Nano enzyme as well as preparation method and application thereof

Abstract

The invention discloses a nano enzyme as well as a preparation method and application thereof. According to the preparation method, firstly, carboxyl microsphere emulsion is synthesized, then the carboxyl microsphere emulsion serves as a carrier to further load metal ions, the reaction can be carried out at normal temperature and normal pressure, water serves as a reaction medium, no stabilizer needs to be added, the nano-enzyme can be prepared through simple reduction reaction, the reaction and aftertreatment time is short, and compared with other methods, the preparation method has the advantages that the cost is low; the reaction time is only 10 minutes. The synthesized carboxyl latex microspheres are selected for loading metal, the loaded nano-enzyme is not prone to falling off, the stability and dispersity of the nano-enzyme can be improved, and the nano-enzyme prepared through the method is high in activity. Experiments show that the enzyme activity of the nano-enzyme can reach 313.27 U / mg, and the enzyme activity is not influenced by 30min of 240W ultrasonic treatment and 30min of 650W ultrasonic crushing, so that effective secondary dispersion of the nano-enzyme after long-term storage is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme preparation, in particular to a nanozyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are a type of enzyme mimics that have both the unique properties of nanomaterials and catalytic functions. Since the discovery of ferroferric oxide (magnetic iron) nanozymes, many metal oxide nanozymes and non-metallic nanozymes have been discovered, such as titanium dioxide nanoparticles, copper oxide nanoparticles, carbon nanodots, and graphene oxide (carbon nanomaterials composed of carbon atoms), all of which have catalytic activity. The reason why nanozymes are considered to have enzyme-like catalytic effects is not only because they have efficient catalytic activity and can catalyze the same chemical reactions as biological enzymes, but also because they can efficiently catalyze chemical reactions at room temperature and pressure like biological enzymes.

[0003] At present, most metal nanozymes are prepared by hydrothermal deposition and incipient wetness impregnation deposition. The hydrothermal method requires high temperature and high pressure, and requires the addition of stabilizers. The incipient wetness impregnation deposition requires the use of templates, the synthesized nanoparticles have a large particle size, and the nanoparticle suspension has poor stability during the deposition process on the template. At the same time, most nanozymes currently have low enzymatic activity and need to be synthesized under high temperature and high pressure conditions, and organic solvents that are harmful to the human body need to be used as reaction media, and the reaction and post-processing time are long. Therefore, further research on the preparation method of nanozymes is still needed. Summary of the invention

[0004] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is how to obtain a nanozyme with good stability, good dispersibility and high enzyme activity under low reaction conditions. Based on this, the present invention provides a nanozyme and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a nanozyme, comprising the following steps:

[0007] (1) Preparation of carboxyl microsphere emulsion:

[0008] Styrene, porogen, emulsifier, initiator and carboxyl donor are mixed, reacted, washed, centrifuged and resuspended in pure water to obtain carboxyl microsphere emulsion;

[0009] (2) Preparation of nanozyme: The carboxyl microsphere emulsion obtained in step (1) is used as a carrier, loaded with metal ions, and prepared by reduction reaction.

[0010] Preferably, in step (1), the porogen is polyvinyl alcohol, the emulsifier is sodium dodecyl sulfate, the initiator is potassium persulfate or sodium persulfate, and the carboxyl donor is citric acid, acrylic acid, aspartic acid, glutamic acid or ethylenediaminetetraacetic acid;

[0011] The reaction temperature is 60-90° C., and the reaction time is 5-10 h.

[0012] Preferably, in the step (1), the preparation method of the carboxyl microsphere emulsion is specifically as follows: styrene, a porogen, and an emulsifier are added to pure water, after ultrasonication, a carboxyl donor is added, after stirring and dissolving, nitrogen is passed through to replace and remove oxygen, an initiator is added, after stirring and dissolving, a reaction is carried out, washing, centrifuging, and resuspending in pure water to obtain the carboxyl microsphere emulsion.

[0013] Preferably, the volume ratio of styrene to pure water is 1:(5-10); the volume ratio of the mass of the porogen to styrene is 1.5g:(6-10)mL; the volume ratio of the mass of the emulsifier to styrene is 0.2g:(6-10)mL; the volume ratio of the mass of the carboxyl donor to styrene is 1g:(2-10)mL; the volume ratio of the mass of the initiator to styrene is 0.1g:(6-12)mL; the power of the ultrasound is 220-260W; and the ultrasound time is 20-40min.

[0014] Preferably, in step (2), the specific preparation method of the nanozyme is:

[0015] 1) dispersing the carboxyl microsphere emulsion obtained in step (1) in a solvent and subjecting it to ultrasonic treatment;

[0016] 2) After ultrasonic treatment, adding the precursor containing metal ions to step 1), pre-stirring, and then ultrasonic treatment;

[0017] 3) After ultrasound, a reducing agent is added to carry out a reaction;

[0018] 4) After the reaction, add acid to the solution in step 3) to adjust the pH value, wash and centrifuge to obtain the product.

[0019] Preferably, the particle size of the carboxyl microspheres in the carboxyl microsphere emulsion is 200-300 nm;

[0020] The metal ion is a platinum ion, an iridium ion, a ruthenium ion, a cerium ion or a gold ion; the precursor containing the metal ion is an inorganic compound or a coordination complex containing the metal ion;

[0021] The solvent is pure water; the reducing agent is NaBH 4 , KBH 4 or LiBH 4 .

[0022] Preferably, in step 1), the volume ratio of the mass of carboxyl microspheres to the solvent in the carboxyl microsphere emulsion is 1 mg: 1 mL;

[0023] In the step 2), the mass ratio of the amount of metal ions in the precursor to the mass ratio of the carboxyl microspheres in the carboxyl microsphere emulsion is (0.1-0.5) mmo l: 40 mg;

[0024] In the step 3), the ratio of the amount of the reducing agent to the amount of the metal ion in the precursor is (1-10):1; the reaction temperature is 25-60° C.; and the reaction time is 5-60 min.

[0025] Preferably, in step 1), the power of the ultrasonic treatment is 220-260W; the ultrasonic treatment time is 5-10min;

[0026] In the step 2), the pre-stirring time is 5-10 min; the ultrasonic treatment power is 220-260 W, and the ultrasonic treatment time is 10-60 min; the ultrasonic treatment temperature is lower than the reaction temperature of step 3);

[0027] In the step 4), the acid is 0.1M HCl solution; the acid is added to adjust the solution pH to 7; the centrifugal speed is 10000-15000 rpm; and the centrifugal time is 5-10 min.

[0028] The present invention provides a nanozyme, and the nanozyme is prepared by the preparation method.

[0029] The present invention provides an application of the nanozyme in preparing an immunochromatography reagent.

[0030] The technical solution of the present invention achieves the following beneficial technical effects:

[0031] The carboxyl latex microspheres prepared by the present invention have hollow mesopores and a porous groove structure on the surface, which is conducive to the loading of the nanozyme by the carrier and the loaded nanozyme is not easy to fall off, which can improve its stability and dispersibility. The activity of the nanozyme prepared by the method of the present invention is high. Experiments show that the enzyme activity of the nanozyme of the present invention can reach 313.27U / mg, and 240W ultrasound for 30min and 650W ultrasonic crushing for 30min have no effect on the enzyme activity. This property will greatly facilitate the effective secondary dispersion of the nanozyme after long-term storage.

[0032] The preparation method of the present invention only needs to be carried out at room temperature and pressure, and water is used as the reaction medium. No stabilizer needs to be added, and nanozymes can be obtained through a simple reduction reaction. The reaction and post-processing time are short. Compared with other methods, the reaction time is only 10 minutes.

[0033] The microspheres used in the present invention have a structure of "latex microspheres as the inner core, and a carboxyl modified layer coated on the outside". The carboxyl-based structure and the high enzyme activity of the nanozyme will be beneficial to the application of the obtained nanozyme in products such as immunochromatography and chemiluminescence. The microspheres of the present invention can alleviate the common problem of easy aggregation of metal nanoparticles to a certain extent because of their low density and charge, and can also improve the stability of nanozyme chromatography on immunochromatography test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 1 of the present invention;

[0035] Figure 2 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 2 of the present invention;

[0036] Figure 3 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 3 of the present invention;

[0037] Figure 4 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 4 of the present invention;

[0038] Figure 5 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 5 of the present invention;

[0039] Figure 6 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 6 of the present invention;

[0040] Figure 7 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 7 of the present invention;

[0041] Figure 8 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 8 of the present invention;

[0042] Fig. 9 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 9 of the present invention;

[0043] Fig.10 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 10 of the present invention;

[0044] Fig.11 Linear fitting diagram of the relative enzyme activity determination of the nanozyme obtained in Comparative Example 1 of the present invention;

[0045] Fig.12 Linear fitting diagram of the relative enzyme activity determination of the nanozyme obtained in Comparative Example 2 of the present invention;

[0046] Fig.13 Linear fitting diagram of the relative enzyme activity determination of the nanozyme obtained in Comparative Example 3 of the present invention;

[0047] Fig.14 Linear fitting diagram of the specific enzyme activity of the nanozyme obtained in Example 2 of the present invention after being treated with different ultrasonic powers;

[0048] Among them, (a) after 240W ultrasound for 30 minutes; (b) after 650W ultrasound for 30 minutes;

[0049] Fig.15 Electron microscopic images of the carboxyl microspheres obtained in Example 1 and Example 6 of the present invention;

[0050] Among them, (a) scanning electron microscopy image of 300nm microspheres obtained in Example 1; (b) transmission electron microscopy image of 300nm microspheres obtained in Example 1; (c) transmission electron microscopy image of 200nm microspheres obtained in Example 6;

[0051] Fig.16 Transmission electron microscopy image of the nanozyme obtained in Example 1 of the present invention;

[0052] Fig.17 Transmission electron microscopy image of the nanozyme obtained in Example 2 of the present invention;

[0053] Fig.18 Transmission electron microscopy image of the nanozyme obtained in Example 5 of the present invention;

[0054] Fig.19 Transmission electron microscopy image of the nanozyme obtained in Example 6 of the present invention;

[0055] Fig. 20 Transmission electron microscopy image of the nanozyme obtained in Example 7 of the present invention;

[0056] Fig.21 Transmission electron microscopy image of the nanozyme obtained in Example 10 of the present invention;

[0057] Fig. 22 Transmission electron microscopy image of the platinum nanozyme obtained in Comparative Example 4 of the present invention;

[0058] Fig.23 The enzyme activity stability of the nanozyme solution and freeze-dried enzyme powder obtained in Example 2 of the present invention; DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and technical effect of the invention of this application clearer, the technical scheme in the embodiment of this application will be clearly and completely described below in conjunction with the drawings in the embodiment of this application. Obviously, the described embodiment is a part of the embodiment of this application, not all the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0060] In view of the technical problems existing in the prior art, the present invention provides a method for preparing a nanozyme, comprising the following steps:

[0061] (1) Preparation of carboxyl microsphere emulsion;

[0062] (2) Preparation of nanozyme: The carboxyl microsphere emulsion obtained in step (1) is used as a carrier, loaded with metal ions, and prepared by reduction reaction.

[0063] In a preferred embodiment of the present invention, in step (1), the preparation method of the carboxyl microsphere emulsion is: styrene, a porogen, an emulsifier, an initiator, and a carboxyl donor are mixed, and after reaction, the mixture is washed, centrifuged, and resuspended in pure water to obtain the carboxyl microsphere emulsion.

[0064] The porogen is polyvinyl alcohol, the emulsifier is sodium dodecyl sulfate, the initiator is potassium persulfate or sodium persulfate, and the carboxyl donor is citric acid, acrylic acid, aspartic acid, glutamic acid or ethylenediaminetetraacetic acid;

[0065] The reaction temperature is 60-90°C and the reaction time is 5-10h;

[0066] In a preferred embodiment of the present invention, in the step (1), the preparation method of the carboxyl microsphere emulsion is specifically as follows: styrene, a porogen, and an emulsifier are added to pure water, after ultrasonication, a carboxyl donor is added, after stirring and dissolving, nitrogen is passed through to replace and remove oxygen, an initiator is added, after stirring and dissolving, a reaction is carried out, washing, centrifugation, and resuspending in pure water to obtain the carboxyl microsphere emulsion.

[0067] The volume ratio of styrene to pure water is 1:(5-10); the volume ratio of the mass of the porogen to styrene is 1.5g:(6-10)mL; the volume ratio of the mass of the emulsifier to styrene is 0.2g:(6-10)mL; the volume ratio of the mass of the carboxyl donor to styrene is 1g:(2-10)mL; the volume ratio of the mass of the initiator to styrene is 0.1g:(6-12)mL;

[0068] The ultrasonic power is 220-260W; the ultrasonic time is 20-40min;

[0069] In a preferred embodiment of the present invention, in step (2), the particle size of the carboxyl microspheres is 200-300 nm;

[0070] The metal ion is platinum ion, iron ion, iridium ion, ruthenium ion, cerium ion or gold ion;

[0071] The reaction temperature of the reduction reaction is 25-60°C, and the reaction time is 5-60min;

[0072] The reducing agent in the reduction reaction is NaBH 4 , KBH 4or Li BH 4 ;

[0073] In a preferred embodiment of the present invention, in step (2), the specific preparation method of the nanozyme is:

[0074] 1) dispersing the carboxyl microsphere emulsion obtained in step (1) in a solvent and subjecting it to ultrasonic treatment;

[0075] 2) After ultrasonic treatment, adding the precursor containing metal ions to step 1), pre-stirring, and then ultrasonic treatment;

[0076] 3) After ultrasound, a reducing agent is added to carry out a reaction;

[0077] 4) After the reaction, add acid to the solution in step 3) to adjust the pH value, wash, and centrifuge to obtain the product.

[0078] In a preferred embodiment of the present invention, in the step 1), the volume ratio of the mass of the carboxyl microspheres in the carboxyl microsphere emulsion to the solvent is 1 mg: 1 mL;

[0079] The mass of carboxyl microspheres in the carboxyl microsphere emulsion (mg) = the concentration of the carboxyl microsphere emulsion (mg / mL) × the volume of the carboxyl microsphere emulsion (mL);

[0080] The solvent is pure water; the power of the ultrasonic treatment is 220-260W; the ultrasonic time is 5-10min;

[0081] In a preferred embodiment of the present invention, in step 2), the precursor containing metal ions is an inorganic compound or coordination complex containing metal ions; the metal ions are platinum ions, iridium ions, ruthenium ions, cerium ions or gold ions;

[0082] In the step 2), the enzyme loading ratio, i.e., the mass ratio of the amount of metal ions in the precursor to the mass ratio of the carboxyl microspheres in the carboxyl microsphere emulsion is (0.1-0.5) mmo l: 40 mg;

[0083] In a preferred embodiment of the present invention, in the step 2), the enzyme loading ratio, i.e., the mass ratio of the amount of metal ions in the precursor to the mass ratio of the carboxyl microspheres in the carboxyl microsphere emulsion is 0.2 mmol:40 mg;

[0084] In a preferred embodiment of the present invention, in the step 2), the pre-stirring time is 5-10 min; the ultrasonic treatment power is 220-260 W, and the ultrasonic treatment time is 10-60 min; the ultrasonic treatment temperature is lower than the reaction temperature of step 3);

[0085] In a preferred embodiment of the present invention, in step 3), the ratio of the amount of the reducing agent to the amount of the metal ion in the precursor is (1-10):1;

[0086] In a preferred embodiment of the present invention, in step 3), the ratio of the amount of the reducing agent to the amount of the metal ions in the precursor is 5:1;

[0087] In a preferred embodiment of the present invention, in the step 3), the reaction temperature is 25-60°C (for example, 25°C, 30°C, 40°C, 50°C or 60°C); the reaction time is 5-60min (for example, 5min, 10min, 20min, 40min or 60min);

[0088] In a preferred embodiment of the present invention, in step 3), the reaction temperature is 30° C. and the reaction time is 10 min;

[0089] In a preferred embodiment of the present invention, in step 4), the acid is a 0.1M HCl solution; the acid is added to adjust the pH of the solution to 7;

[0090] In the step 4), the centrifugal speed is 10000-15000 rpm; the centrifugal time is 5-10 min;

[0091] The present invention provides a nanozyme, and the nanozyme is prepared by the preparation method.

[0092] In a preferred embodiment of the present invention, the nanozyme is dispersed in pure water and stored.

[0093] The present invention provides an application of the nanozyme in preparing an immunochromatography reagent.

[0094] The following is a detailed description of a nanozyme of the present invention and its preparation method and application through specific examples.

[0095] Example 1

[0096] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0097] (1) Preparation of carboxyl microsphere emulsion;

[0098] To 50 mL of pure water, 10 mL of styrene, 1.5 g of porogen (polyvinyl alcohol), and 0.2 g of emulsifier (sodium dodecyl sulfate) were added, and after 240 W ultrasound for 30 min, 1.0 g of carboxyl donor (citric acid) was added, and after stirring and dissolving, nitrogen was passed through to replace and remove oxygen, and then 0.1 g of initiator (potassium persulfate) was added. After stirring and dissolving, the mixture was reacted at 70° C. for 5 h, washed with 50% (v / v) ethanol solution, centrifuged 3 times, and the precipitate was resuspended in pure water to obtain a carboxyl microsphere emulsion with a concentration of 40 mg / mL and a particle size of the carboxyl microspheres of about 300 nm; wherein the concentration of the carboxyl microsphere emulsion was determined according to the solid content.

[0099] (2) Preparation of nanozymes:

[0100] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0101] 2) After ultrasound, add 52.8 mg (0.2 mmol) of RuCl 3 ·3H 2 O solid, pre-stir for 5 minutes, then ultrasonically treat at 240W for 5 minutes to dissolve; the temperature during the ultrasonic treatment is less than 30°C, completing the pre-adsorption;

[0102] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0103] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0104] Example 2

[0105] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0106] (1) Preparation of carboxyl microsphere emulsion;

[0107] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 1, and the concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL.

[0108] (2) Preparation of nanozymes:

[0109] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0110] 2) After ultrasound, 103.5 mg (0.2 mmol) of H 2 PtCl 6 6H 2 O solid, pre-stir for 5 minutes, then ultrasonically treat at 240W for 5 minutes to dissolve; the temperature during the ultrasonic treatment is less than 30°C, completing the pre-adsorption;

[0111] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0112] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0113] Example 3

[0114] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0115] (1) Preparation of carboxyl microsphere emulsion;

[0116] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 1, and the concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL.

[0117] (2) Preparation of nanozymes:

[0118] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0119] 2) After ultrasound, 59.7 mg (0.2 mmol) IrCl was added to step 1). 3 The solid was pre-stirred for 5 minutes, and then ultrasonically treated at a power of 240 W for 5 minutes to dissolve; the temperature during the ultrasonic treatment was less than 30°C, and the pre-adsorption was completed;

[0120] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0121] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0122] Example 4

[0123] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0124] (1) Preparation of carboxyl microsphere emulsion;

[0125] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 1, and the concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL.

[0126] (2) Preparation of nanozymes:

[0127] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0128] 2) After ultrasound, add 49.3 mg (0.2 mmol) CeCl 3 , pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240W; the temperature during the ultrasonic treatment process is less than 30°C, completing the pre-adsorption;

[0129] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0130] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0131] Example 5

[0132] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0133] (1) Preparation of carboxyl microsphere emulsion;

[0134] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 1, and the concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL.

[0135] (2) Preparation of nanozymes:

[0136] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0137] 2) After ultrasound, add 67.9 mg (0.2 mmol) of tetrachloroauric acid (AuCl 4 H), pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240 W; the temperature during the ultrasonic treatment process is less than 30° C., and the pre-adsorption is completed;

[0138] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0139] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0140] Example 6

[0141] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0142] (1) Preparation of carboxyl microsphere emulsion;

[0143] The preparation method of the carboxyl microsphere emulsion in this embodiment is different from that in Example 1 in that the added carboxyl donor is acrylic acid, the added amount is 1.0 g, and the other raw materials and parameters are the same as those in Example 1; the concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL, and the particle size of the carboxyl microspheres is about 200 nm;

[0144] (2) Preparation of nanozymes:

[0145] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0146] 2) After ultrasound, add 52.8 mg (0.2 mmol) of RuCl 3 ·3H 2 O, pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240 W; the temperature during the ultrasonic treatment process is less than 30°C, completing the pre-adsorption;

[0147] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0148] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0149] Example 7

[0150] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0151] (1) Preparation of carboxyl microsphere emulsion;

[0152] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 6. The concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL, and the particle size of the carboxyl microspheres is about 200 nm.

[0153] (2) Preparation of nanozymes:

[0154] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0155] 2) After ultrasound, 103.5 mg (0.2 mmol) of H 2 PtCl 6 6H 2 O, pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240 W; the temperature during the ultrasonic treatment process is less than 30°C, completing the pre-adsorption;

[0156] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0157] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0158] Example 8

[0159] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0160] (1) Preparation of carboxyl microsphere emulsion;

[0161] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 6. The concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL, and the particle size of the carboxyl microspheres is about 200 nm.

[0162] (2) Preparation of nanozymes:

[0163] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0164] 2) After ultrasound, 59.7 mg (0.2 mmol) IrCl was added to step 1). 3 , pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240W; the temperature during the ultrasonic treatment process is less than 30°C, completing the pre-adsorption;

[0165] 3) After ultrasound, add 37.8 mg (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0166] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0167] Example 9

[0168] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0169] (1) Preparation of carboxyl microsphere emulsion;

[0170] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 6. The concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL, and the particle size of the carboxyl microspheres is about 200 nm.

[0171] (2) Preparation of nanozymes:

[0172] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0173] 2) After ultrasound, add 49.3 mg (0.2 mmol) CeCl 3 , pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240W; the temperature during the ultrasonic treatment process is less than 30°C, completing the pre-adsorption;

[0174] 3) After ultrasound, add 37.8 g (1 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0175] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0176] Example 10

[0177] This embodiment relates to a method for preparing a nanozyme, comprising the following steps:

[0178] (1) Preparation of carboxyl microsphere emulsion;

[0179] The preparation method of the carboxyl microsphere emulsion in this example is the same as that in Example 1. The concentration of the obtained carboxyl microsphere emulsion is 40 mg / mL, and the particle size of the carboxyl microspheres is about 200 nm.

[0180] (2) Preparation of nanozymes:

[0181] 1) using the carboxyl microsphere emulsion obtained in step (1) as a carrier, dispersing 1 mL of the carboxyl microsphere emulsion in 40 mL of pure water, and subjecting the mixture to ultrasonic treatment at a power of 240 W for 5 min;

[0182] 2) After ultrasound, add 67.9 mg (0.2 mmol) of tetrachloroauric acid (AuCl 4 H), pre-stir for 5 minutes, and then ultrasonically treat for 5 minutes at a power of 240 W; the temperature during the ultrasonic treatment process is less than 30° C., and the pre-adsorption is completed;

[0183] 3) After ultrasound, add 37.8 (1.0 mmol) NaBH 4 , heat to 30°C and react for 10 min;

[0184] 4) After the reaction, 0.1 M HCl solution was added to step 3) to adjust the solution pH to 7, washed with pure water, centrifuged 3 times (centrifugal speed was 15000 rpm; centrifugal time was 10 min), and then the precipitate was resuspended in pure water to obtain.

[0185] Comparative Example 1

[0186] This comparative example relates to a method for preparing a nanozyme. The difference between this comparative example and Example 2 is that in step (2) of this comparative example, the enzyme loading ratio, that is, the mass ratio of the amount of metal ions in the precursor to the mass ratio of carboxyl microspheres in the carboxyl microsphere emulsion is 0.1 mmol: 40 mg; that is, in step 1) of this comparative example, 1 mL of carboxyl microsphere emulsion is dispersed in 40 mL of pure water; in step 2), 51.75 mg of

[0187] (0.1mmol)H 2 PtCl 6 6H 2 O;

[0188] The other steps and parameters are the same as those in Example 2.

[0189] Comparative Example 2

[0190] This comparative example relates to a method for preparing a nanozyme. The difference between this comparative example and Example 2 is that in step 3) of step (2) of this comparative example, the reaction time is 30 min; that is, after ultrasound in step 3) of this comparative example, 37.8 mg (1.0 mmol) of NaBH 4 , heat to 30°C and react for 30 minutes;

[0191] The other steps and parameters are the same as those in Example 2.

[0192] Comparative Example 3

[0193] This comparative example relates to a method for preparing a nanozyme. The difference between this comparative example and Example 2 is that in step 3) of step (2) of this comparative example, the reaction temperature is 50°C; that is, after ultrasound in step 3) of this comparative example, 37.8 mg (1.0 mmol) of NaBH 4 , heat to 50°C and react for 10 min;

[0194] The other steps and parameters are the same as those in Example 2.

[0195] Comparative Example 4

[0196] This comparative example relates to a method for preparing a nanozyme. The difference between this comparative example and Example 2 is that carboxyl microspheres are not involved. The method for preparing the nanozyme in this comparative example is:

[0197] 1) At room temperature, add 103.5 mg (0.2 mmol) H 2 PtCl 6 6H 2 O, stir for 5 min to dissolve.

[0198] 2) Raise the temperature to 30°C and add 37.8 mg (1.0 mmol) NaBH 4 , react for 10 minutes;

[0199] 3) After the reaction, add 0.1M HCl solution to step 2) to adjust the solution pH to 7, wash with pure water, and centrifuge for 3 times (centrifugal speed is 15000 rpm; centrifugal time is 10 min) to obtain. Performance measurement and analysis:

[0200] (1) Enzyme activity detection

[0201] The products obtained in the embodiment of the present invention and the comparative example were respectively dispersed in pure water to obtain the sample enzyme solution to be tested, and the enzyme activity of the obtained nanozyme was detected according to the standard GB / T 37966-2019 nanotechnology iron oxide nanoparticle peroxidase activity measurement method.

[0202] At the same time, the effect of ultrasonic treatment on the activity of nanozymes was investigated: 5 mL of the nanozyme solution obtained in Example 2 was taken, and the enzyme activity was measured after 240 W ultrasound for 30 min and 650 W ultrasound for 30 min.

[0203] 2 mL of 0.2 M acetate buffer (pH 3.6), 100 μL of 30% (w / w) H 2 O 2 , 100 μL 10 mg / mL TMBDMSO solution, and mixed with 100 μL enzyme solution of the sample to be tested with different concentrations, respectively. The absorbance value at 652 nm was measured at 37°C.

[0204] The peroxidase activity was calculated as follows: Where:

[0205] B nano : The number of nanozyme peroxidase activity units, unit is U;

[0206] V: total volume of reaction solution, in μL;

[0207] ε: molar absorption coefficient of TMB derivative, unit is 39000 mol -1 ·L·cm -1 ;

[0208] l: cuvette optical path, in cm;

[0209] ΔA / Δt: The rate of change of absorbance of reaction solutions of different concentrations over time, in cm -1 .

[0210] Then, according to the B corresponding to the enzyme solution of the sample with different concentrations nano The value was linearly fitted with the corresponding nanozyme mass, and the slope of the fitting line (k) was the measured peroxidase specific activity of the nanozyme, in U / mg. Figure 1-14 As shown in Table 1.

[0211] Table 1 Enzyme activity of nanozymes obtained in the examples of the present invention

[0212]

[0213] As can be seen from Table 1, there are large differences in the activity of nanozymes prepared after microspheres of the same particle size adsorb different metal ions; microspheres of the same particle size adsorb the same metal ions, but different ratios of microspheres to metal ions will also lead to differences in enzyme activity. Among them, the platinum nanozyme in Comparative Example 4 was severely agglomerated and could not be effectively dispersed in water, so the enzyme activity was not detected.

[0214] ① According to the comparison between Examples 1-5 and Examples 6-10, it can be seen that the enzymatic activity of the latex nanozymes prepared by using microsphere emulsions of different particle sizes is significantly different. Compared with 200nm microspheres, the nanozymes prepared under the same conditions generally have higher enzymatic activity. The main reason is that the thickness of the carboxyl modified layer contained in the 300nm microspheres is much greater than that contained in the 200nm microspheres ( Fig.15 ), the carboxyl modified layer can improve the dispersibility of the microspheres themselves, so that they can load the nanozyme precursor metal ions to the greatest extent in the pre-dispersion stage, thereby increasing the loading amount of the microspheres on the nanozyme after the reaction. Figure 16-21The loading amount of the microspheres can be verified on the TEM image; on the other hand, after the microspheres are loaded with nanozymes, the prepared microsphere nanozymes have better dispersibility, so that the active sites of the nanozymes are fully exposed, thereby exhibiting higher enzyme activity.

[0215] ② According to the comparative tests under different conditions in the best embodiment 2 (Comparative Examples 1-3), it can be seen that reducing the molar amount of platinum precursor metal ions, prolonging the reaction time and increasing the reaction temperature will lead to a decrease in the enzyme activity of the nanozyme. This is because reducing the molar amount of platinum precursor metal ions will lead to a decrease in the loading amount of the microspheres on the nanozyme; prolonging the reaction time will further increase the particle size of the nanozyme; NaBH 4 It is a strong reducing agent. Raising the temperature will make the reaction more intense, resulting in uneven particle size and the growth of nanoparticles before they are fully nucleated, which reduces the stability of the nanozyme. We found that during the reaction at 50°C, as time went by, a large amount of precipitation aggregated in the system and the solvent gradually became clear, indicating that the nanozyme under this condition was severely aggregated.

[0216] ③ According to Examples 1-5 or 6-10, for microspheres of the same particle size, when the adsorbed metal ions are different, due to the differences in the active site density and microstructure of each metal ion, the prepared latex nanozyme has significant differences in enzymatic activity. For example, in the present invention, the microspheres loaded with platinum nanozymes have the highest enzyme activity, followed by ruthenium nanozymes and gold nanozymes, and the lowest activity is cerium nanozymes. This is mainly because the metal centers and outer electron distribution of platinum and ruthenium atoms can maximize the atomic utilization efficiency and active site density, and the catalytic activity is 10 to 100 times higher than most traditional nanozymes; and cerium nanozymes are easily oxidized to cerium oxide (CeO in the air) due to their high sensitivity to the environment. 2 ), which makes its enzyme activity and stability inferior to those of several other nanozymes. Most of the cerium nanozymes reported so far exist in their oxidized state.

[0217] ④According to Fig.14 It can be seen that compared with the initial enzyme activity of 313.27U / mg of the microsphere nanozyme prepared in Example 2, the enzyme activity was 309.25U / mg after 240W ultrasound for 30min; after 650W ultrasound for 30min, the enzyme activity was 320.22U / mg. Increasing the ultrasonic power had no significant effect on the enzyme activity of the prepared microsphere nanozyme, and its value was within the fluctuation range of 5%, which is a normal phenomenon. This property will greatly facilitate the effective secondary dispersion of the enzyme solution after long-term storage.

[0218] (2) Transmission electron microscopy analysis

[0219] ① The carboxyl microspheres obtained in Example 1 and Example 6 were analyzed by scanning electron microscopy and transmission electron microscopy. The results are as follows: Fig.15(a) is a scanning electron microscope image of 300 nm microspheres prepared in Example 1, (b) is a transmission electron microscope image of 300 nm microspheres prepared in Example 1, and (c) is a transmission electron microscope image of 200 nm microspheres prepared in Example 6.

[0220] As can be seen from the figure, the prepared microspheres are spherical, with uniform particle size and good dispersibility; they are core-shell structures with "latex microspheres as the inner core and a carboxyl modification layer on the outside", and the thickness of the carboxyl modification layer of the 300nm microspheres is much greater than that of the 200nm microspheres. This is because the 300nm microspheres use citric acid as the carboxyl donor, and citric acid contains 3 -COOHs and one -OH, of which two -COOHs are located at the ends of the carbon chain; while the 200nm microspheres use acrylic acid as the carboxyl donor, which contains only one -COOH. In addition, since the molecular structure of acrylic acid is composed of a vinyl group and a carboxyl group, this chemical structure gives acrylic acid active chemical properties, allowing it to polymerize at room temperature in the air, and its polymerization reaction is a spontaneous process, which is also the main reason why the dispersibility and particle size uniformity of the 200nm microspheres are not as good as those of the 300nm microspheres.

[0221] ② The products obtained from Examples 1, 2, 5 (300 nm microspheres) and Examples 6, 7, 10 (200 nm microspheres) with higher enzyme activity of the present invention were analyzed by transmission electron microscopy. The results are as follows Figure 16-21 shown.

[0222] As can be seen from the figure, the loading amount of nanozymes on 300nm microspheres is much greater than that on 200nm microspheres, and the loading amount of platinum, ruthenium and gold nanozymes on microspheres of both particle sizes tends to decrease in sequence, which is consistent with the difference in their enzyme activities. This may be because the combination of platinum ions and the carboxyl modification layer is more stable.

[0223] ③ In addition, the monomeric platinum nanozyme prepared in Comparative Example 4 was subjected to transmission electron microscopy analysis, and the results were as follows: Fig. 22 The average particle size was measured to be 3.42±0.5nm, and the agglomeration was serious and could not be effectively dispersed, so the enzyme activity was not tested. However, after being loaded with microspheres, the dispersibility of the nanozyme was significantly improved, which would be beneficial to the exposure of the enzyme activity sites and further improve the enzyme activity.

[0224] (3) Stability analysis

[0225] The nanozyme obtained by the present invention can be dispersed in pure water for storage (enzyme solution), or can be freeze-dried for storage (enzyme powder).

[0226] The nanozyme obtained in Example 2 of the present invention was: ① dispersed in pure water to obtain 5 mg / mL enzyme solution; ② freeze-dried for 24 hours to obtain freeze-dried enzyme powder; compared the specific enzyme activity of the enzyme solution and the freeze-dried enzyme powder within 0-15 weeks, and analyzed the stability of the nanozyme. The results are as follows Fig.23shown.

[0227] according to Fig.23 It can be seen that after freeze-drying, the initial enzyme activity of the nanozyme is significantly reduced, and the enzyme activity is reduced to the maximum within 1 week after freeze-drying, and then basically remains stable; while the enzyme activity of the enzyme solution is lost to the maximum after 4 weeks, and then basically remains stable, and its value is higher than the activity of the freeze-dried nanozyme after stabilization. This is because the nanozyme is easier to aggregate after drying, and the aggregation is alleviated during the storage of the enzyme solution due to its hydrophobic interaction, and the enzyme activity loss is slower.

[0228] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing a nanozyme, characterized in that: The following steps are involved: (1) Preparation of carboxyl microsphere emulsion: Styrene, porogen, emulsifier, initiator and carboxyl donor are mixed, reacted, washed, centrifuged and resuspended in pure water to obtain carboxyl microsphere emulsion; (2) Preparation of nanozyme: The carboxyl microsphere emulsion obtained in step (1) is used as a carrier, loaded with metal ions, and prepared by reduction reaction.

2. The preparation method according to claim 1, characterized in that In step (1), the porogen is polyvinyl alcohol, the emulsifier is sodium dodecyl sulfate, the initiator is potassium persulfate or sodium persulfate, and the carboxyl donor is citric acid, acrylic acid, aspartic acid, glutamic acid or ethylenediaminetetraacetic acid; The reaction temperature is 60-90° C., and the reaction time is 5-10 h.

3. The preparation method according to claim 2, characterized in that: In the step (1), the preparation method of the carboxyl microsphere emulsion is specifically as follows: styrene, a porogen, and an emulsifier are added to pure water, after ultrasonication, a carboxyl donor is added, after stirring and dissolving, nitrogen is passed to replace and remove oxygen, an initiator is added, after stirring and dissolving, a reaction is carried out, washing, centrifugation, and resuspending in pure water to obtain the carboxyl microsphere emulsion.

4. The preparation method according to claim 3, characterized in that: The volume ratio of styrene to pure water is 1:(5-10); the volume ratio of the mass of the porogen to styrene is 1.5 g:(6-10) mL; the volume ratio of the mass of the emulsifier to styrene is 0.2 g:(6-10) mL; the volume ratio of the mass of the carboxyl donor to styrene is 1 g:(2-10) mL; the volume ratio of the mass of the initiator to styrene is 0.1 g:(6-12) mL; the power of the ultrasound is 220-260 W; and the ultrasound time is 20-40 min.

5. The preparation method according to claim 1, characterized in that: In the step (2), the specific preparation method of the nanozyme is: 1) dispersing the carboxyl microsphere emulsion obtained in step (1) in a solvent and subjecting it to ultrasonic treatment; 2) After ultrasonic treatment, adding the precursor containing metal ions to step 1), pre-stirring, and then ultrasonic treatment; 3) After ultrasound, a reducing agent is added to carry out a reaction; 4) After the reaction, add acid to the solution in step 3) to adjust the pH value, wash and centrifuge to obtain the product.

6. The preparation method according to claim 5, characterized in that: The particle size of the carboxyl microspheres in the carboxyl microsphere emulsion is 200-300 nm; The metal ion is a platinum ion, an iridium ion, a ruthenium ion, a cerium ion or a gold ion; the precursor containing the metal ion is an inorganic compound or a coordination complex containing the metal ion; The solvent is pure water; the reducing agent is NaBH4, KBH4 or LiBH4.

7. The preparation method according to claim 5, characterized in that: In the step 1), the volume ratio of the mass of the carboxyl microspheres to the solvent in the carboxyl microsphere emulsion is 1 mg: 1 mL; In the step 2), the mass ratio of the amount of metal ions in the precursor to the mass ratio of the carboxyl microspheres in the carboxyl microsphere emulsion is (0.1-0.5) mmol: 40 mg; In the step 3), the ratio of the amount of the reducing agent to the amount of the metal ion in the precursor is (1-10):1; the reaction temperature is 25-60° C.; and the reaction time is 5-60 min.

8. The preparation method according to claim 5, characterized in that: In the step 1), the power of the ultrasonic treatment is 220-260W; the ultrasonic treatment time is 5-10min; In the step 2), the pre-stirring time is 5-10 min; the ultrasonic treatment power is 220-260 W, and the ultrasonic treatment time is 10-60 min; the ultrasonic treatment temperature is lower than the reaction temperature of step 3); In the step 4), the acid is 0.1M HCl solution; the acid is added to adjust the solution pH to 7; the centrifugal speed is 10000-15000 rpm; and the centrifugal time is 5-10 min.

9. A nanozyme, characterized in that: The nanozyme is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nanozyme as claimed in claim 9 in the preparation of immunochromatography reagents.

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