Preparation method and application of an amorphous / crystalline heterogeneous nanozyme
A DNA-mediated self-assembly method at 95°C synthesizes Fe-DNA nanozymes with enhanced stability and catalytic activity, addressing the complexity and harshness of traditional synthesis methods and enabling effective biosensor applications.
Patent Information
- Application Number
- CN202211335075.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-28
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2042-10-28
AI Technical Summary
Most of the existing nanoenzymes are traditional crystal phase materials, with harsh synthesis conditions and lack of research on amorphous materials, resulting in the underexploration of their activities and functions.
Through DNA coordination, Fe2+ self-assembly is driven to form Fe-DNA nanoenzymes, a simple and controllable synthesis method is adopted to avoid harsh conditions, and amorphous/crystalline heterophase nanoenzymes are prepared.
The obtained Fe-DNA nanoenzyme has excellent pH and thermal stability, good room temperature storage, significant peroxidase activity, negligible oxidase activity and degradability, and is suitable for the establishment of biosensors.
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Figure CN116003498B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of nanozymes, and particularly relates to a preparation method and application of amorphous / crystalline heterogeneous nanozymes. Background Art
[0002] Nanozymes refer to nanomaterials with enzyme-like properties. Existing studies have demonstrated that the composition, morphology, structure, crystal plane, size, and dimension of nanomaterials are closely related to the activity and function of nanozymes. However, as an important structural parameter of nanomaterials, the phase has received little attention. Generally, most of the reported nanozymes are nanomaterials with traditional crystal phases, which are composed of ordered and periodic atomic arrangements. Different from crystal phases, amorphous materials exhibit random atomic arrangements or short-range order on several atoms, resulting in distorted lattices and / or dangling bonds. Perhaps, amorphous materials can change the activity of nanozymes or bring more interesting properties to them. In recent years, amorphous nanomaterials, as a new type of functional materials, have obtained certain applications in fields such as catalysis due to their unique physical and chemical properties. So far, the synthesis of amorphous nanomaterials has only been achieved in limited materials, and their synthesis conditions are very harsh. Therefore, it is highly necessary to develop a simple synthesis strategy for the synthesis of low-crystallinity nanozymes, including amorphous or amorphous / crystalline heterogeneous nanozymes. Summary of the Invention
[0003] The purpose of the present invention is to provide a preparation method and application of amorphous / crystalline heterogeneous nanozymes.
[0004] To achieve the purpose of the present invention, at 95 °C, Fe 2+ self-assembles through DNA coordination-driven to form Fe-DNA nanozymes. This strategy is a simple and controllable synthesis method, avoiding the harsh conditions required in the traditional synthesis methods of amorphous materials or nanozymes. The obtained Fe-DNA nanozymes have excellent pH and thermal stability, good room-temperature storage, significant peroxidase activity, negligible oxidase activity, and degradability, etc., and have been confirmed in biosensing. The method for preparing amorphous / crystalline heterogeneous nanozymes of the present invention is simple, controllable, and low-cost, very suitable for the preparation of amorphous / crystalline heterogeneous nanozymes, and the obtained nanozymes can be used for the establishment of biosensors.
[0005] In the first aspect, the present invention provides a preparation method of amorphous / crystalline heterogeneous nanozymes, and the DNA solution and Fe 2+ solution are synthesized through a DNA coordination-driven self-assembly strategy under specific conditions. The prepared amorphous / crystalline heterogeneous nanozymes have significant POD activity, negligible oxidase activity, and degradability;
[0006] The specific steps are to mix the DNA solution and Fe 2+The solution reacts for 100 - 300 min under the condition of 65°C - 125°C.
[0007] The described DNA solution is single-stranded DNA dissolved in ultrapure water, with an oligonucleotide sequence of 5 - 22 bases in length.
[0008] The described Fe 2+ solution is a FeSO4 solution.
[0009] The concentration range of the described DNA is 0 - 50 μM; the concentration range of the described Fe 2+ solution is 1 - 100 mM.
[0010] The described DNA coordination-driven self-assembly strategy can be controlled depending on the synthesis conditions;
[0011] The described synthesis conditions include DNA length, DNA base composition, the molar ratio of Fe 2+ to DNA concentration, and the concentration of DNA and Fe 2+ concentration.
[0012] The described DNA length is such that the nanozyme particle size increases with the increase of DNA length;
[0013] The described DNA base composition is that only in the DNA composed of A or / and G bases, the synthesized nanozyme is spherical;
[0014] The described Fe 2+ to DNA concentration molar ratio is such that the nanozyme particle size increases with the increase of the molar ratio;
[0015] The described dependence on the concentration of DNA and Fe 2+ is such that the nanozyme particle size increases with the increase of the concentration of DNA and Fe 2+ concentration.
[0016] Preferably, the DNA length is 11 nucleotide bases, the DNA base composition is adenine deoxyribonucleotide, the molar ratio of Fe 2+ to DNA concentration is 160:1, the DNA concentration is 25 μM, the Fe 2+ concentration is 4 mM, the self-assembly temperature is 95°C, and the self-assembly time is 200 min.
[0017] Subsequently, centrifuge at 13000 rpm / min for 10 min at room temperature, discard the supernatant; add 500 μL of deionized water for washing; repeat the above centrifugation - washing 3 times, and finally disperse in 50 μL of deionized water and store at room temperature.
[0018] On the other hand, the present invention provides an amorphous / crystalline heterogeneous nanozyme with excellent pH and thermal stability, good room-temperature storage stability, remarkable peroxidase activity, negligible oxidase activity, and degradability.
[0019] The excellent pH and thermal stability means that, compared with other reported nanozymes, the amorphous / crystalline heterogeneous nanozyme can tolerate a wide range of pH and temperature while maintaining high activity.
[0020] Specifically, the amorphous / crystalline heterogeneous nanozyme still maintains high activity after being treated at different pH values (3.0 - 11.0) and temperatures (-80 °C - 110 °C) for 30 min.
[0021] The good room-temperature storage stability means that the amorphous / crystalline heterogeneous nanozyme can still maintain high activity after being stored at room temperature for a long time.
[0022] Specifically, the amorphous / crystalline heterogeneous nanozyme can be stably stored in aqueous solution and HEPES buffer at room temperature for 5 months while maintaining high activity.
[0023] The remarkable peroxidase activity means that the amorphous / crystalline heterogeneous nanozyme has remarkable peroxidase activity similar to natural enzymes.
[0024] Specifically, the amorphous / crystalline heterogeneous nanozyme can catalyze the significant oxidation of TMB-H2O2 in 25 mM sodium acetate buffer at pH 4.0.
[0025] The negligible oxidase activity means that, compared with the remarkable peroxidase activity, the amorphous / crystalline heterogeneous nanozyme has negligible oxidase activity.
[0026] Specifically, the amorphous / crystalline heterogeneous nanozyme cannot catalyze the obvious oxidation of TMB when air, N2, and O2 are respectively introduced into 25 mM sodium acetate buffer at pH 4.0.
[0027] The degradability means that phosphate buffer (PB) can cause the degradation of the amorphous / crystalline heterogeneous nanozyme.
[0028] On the other hand, the present invention provides a method for detecting H2O2. When H2O2 is present, it catalyzes the oxidation of the enzyme substrate TMB to cause a color change, thereby realizing the detection of H2O2;
[0029] The specific steps are as follows: the H2O2 to be detected is added to 25 mM sodium acetate solution, mixed evenly, reacted at 50 - 70 °C, and then the absorbance value at 655 nm is recorded by an enzyme-labeling instrument;
[0030] The sodium acetate solution contains 40 mM TMB and nanozyme, with a pH of 4.0.
[0031] Preferably, 10 μL of H2O2 is added to 240 μL of 25 mM sodium acetate solution, mixed well, reacted at 60 °C for 20 min, and then the absorbance at 655 nm is recorded by an enzyme-linked immunosorbent assay (ELISA) reader.
[0032] The sodium acetate solution contains 20 μL of 40 mM TMB and 5 μL of nanozyme, with a pH of 4.0.
[0033] On the other hand, the present invention provides a method for detecting glucose. When glucose is present, glucose oxidase first oxidizes glucose to produce gluconic acid and H2O2, and then uses nanozyme to catalyze the oxidation of the enzyme substrate TMB to cause a color change, thereby realizing the detection of glucose.
[0034] The specific steps are as follows: The glucose to be measured is added to 10 mM sodium acetate solution I, reacted at 37 °C for 60 min, then the above reaction solution is taken and added to 25 mM sodium acetate solution II, mixed well, and reacted at 60 °C for another 20 min. Finally, the absorbance at 655 nm is recorded by an ELISA reader.
[0035] The sodium acetate solution I contains 13 mg / mL -1 glucose oxidase, with a pH of 5.0;
[0036] The sodium acetate solution II contains 40 mM TMB and nanozyme, with a pH of 4.0.
[0037] Preferably, 100 μM of glucose is added to 190 μL of 10 mM sodium acetate solution I (containing 5 μL of 13 mg / mL -1 glucose oxidase, with a pH of 5.0), reacted at 37 °C for 60 min, then 10 μL of the above reaction solution is taken and added to 240 μL of 25 mM sodium acetate solution II (containing 20 μL of 40 mM TMB and 5 μL of nanozyme, with a pH of 4.0), mixed well, and reacted at 60 °C for another 20 min. Finally, the absorbance at 655 nm is recorded by an ELISA reader.
[0038] By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:
[0039] The present invention discloses a preparation method and application of an amorphous / crystalline heterogeneous nanozyme. At 95 °C, Fe-DNA nanozyme is formed by DNA coordination-driven self-assembly of Fe2+. This strategy is a simple and controllable synthesis method, avoiding the harsh conditions required in the traditional synthesis methods of amorphous materials or nanozymes. The obtained Fe-DNA nanozyme has excellent pH and thermal stability, good storage stability at room temperature, significant peroxidase activity, negligible oxidase activity, and degradability, etc., and has been verified in biosensing. The method for preparing the amorphous / crystalline heterogeneous nanozyme of the present invention is simple, controllable and low-cost, very suitable for the preparation of amorphous / crystalline heterogeneous nanozymes, and the obtained nanozymes can be used for the establishment of biosensors.
[0040] 1. The present invention discloses a preparation method of an amorphous / crystalline heterogeneous nanozyme, in which a DNA solution and an Fe 2+ solution are synthesized by a DNA coordination-driven self-assembly strategy under specific conditions. This strategy is simple and low-cost, avoiding the harsh conditions required in the traditional synthesis methods of amorphous materials or nanozymes.
[0041] 2. This synthesis method is a controllable synthesis strategy, which can be regulated by the DNA base length, base composition, molar ratio of Fe 2+ to DNA concentration, and the concentration of Fe 2+ to DNA.
[0042] 3. The prepared amorphous / crystalline heterogeneous nanozyme has significant POD activity and negligible oxidase activity, and its affinity (K m = 0.81 mM) for H2O2 is superior to that of horseradish peroxidase (K m = 3.70 mM).
[0043] 4. The prepared amorphous / crystalline heterogeneous nanozyme has excellent pH and thermal stability, good storage stability at room temperature, and degradability.
[0044] 5. The present invention demonstrates the advantages of the amorphous / crystalline heterogeneous nanozyme in the construction of H2O2 and glucose biosensors, providing a reference for the application of the amorphous / crystalline heterogeneous nanozyme in biosensors. Description of the Drawings
[0045] Figure 1 Schematic diagram of the synthesis of an amorphous / crystalline heterogeneous nanozyme based on a DNA coordination-driven self-assembly strategy.
[0046] Figure 2Synthesis and Characterization of Amorphous / Crystalline Heterophase Nanozymes, including a) SEM images, b) TEM images, c) High-resolution TEM (HRTEM) images, d) Selected Area Electron Diffraction (SAED) patterns, e) High-angle Annular Dark-field Scanning TEM (HAADF-STEM) images and elemental mapping, f) Linear sweep voltammograms, g) Energy Dispersive X-ray Spectroscopy (EDS) spectra, and h) X-ray Photoelectron Spectroscopy (XPS) spectra.
[0047] Figure 3 SEM images in the controllable synthesis of amorphous / crystalline heterophase nanozymes, including different sequence lengths (a), different base compositions (b), different Fe:DNA molar ratios (c), and different DNA concentrations (d).
[0048] Figure 4 Particle size statistical graphs during the controllable synthesis process of amorphous / crystalline heterophase nanozymes, including different sequence lengths (a), different Fe:DNA molar ratios (b), and different DNA concentrations (c).
[0049] Figure 5 Verification of peroxidase activity of amorphous / crystalline heterophase nanozymes. (a) Schematic diagram showing that the nanozyme has significant peroxidase activity and negligible oxidase activity; (b) The nanozyme catalyzes the oxidation and color development of the TMB-H2O2 system; (c) The nanozyme catalyzes the oxidation and color development of the OPD-H2O2 system.
[0050] Figure 6 Verification of oxidase activity of amorphous / crystalline heterophase nanozymes. The nanozyme catalyzes the oxidation of enzyme substrates TMB (a) and OPD (b) in air, N2, O2, and H2O2 respectively.
[0051] Figure 7 Optimization of catalytic conditions of amorphous / crystalline heterophase nanozymes, including buffer pH (a), reaction temperature (b), and buffer ion concentration (c).
[0052] Figure 8 pH (a) and thermal stability (b) experiments of amorphous / crystalline heterophase nanozymes.
[0053] Figure 9 Storage stability (a) and batch stability (b) of amorphous / crystalline heterophase nanozymes.
[0054] Figure 10Steady-state kinetics and catalytic mechanism. (a) Michaelis–Menten equation curve fitting at a constant TMB concentration (1 mM) with varying H2O2 concentrations (0.1 - 0.6 mM) and (b) at a constant H2O2 concentration (25 mM) with varying TMB concentrations (0 - 1.0); (c) Lineweaver–Burk plots at different TMB concentrations (2.0, 2.5, 3.0 mM) and (d) at different H2O2 concentrations (25, 30, 40 mM); (e) Verification of O2 by the DHE method ·- ; (f) Schematic summary of the peroxidase-like catalytic mechanism of the amorphous / crystalline heterophase nanozyme.
[0055] Figure 11 Verification of the degradability of the amorphous / crystalline heterophase nanozyme. a) TEM images of the nanozyme during the degradation process; b) Peroxidase-like activity of the nanozyme at different time points during the degradation process, and photos of the catalytic substrate oxidation and color development; c) Generation of O2 by the nanozyme catalyzing H2O2 at different time points during the degradation process ·- conditions.
[0056] Figure 12 Feasibility analysis of H2O2 detection.
[0057] Figure 13 Establishment of H2O2 and glucose colorimetric sensors using the amorphous / crystalline heterophase nanozyme. a) Schematic diagram of the detection principle; b - d) Sensitivity graph, time-dependent detection kinetics graph, and linear graph of H2O2 detection; e - g) Sensitivity graph, time-dependent detection kinetics graph, and linear graph of glucose detection.
[0058] Figure 14 Feasibility analysis of glucose detection.
[0059] Figure 15 Specificity analysis of glucose detection. Detailed implementation methods
[0060] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps, or conditions of the present invention fall within the scope of the present invention.
[0061] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0062] Example 1 Synthesis and controllable synthesis of the amorphous / crystalline heterophase nanozyme
[0063] (1) Synthesis
[0064] As Figure 1 shown, the amorphous / crystalline heterophase nanozyme was synthesized through a DNA coordination-driven self-assembly strategy.
[0065] That is, 15 μL of 20 mM FeSO4 solution and 285 μL of 25 μM DNA solution were taken in a 1.5 mL centrifuge tube. After thorough mixing, the mixture was placed at 95 °C for reaction for 200 min. After the reaction was completed, it was naturally cooled to room temperature, centrifuged at 13,000 rpm for 10 min to remove the supernatant, and washed with deionized water. The centrifugation and washing were repeated 3 times, and finally it was dispersed in 50 μL of deionized water. The characterization results are as Figure 2 shown. The obtained nanozyme is spherical nanoparticles with an amorphous / crystalline heterogeneous structure. The lattice spacing of the crystalline region is 0.314 nm. The periphery of the spherical nanoparticles is an amorphous structure, which is composed of Fe, O, N, C, and P elements.
[0066] (2) Controllable synthesis
[0067] For the controllable synthesis of amorphous / crystalline heterogeneous nanozymes, in addition to adding different DNA lengths, different DNA base compositions (Table 1), different molar ratios of Fe 2+ to DNA concentration (10:1, 40:1, 80:1, and 160:1), and different Fe 2+ to DNA concentration (25, 50, and 100 μM, where Fe:DNA = 160:1), the remaining steps were the same as (1). The results are as Figure 3 and Figure 4 shown. The specific sizes are shown in Table 2 below. The optimal parameters are A11, Fe:DNA = 160:1, 25 μM DNA. The synthesis process is the same as in (1) above, and the nanozymes used later are all nanozymes synthesized with the optimal parameters.
[0068] Table 1 DNA sequences involved in controllable synthesis
[0069] Name Sequence (5' to 3') SEQ ID NO. A11 AAAAAAAAAAA SEQ ID NO.1 A16 AAAAAAAAAAAAAAAA SEQ ID NO.2 A22 AAAAAAAAAAAAAAAAAAAAAA SEQ ID NO.3 A3T8 AAATTTTTTTT SEQ ID NO.4 A8T3 AAAAAAAATTT SEQ ID NO.5 A3C8 AAACCCCCCCC SEQ ID NO.6 A8C3 AAAAAAAACCC SEQ ID NO.7 A3G8 AAAGGGGGGGG SEQ ID NO.8 A8G3 AAAAAAAAGGG SEQ ID NO.9
[0070] Table 2 Size statistics in controllable synthesis
[0071]
[0072]
[0073] Example 2 Enzyme activity study experiment of amorphous / crystalline heterogeneous nanozyme
[0074] (1) Peroxidase activity study
[0075] As Figure 5As shown in the figure, the peroxidase activity of the amorphous / crystalline heterogeneous nanozyme was evaluated by catalyzing the TMB-H2O2 system. That is, 5 μL of the nanozyme was added to 245 μL of 25 mM sodium acetate buffer (pH 4.0, containing 20 μL of 40 mM TMB and 20 μL of 120 mM H2O2). After incubation at room temperature for 5 min, the absorbance at 655 nm was recorded by an enzyme-linked immunosorbent assay (ELISA) reader. It can be seen that in the TMB-H2O2 system, the amorphous / crystalline heterogeneous nanozyme can catalyze the oxidation of the substrate, indicating that it has peroxidase activity.
[0076] For the catalyzed OPD-H2O2 system, except for adding 20 μL of 250 mM OPD, the rest was the same as above. The results were the same as above. In the OPD-H2O2 system, the amorphous / crystalline heterogeneous nanozyme can catalyze the oxidation of the substrate, indicating that it has peroxidase activity.
[0077] (2) Oxidase activity study
[0078] As Figure 6 shown, the oxidase activity of the amorphous / crystalline heterogeneous nanozyme was studied by catalyzing the TMB or OPD system. That is, 5 μL of the nanozyme was added to 245 μL of 25 mM sodium acetate buffer (pH 4.0, containing 20 μL of 40 mM TMB or 20 μL of 250 mM OPD). Under the conditions of respectively introducing air, N2, O2 and adding 20 μL of 120 mM H2O2, after incubation at room temperature for 5 min, the absorbance at 655 nm was recorded by an ELISA reader. The results showed that the nanozyme could only catalyze the oxidation of the substrate in the presence of H2O2, and on the contrary, it could not catalyze the oxidation of the substrate under other conditions, indicating that the amorphous / crystalline heterogeneous nanozyme has peroxidase activity but does not have oxidase activity.
[0079] (3) Optimization of catalytic conditions
[0080] The optimal catalytic pH, reaction temperature, and buffer ion concentration of the amorphous / crystalline heterogeneous nanozyme were studied by catalyzing the TMB-H2O2 system. As Figure 7 shown, its optimal catalytic conditions were pH 3.6, 60 °C, and 25 mM.
[0081] (4) Stability study of the amorphous / crystalline heterogeneous nanozyme
[0082] pH stability: After treating the amorphous / crystalline heterogeneous nanozyme with sodium acetate solution at different pH values (3.0 - 11.0) for 30 min, the peroxidase activity of the nanozyme was tested by an ELISA reader. The results were as Figure 8 shown. The nanozyme was very stable between pH 4.0 and 9.0, and the enzyme activity increased at pH 3.0 or pH 11.0.
[0083] Temperature stability: The amorphous / crystalline heterogeneous nanozyme was first treated at different temperatures for 1 h, and then the peroxidase activity of the nanozyme was measured by a microplate reader. The results are as Figure 8 shown. The nanozyme is basically stable at -80 to 80 °C, and its activity increases at 110 °C.
[0084] Storage stability: The amorphous / crystalline heterogeneous nanozyme was first stored in aqueous solution and HEPES solution at room temperature for different times (0 - 5 months), and then the peroxidase activity of the nanozyme was measured by a microplate reader. The results are as Figure 9 shown. The nanozyme can maintain its unchanged enzyme activity after being stored in deionized water at room temperature for 5 months, and can maintain its unchanged enzyme activity after being stored in HEPES buffer at room temperature for 3 months.
[0085] Batch stability: Four different batches of nanozymes were synthesized according to the synthesis steps of the amorphous / crystalline heterogeneous nanozyme in Example 1, and the peroxidase activity of the nanozymes was measured by a microplate reader. The results are as Figure 9 shown. The enzyme activities of the nanozymes synthesized in four different batches are almost the same.
[0086] (5) Steady-state kinetics and catalytic mechanism studies
[0087] Steady-state kinetics: According to the steps of the peroxidase activity study in (1) above, under the conditions of keeping the H2O2 concentration constant (25, 30, 40 mM) and changing the TMB concentration (0 - 1.0 mM) or keeping the TMB concentration constant (2.0, 2.5, 3.0 mM) and changing the H2O2 concentration (0.1 - 0.6 mM) respectively, the initial reaction rates of each group were measured, and the Michaelis equation curve was fitted (as Figure 10 a - d), and the Michaelis kinetic parameters were calculated: The Km m and Vmax max of TMB are 1.68 mM and 1.08×10 -8 Ms -1 respectively, and the Km m and Vmax max of H2O2 are 0.81 mM and 1.78×10 -8 M s -1 respectively. It can be seen that its affinity for H2O2 is better than that of horseradish peroxidase (Km m = 3.70 mM).
[0088] Catalytic mechanism: Using dihydroethidium (DHE) as the O2 ·- capture probe to verify whether O2 ·-。20 μL of nanozyme was added to 875 μL of 25 mM sodium acetate buffer (pH 4.0, containing 5 μL of 2 mM DHE and 20 μL of 120 mM H2O2), and the absorption spectrum of the solution was measured by a UV-visible absorption spectrometer. The results are as Figure 10 e. As the reaction time increased, the absorption peaks in the 200 nm - 300 nm wavelength range increased, indicating that O2 was generated during the reaction ·- 。Therefore, the catalytic mechanism of the amorphous / crystalline heterophase nanozyme is as follows: H2O2 adsorbed on the surface of the nanozyme is first catalyzed to generate O2 ·- , which then oxidizes TMB to realize the peroxidase activity of the amorphous / crystalline heterophase nanozyme ( Figure 10 f).
[0089] Example 3 Degradability experiment of amorphous / crystalline heterophase nanozyme
[0090] The degradability of the amorphous / crystalline heterophase nanozyme was studied by TEM characterization, peroxidase activity test, and the generation of O2 ·- . Specifically, 20 μL of nanozyme was added to 50 mM PB solution and reacted at room temperature for different times (0, 1, 2, 2.5, 3, 5, 6 h). After centrifuging to remove the PB solution, TEM characterization, peroxidase activity test (performed according to (1) in Example 2), and O2 ·- test (performed according to (5) in Example 2) were carried out. The results are as Figure 11 shown. As the reaction time increased, the nanozyme was gradually degraded, the peroxidase activity gradually decreased, and the generation of O2 ·- gradually decreased.
[0091] Example 4 Establishment of H2O2 and glucose colorimetric sensors using amorphous / crystalline heterophase nanozyme
[0092] Establishment of H2O2 colorimetric sensor:
[0093] ① Feasibility: A sensor was established by catalyzing the TMB-H2O2 system. As Figure 12 shown, 160 μM H2O2 was added to 240 μL of 25 mM sodium acetate solution (pH 4.0, containing 20 μL of 40 mM TMB and 5 μL of nanozyme). After repeated mixing, the reaction was carried out at 60 °C for 20 min, and then the absorbance value at 655 nm was recorded by a microplate reader.
[0094] ② Sensitivity: As Figure 13As shown in b-d, 10 μL of H2O2 with different concentrations (0 - 160 μM) was added to 240 μL of 25 mM sodium acetate solution (pH 4.0, containing 20 μL of 40 mM TMB and 5 μL of nanozyme). After repeated mixing, the reaction was carried out at 60 °C for 20 min. Then, the absorbance at 655 nm was recorded by a microplate reader. The detection limit of this sensor was 1.71 μM, and the linear range was 0 - 72 μM.
[0095] ③ Detection of actual samples: Commercially available pre-packaged milk, dried tofu, and chicken feet were used as actual samples, and the actual sample detection experiment was carried out by the standard addition method. The operation was carried out according to the steps in ② above, and the results are shown in Table 3 below:
[0096] Table 3 Analysis of H2O2 sensor for actual samples
[0097]
[0098]
[0099] Establishment of glucose colorimetric sensor:
[0100] ① Feasibility: In combination with glucose oxidase, a sensor was established by catalyzing the TMB-H2O2 system. As Figure 14 shown, 100 μM of glucose was added to 190 μL of 10 mM sodium acetate solution (pH 5.0, containing 5 μL of 13 mg mL -1 glucose oxidase). After reacting at 37 °C for 60 min, 10 μL of the above reaction solution was taken and added to 240 μL of 25 mM sodium acetate solution (pH 4.0, containing 20 μL of 40 mM TMB and 5 μL of nanozyme). After sufficient mixing, the reaction was carried out at 60 °C for another 20 min. Finally, the absorbance at 655 nm was recorded by a microplate reader.
[0101] ② Sensitivity: As Figure 13 shown in e-g, different concentrations of glucose were added to 190 μL of 10 mM sodium acetate solution (pH 5.0, containing 5 μL of 13 mg mL -1 glucose oxidase). After reacting at 37 °C for 60 min, 10 μL of the above reaction solution was taken and added to 240 μL of 25 mM sodium acetate solution (pH 4.0, containing 20 μL of 40 mM TMB and 5 μL of nanozyme). After sufficient mixing, the reaction was carried out at 60 °C for another 20 min. Finally, the absorbance at 655 nm was recorded by a microplate reader. The detection limit of this sensor was 12.77 μM, and the linear range was 0 - 300 μM.
[0102] ③ Specificity: As Figure 15As shown, 200 μM β-lactose, fructose, maltose, and trehalose were respectively added to the reaction solution in place of glucose, and the experiment was carried out according to the steps in ② above. The results showed that the sensor had good specific responsiveness to glucose.
[0103] ④ Detection of actual samples: Honey, litchi juice, watermelon juice, and fetal bovine serum were used as actual samples, and the actual sample detection experiment was carried out by the standard addition method. The operation was carried out according to the steps in ③ above, and the results are shown in Table 4 below:
[0104] Table 4 Glucose sensor for actual sample analysis
[0105]
[0106]
[0107] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.
Claims
1. A preparation method of an amorphous / crystalline heterogeneous nanozyme, characterized in that: The DNA solution and Fe 2+ solution are synthesized through a DNA coordination-driven self-assembly strategy under high-temperature conditions; The DNA solution is single-stranded DNA dissolved in ultrapure water, with an oligonucleotide sequence of 5 to 22 bases in length, and the DNA concentration ranges from 25 to 50 μM; The DNA sequence is one or more of SEQ ID NO.1 to SEQ ID NO.9; The described Fe 2+ solution is an FeSO4 solution, and the Fe 2+ solution has a concentration range of 1 to 4 mM.
2. The preparation method of the amorphous / crystalline heterogeneous nanozyme according to claim 1, wherein including the following steps: reacting a DNA solution and an Fe 2+ solution at 65 °C to 125 °C for 100 to 300 minutes.
3. The preparation method of the amorphous / crystalline heterogeneous nanozyme according to claim 1, wherein The DNA coordination-driven self-assembly strategy can be controlled depending on the synthesis conditions; The synthesis conditions include DNA length, DNA base composition, the molar ratio of Fe 2+ to DNA concentration, and the concentration of DNA and Fe 2+ .
4. An amorphous / crystalline heterogeneous nanozyme, characterized in that, Synthesize the nanozyme using the preparation method described in claim 1, with specific conditions being the mixing of a 25 μM A11 solution and a 4 mM Fe 2+ solution, and incubating for 200 min at 95 °C.
5. The amorphous / crystalline heterogeneous nanozyme according to claim 4, wherein The nanozyme remains stable in the pH range of 3.0 to 11.0; remains stable in the temperature range of -80 to 110 °C; maintains its enzyme activity unchanged when stored at room temperature in an aqueous solution for 0 to 5 months; and is degradable in PB phosphate buffer.
6. A method for detecting H2O2, characterized in that, Using the nanozyme described in claim 4, when H2O2 is present, it catalyzes the oxidation of the enzyme substrate TMB to cause a color change, thereby realizing the detection of H2O2; The specific steps are as follows: The H2O2 to be measured is added to a 25 mM sodium acetate solution, mixed well, and reacted at 50 to 70 °C, and then the absorbance value at 655 nm is recorded by an enzyme-linked immunosorbent assay (ELISA) reader; The sodium acetate solution contains 40 mM TMB and the nanozyme, with a pH of 4.
0.
7. A method for detecting glucose, wherein the detection method does not include methods for diagnosing and treating diseases, and is characterized in that, Using the nanozyme described in claim 4, when glucose is present, glucose oxidase first oxidizes glucose to produce gluconic acid and H2O2, and then the nanozyme is used to catalyze the oxidation of the enzyme substrate TMB to cause a color change, thereby realizing the detection of glucose; The specific steps are as follows: The glucose to be measured is added to a 10 mM sodium acetate solution I, reacted at 37 °C for 60 min, then the above reaction solution is added to a 25 mM sodium acetate solution II, mixed well, and reacted at 60 °C for another 20 min, and finally the absorbance value at 655 nm is recorded by an enzyme-linked immunosorbent assay (ELISA) reader; The sodium acetate solution I contains 13 mg / mL glucose oxidase, with a pH of 5.0; The sodium acetate solution II contains 40 mM TMB and the nanozyme, with a pH of 4.
0.
8. Use of the preparation method according to claim 1 in the preparation of nanozymes.
9. Use of the amorphous / crystalline heterogeneous nanozyme according to claim 4 in the construction of H2O2 biosensors.
10. Use of the amorphous / crystalline heterogeneous nanozyme according to claim 4 in the detection methods of H2O2 and glucose, and the use does not include the diagnosis and treatment of diseases.