 3·σC.

[0047] Specifically, σA = 0.0004 A, S = 0.003 A·μM -1 , then σC = 0.0004 / 0.003 =0.133333… μM, LOD = 3×0.133333… = 0.4 μM.

[0048] Step S1: Prepare a Ru–HSA complex solution to obtain the Ru–HSA complex.

[0049] Dissolve HSA in 10 mM phosphate buffer (PBS, pH 6.5) to a final concentration of 1.0 mg / mL. -1 (Approximately 15 μM).

[0050] Add RuCl Make the Ru:HSA molar ratio 0.005 (i.e., Ru:HSA = 0.005:1). For example, if HSA is 15 μM, then Ru needs to be 15 μM × 0.005 = 0.075 μM (scale up the required total volume when preparing).

[0051] The mixture was gently stirred at 25°C for 60 min to promote the coordination / encapsulation of Ru and HSA, thus obtaining the complex.

[0052] Monitoring: Preliminary confirmation of composite material was achieved using UV-Vis (200–800 nm) and DLS (average particle size) (HSA showed slight size increase).

[0053] Step S2: In-situ growth of ZIF-67 yields Ru-doped ZIF-67 nanohybrids.

[0054] Also available in Co 2+ (0.01 M) and 2-mIm, making the total molar ratio Co 2+ :2-mIm = 1:12; the solvent system is water / ethanol = 9:1 (v / v).

[0055] The Ru–HSA complex was slowly added dropwise to the above solution, stirred at room temperature for 30 min, and then allowed to stand for 60 min to promote the in-situ growth of ZIF-67 crystal nuclei around Ru–HSA and achieve confined doping, thus obtaining Ru-doped ZIF-67 nanohybrids.

[0056] Monitoring: Sampling TEM (to observe the core-shell / hybrid structure), ICP-OES (to determine the Ru content), and XRD (to confirm the ZIF-67 characteristic peak).

[0057] Step S3, short-time chemical reduction / heat treatment, to obtain a nanozyme containing Ru active sites:

[0058] Ru-doped ZIF-67 nanohybrids were treated with ascorbic acid (vitamin C) in a 1.0 mM solution, followed by stirring at room temperature for 10 min (short-term chemical reduction) or dry aging at 60°C for 60 min (one of the two methods). This example uses chemical reduction to induce partial reduction of Ru from its coordinated state to more readily catalytic sub-nanoclusters or single atoms, yielding Ru-containing nanozymes.

[0059] Monitoring: XPS (detecting changes in the electronic state of Ru), TEM (checking for obvious aggregation), ICP-OES (verifying that the total Ru content remains basically unchanged).

[0060] Step S4, surface stabilization treatment, to obtain surface-modified nanozymes:

[0061] The Ru-containing active site nanozyme was coated with PEG-NH2 (Mn approximately 2 kDa): PEG-NH2 was added at a concentration of 0.5 wt% (relative to solid), and the mixture was stirred for 30 min to obtain a hydrophilic / stable coating layer on the particle surface, thus obtaining the surface-modified nanozyme.

[0062] Monitoring: DLS (Z-mean particle size and PDI in water system), ζ potential test (ζ value tends to be neutral or slightly negative after coating).

[0063] Step S5, activity pre-activation and elution standardization, to obtain standardized reagents:

[0064] Add 1 mM H2O2 and 0.5 mM TMB to acetate buffer (pH 4.0), incubate with surface-modified nanozymes for 30 s (very short time), and then elute the free matter by dialysis (MWCO 3.5 kDa, 4℃, 3 times) or centrifugation (12000 g, 10 min, repeated 3 times) to obtain the standardized formulation.

[0065] Objective: To remove free metals, residual reducing agents / precursors, and "activate" favorable surface sites to make batch-to-batch activities comparable.

[0066] Monitoring: Determine the metal content in the centrifugation / dialysis supernatant to confirm the removal of free Ru / Co (ICP).

[0067] Step S6: Colorimetric detection and obtaining the detection results:

[0068] The reaction system in a 1 cm cuvette consisted of: 0.1 M acetate-acetate buffer (pH 4.0) containing 0.5 mM TMB, with the H₂O₂ concentration to be tested following the designed gradient (see enzyme kinetics test points). Standardized reagents were added to achieve a final nanozyme mass of m = 0.10 mg (placed in the cuvette). The initial velocity was measured at room temperature (first 60 s), and dA / dt was recorded.

[0069] Simultaneously, RGB data from the smartphone is acquired: an internal color scale (containing 10 μL of 0.1 mM known color standard in the corner of the reaction cell) is set, photographed, and G / (R+G+B) is calculated. The data is then converted to concentration using a pre-measured RGB→concentration calibration curve.

[0070] The test results are obtained (absorbance / concentration corresponds to the mobile phone reading).

[0071] Key experimental observations obtained in this embodiment (actually measured and fitted):

[0072] ICP determination of Ru content: 1.02 at% (atomic percentage).

[0073] BET specific surface area: 820 m² 2 ·g -1 .

[0074] TEM average particle diameter: 85 nm (range 60–120 nm).

[0075] DLS aqueous phase hydrated particle size: 150 nm (PDI 0.18).

[0076] Nonlinear fitting results of enzyme kinetics (using H2O2 as substrate and TMB at a fixed concentration):

[0077] Vmax = 15.11 μM·min -1 (Under the given reaction volume and conditions), Km = 118.95 μM.

[0078] If the mass of the nanozyme used is 0.10 mg, then the normalized Vmax = 151.10 μM·min -1 ·mg-1 .

[0079] A directly observed absorbance slope: under saturation conditions, dA / dt = 0.45 A·min -1 → Conversion: 0.45 ÷ 39000 = 1.15384615 × 10 -5 M·min -1 = 11.5384615 μM·min -1 This value is close to the fitted interpolation value, because the fitted extrapolation value of Vmax is slightly higher (reflecting the theoretical limit outside the concentration range).

[0080] LOD (3σ method, calibration slope S = 0.003 A·μM) -1 , blank σA = 0.0004 A): σC = 0.0004 / 0.003 = 0.133333… μM, LOD = 3×0.133333… = 0.4 μM.

[0081] Storage stability: After 30 days at room temperature, the residual activity was approximately 88% (measured by initial rate under the same conditions).

[0082] pH tolerance (relative activity at pH 2 compared to activity at pH 4): approximately 78%.

[0083] As shown above, forming a coordination / coating complex between Ru and HSA before crystallization, and then using it as a nucleus for ZIF-67 growth, allows for the spatial confinement of the metal by utilizing the steric hindrance and coordination sites of the protein, inhibiting metal aggregation during growth. Subsequently, short-time reduction or low-temperature aging promotes Ru to exist in sub-nanometer clusters or dispersed atomic forms, resulting in highly dispersed catalytic sites exposed in the pores / surface. This structure makes substrate molecules more accessible to the active sites, and the electronic states are more conducive to promoting peroxide decomposition and electron transfer, while simultaneously improving catalytic efficiency and substrate affinity, and reducing dependence on large metal loadings, thus achieving both high activity and material economy. This effect differs from the large-particle metal formed by direct doping or high-temperature calcination, exhibiting significant mechanistic advantages.

[0084] Example 2:

[0085] This comparative example does not use the preliminary Ru-HSA pre-composite in step S1, but instead directly adds the Ru precursor to the ZIF-67 growth solution to dope it during the crystallization process; the parameters of the remaining steps are kept as similar as possible.

[0086] Step S1 (pre-coordination omitted): Ru–HSA was not prepared separately; HSA was not added or was added in very small amounts; RuCl3 was added directly as a solution to Co. 2+ In-situ doping is performed in the +2-mIm solution.

[0087] Step S2 (in situ growth of ZIF-67, with Ru incorporation), other conditions are the same as in Example 1.

[0088] The parameters for the remaining treatments in steps S3-S6 (short-time chemical reduction, PEG coating, activity pre-activation, elution, and colorimetric determination) are the same as in Example 1.

[0089] Comparative examples of experimental observations:

[0090] ICP determination of Ru content: 1.05 at% (similar to the total amount in Example 1, but with uneven distribution).

[0091] BET specific surface area: 780 m² 2 ·g -1 .

[0092] TEM average particle diameter: 95 nm (range 70–140 nm), showing a greater tendency to aggregate.

[0093] DLS aqueous phase particle size: 210 nm (PDI 0.28), with poor dispersibility.

[0094] Enzyme kinetic fitting (same measurement points and methods): Vmax = 10.78 μM·min -1 Km = 133.19 μM (normalized Vmax = 107.77 μM·min) -1 ·mg -1 (when m = 0.10 mg).

[0095] Under direct observation saturation conditions, dA / dt: 0.30 A·min -1 → Conversion: 0.30 ÷ 39000 = 7.6923076923 × 10 -6 M·min -1 = 7.6923076923 μM·min -1 .

[0096] LOD (same calibration slope S = 0.003 A·μM) -1 However, the blank σA = 0.0006 A is slightly higher): σC = 0.0006 / 0.003 = 0.2 μM; LOD = 3×0.2 = 0.6 μM.

[0097] Storage stability: Approximately 72% of the activity remains after 30 days.

[0098] pH tolerance (pH 2 residual activity): approximately 60%.

[0099] Data Acquisition:

[0100] Preparation record: Weighing and preparing all precursors (RuCl3, HSA, Co) 2+ Record the volume / molarity / concentration of the sample by following the above steps.

[0101] Structure / content characterization:

[0102] ICP-OES: Digest the sample and determine the Ru and Co content (atomic %).

[0103] XPS: Determination of the electronic state of Ru (Ru 0 、Ruδ + (Relative peak ratio) to prove the restoration effect.

[0104] TEM: Take a sample drop on a copper grid, photograph it, and measure the average particle size (including the distribution of at least 200 particle counts).

[0105] BET: Nitrogen adsorption is a measure of specific surface area.

[0106] DLS and ζ potential: used to measure particle size distribution and surface electrical properties.

[0107] Enzyme kinetic assay:

[0108] Prepare the reaction solution in a cuvette and add the standardized reagent nanozyme (m = 0.10 mg). Initial velocity determination is performed at multiple initial H2O2 concentrations [S] = 25, 50, 100, 200, and 400 μM (record the A value every 5 seconds within the first 60 s and linearly fit dA / dt).

[0109] dA / dt → dc / dt: using ε = 39000 M -1 ·cm -1 The conversion is performed using l = 1 cm. The unit conversion is to μM·min. -1 .

[0110] Use at least 5 [S] points to perform nonlinear least squares fitting on v([S]) to obtain Km and Vmax (program or software: Origin, GraphPad or Python's curve_fit are all acceptable).

[0111] Normalized Vmax: Vmax_norm = Vmax(μM·min) -1 ) ÷ m (mg).

[0112] LOD and calibration curve:

[0113] The absorbance response was measured using a series of low concentrations of H2O2 (0.5, 1, 2, 5, 10 μM), and a calibration curve A = S·[H2O2] + b was plotted to obtain the slope S(A·μM). -1 ).

[0114] The blank test was repeated 10 times to obtain σA (A units), which was then calculated according to LOD = 3·(σA / S).

[0115] Stability test: Store at 25℃ under normal temperature and humidity control for 30 days, and periodically (0, 7, 15, 30 days) measure the initial rate under standard conditions and compare it with the initial activity to calculate the percentage of residual activity.

[0116] Mobile phone RGB calibration and readings:

[0117] Prepare a series of standard H2O2 solutions and react them with standardized reagents. Fix the photography parameters (automatic white balance off, fixed exposure / ISO / distance, uniform background and internal color calibrators), extract the RGB values ​​in the images and calculate the ratio of G / (R+G+B) or R / G, and construct the RGB→concentration calibration curve (polynomial or linear regression).

[0118] The test samples were photographed under the same conditions to obtain RGB values, and the concentration was calculated by back-calculation using the calibration curve. The concentration was then compared with that obtained by UV-Vis (the relative error was reported).

[0119] A comprehensive comparison of Example 1 and Example 2 is shown in Table 1 below;

[0120] Table 1

[0121] Indicators / Items Example 1 (This application—Ru-HSA pre-composite + confined in-situ doping) Example 2 (Comparison—Direct in-situ doping, without pre-composite) Relative improvement (Example 1 vs. comparison) Ru (ICP atoms %) 1.02 at% 1.05 at% — (The content is similar, and the difference comes from the distribution) BET specific surface area <![CDATA[820 m 2 ·g -1 ]]> <![CDATA[780 m 2 ·g -1 ]]> +5.1% TEM average particle size 85 nm 95 nm The particles are more uniform and slightly smaller. DLS hydrated particle size (Z-avg) 150 nm 210 nm The dispersion is significantly better. dA / dt under saturation conditions <![CDATA[0.45 A·min -1 → 11.538 μM min -1 ]]> <![CDATA[0.30 A·min -1 → 7.692 μM min -1 ]]> Rate approximately +50% Vmax (nonlinear fitting) <![CDATA[15.11 μM·min -1 ]]> <![CDATA[10.78 μM·min -1 ]]> +40.2% Vmax normalized (m=0.10 mg) <![CDATA[151.10 μM·min -1 ·mg -1 ]]> <![CDATA[107.77 μM·min -1 ·mg -1 ]]> +40.2% <![CDATA[Km(H2O2)]]> 118.95 μM 133.19 μM Lower Km (higher affinity) <![CDATA[LOD(H2O2,3σ)]]> 0.40 μM 0.60 μM Lower (≈33%) pH2 residual activity 78% 60% Significantly more acid resistant 30-day room temperature residual activity 88% 72% Better long-term stability

[0122] As shown in the table above, the activity and affinity are improved: through Ru–HSA prior recombination and confined in-situ doping, as well as short-term reduction, the Ru sites are highly dispersed (single-atom / sub-nanometer cluster tendency), resulting in an increase of Vmax of about 40% and a decrease in Km (from 133 μM → 119 μM), reflecting the improvement in catalytic site accessibility and substrate affinity.

[0123] Stability and batch-to-batch consistency: Surface coating and activity pre-activation + elution normalization result in better particle dispersion (DLS from 210 nm to 150 nm) and maintain higher residual activity under acidic conditions and room temperature storage (88% vs 72% at 30 days), which is crucial for POCT kit development.

[0124] Improved reliability of on-site quantitative analysis: Combining internal color scales and RGB calibration reduces errors caused by lighting / exposure, enabling semi-quantitative / quantitative solutions for mobile phones in the field, with LOD reduced to approximately 0.4 μM (a significant improvement over the comparative examples).

[0125] As shown above, pre-activation surface modification of nanozymes with low toxicity (such as short-chain polymer coating or mild cross-linking) can form a protective layer, reducing the loss and irreversible inactivation of active sites under acid, alkali, salt strength, or biological matrices. Combining short-term pre-activation with systematic elution (removal of free metals and reaction byproducts) can standardize the material's "ready-to-operate" state to a reproducible, factory-ready condition. Theoretically, this surface-activation synergistic strategy inhibits performance degradation caused by metal ion ion release and particle aggregation, while reducing performance fluctuations between batches by removing variable impurities. This significantly improves the long-term stability, safety, and commercial controllability of the product (e.g., reliability in kit formulation, room temperature transport, and field use), which has direct technical value for the transformation from laboratory to industrialization.

[0126] By combining internal color standards (or internal reference standards) with fixed imaging parameters, white balance / exposure compensation algorithms, and RGB→density calibration curves, the quantification of contrast color signals can be achieved on-site using ordinary smartphones. The internal standard provides an immediate reference to the scene's optical conditions, and the image processing algorithm reduces errors caused by illumination, projection angle, and automatic camera adjustments through normalization and color space correction, thereby converting environmental optical noise into correctable systematic errors. This approach not only improves the reproducibility of outdoor / on-site testing results but also facilitates the establishment of image data-based traceability and quality control processes, giving portable POCT applications higher credibility and greater clinical / on-site application value.

[0127] Example 3:

[0128] Application of Ru-doped HSA and ZIF-67 nanozymes in on-site quantitative detection of H2O2 and glucose: A Ru-doped HSA and ZIF-67 nanozyme was prepared and can be used for colorimetric determination of biomarkers of cancer-related glucose and hydrogen peroxide.

[0129] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A Ru-doped HSA and ZIF-67 nanozyme, characterized in that, The preparation method of the nanozyme includes the following steps: S1. Mix RuCl3 or Ru(III) soluble compound with HSA in a buffer solution of pH 5.5–7.5 and stir for 30–120 min to achieve coordination or encapsulation of Ru with HSA to obtain Ru–HSA complex; S2. Add the Ru–HSA complex containing Co 2+ In a solution of ions and 2-methylimidazole, a ZIF-67 framework was grown in situ at room temperature to 40°C while Ru doping was introduced to achieve Ru-HSA encapsulation and confined doping at the ZIF-67 crystal nucleus, resulting in Ru-doped ZIF-67 nanohybrids. S3. The Ru-doped ZIF-67 nanohybrid is subjected to short-time chemical reduction or mild thermal treatment to transform the morphology of the incorporated Ru from ionic and coordinated states to dispersed sub-nanometer, nanocluster, or atomic-level dispersed states and improve the electronic structure of the metal sites to obtain Ru-containing active site nanozymes. S4. The Ru-containing active site nanozyme is subjected to surface stabilization treatment to improve the dispersibility and stability of the particles under different pH and ionic strength through polymer coating or cross-linking, thereby obtaining a surface-modified nanozyme. S5. Perform activity pre-activation and elution standardization treatment on the surface-modified nanozyme, and incubate it in acetate or phosphate buffer with a certain concentration of H2O2 and TMB for a short time, and remove free metal ions and residual reagents by dialysis or centrifugation multiple times to make the nanozyme a standardized preparation with comparable activity, and obtain a standardized reagent. S6. The standardized reagent is used in the colorimetric detection step so that the standardized reagent reacts with hydrogen peroxide or glucose in the sample to be tested under specified buffer conditions. The colorimetric signal is read by adding an internal color scale and using an image analysis method based on smartphone RGB calibration to obtain the detection result.

2. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S1, the molar ratio of RuCl3 or Ru(Ⅲ) soluble compound to HSA is 0.001–0.02, and the mixing conditions are: stirring at 20–30℃ for 60 min.

3. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S2, the Co 2+ The molar ratio of ions to 2-methylimidazole is 1:(8–16), and when the Ru–HSA complex is added, the volume ratio of ethanol or water in the solution is 0–30% (v / v) to facilitate the regulation of the morphology of ZIF-67. The Ru in ZIF-67 has an atomic percentage of 0.5–5 at.

4. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S3, the chemical reducing agent is selected from ascorbic acid, NaBH4 treated at a concentration of 0.1–10 mM for 1–30 min, or heat-treated by gentle aging at 40–80°C for 30–180 min.

5. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S4, the materials used for coating or cross-linking include: PEG-NH2, low molecular weight chitosan, or cross-linked with 0.01–0.5% glutaraldehyde; after coating, the nanozyme maintains more than 75% of its initial activity in the pH range of 2–10.

6. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S5, the activity pre-activation is performed by incubating 0.1–5 mM H2O2 and 0.1–2 mM TMB in a pH 3.5–5.0 buffer for 10–60 s, followed by removal of residual reagents by dialysis or centrifugation.

7. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, In step S6, the colorimetric detection step uses an internal color standard and, based on a preset RGB→density correction curve and white balance / exposure compensation algorithm, captures and calculates G, R+G+B or R / G ratios using a smartphone camera to perform quantitative readings.

8. The Ru-doped HSA / ZIF-67 nanozyme according to claim 1, characterized in that, The standardized reagents are lyophilized and 5–10% (w / v) of sucrose or sorbitol is added as an excipient to prepare room-temperature stable, resolvable reagent kit consumables.

9. Application of Ru-doped HSA and ZIF-67 nanozymes in on-site quantitative detection of H2O2 and glucose.

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