Preparation method of MnO2 nanoflower-based composite hybrid nanoflower and application of MnO2 nanoflower-based composite hybrid nanoflower in cyanoglycoside detection

Abstract

The invention relates to a preparation method of a composite hybrid nanoflower based on MnO2 nanoflower and application of the composite hybrid nanoflower to cyanoglycoside detection. MnO2NFs are loaded on the surface of HNFs, the peroxidase activity is remarkably improved through the synergistic effect of MnO2 and Cu < 2 + >, and MnO2NFs form a protective barrier on the surface of HNFs, so that the stability of HNFs is effectively improved; on one hand, MnO2NFs with a large number of branches and high porosity are loaded on HNFs, more catalytic sites are provided, a reaction interface is enhanced, and the overall catalytic efficiency is improved; on the other hand, the nanostructure of MnO2NFs improves the electron transfer efficiency of the system, enhances electron transfer, and improves the peroxidase activity and stability of the nanomaterial.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial preparation and biological detection, and in particular to a preparation method of MnO2 nanoflower composite hybrid nanoflower and its application in detecting cyanogenic glycosides. Background Art

[0002] Hybrid nanoflowers (HNFs) are compounds composed of organic and inorganic components, with a flower-like hierarchical three-dimensional nanostructure. The hierarchical structure endows HNFs with a large specific surface area, reduces the mass transfer resistance between enzymes, substrates and products, and greatly improves their catalytic activity; the inorganic components form a protective structure for the organic components, thus providing high stability for HNFs. Compared with inorganic nanoflowers, HNFs have advantages such as better biocompatibility, stronger catalytic activity and label-free properties. Therefore, biosensors based on HNFs have become an important part of the development of biosensors.

[0003] Cyanogenic glycosides are natural toxins of plants widely distributed in nature and are important chemical weapons for plants to defend against herbivores. When plant tissues are crushed or damaged in other ways, cyanogenic glycosides come into contact with endogenous enzymes (β-glucosidase and α-hydroxynitrile lyase), catalyzing the degradation of cyanogenic glycosides into benzaldehyde and hydrogen cyanide. It is reported that the lethal dose of acute oral cyanide is 0.5 - 3.5 mg / kg body weight, and its poisoning symptoms are similar to those of cyanide, including vomiting, diarrhea, confusion, paralysis and coma. Therefore, it is particularly important to determine the content of cyanogenic glycosides in food in actual monitoring.

[0004] However, the synthesis of composite nanoflowers and the detection methods for cyanogenic glycosides at the present stage mainly have the following technical problems:

[0005] 1. The combination of HNFs and metals mostly relies on physical adsorption or sol-gel methods. The non-covalent binding force is weak and is easily affected by factors such as pH and ionic strength, resulting in the shedding of enzymes and affecting the exertion of activity. And sol-gel methods etc. require harsh conditions such as high temperature, high pressure and toxic reagents;

[0006] 2. Due to the differences in electronic structure, hydrophilicity-hydrophobicity and charge distribution between the organic and inorganic components in HNFs, incomplete pairing or defects occur at their interfaces, reducing the stability and enzyme activity of HNFs;

[0007] 3. At present, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) etc. are mostly used to determine the content of cyanogenic glycosides in food. However, using the above-mentioned large-scale instruments to achieve quantitative detection usually takes several hours, the operation process is complex, and regular maintenance and calibration are required, and the equipment cost is high;

[0008] 4. Cyanogenic glycosides usually have low concentrations in actual samples. Currently, most detection materials have low sensitivity, are vulnerable to background noise and environmental changes, and have low stability, failing to meet the requirements for detecting low-concentration cyanogenic glycosides.

[0009] Chinese Patent No.: CN113720820A discloses a sensor composed of protein-inorganic hybrid nanoflowers with mimetic enzyme activity, fluorescent red dye, cyanogenic glycoside, and H2O2 solution for the detection of β-glucosidase. The synthesized protein-inorganic hybrid nanoflowers rely only on copper nodes as the main catalytic sites and have low enzyme activity.

[0010] Chinese Patent Publication No.: CN110237865A discloses a keratin nanoflower material loaded with silver phosphate and its preparation and application. In-situ precipitation reaction of silver nitrate and phosphate is carried out on the surface of the nanoflowers, and silver phosphate particles are loaded through physical adsorption and ion exchange. The formed composite nanoflowers have low stability, and the random deposition of silver salts results in poor material homogeneity, affecting the material performance. Summary of the Invention

[0011] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a preparation method of MnO2 nanoflower composite hybrid nanoflowers and their application in detecting cyanogenic glycosides, so as to solve the problems of poor stability and low enzyme activity existing in the composite of existing HNFs and metals. At the same time, the prepared composite material is applied to the detection of cyanogenic glycosides to achieve low-cost, portable, rapid, and highly sensitive detection.

[0012] The present invention solves its technical problems by adopting the following technical solutions:

[0013] A preparation method of MnO2 nanoflower composite hybrid nanoflowers includes the following steps:

[0014] S1. Preparation of Cu nanoflowers: Add an aqueous solution of copper salt to a buffer solution containing papain, incubate with shaking at room temperature for 1 - 24 h. After self-assembly is completed, centrifuge, wash, and dry to obtain Cu nanoflowers.

[0015] S2. Preparation of MnO2 nanoflowers: Add an aqueous solution of citric acid to an aqueous hydrochloric acid solution of potassium permanganate, stir, centrifuge, precipitate, wash, and dry to obtain MnO2 nanoflowers.

[0016] S3. Preparation of amine-functionalized MnO2 nanoflowers: Add γ-aminopropyltriethoxysilane to the dispersed solution of MnO2 nanoflowers, continuously stir, centrifuge, precipitate, wash, and dry to obtain amine-functionalized MnO2 nanoflowers.

[0017] S4. Preparation of Cu / MnO₂ nanoflowers: An activator was added to the Cu nanoflower dispersion and activated to obtain a dispersion containing activated Cu nanoflowers; the dispersion containing activated Cu nanoflowers was mixed with the dispersion of amine-functionalized MnO₂ nanoflowers, and co-incubated at room temperature for 1 - 8 h, followed by centrifugation, precipitation, washing, and drying to obtain Cu / MnO₂ nanoflowers.

[0018] Moreover, in step S1, the copper salt is at least one of copper sulfate, copper chloride, and copper nitrate.

[0019] Moreover, in step S1, the concentration of the copper salt is 80 - 150 mM, and the concentration of papain is 0.25 - 10 mg / ml.

[0020] Moreover, in step S4, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0021] Moreover, the molar ratio of copper ions: potassium permanganate: γ-aminopropyltriethoxysilane: 1-ethyl-(3-dimethylaminopropyl)carbodiimide: N-hydroxysuccinimide is 80 - 150: 1 - 5: 0.005 - 0.02: 0.1 - 50: 0.1 - 25.

[0022] Moreover, in step S1, the centrifugation speed is 4000 - 10000 rpm, the centrifugation time is 5 - 15 min, and the drying temperature is 25 - 60 °C.

[0023] Moreover, in step S4, the activation time is 15 - 60 min, the co-incubation speed is 0 - 500 rpm, the centrifugation speed is 4000 - 8000 rpm, the centrifugation time is 5 - 10 min, and the drying temperature is 25 - 60 °C.

[0024] Application of Cu / MnO₂ nanoflowers prepared by a preparation method of MnO₂ nanoflower composite hybrid nanoflowers in the preparation of cyanogenic glycoside biosensors.

[0025] A detection method for a cyanogenic glycoside biosensor based on Cu / MnO₂ nanoflowers, comprising the following steps:

[0026] (1) Mix the sample to be tested with an acid solution, and release cyanide ions through hydrolysis reaction;

[0027] (2) Transfer the hydrolysate to an alkaline solution, mix and let stand;

[0028] (3) Add a sodium acetate - acetic acid buffer solution with a pH of 4, a 3,3',5,5'-tetramethylbenzidine color reagent, hydrogen peroxide, and Cu / MnO₂ nanoflowers to the neutralized solution to trigger a color reaction;

[0029] (4)Quantitatively detect the cyanogenic glycoside concentration through color signal changes, where CN- complexes with the active centers Cu 2+ and Mn 4+ of the Cu / MnO2 nanoflowers, inhibiting their peroxidase activity and resulting in a decrease in the color development intensity;

[0030] (5)Use a smartphone device to collect color signals, and establish a linear relationship curve between R / R0 and the cyanogenic glycoside concentration by analyzing the RGB values or absorbance values of the color reaction to achieve quantitative detection.

[0031] The advantages and positive effects of the present invention are as follows:

[0032] 1. In the present invention, amide bonds are formed by activating the carboxyl groups on the surface of Cu NFs and the amino groups on the surface of MnO2 NFs, and covalent binding is achieved through three steps of "activation - mixing - washing" without the need for harsh conditions such as complex templates, high temperature and high pressure. Moreover, the precise regulation of functional groups makes Cu / MnO2 NFs have high stability and uniform distribution.

[0033] 2. The prepared Cu / MnO2 NFs in the present invention have excellent peroxidase activity. After loading MnO2 NFs, the enzyme activity of Cu / MnO2 NFs is increased by 220.11% compared with Cu NFs and by 648.57% compared with papain.

[0034] 3. The Cu / MnO2 NFs in the present invention have high stability in a wide temperature and pH range, and still retain 72% of the enzyme activity after storage at 4°C for 60 days.

[0035] 4. The present invention uses a smartphone to collect the color information of the solution. By converting the color change into RGB information, analyzing the relationship between the inhibition of the enzyme activity of Cu / MnO2 NFs by cyanogenic glycosides and R / R0, and accurately measuring the content of cyanogenic glycosides through the RGB detection mode, on-site quantitative detection is achieved. In addition, the application of the smartphone eliminates the visual judgment error.

[0036] 5. The present invention is the first to develop and successfully apply a biosensor based on MnO2 NFs composite HNFs for quantitative detection of cyanogenic glycosides, which has the advantages of high sensitivity, real-time, fast, portable, good stability and strong specificity.

[0037] 6. The present invention adopts a covalent coupling strategy to achieve the covalent composite of metal oxide nanoflowers on the surface of HNFs. The carboxyl groups on the surface of Cu NFs are activated by EDC and NHS, improving the binding efficiency between Cu NFs and MnO2 NFs, avoiding the instability of physical mixing, and only through simple liquid-phase stirring of the two nanoflowers, a covalent interface with high stability is formed.

[0038] 7. The present invention loads MnO2 NFs on the surface of HNFs. The synergistic effect between MnO2 and Cu 2+ significantly improves the peroxidase activity, and the MnO2 NFs form a protective barrier on the surface of HNFs, effectively improving the stability of HNFs. On the one hand, the MnO2 NFs with a large number of branches and high porosity are loaded on HNFs, providing more catalytic sites, enhancing the reaction interface, and improving the overall catalytic efficiency. On the other hand, the nanostructure of MnO2 NFs improves the electron transfer efficiency of the system, enhances electron transfer, and improves the peroxidase activity and stability of the nanomaterials.

[0039] 8. The present invention first applies the biosensor based on MnO2 NFs composite HNFs to the detection of cyanogenic glycosides in food, realizing the low-cost, portable and rapid detection of cyanogenic glycosides. At the same time, the present invention uses the peroxidase activity of Cu / MnO2 nanoflowers as the signal output, and realizes highly sensitive detection through the complexation of CN- with the active centers Cu2+ and Mn4+.

[0040] 9. The present invention selects low-cost Papain as the organic component to synthesize HNFs, and uses the amide groups in Papain to regulate the kinetic nucleation and growth of copper phosphate crystals, promoting their self-assembly into a flower-like structure. The synthesis cost is low, and the synthesized Cu NFs have excellent peroxidase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 are the scanning electron microscope (SEM) images of Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs in the present invention. Among them, Figures A and B are the SEM images of Cu NFs at 3k and 8k respectively; Figures C and D are the SEM images of amine-functionalized MnO2 NFs at 20k and 50k respectively; Figures E and F are the SEM images of Cu / MnO2 NFs at 3k and 15k respectively.

[0042] Figure 2 are the FT-IR images of Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs in the present invention.

[0043] Figure 3 are the color reaction images of Papain, Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs in the TMB-H2O2 system in the present invention.

[0044] Figure 4 are the stability determination images of Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs at 30 - 80 °C in the present invention.

[0045] Figure 5This is the stability determination diagram of Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs in the present invention at pH 3 - 11.

[0046] Figure 6 This is the stability determination diagram of Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs in the present invention after storage for 5 - 60 days.

[0047] Figure 7 This is the schematic diagram of qualitative colorimetric detection of Cu / MnO2 NFs solution under different concentrations of amygdalin in the present invention.

[0048] Figure 8 This is the standard curve determination diagram of amygdalin standard in the present invention. Detailed implementation manners

[0049] The present invention will be further described in detail through specific embodiments below. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0050] A preparation method of a composite hybrid nanoflower based on MnO2 nanoflower, comprising the following steps:

[0051] S1. Preparation of Cu nanoflowers: Add an aqueous solution of copper salt to a phosphate buffer solution containing papain, incubate with shaking at room temperature for 1 - 24 h. After self-assembly is completed, centrifuge at 4000 - 10000 rpm for 5 - 15 min, wash, dry at 25 - 60 °C, and precipitate to obtain Cu nanoflowers;

[0052] S2. Preparation of MnO2 nanoflowers: Add an aqueous solution of citric acid to an aqueous hydrochloric acid solution of potassium permanganate, stir at room temperature for 30 min, centrifuge at 1000 rpm for 5 min, precipitate, wash with distilled water, and dry at 50 °C to obtain MnO2 nanoflowers;

[0053] S3. Preparation of amine-functionalized MnO2 nanoflowers: Disperse MnO2 nanoflowers in absolute ethanol, ultrasonicate, add γ-aminopropyltriethoxysilane, continuously stir, centrifuge, precipitate, wash with ethanol, and dry to obtain amine-functionalized MnO2 nanoflowers;

[0054] S4. Preparation of Cu / MnO₂ nanoflowers: Disperse Cu nanoflowers in 2-morpholinoethanesulfonic acid buffer solution, add an activator, stir and activate at room temperature for 15 - 60 min to obtain a solution containing activated Cu nanoflowers; subsequently, disperse amine-functionalized MnO₂ nanoflowers in phosphate buffer solution to obtain a dispersion of amine-functionalized MnO₂ nanoflowers; mix the solution containing activated Cu nanoflowers with the dispersion of amine-functionalized MnO₂ nanoflowers, co-incubate at room temperature for 1 - 8 h, with an incubation rotation speed of 0 - 500 rpm, centrifuge at 4000 - 8000 rpm for 5 - 10 min, precipitate, wash with distilled water, and dry at 25 - 60 °C to prepare Cu / MnO₂ nanoflowers.

[0055] Example 1

[0056] A preparation method of composite hybrid nanoflowers based on MnO₂ nanoflowers, comprising the following steps:

[0057] S1. Add 120 mM CuSO₄ aqueous solution to PBS (0.2 mM, pH 8.0) containing 0.5 mg / mL Papain, with the volume ratio of CuSO₄ aqueous solution to Papain being 1:150, shake and incubate at room temperature for 12 h. After self-assembly is completed, centrifuge the obtained solution at 10000 rpm for 8 min, collect the precipitate, wash it three times with distilled water, and then dry it at 50 °C to obtain Cu NFs.

[0058] S2. Add 2 mL of 100 mM citric acid aqueous solution to 80 mL of HCl aqueous solution containing 2 mg / mL KMnO₄, stir at room temperature for 30 min. At this time, the solution changes from purple-red to brown. After centrifuging at 10000 rpm for 5 min, collect the precipitate, wash it three times with distilled water, and then dry it at 50 °C to obtain MnO₂ NFs.

[0059] S3. Take 150 mg of the obtained MnO₂ NFs, dissolve them in 300 mL of absolute ethanol, ultrasonicate for 10 min, then add 0.6 mL of APTES, continuously stir for 7 h, centrifuge at 10000 rpm for 5 min, collect the precipitate, wash it three times with ethanol, and then dry it at 50 °C to obtain amine-functionalized MnO₂ NFs.

[0060] S4. Disperse the obtained Cu NFs in MES (pH 5.5), add 20 mM EDC and 10 mM NHS with a volume ratio of 1:1, and stir and activate at room temperature for 30 min. Then disperse the amine-functionalized MnO2 NFs in PBS (0.2 M, pH 7.2), and mix the above two solutions. The volume ratio of the Cu NFs and MnO2 NFs dispersions is 5:1, and co-incubate at 200 rpm at room temperature for 6 h. Then centrifuge at 5000 rpm for 5 min, collect the precipitate, wash it three times with distilled water, and dry it at 50 °C to obtain Cu / MnO2 NFs.

[0061] The morphology and physicochemical properties of NFs were studied by SEM and FT-IR, such as Figure 1 、 Figure 2 shown. Papain and copper salts will form flaky petals at room temperature and self-assemble into hydrangea-like nanoflowers with a particle size of 8.55 μm. The synthesized MnO2 NFs have a particle size of 0.42 μm. After co-incubating the two, the activated carboxyl groups on the surface of Cu NFs and the amino groups of Cu / MnO2 NFs form amide bonds to achieve covalent conjugation.

[0062] Figure 2 In the FT-IR results of -1 、1050 cm -1 、1148 cm -1 , the absorption peaks are caused by P-O and P=O vibrations, proving the existence of phosphate groups; the absorption peak of MnO2 NFs at 1504 cm -1 is the symmetric vibration peak of -NH3 + , indicating that the amino group was successfully modified on the surface of MnO2 NFs. The intensity of the N-H bending peak of Cu / MnO2 NFs at 1480 cm -1 significantly weakens because amide bonds are formed with the -COOH of Cu NFs; in addition, the vibration peaks of amide I band (stretching vibration of C=O) and amide II band (N-H bending and C-N stretching vibration) appear at 1620 cm -1 and 1513 cm -1 for Cu / MnO2 NFs, indicating that HNFs are covalently bonded to MnO2 to form Cu / MnO2 NFs.

[0063] Example 2

[0064] A preparation method of a composite hybrid nanoflower based on MnO2 nanoflower. This example provides a study on the peroxidase-like activity of Cu / MnO2 nanoflowers, including the following steps:

[0065] TMB and H2O2 solutions with a volume ratio of 1:1 were respectively added to Papain, Cu NFs, amine-functionalized MnO2 NFs, and Cu / MnO2 NFs for catalytic reactions. The results are as Figure 3 shown. The experiments showed that after the immobilization of Papain, its peroxidase activity increased by 294.66%. The MnO2 NFs prepared in this invention also had excellent enzyme activity. When loaded onto Cu NFs, the enzyme activity of Cu / MnO2 NFs increased by 220.11% compared to Cu NFs and by 648.57% compared to Papain. This indicates that the NFs of this invention significantly improved the peroxidase activity of Papain.

[0066] Example 3

[0067] A preparation method of a composite hybrid nanoflower based on MnO2 nanoflowers. This example provides a study on the stability of Cu / MnO2 nanoflowers at different pH values, different temperatures, and different storage times, including the following steps:

[0068] First, Cu / MnO2 NFs were pre-incubated in PBS (20 mM, pH 8.0) at 30 - 80 °C for 4 h, and the thermal stability of Cu / MnO2 NFs was evaluated by measuring the remaining activity.

[0069] Subsequently, Cu / MnO2 NFs were pre-incubated in PBS buffer at pH 3.0 - 11.0 for 4 h, and the pH stability of Cu / MnO2 NFs was evaluated by measuring the remaining activity.

[0070] Cu / MnO2 NFs were dissolved in PBS (20 mM, pH 8.0) and then stored in a 4 °C refrigerator. The relative enzyme activity was measured at regular intervals. The results are as Figures 4 - 6As shown, under temperature-dependent conditions, both the enzymatic activity and stability of Cu / MnO2 NFs are significantly higher than those of Cu NFs and Papain. The trend of relative activity decline is significantly lower than that of free Papain. A similar phenomenon was also observed in the pH-dependent experiment. When the culture pH is lower than 5.0, the catalytic ability of free Papain drops rapidly, and the decline trend of Cu NFs stability also increases significantly, while Cu / MnO2 NFs only decreases by 25% compared to its highest enzymatic activity. In terms of the effect of storage time on enzymatic activity, free Papain almost completely loses its activity after being stored at 4°C for 15 days, the activity of Cu NFs drops significantly after 30 days, while the enzymatic activity of Cu / MnO2 NFs is still 95% and 82% of its maximum activity after being stored for 15 days and 30 days, respectively, and still retains 72% of its enzymatic activity after being stored for 60 days. It can be seen that the Cu / MnO2 NFs prepared by the present invention have excellent temperature, pH, and storage stability.

[0071] Example 4

[0072] A preparation method of a composite hybrid nanoflower based on MnO2 nanoflowers. This example provides a Cu / MnO2 nanoflower for the detection of amygdalin standard, including the following steps:

[0073] Weigh 0.1 g of amygdalin, add 2 mL of 5% sulfuric acid, mix well, and then place it in a boiling water bath for hydrolysis for 50 min. After the hydrolysis is completed, cool it in an ice-water bath, and then transfer it to 4 mL of cold 5 M sodium hydroxide solution, and mix and let it stand for 10 min. Add 0.2 mM pH 4.0 NaAc-HAc, 2.2 mM TMB, 0.1% H2O2, and Cu / MnO2 NFs according to the volume ratio of 1:1:1:1:1, and the color reaction is triggered. Incubate it in a dark environment for 5 min, and it can be observed that the color of the solution shows a blue gradient becoming lighter as the content of amygdalin increases.

[0074] Use Color Picker software to record the color change, convert the color information into an R value, select the R value of the solution when the amygdalin content is 0 as R0, and establish a relationship curve between R / R0 and the amygdalin concentration, so as to obtain the amygdalin quantitative real-time detection platform described in the present invention. Figure 7 、 8 The results show that as the amygdalin concentration increases, the color development intensity of the solution decreases, and there is a good linear relationship between R / R0 and the amygdalin concentration (R 2 = 0.9974). The calibration data is linearly fitted, and R / R0 = 0.23×C Amygdalin + 1.00. The Cu / MnO2 NFs sensor combined with Color Picker software can realize the rapid quantitative monitoring of amygdalin.

[0075] The detection principle is as follows:

[0076] The Cu / MnO2 NFs of the present invention have excellent peroxidase activity and can catalyze the oxidation of colorless TMB to blue oxTMB in the presence of H2O2. After cyanogenic glycoside hydrolysis, cyanide ions (CN - ) are generated. CN - forms a complex with Cu 2+ in Cu NFs to form [Cu(CN)4] 3- , occupying the active sites of Cu NFs and hindering the binding to substrates (TMB and H2O2), thereby reducing the peroxidase activity of Cu NFs; in addition, MnO2 in MnO2 NFs can oxidize CN - to generate cyanate ions under acidic or neutral conditions, while Mn 4+ is reduced to Mn 2+ , and the oxidation state of MnO2 decreases, resulting in a decrease in the activity of the catalytic center of MnO2 NFs, thereby affecting the peroxidase activity of MnO2 NFs. In summary, in the presence of CN - , the color development of the TMB-H2O2 system weakens. In practical applications, the Color Picker software is used to convert the color change of the solution into digital information (this part is not shown in the examples), and the obtained R value data is substituted into the previously obtained linear relationship curve of "R / R0 - cyanogenic glycoside concentration" to calculate the concentration of cyanogenic glycoside, realizing the on-site rapid detection of cyanogenic glycoside.

[0077] Comparative Example 1

[0078] The raw materials and preparation method are the same as those in Example 1, except that 0.5 mg / mL Papain is replaced with 0.1 mg / mL Papain, and scattered petal fragments are obtained. At this time, the enzyme activity of the obtained Cu NFs is only 132.69% of that of Papain.

[0079] Comparative Example 2

[0080] The raw materials and preparation method are the same as those in Example 1, except that the co-incubation time of Cu NFs and MnO2 NFs is reduced from 6 h to 2 h. The synthesized Cu / MnO2 NFs is only 286.21% of that of Papain, and its enzyme activity is only 35% of its maximum activity after storage for 60 d.

[0081] Comparative Example 3

[0082] The raw materials and preparation method are the same as those in Example 1, except that in step S1, Papain is not added, and the CuSO4 aqueous solution is directly added to PBS, and irregular agglomerated particles are obtained. At this time, the obtained particles have no peroxidase activity.

[0083] Comparative Example 4

[0084] The raw materials and preparation method were the same as those in Example 1, except that: in step S4, the dispersion liquid of Cu NFs and amine-functionalized MnO2 NFs was directly blended and incubated. The SEM results showed that there were many separate Cu NFs and MnO2 NFs. The infrared peak of the amide group was hardly present in FT-IR, and the enzyme activity of the obtained Cu / MnO2 NFs ratio was only 63.27% of that of the Cu / MnO2 NFs synthesized in the present invention.

[0085] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A preparation method of a composite hybrid nanoflower based on MnO2 nanoflowers, characterized in that: It includes the following steps: S1. Preparation of Cu nanoflowers: Add an aqueous solution of copper salt into a buffer solution containing papain, shake and incubate at room temperature for 1 - 24 h. After self-assembly is completed, centrifuge, wash, and dry to obtain Cu nanoflowers. S2. Preparation of MnO₂ nanoflowers: Add an aqueous solution of citric acid into an aqueous hydrochloric acid solution of potassium permanganate, stir, centrifuge, precipitate, wash, and dry to obtain MnO₂ nanoflowers. S3. Preparation of amine-functionalized MnO₂ nanoflowers: Add γ-aminopropyltriethoxysilane into the MnO₂ nanoflower dispersion solution, continuously stir, centrifuge, precipitate, wash, and dry to obtain amine-functionalized MnO₂ nanoflowers. S4. Preparation of Cu / MnO₂ nanoflowers: Add an activator into the Cu nanoflower dispersion, activate to obtain a dispersion containing activated Cu nanoflowers. Mix the dispersion containing activated Cu nanoflowers with the dispersion of amine-functionalized MnO₂ nanoflowers, co-incubate at room temperature for 1 - 8 h, centrifuge, precipitate, wash, and dry to obtain Cu / MnO₂ nanoflowers.

2. The preparation method of the composite hybrid nanoflower based on MnO2 nanoflower according to claim 1, characterized in that: In step S1, the copper salt is at least one of copper sulfate, copper chloride, and copper nitrate.

3. The preparation method of the MnO2 nanoflower composite hybrid nanoflower according to claim 2, characterized in that: In step S1, the concentration of the copper salt is 80 - 150 mM, and the concentration of papain is 0.25 - 10 mg / ml.

4. The preparation method of the composite hybrid nanoflower based on MnO2 nanoflower according to claim 3, wherein: In step S4, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

5. The preparation method of the MnO2 nanoflower composite hybrid nanoflower according to claim 4, characterized in that: The molar ratio of copper ion: potassium permanganate: γ-aminopropyltriethoxysilane: 1-ethyl-(3-dimethylaminopropyl)carbodiimide: N-hydroxysuccinimide is 80 - 150: 1 - 5: 0.005 - 0.02: 0.1 - 50: 0.1 - 25.

6. The preparation method of the MnO2 nanoflower composite hybrid nanoflower according to claim 1, characterized in that: In step S1, the centrifugation speed is 4000 - 10000 rpm, the centrifugation time is 5 - 15 min, and the drying temperature is 25 - 60 °C.

7. The preparation method of the composite hybrid nanoflower based on MnO2 nanoflower according to claim 5, characterized in that: In step S4, the activation time is 15 - 60 min, the co-incubation speed is 0 - 500 rpm, the centrifugation speed is 4000 - 8000 rpm, the centrifugation time is 5 - 10 min, and the drying temperature is 25 - 60 °C.

8. Application of the Cu / MnO₂ nanoflowers prepared by the preparation method of the composite hybrid nanoflowers based on MnO₂ nanoflowers according to any one of claims 1 - 7 in the preparation of a cyanogenic glycoside biosensor.

9. A detection method for the cyanogenic glycoside biosensor based on Cu / MnO2 nanoflowers according to claim 8, characterized in that, It includes the following steps: (1) Mix the sample to be tested with an acid solution, and release cyanide ions through hydrolysis reaction. (2) Transfer the hydrolyzate to an alkaline solution, mix and let stand. (3) Add a sodium acetate - acetic acid buffer solution with a pH of 4, a 3,3',5,5'-tetramethylbenzidine color reagent, hydrogen peroxide, and Cu / MnO₂ nanoflowers to the neutralized solution to trigger a color reaction. (4)Quantitatively detect the concentration of cyanogenic glycoside through the change of color signal, where CN- complexes with the active centers Cu2 + and Mn 4+ of the Cu / MnO2 nanoflowers, inhibiting their peroxidase activity and resulting in a decrease in the color development intensity; (5) Use a smartphone device to collect color signals, and establish a linear relationship curve of R / R₀ vs. cyanogenic glycoside concentration by analyzing the RGB value or absorbance value of the color reaction to achieve quantitative detection.

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