Metal catalyst based on biological carrier loading, preparation method and application thereof

Abstract

The invention provides a metal catalyst loaded on the basis of a biological carrier, a method and application of the metal catalyst. The preparation method of the catalyst comprises the following steps: adding one or more than two metal ion-containing solutions into the biological carrier to obtain a mixed solution; a reducing agent is added into the mixed solution for a reduction reaction, the reducing agent comprises but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate so as to form a metal catalyst with metal nanoparticles loaded on the surface of the biological carrier, the obtained metal catalyst has catalytic activity of multiple enzymes, the synthesis process is green and environmentally friendly, and the method is suitable for industrial production. The obtained catalyst has high activity, can be used for prediction and diagnosis of disease markers such as prostatic cancer, gout and diabetes, and has wide application prospects in biological medicines and diagnostic reagents.

Description

Technical Field

[0001] The present application relates to the field of bio-mediated green nano-synthesis technology, and in particular to a metal catalyst supported on a bio-carrier, a preparation method and use thereof. Background Art

[0002] Bio-mediated green nanosynthesis is an environmentally friendly method to synthesize nanomaterials using organisms or biomolecules (such as plant extracts, microorganisms, enzymes, etc.). Compared with traditional physical or chemical synthesis methods, bio-mediated synthesis has the characteristics of low cost, low energy consumption, no toxic byproducts and sustainable development, and is regarded as a green technology in the field of nanomaterial preparation. Nanomaterials prepared by bio-mediated methods have broad application prospects in medicine, environmental governance, catalysis and other fields due to their unique physical and chemical properties.

[0003] Nanometal catalysts synthesized by bio-mediated methods have better dispersibility and higher catalytic activity. These catalysts not only retain the efficient catalytic ability of natural enzymes, but also overcome the shortcomings of natural enzymes such as easy inactivation and difficulty in large-scale production. By developing colorimetric detection methods based on new nanocatalysts, rapid detection of a variety of disease markers can be achieved. This method not only improves the sensitivity and specificity of detection, but also significantly reduces the cost of detection, making it suitable for large-scale screening and early diagnosis. For example, designing nanometal catalysts that specifically recognize acid phosphatase or uric acid and applying them to colorimetric detection can achieve early detection of diseases such as prostate cancer or gout, thereby improving the treatment effect and survival rate of patients. Therefore, the development of colorimetric detection methods for different disease markers using new nanocatalysts is of great significance for the rapid detection of disease markers. Summary of the invention

[0004] Based on this, the present application provides a metal catalyst supported on a biological carrier, a preparation method and its use, wherein the catalyst has the catalytic activity of multiple enzymes.

[0005] The specific technical solutions of this application are as follows:

[0006] 1. A metal catalyst based on a bio-carrier:

[0007] The metal catalyst has one or more enzyme-like activities of peroxidase-like, ascorbate oxidase-like, laccase-like or oxidase-like catalytic activities;

[0008] The metal catalyst is prepared by the following method:

[0009] adding one or more solutions containing metal ions to the biological carrier to obtain a mixed solution;

[0010] A reducing agent is added to the mixed solution of the biocarrier and metal ions for reduction reaction, wherein the reducing agent includes but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate to form a metal catalyst in which metal nanoparticles are loaded on the surface of the biocarrier.

[0011] 2. The metal catalyst according to claim 1, wherein the addition rate of the sodium borohydride is 0.01-10 mL / min; and / or

[0012] The mass ratio of sodium borohydride to sodium hydroxide is 1:1-4;

[0013] The mass ratio of sodium borohydride to ascorbic acid is 1:4-9;

[0014] The mass ratio of the sodium borohydride to sodium citrate is 1:4-15.

[0015] 3. The metal catalyst according to item 1 or 2, wherein the metal ion comprises one or more selected from Fe, Al, Cu, Au, Pt, Pd, Ni, Ti and Ag; and / or

[0016] The mass ratio of the two metal ions is 1-9:9-1.

[0017] 4. The metal catalyst according to any one of items 1 to 3, wherein the biological carrier comprises a polypeptide, preferably, the polypeptide comprises a bacteriophage, preferably an M13 bacteriophage;

[0018] Further preferably, the concentration of the biological carrier is 0.5×10^11~10×10^11 pfu / L.

[0019] 5. The metal catalyst according to any one of items 1 to 4, wherein the D90 of the metal nanoparticles is 0.1 nm to 100 nm, and the metal nanoparticles are distributed on the surface of the biological carrier;

[0020] Preferably, the particle morphology of the metal catalyst is one of flower-shaped, spherical, rod-shaped or cluster-shaped.

[0021] 6. A method for preparing a metal catalyst supported on a bio-carrier, comprising:

[0022] adding one or more solutions containing metal ions to the biological carrier to obtain a mixed solution;

[0023] A reducing agent is added to the mixed solution of the biocarrier and metal ions for reduction reaction, wherein the reducing agent includes but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate to form a metal catalyst in which metal nanoparticles are loaded on the surface of the biocarrier.

[0024] 7. The method according to claim 6, wherein the addition rate of the sodium borohydride is 0.01-10 mL / min; and / or

[0025] The mass ratio of sodium borohydride to sodium hydroxide is 1:1-4;

[0026] The mass ratio of sodium borohydride to ascorbic acid is 1:4-9;

[0027] The mass ratio of the sodium borohydride to sodium citrate is 1:4-15.

[0028] 8. The method according to item 6 or 7, wherein the metal ions comprise one or more selected from Fe, Al, Cu, Au, Pt, Pd, Ni, Ti and Ag; and / or

[0029] The mass ratio of the two metal ions is 1-9:9-1; and / or

[0030] The biological carrier comprises a polypeptide, preferably, the polypeptide comprises a bacteriophage, preferably an M13 bacteriophage;

[0031] Further preferably, the concentration of the biological carrier is 0.5×10^11 to 10×10^11 pfu / L;

[0032] Preferably, the metal catalyst is the metal catalyst involved in any one of items 1-5.

[0033] 9. Use of the metal catalyst described in any one of Items 1 to 5 or the metal catalyst described in any one of Items 6 to 8 in a kit for diagnosing prostate cancer, gout or diabetes.

[0034] 10. A kit for diagnosing prostate cancer, gout or diabetes, comprising the metal catalyst described in any one of items 1 to 5 or the metal catalyst described in any one of items 6 to 8.

[0035] Effects of the Invention

[0036] The metal catalyst described in the present application can be prepared under mild conditions at room temperature and pressure. This synthesis method is not only simple and easy, but also suitable for large-scale production. By adjusting various variables in the synthesis process, such as the type of metal ions, the amount of biological carrier added, the selection of reducing agents and the order of addition, the particle size, morphology, type of precious metals, loading rate of precious metals and final yield of the nanoparticles can be accurately controlled. This multifunctional controllability enables the metal catalyst to have the catalytic activity of multiple enzymes and can adapt to different biomedical application requirements.

[0037] The obtained catalyst exhibits high catalytic activity and has broad application prospects in the biomedical field, especially in the predictive diagnosis of disease markers. For example, the catalyst can be used to detect markers of major diseases such as prostate cancer, gout, and diabetes, providing an efficient, economical and accurate early diagnosis tool.

[0038] Compared with single metal catalysts, the combined metal catalysts in this application show higher catalytic activity and higher particle yield. This means that under the same reaction conditions, the combined catalyst can more effectively catalyze the target reaction and generate more nanoparticles, showing better industrial application potential. This superiority not only helps to improve the sensitivity and accuracy of detection, but also reduces the overall cost, making it more competitive in large-scale screening and clinical applications.

[0039] In summary, the metal catalyst in this application provides a nanocatalyst with high activity, high stability and environmental friendliness by optimizing the synthesis process and material combination. The application of this catalyst in biomedicine and diagnostic reagents, especially in the detection of disease markers such as prostate cancer, gout, and diabetes, shows broad market potential and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A This is a transmission electron micrograph of a metal catalyst obtained using sodium borohydride and sodium hydroxide as reducing agents.

[0041] Figure 1B This is a transmission electron micrograph of the metal catalyst obtained using sodium borohydride and ascorbic acid as reducing agents.

[0042] Figure 1C This is a transmission electron micrograph of a metal catalyst obtained using sodium borohydride and sodium citrate as reducing agents.

[0043] Figure 1D This is a transmission electron micrograph of a metal catalyst obtained using sodium borohydride as a reducing agent.

[0044] Figure 1E This is an EDS schematic diagram of the metal catalyst obtained in Example 1.

[0045] Figure 2 is a schematic diagram of the verification of catalase activity of metal catalysts

[0046] Figure 3 Schematic diagram of the verification of ascorbate oxidase activity using metal catalysts

[0047] Figure 4 The effect of different addition rates of sodium borohydride on the enzyme activity of metal catalysts.

[0048] Figure 5AThis is a linear relationship diagram between the concentration and absorption peak of ACP detected by metal catalysts.

[0049] Figure 5B Schematic diagram of the fluorescence intensity of other interferents and ACP detected using metal catalysts.

[0050] Figure 6 This is the linear relationship diagram between the concentration and absorption peak of uric acid detected by metal catalyst.

[0051] Figure 7 This is the linear relationship diagram between the concentration and absorption peak of glucose detected by metal catalyst. DETAILED DESCRIPTION

[0052] The present application is described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0053] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0054] The present application provides a method for preparing a metal catalyst supported on a bio-carrier, which comprises: adding one or more solutions containing metal ions to the bio-carrier to obtain a mixed solution;

[0055] A reducing agent is added to the mixed solution to carry out a reduction reaction, wherein the reducing agent includes but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate to form a metal catalyst in which metal nanoparticles are loaded on the surface of the biological carrier.

[0056] The metal catalyst obtained by reduction using the above reducing agent in the present application has a higher catalytic activity, which is above 850 U / mg.

[0057] Preferably, the reducing agent is a reducing agent consisting of sodium borohydride and one selected from the group consisting of sodium hydroxide, ascorbic acid and sodium citrate.

[0058] In some embodiments, the present application does not impose any restrictions on the manner in which the reducing agent is added to the mixed solution, and those skilled in the art can make routine selections as needed, for example, the reducing agent is directly added to the mixed solution, preferably sodium borohydride is added first, and then one selected from sodium hydroxide, ascorbic acid and sodium citrate is added; the reducing agent can also be added to the mixed solution by dropwise addition, for example, sodium borohydride is first added to the mixed solution by dropwise addition, and then one selected from sodium hydroxide, ascorbic acid and sodium citrate is directly added; preferably, sodium borohydride is first added to the mixed solution by dropwise addition, and then one selected from sodium hydroxide, ascorbic acid and sodium citrate is directly added.

[0059] In some embodiments, the addition rate of the sodium borohydride is 0.01-10 mL / min. For example, the addition rate of the sodium borohydride can be 0.01 mL / min, 0.05 mL / min, 0.1 mL / min, 0.5 mL / min, 1 mL / min, 1.5 mL / min, 2 mL / min, 2.5 mL / min, 3 mL / min, 3.5 mL / min, 4 mL / min, 4.5 mL / min, 5 mL / min, 5.5 mL / min, 6 mL / min, 6.5 mL / min, 7 mL / min, 7.5 mL / min, 8 mL / min, 8.5 mL / min, 9 mL / min, 9.5 mL / min, 10 mL / min, etc.

[0060] In the present application, there is no limitation on the amount of sodium borohydride used. A person skilled in the art can select the amount of sodium borohydride based on the amount of metal ions so that all metal ions are reduced. In the present application, there is no limitation on the concentration of sodium borohydride. A person skilled in the art can make a conventional selection based on needs.

[0061] In some embodiments, the mass ratio of sodium borohydride to sodium hydroxide is 1:1-4;

[0062] The mass ratio of sodium borohydride to ascorbic acid is 1:4-9;

[0063] The mass ratio of the sodium borohydride to sodium citrate is 1:4-15.

[0064] In the present application, the mass ratio of sodium borohydride to sodium hydroxide refers to the mass ratio of the total amount of sodium borohydride used to sodium hydroxide.

[0065] For example, the mass ratio of sodium borohydride to sodium hydroxide (m 硼氢化钠 :m 氢氧化钠 ) can be 1:1, 1:2, 1:3, 1:4, etc.;

[0066] The mass ratio of sodium borohydride to ascorbic acid (m 硼氢化钠 :m 抗坏血酸 ) can be 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.;

[0067] The mass ratio of sodium borohydride to sodium citrate (m 硼氢化钠 :m 柠檬酸钠 ) can be 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0068] In the present application, there is no limitation on the concentrations of sodium hydroxide, ascorbic acid and sodium citrate. Those skilled in the art can select commonly used concentrations based on actual needs, as long as the mass ratio of sodium borohydride to sodium hydroxide or ascorbic acid or sodium citrate is within the above range.

[0069] In some embodiments, the metal ion comprises one selected from Fe, Al, Cu, Au, Pt, Pd, Ni and Ag.

[0070] In the present application, the valence of the metal ions is not limited, and they are all valences commonly used in the art. For example, for iron ions, it can be a ferrous chloride solution containing iron ions;

[0071] For gold ions, it can be a chloroauric acid solution or a gold trichloride solution containing gold ions;

[0072] For aluminum ions, it can be an aluminum chloride solution, an aluminum sulfate solution or an aluminum nitrate solution containing aluminum ions;

[0073] For copper ions, it can be a copper nitrate solution, a copper chloride solution or a copper sulfate solution containing copper ions;

[0074] For platinum ions, it can be a platinum chloride solution, a chloroplatinic acid solution, or a sodium chloroplatinate solution containing platinum ions;

[0075] For palladium ions, it can be a palladium chloride solution, a palladium nitrate solution or a sodium chloropalladate solution containing palladium ions;

[0076] For nickel ions, it may be a nickel chloride hexahydrate solution containing nickel ions;

[0077] As for silver ions, it may be a silver nitrate solution containing silver ions.

[0078] In some embodiments, the mass ratio of the two metal ions is 1-9:9-1, for example, the mass ratio of the two metal ions can be 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, etc.

[0079] In some embodiments, the biological vector comprises a polypeptide, preferably the polypeptide comprises a bacteriophage, more preferably an M13 bacteriophage;

[0080] Preferably, the concentration of the biological carrier is 0.5×10^11 to 10×10^11 pfu / L.

[0081] In the present application, when the biological carrier is a bacteriophage, the concentration of the biological carrier refers to the concentration in the biological carrier, the reducing agent and the solution containing metal ions. For example, the concentration of the biological carrier can be 0.5×10^11pfu / L, 1×10^11pfu / L, 2×10^11pfu / L, 3×10^11pfu / L, 4×10^11pfu / L, 5×10^11pfu / L, 6×10^11pfu / L, 7×10^11pfu / L, 8×10^11pfu / L, 9×10^11pfu / L, 10×10^11pfu / L, etc.

[0082] The catalyst prepared by the method described in the present application has the catalytic activity of multiple enzymes. The type of metal ions, the type of reducing agent, the order of adding the reducing agent and the amount of addition can be controlled during the preparation process to control the particle size, morphology, type of metal catalyst, loading rate and yield of the obtained catalyst. The loading rate is above 70%, and the preparation process is green and environmentally friendly.

[0083] The present application provides a metal catalyst supported on a biological carrier, which is prepared by any of the methods described above.

[0084] The metal catalyst described in the present application has high activity and can be used for predictive diagnosis of disease markers such as prostate cancer, gout and diabetes. It has broad application prospects in biomedicine and diagnostic reagents and is of great significance for early diagnosis and prognosis detection of complex diseases.

[0085] In some embodiments, the particle size D90 of the metal catalyst is 0.1-50 nm.

[0086] In the present application, the method for determining the particle size D90 of the metal catalyst is not limited in any way. A person skilled in the art can make a conventional selection based on the needs. For example, the particle size distribution diagram can be obtained by analyzing and calculating the particle size based on the spherical aberration transmission electron microscope image and using NanoMeasurer to obtain the particle size distribution diagram, thereby obtaining the particle size D90. The particle size D90 of the metal catalyst can be 0.1 nm, 1 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, etc.

[0087] In some embodiments, the particle morphology of the metal catalyst is one of flower-shaped, spherical, rod-shaped or cluster-shaped. Preferably, the metal catalyst has a peroxidase-like, ascorbate oxidase-like, laccase-like or oxidase-like catalytic activity.

[0088] The present application provides the use of the above-mentioned metal catalyst in a kit for diagnosing prostate cancer, gout or diabetes.

[0089] The metal catalyst described in the present application can be used to detect markers of prostate cancer, gout or diabetes, for example, it can be used to detect the prostate cancer marker acid phosphatase, the gout marker uric acid and the diabetes marker glucose, and thus can be used to detect these diseases.

[0090] The present application provides a kit for diagnosing prostate cancer, gout or diabetes, which comprises the metal catalyst described above or the metal catalyst prepared by the above method.

[0091] Example

[0092] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0093] Example 1-3 Preparation of metal catalyst based on biological carrier

[0094] (1) Phage culture:

[0095] A. Weigh 10g of peptone, 5g of yeast extract powder, and 10g of sodium chloride, dissolve in 950mL of deionized water, add water to make up to 1L, and dispense into 500mL conical flasks, 100mL per bottle, sterilize at 121℃ for 20min, and set aside.

[0096] B. In the clean bench, inoculate 200 μL of E. coli seed solution into 20 mL of LB medium according to standard operating procedures, and culture at 37°C and 200 rpm in a shaking incubator for 12 to 16 hours to obtain seed solution for subsequent experiments.

[0097] C. Add 1% volume fraction of the seed solution in step 2, 1‰ volume of tetracycline, and 1‰ phage (from Ph.D.-7) to the LB medium. TM Phage display peptide library kit), cultured at 37°C, 200 rpm shaking for 8 hours to obtain M13 phage culture fluid.

[0098] D. Centrifuge and purify the M13 phage culture fluid obtained in step C to obtain M13 phage.

[0099] (2) The M13 phage was diluted to 400 μL with TBS buffer solution (pH = 7.5) to form a mixed solution 1; 200 μL of 2 mM FeCl2 and 2 mM CuCl2 were added to the mixed solution 1 in a volume ratio of 1:1, and each solution was allowed to stand for 2-5 min to form a mixed solution 2;

[0100] (3) 400 μL of 4 mM reducing agent (the reducing agent is first sodium borohydride and then sodium hydroxide in Example 1; first sodium borohydride and then ascorbic acid in Example 2; first sodium borohydride and then sodium citrate in Example 3) is added to the mixed solution 2, wherein the concentration of the bacteriophage is 1×10^11 pfu / L, and the mixture is reduced at room temperature for 10 min and centrifuged at 8000 rpm for 5 min. The supernatant is removed to obtain a metal catalyst, which is denoted as Fe-Cu@M13. Its transmission electron microscopy images are shown as follows: Figures 1A to 1C As shown, Figure 1A This is a transmission electron microscope image of a metal catalyst obtained using sodium borohydride and sodium hydroxide as reducing agents. Figure 1B This is a transmission electron microscope image of a metal catalyst obtained using sodium borohydride and ascorbic acid as reducing agents. Figure 1C FIG. 1 is a transmission electron microscope image of a metal catalyst obtained by using sodium borohydride and sodium citrate as reducing agents. In addition, an energy dispersive X-ray spectrometer (EDS) analysis was performed on the obtained catalyst. The results are as follows: Figure 1E shown.

[0101] Embodiment 4-6

[0102] The difference between Example 4-6 and Example 1 is that the two metal solutions are FeCl2 and Na2PtCl4 aqueous solutions to prepare the metal catalysts.

[0103] Embodiment 7-11

[0104] The difference between Examples 7-11 and Example 4 is that the addition rates of sodium borohydride are 0.03 ml / min, 0.05 ml / min, 0.1 ml / min, 0.2 ml / min, and 0.5 ml / min, respectively, to obtain different metal catalysts.

[0105] Example 12

[0106] The difference between Example 12 and Example 1 is that the metal catalyst is obtained by using a mass ratio of iron ions to copper ions of 1:9.

[0107] Example 13

[0108] The difference between Example 13 and Example 1 is that the metal catalyst is obtained by using a mass ratio of iron ions to copper ions of 9:1.

[0109] Embodiment 14

[0110] The difference between Example 14 and Example 1 is that the metal catalyst is obtained by using a mass ratio of iron ions to copper ions of 1:1.

[0111] Embodiment 15

[0112] The difference between Example 15 and Example 1 is that the concentration of bacteriophage is 0.5×10^11 pfu / L to obtain the metal catalyst.

[0113] Example 16

[0114] The difference between Example 16 and Example 1 is that the concentration of bacteriophage is 10×10^11 pfu / L to obtain the metal catalyst.

[0115] Embodiment 17

[0116] The difference between Example 17 and Example 1 is that the mass ratio of sodium borohydride to sodium hydroxide is 1:1 to obtain the metal catalyst.

[0117] Embodiment 18

[0118] The difference between Example 18 and Example 1 is that the mass ratio of sodium borohydride to sodium hydroxide is 1:4 to obtain the metal catalyst.

[0119] Embodiment 19

[0120] The difference between Example 19 and Example 2 is that the metal catalyst is obtained by using a mass ratio of sodium borohydride to ascorbic acid of 1:4.

[0121] Embodiment 20

[0122] The difference between Example 20 and Example 2 is that the mass ratio of sodium borohydride to ascorbic acid is 1:9 to obtain the metal catalyst.

[0123] Embodiment 21

[0124] The difference between Example 21 and Example 3 is that the mass ratio of sodium borohydride to sodium citrate is 1:4 to obtain the metal catalyst.

[0125] Embodiment 22

[0126] The difference between Example 22 and Example 3 is that the mass ratio of sodium borohydride to sodium citrate is 1:15 to obtain the metal catalyst.

[0127] Comparative Example 1

[0128] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses only sodium borohydride to prepare the metal catalyst, and its transmission electron microscope image is as follows: Figure 1D shown.

[0129] Comparative Example 2

[0130] The difference between Comparative Example 2 and Example 4 is that Comparative Example 2 uses only sodium borohydride to prepare the metal catalyst.

[0131] Table 1

[0132]

[0133]

[0134] Experimental Example 1 Determination of Activity and D90

[0135] The metal catalysts obtained in Examples 1-22 and Comparative Examples 1-2 were subjected to activity measurements, and the results are shown in Table 2. The method for measuring the activity is as follows:

[0136] The metal catalyst (1 mg / mL) prepared in the example, 100 μL of 4 mM 3,3',5,5'-tetramethylbenzidine (TMB) and 200 μL of 10% H2O2 solution were prepared with acetic acid-sodium acetate buffer solution (0.2 M, pH = 5.0). The above solutions were mixed evenly and water was added to 2 mL. The solution was placed in a constant temperature metal bath at 37°C and reacted for 10 min. The absorbance at 652 nM was measured.

[0137] Enzyme activity was defined as the amount of enzyme that produced 1 μmol of ox-TMB within 1 min.

[0138] Enzyme activity formula = (A*331.3*V / 1000) / (t*331.3*m)(U / mg)

[0139] A is the absorbance at 652 nM, V is the reaction volume, t is the reaction time, and m is the mass of metal catalyst added (mg).

[0140] The particle size D90 is calculated by obtaining the spherical aberration transmission electron microscope image of the particles and analyzing the particle size D90 using Nano Measurer software.

[0141] Table 2 Activity data

[0142]

[0143]

[0144] It can be seen from Table 2 that the metal catalyst is prepared by the method described in this application with high yield and simple method, and the obtained catalyst has high activity.

[0145] Experimental Example 2 Verification of peroxidase activity

[0146] The metal catalyst obtained in Example 1 and 10 μL Fe-Cu@M13 (1 mg / mL) obtained in Comparative Example 1 were prepared with acetic acid-sodium acetate buffer solution (0.2 M, pH = 5.0), 100 μL 4 mM 3,3',5,5'-tetramethylbenzidine (TMB), 2,2-azo-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) or o-phenylenediamine (OPD) solution and 200 μL 10% H2O2 solution. The above solutions were mixed evenly and then water was added to 2 mL. After being placed in a constant temperature metal bath at 37°C and fully reacted for 10 minutes, the ultraviolet-visible absorption spectrum of the solution in the wavelength range of 400-800 nm was measured. Fe-Cu@M13 was replaced with an equal volume of 0.2 M acetic acid-sodium acetate buffer solution with a pH of 5.0. The remaining steps were the same as the above system. A blank experiment was performed, and the results are shown as follows. Figure 2A and Figure 2B As shown, Figure 2A is a schematic diagram of the absorption spectrum of the metal catalyst used in Comparative Example 1, Figure 2B It is a schematic diagram of the absorption spectrum of the metal catalyst used in Example 1.

[0147] from Figure 2A and Figure 2B It can be seen that in the presence of H2O2, although the metal catalysts obtained in Comparative Example 1 and Example 1 can catalyze the peroxidase substrate molecule TMB to produce absorption peaks at 652nm, 405nm and 450nm, accompanied by obvious changes in the color of the system, the characteristic peak of Example 1 is stronger, indicating that the metal catalyst obtained in the example has better enzyme simulation properties and can achieve catalytic oxidation of the enzyme substrate.

[0148] Experimental Example 3 Verification of ascorbate oxidase activity

[0149] The ascorbate oxidase-like activity of the metal catalyst was verified by measuring the reduction of the ultraviolet absorption of the substrate ascorbic acid (AA). 50 μL of Fe-Cu@M13 (1 mg / mL) obtained in Example 1 and the metal catalyst obtained in Comparative Example 1 were added to 100 μL of AA (100 μM) to obtain a mixed solution of the experimental group and a mixed solution of the control group. After 1 minute, the wavelength in the range of 200-400 nm was immediately measured to observe the change in absorbance at 285 nm. The results are shown in FIG. Figure 3 As shown, from Figure 3 It can be seen that after the Fe-Cu@M13 prepared in Example 1 was added, the absorbance at 285 nm decreased significantly, indicating that the metal catalyst obtained in Example 1 can better simulate the activity of ascorbic acid oxidase.

[0150] Experimental Example 4 Effect of different addition rates on the enzyme activity of metal catalysts

[0151] The metal catalysts obtained in Examples 7-11 were subjected to enzyme activity assays in the same manner as in Experimental Example 1, with natural enzyme horseradish peroxidase (HRP) used as a reference. The results are shown in Table 1. Figure 4 shown.

[0152] from Figure 4 It can be seen that the enzyme activity of the metal catalyst can be improved by adjusting the different addition rates of the reducing agent sodium borohydride.

[0153] Experimental Example 5 Detection of prostate cancer marker acid phosphatase (ACP) based on metal catalyst

[0154] Add acetic acid-sodium acetate buffer with pH=5 to a mixed solution of 100μL 8mmol / L sodium ascorbyl phosphate (AAP) and 50μL ACP (1-0.05U / L) of different concentrations to 200μL, place in a preheated 37°C constant temperature metal bath, and react for 10 minutes. Then, add 10μL 1mg / mL metal catalyst Fe-Cu@M13 prepared in Example 1 to the above reaction system, incubate at room temperature for 5 minutes, then centrifuge at 8000rpm for 1 minute, take the supernatant and mix it with 200μL 4mM o-phenylenediamine, react at room temperature for 10 minutes, and observe the fluorescence generation by ultraviolet light irradiation, and measure the fluorescence intensity of ACP at 440nm at different concentrations. The results are as follows: Figure 5A As shown, interfering substances (tyrosine, alanine, horseradish peroxidase and glucose oxidase) were used to replace ACP respectively. The other steps were the same as above. The results are shown in Figure 5B shown.

[0155] right Figure 5AThe linear equation of the standard curve is A=3535C. ACP +2138.7 (R2=0.9899), and the linear range is 0.016-20U / L. The detection limit of this method for ACP is 0.016U / L, indicating that the metal catalyst has a wide detection range and a low detection limit for ACP.

[0156] from Figure 5B It can be seen that the fluorescence intensity of ACP is much higher than that of the interferent, indicating that the metal catalyst described in Example 1 can be used to detect ACP.

[0157] Experimental Example 6 Detection of gout marker uric acid based on metal catalyst

[0158] 150 μL of uric acid of different concentrations (1-800 μM) and an equal volume of TMB (5 mM) were added to acetic acid-sodium acetate buffer (0.2 M, pH = 5) to a total volume of 500 μL, and placed in a preheated 30°C, 200 rpm metal bath shaker for a timed reaction of 10 minutes. Then, 10 μL of 1 mg / mL metal catalyst Fe-Cu@M13 prepared in Example 1 was added to the above reaction system and mixed evenly, and finally added to 200 μL 50 mM H2O2, mixed evenly, and placed in a preheated 30°C, 200 rpm metal bath shaker for a timed reaction of 10 minutes, observing the color change of the system and taking the supernatant to measure the absorbance of different concentrations of uric acid at 652 nm. The uric acid concentration and absorbance curve is shown in the figure. Figure 6 As shown, the linear equation of the standard curve is obtained by fitting: 1 / A = 18.36C 尿酸 +1.115 (R2=0.9729), the linear range is 50-800 μM, the detection limit of uric acid by this method is 50 μM, the color of solutions with different concentrations of uric acid (the concentration of uric acid from left to right corresponds to the concentration on the coordinate) is shown in the figure below. Figure 6 Shown in the upper left corner.

[0159] from Figure 6 It can be seen that the metal catalyst has a wider detection range and lower detection limit for uric acid.

[0160] Example 7 Detection of diabetes marker glucose based on metal catalyst

[0161] 50 μL of glucose of different concentrations (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 mM) and the metal catalyst Fe-Cu@M13 (10 μL, 1 mg / mL) obtained in Example 1 were added to a PBS buffer solution (0.2 M, 140 μL, pH = 5), mixed evenly, and placed in a preheated 40°C, 200 rpm metal bath shaker for a timed reaction of 10 min. The above mixture was added to an equal volume of 6 mM dimethylphenol orange solution, mixed thoroughly, incubated at 37°C for 5 min in the dark, and the color change of the system was observed and the absorbance at 580 nm was measured. The glucose concentration and absorbance curve is shown in FIG. Figure 7 , and the linear equation of the standard curve is obtained by fitting: A = 2.201C 葡萄糖 +0.0438 (R2=0.982), the linear range is 0-0.2mM, and the color response of solutions with different concentrations of glucose (the concentration of glucose from left to right corresponds to the concentration on the coordinate) is as follows Figure 7 As shown in the upper left corner, it shows that the metal catalyst has a wide detection range for glucose.

[0162] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A metal catalyst based on a bio-carrier: The metal catalyst has one or more enzyme-like activities of peroxidase-like, ascorbate oxidase-like, laccase-like or oxidase-like catalytic activities; The metal catalyst is prepared by the following method: adding one or more solutions containing metal ions to the biological carrier to obtain a mixed solution; A reducing agent is added to the mixed solution of the biocarrier and metal ions for reduction reaction, wherein the reducing agent includes but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate to form a metal catalyst in which metal nanoparticles are loaded on the surface of the biocarrier.

2. The metal catalyst according to claim 1, wherein The addition rate of the sodium borohydride is 0.01-10 mL / min; and / or The mass ratio of sodium borohydride to sodium hydroxide is 1:1-4; The mass ratio of sodium borohydride to ascorbic acid is 1:4-9; The mass ratio of the sodium borohydride to sodium citrate is 1:4-15.

3. The metal catalyst according to claim 1 or 2, wherein the metal ion comprises one or more selected from Fe, Al, Cu, Au, Pt, Pd, Ni, Ti and Ag; and / or The mass ratio of the two metal ions is 1-9:9-1.

4. The metal catalyst according to any one of claims 1 to 3, wherein the biological carrier comprises a polypeptide, preferably, the polypeptide comprises a bacteriophage, preferably an M13 bacteriophage; Further preferably, the concentration of the biological carrier is 0.5×10^11~10×10^11 pfu / L.

5. The metal catalyst according to any one of claims 1 to 4, wherein the D90 of the metal nanoparticles is 0.1 nm to 100 nm, and the metal nanoparticles are distributed on the surface of the biological carrier; Preferably, the particle morphology of the metal catalyst is one of flower-shaped, spherical, rod-shaped or cluster-shaped.

6. A method for preparing a metal catalyst supported on a bio-carrier, comprising: adding one or more solutions containing metal ions to the biological carrier to obtain a mixed solution; A reducing agent is added to the mixed solution of the biocarrier and metal ions for reduction reaction, wherein the reducing agent includes but is not limited to sodium borohydride, sodium hydroxide, ascorbic acid and sodium citrate to form a metal catalyst in which metal nanoparticles are loaded on the surface of the biocarrier.

7. The method according to claim 6, wherein the addition rate of the sodium borohydride is 0.01-10 mL / min; and / or The mass ratio of sodium borohydride to sodium hydroxide is 1:1-4; The mass ratio of sodium borohydride to ascorbic acid is 1:4-9; The mass ratio of the sodium borohydride to sodium citrate is 1:4-15.

8. The method according to claim 6 or 7, wherein the metal ions comprise one or more selected from Fe, Al, Cu, Au, Pt, Pd, Ni, Ti and Ag; and / or The mass ratio of the two metal ions is 1-9:9-1; and / or The biological carrier comprises a polypeptide, preferably, the polypeptide comprises a bacteriophage, preferably an M13 bacteriophage; Further preferably, the concentration of the biological carrier is 0.5×10^11 to 10×10^11 pfu / L; Preferably, the metal catalyst is the metal catalyst according to any one of claims 1 to 5.

9. Use of the metal catalyst according to any one of claims 1 to 5 or the metal catalyst according to any one of claims 6 to 8 in a kit for diagnosing prostate cancer, gout or diabetes.

10. A kit for diagnosing prostate cancer, gout or diabetes, comprising the metal catalyst according to any one of claims 1 to 5 or the metal catalyst according to any one of claims 6 to 8.

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