Prussian blue nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a Prussian blue nano-enzyme and a preparation method and application thereof.The preparation method comprises the steps that a water / ethylene glycol mixture serves as a solvent, a mixture of ferric chloride and potassium hexacyanoferrate (III) is reduced through hydrogen peroxide, and nano-particles, namely PBNPs, with the average particle size being about 15 nm are successfully synthesized; the test strip is applied to a vertical flow detection test strip for detecting triglyceride. According to the method disclosed by the invention, low-cost and high-yield preparation of the Prussian blue nano-enzyme can be realized, and the PBNPs have uniform size and good peroxidase activity.

Description

Technical Field

[0001] The invention belongs to the field of chemical synthesis and biological detection, and specifically relates to a Prussian blue nanozyme and a preparation method and application thereof. Background Art

[0002] Chronic diseases refer to health problems that exist for a long time and require continuous treatment or management. Unlike acute diseases, chronic diseases usually last for months or even years, significantly affecting the quality of life and increasing morbidity and mortality, which has become a global challenge. As a rapidly developing country, China faces severe challenges of chronic diseases, especially in urban areas, where the prevalence of cardiovascular diseases, diabetes and chronic respiratory diseases continues to rise. In this context, the introduction of rapid testing technology (Point-of-Care Testing, POCT) is particularly important. POCT allows immediate testing at the patient's side, reducing dependence on laboratories. POCT provides healthy people with convenient self-testing methods, such as blood sugar and cholesterol monitoring, to enhance health management awareness. For patients, POCT enables rapid diagnosis and treatment, reduces the risk of complications, and reduces medical costs. At the clinic level, POCT improves service efficiency, optimizes work processes, and improves patient satisfaction. In terms of public health management, POCT helps the government to carry out effective public health intervention and emergency response, and improve medical services and emergency response capabilities.

[0003] Prussian Blue Nanoparticles (PBNPs) are a porous coordination polymer composed of Fe 2+ and Fe 3+ Ions are alternately connected to cyanide ligands. It was originally used as a dye, but was later approved by the US Food and Drug Administration for the treatment of radioactive cesium ion and thallium ion poisoning due to its excellent biocompatibility and biosafety. The large surface area and high porosity of PBNPs make its internal environment similar to the active site of natural enzymes, which is very suitable for building a biosensor platform with self-testing advantages to detect important analytes with high confidence. The unique structure of PBNPs gives it a rich redox potential, thereby exhibiting a variety of enzyme activities, including superoxide dismutase, peroxidase and catalase. Among them, the peroxidase activity of PBNPs makes it one of the most promising alternatives to horseradish peroxidase (HRP).

[0004] The synthesis methods of PBNPs include hydrothermal synthesis, microwave-assisted synthesis, self-assembly, and biosynthesis. Although the traditional chemical reduction method has been widely used, the reaction conditions need to be strictly controlled during the synthesis process to ensure the quality of the product. Although other synthesis methods such as hydrothermal synthesis, microwave-assisted synthesis, and self-assembly have excellent performance in some aspects, they also face challenges such as equipment investment and operational complexity. Summary of the invention

[0005] The purpose of the present invention is to provide a Prussian blue nanozyme and its preparation method and application in view of the deficiencies of the prior art. The present application successfully synthesized PBNPs using the water-alcohol method, and the synthesis process has the characteristics of high yield and uniform and stable products. In addition, the present invention also innovatively developed a vertical flow test strip, using PBNPs to replace HRP for efficient detection of triglycerides (TG), a key biomarker of chronic diseases.

[0006] The object of the present invention is achieved through the following technical solutions: In the first aspect, a method for preparing a Prussian blue nanozyme comprises:

[0007] Potassium hexacyanoferrate (III) is dissolved in water to prepare solution A;

[0008] Dissolve ferric chloride in water to make solution B;

[0009] Dilute hydrogen peroxide and ethylene glycol with water to form solution C and solution D respectively; wherein ethylene glycol makes the particle size of the synthesized Prussian blue nanozyme uniform;

[0010] Solutions A, B, and C are added to solution D in sequence, so that solutions A and B continue to react under constant temperature and constant speed stirring; the reaction solution is dialyzed, ultrafiltered, and then freeze-dried to obtain the Prussian blue nanozyme product.

[0011] Furthermore, solutions A, B, and C are added to solution D in sequence, and the final concentration of potassium hexacyanoferrate (III) is 0.5-1.0 M, the final concentration of ferric chloride is 0.5-1.0 M, the final concentration of hydrogen peroxide is 1.5-2.0 M, and the final concentration of ethylene glycol is 75-80% (v / v).

[0012] Furthermore, solutions A, B, and C are added to solution D in sequence, and the final concentrations of potassium hexacyanoferrate (III) are 0.444 M, the final concentration of ferric chloride is 0.444 M, the final concentration of hydrogen peroxide is 1.46 M, and the final concentration of ethylene glycol is 79.4% (v / v).

[0013] Furthermore, the reaction is continuously carried out under constant temperature and constant speed stirring, specifically: the constant temperature is 30-40° C., the constant speed is 800-1000 rpm, and the reaction time is 60-80 min.

[0014] Furthermore, the constant temperature is 35° C., the constant rotation speed is 1000 rpm, and the reaction time is 60 min.

[0015] In the second aspect, the Prussian blue nanozyme is prepared according to the above-mentioned preparation method.

[0016] Furthermore, the average particle size of Prussian blue nanozyme is 15nm, showing the same crystal structure and absorption characteristics as Prussian blue; Prussian blue nanozyme exhibits significant peroxidase-like activity, and its activity remains at a high level in the temperature range of 15-45°C under the condition of pH 4; when hydrogen peroxide is used as the substrate, the maximum reaction rate is 2.33×10 -7 M.s -1 , the Michaelis constant is 2.37mM; when tetramethylbenzidine is used as substrate, the maximum reaction rate is 1.51×10 - 6 M.s -1 , the Michaelis constant is 0.84mM.

[0017] In a third aspect, the use of the Prussian blue nanozyme prepared according to the above preparation method or the above Prussian blue nanozyme in a dry chemical test paper; the dry chemical test paper is used to detect triglyceride concentration

[0018] Furthermore, the dry chemical test paper is prepared as follows:

[0019] (1) dissolving potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, ethylenediaminetetraacetic acid, sodium benzoate and bovine serum albumin in water to ensure that their final concentrations are 25-30 mM, 55-60 mM, 3-4 mM, 6-7 mM and 35-40 μM, respectively, and then adding poly(methyl vinyl ether-alt-maleic anhydride) to a final concentration of 5% (v / v), and mixing well;

[0020] (2) dissolving Prussian blue nanozyme, lipoprotein lipase, glycerol kinase, phosphoglycerol oxidase, 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate in the solution prepared in step (1) in sequence to ensure that their final concentrations are 1-1.8 mg / mL, 45-65 U / mL, 20-60 U / mL, 20-60 U / mL, 0.5-6 mg / mL and 1-8 mg / mL, respectively;

[0021] (3) spraying the reaction solution of step (2) onto the reaction layer at a spray rate of 5 to 9 μL / cm;

[0022] (4) placing the reaction layer obtained in step (3) in an oven at 37° C. for 60 min and then taking it out;

[0023] (5) The reaction layer obtained in step (4) is sequentially pasted onto a bottom plate together with the filter layer and the diffusion layer to obtain a dry chemical test paper.

[0024] Further, the concentration of triglycerides is detected by:

[0025] Several triglyceride simulation samples of different concentrations were prepared, 15 μL of sample was aspirated and dripped vertically, and the RGB value was read through an APP on a smartphone. The test results were linearly fitted by plotting B / (R+G+B) and triglyceride concentration, and the standard curve y=0.003x+0.339 was obtained, with a standard deviation of 0.996 and a detection limit of 0.425 mM.

[0026] In summary, this application uses a water / ethylene glycol (EG) mixture as a solvent and hydrogen peroxide (H 2 O 2 ) reduces ferric chloride (FeCl 3 ) and potassium hexacyanoferrate (III) (K 3 [Fe(CN) 6 ]) of the mixture, successfully prepared small-sized nanoparticles with a particle size of about 15nm, and about 200mL of the reaction stock solution can produce about 1mg of nanoparticles; the obtained PBNPs particles were applied to dry chemical test strips for the detection of triglycerides.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention creatively proposes to use a water / ethylene glycol (EG) mixture as a solvent, using H 2 O 2 Reduction of FeCl 3 With K 3 [Fe(CN) 6 ] mixture, successfully prepared small-sized nanoparticles with a particle size of about 15nm. The method is simple to operate, cost-effective, the prepared nanoparticles have high yield and excellent performance, and the synthesis process is easy to replicate, showing good commercial potential.

[0029] 2. The present invention utilizes the peroxidase-like activity of PBNPs to build a detection platform for efficient determination of TG content. The platform has the following advantages: fast detection speed, wide detection concentration range, low detection sensitivity (i.e., low detection limit), and strong specificity. These characteristics make the detection platform have important application value in clinical and scientific research fields.

[0030] 3. The PBNPs prepared by the present invention have the characteristics of low cost, high yield and simple synthesis. In addition, the PBNPs have uniform size and good peroxidase activity, and show good performance characteristics in TG detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0032] Figure 1 This is a transmission electron microscopy image of Prussian blue nanozyme;

[0033] Figure 2 is the X-ray diffraction pattern of Prussian blue nanozyme;

[0034] Figure 3 This is the UV absorption graph of Prussian blue nanozyme;

[0035] Figure 4 This is a diagram to verify the peroxidase-like activity of Prussian blue nanozyme;

[0036] Figure 5 is the pH dependence diagram of Prussian blue nanozyme;

[0037] Figure 6 is the temperature dependence diagram of Prussian blue nanozyme;

[0038] Figure 7 Prussian blue nanozyme 2 O 2 Steady-state kinetic analysis diagram of

[0039] Figure 8 This is the steady-state kinetic analysis diagram of Prussian blue nanozyme on TMB;

[0040] Fig. 9 It is a schematic diagram of a vertical flow test strip;

[0041] Fig.10 This is a graph of the specificity of the vertical flow test strip test;

[0042] Fig.11 This is the standard curve for triglyceride detection. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below in conjunction with the accompanying drawings. Under the premise of no conflict, the technical features disclosed in each embodiment and implementation method can be used in combination with each other.

[0044] A method for preparing a Prussian blue nanozyme of the present invention comprises:

[0045] Potassium hexacyanoferrate (III) is dissolved in water to prepare solution A;

[0046] Dissolve ferric chloride in water to make solution B;

[0047] Dilute hydrogen peroxide and ethylene glycol with water to form solution C and solution D respectively;

[0048] Solutions A, B, and C are added to solution D in sequence, so that solutions A and B continue to react under constant temperature and constant speed stirring; the reaction solution is dialyzed, ultrafiltered, and then freeze-dried to obtain the Prussian blue nanozyme product.

[0049] In some embodiments, the solutions A, B, and C are added to the solution D in order, and the final concentration of potassium hexacyanoferrate (III) is any value in the range of 0.5 to 1.0 M, the final concentration of ferric chloride is any value in the range of 0.5 to 1.0 M, the final concentration of hydrogen peroxide is any value in the range of 1.5 to 2.0 M, and the final concentration of ethylene glycol is any value in the range of 75 to 80% (v / v). For example, the final concentration of potassium hexacyanoferrate (III) is 0.5 M, the final concentration of ferric chloride is 0.5 M, the final concentration of hydrogen peroxide is 1.5 M, and the final concentration of ethylene glycol is 75% (v / v); or the final concentration of potassium hexacyanoferrate (III) is 1.0 M, the final concentration of ferric chloride is 1.0 M, the final concentration of hydrogen peroxide is any value in the range of 2.0 M, and the final concentration of ethylene glycol is 75 to 80% (v / v), and so on.

[0050] Preferably, the final concentration of potassium hexacyanoferrate (III) is 0.444 M, the final concentration of ferric chloride is 0.444 M, the final concentration of hydrogen peroxide is 1.46 M, and the final concentration of ethylene glycol is 79.4% (v / v).

[0051] In some embodiments, the reaction is continuously carried out under constant temperature and constant speed stirring, the constant temperature is any value between 30 and 40° C., and the constant speed is any value between 800 and 1000 rpm.

[0052] Preferably, the constant temperature is 35° C., the constant speed is 1000 rpm, and the reaction time is 60 min.

[0053] It should be noted that in other embodiments of the present invention, other different schemes obtained by making specific selections within the scope of the steps, components, ratios, and process parameters recorded in the present invention can all achieve the technical effects recorded in the present invention, so the present invention will no longer list them one by one.

[0054] The present invention also provides an application of Prussian blue nanozyme in detecting triglyceride concentration in dry chemical test paper.

[0055] Specifically, the preparation method of dry chemical test paper is as follows:

[0056] (1) Dissolve potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, ethylenediaminetetraacetic acid, sodium benzoate and bovine serum albumin in water to ensure that their final concentrations are 25-30 mM, 55-60 mM, 3-4 mM, 6-7 mM and 35-40 μM, respectively, then add poly(methyl vinyl ether-alt-maleic anhydride) to a final concentration of 5% (v / v), mix well, and store the reaction mother solution at 4° C. for use;

[0057] (2) dissolving Prussian blue nanozyme, lipoprotein lipase, glycerol kinase, phosphoglycerol oxidase, 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate in the solution prepared in step (1) in sequence to ensure that their final concentrations are 1-1.8 mg / mL, 45-65 U / mL, 20-60 U / mL, 20-60 U / mL, 0.5-6 mg / mL and 1-8 mg / mL, respectively;

[0058] (3) spraying the reaction solution of step (2) onto the reaction layer at a spray rate of 5 to 9 μL / cm;

[0059] Specifically, a film-spraying gold spraying device is used to spray the gold onto the surface of the reaction layer.

[0060] (4) placing the reaction layer obtained in step (3) in an oven at 37° C. for 60 min and then taking it out;

[0061] (5) The reaction layer obtained in step (4) is sequentially pasted onto the bottom plate together with the filter layer and the diffusion layer, and is cut into 50*6 mm dry chemical test papers using a programmable strip cutter.

[0062] In one embodiment, the pretreatment of the test strip consumables is as follows: the PCV bottom plate of Shanghai Jinbiao Biotechnology Co., Ltd., model SM31-40 and specification 300*50mm, is cut from top to bottom into 300*19mm, 300*3mm, 300*2mm, 9300*6mm, 300*2mm, 300*3mm, and 300*5mm. Use a small manual puncher to punch holes in the 300*6mm area, with a hole diameter of 4mm and a hole spacing of 2mm. Cut the reaction layer into strips of 100*6mm, the filter layer into strips of 100*10mm, and the diffusion layer into strips of 100*16mm. Among them, the filter layer needs to be pretreated with a bubble film solution containing 0.9% sodium chloride (NaCl) and 1% sorbitol.

[0063] Specifically, the method for detecting the concentration of triglycerides is:

[0064] Several triglyceride simulation samples of different concentrations were prepared, 15 μL of sample was aspirated and dripped vertically, and the RGB value was read through an APP on a smartphone. The test results were linearly fitted by plotting B / (R+G+B) and triglyceride concentration, and the standard curve y=0.003x+0.339 was obtained, with a standard deviation of 0.996 and a detection limit of 0.425 mM.

[0065] Specifically, the detection mechanism is that TG is hydrolyzed into glycerol and free fat under the action of LPL. Subsequently, the generated glycerol is phosphorylated under the action of ATP and GK to form 3-phosphoglycerol, which is then oxidized under the action of GPO to generate dihydroxyacetone phosphate and H 2 O 2 , H 2 O 2 Under the catalysis of PBNPs, 4-AAP is oxidized and combined with MAOS to generate a blue product. Finally, the blue change of the reaction layer is detected by the detection platform to reflect the TG content. The detection platform includes a smartphone, a smart application, and a mobile phone magnifier. Among them, the smartphone model is iPhone 13, the iOS version is 17.1.1, the smart application is ColorAssist, and the magnification of the mobile phone magnifier is 200 times.

[0066] Example 1: Efficient preparation of Prussian blue nanozyme

[0067] This implementation uses H 2 O 2 Reduction of FeCl in water / ethylene glycol 3 With K 3 [Fe(CN) 6 ] mixture, the water used is ultrapure water, and the chemicals are all analytical grade and can be used without further purification. The specific preparation steps are as follows:

[0068] (1) Take 144 mL of ethylene glycol stock solution and 3.28 mL of water to make solution D. Place solution D on a magnetic heating stirrer and keep it at 35°C for 30 min.

[0069] (2) Take 1462 mg K 3 [Fe(CN) 6 ] was dissolved in 10 mL of water to prepare solution A for later use.

[0070] (3) Take 720.2 mg FeCl 3 Dissolve in 10 mL of water to prepare solution B for later use.

[0071] (4) Take 2.92 mL of 30% H 2 O 2 Add 17.08 mL of water to prepare solution C for later use.

[0072] (5) Add 8.18 mL of each of the solutions obtained in step (2) and step (3) and 18.18 mL of the solution obtained in step (4) to the solution in step (1), maintain a constant stirring speed of 1000 rpm, and continue the reaction at 35° C. for 60 min.

[0073] (6) The solution obtained in step (5) was transferred into a dialysis bag (regenerated cellulose membrane, 10000 MWCO, from Chinese source leaves), and dialyzed with ultrapure water at room temperature for 5 days, during which the water was changed 5 times. The dialyzed nanoparticles were placed in an ice-water mixture at 0°C for 40 minutes of ultrasound. The ultrasound-treated nanoparticles were then freeze-dried (the specific freeze-drying time depends on the volume) to obtain blue Prussian blue nanozyme powder.

[0074] The Prussian blue nanozyme obtained in this example was characterized by transmission electron microscopy for morphology, X-ray diffractometer for crystal structure, and microplate reader for absorbance. Figure 1 As shown in FIG. 1 , transmission electron microscopy shows that the Prussian blue synthesized by this method is a nanoscale particle with a diameter of about 15 nm. The particle shape is irregular, but overall it is relatively uniform. Figure 2 As shown, X-ray diffractometer shows that the nanoparticles have a crystalline form consistent with Prussian blue. Figure 3 As shown, the microplate reader shows that the nanoparticles have an absorption peak consistent with that of Prussian blue nanozyme.

[0075] Example 2: Enzyme kinetics of Prussian blue nanozyme

[0076] (1) Verification of peroxidase activity. Four reaction systems were set up, A (containing PBNPs, TMB and H 2 O 2 ), B (containing PBNPs and TMB), C (containing TMB and H 2 O 2 ), D (containing only TMB), where the reaction buffer was acetic acid-sodium acetate (HAc-NaAc, pH 4.0), PBNPs, TMB and H 2 O 2 The final concentrations were 2.13 μM, 1.42 mM and 37.6 mM, respectively. After reacting at room temperature for 10 min, the plates were transferred to 96-well plates and the absorbance at 625 nm was measured using an ELISA reader. Figure 4 As shown in the figure, system A has a strong absorption peak at 652nm, systems B and D have no absorption peak at 652nm, and system C has a weak absorption peak at 652nm, which indicates that when the reaction system contains PBNPs, TMB and H 2 O 2 When PBNPs can catalyze H 2 O2 Oxidation of TMB verified the peroxidase-like activity of the PBNPs prepared in the present application.

[0077] (2) pH dependence study. HAc-NaAc buffers with pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 were prepared respectively. The reaction buffers with different pH values ​​were added to centrifuge tubes. PBNPs, TMB, and H were then added to the reaction buffers in sequence. 2 O 2 , ensuring that their final concentrations were 2.13μM, 1.42mM, and 37.6mM, respectively. After reacting at room temperature for 10 minutes, the plates were transferred to 96-well plates and the absorbance at 625nm was measured using an ELISA reader. Figure 5 As shown, when pH = 4.0, the absorbance of the reaction solution at 652 nm is the highest, showing the highest peroxidase-like activity.

[0078] (3) Temperature dependence study. Five reaction temperatures were set at 15°C, 25°C, 35°C, 45°C, 55°C, and 65°C. A pH 4.0 reaction buffer was added to a centrifuge tube. PBNPs, TMB, and H 2 O 2 , ensuring that the final concentrations were 2.13μM, 1.42mM, and 37.6mM, respectively. After 10 minutes of reaction, the reaction solution was transferred to a 96-well plate and the absorbance at 625nm was measured using an ELISA reader. Figure 6 As shown, between 15-45°C, the absorbance of the reaction solution at 652nm was good, and it had a high peroxidase-like activity.

[0079] (4) Study on the kinetics of enzyme-catalyzed reactions. 2 O 2 As substrate, take 7 600 μL centrifuge tubes and label them as 0, 1, 2, 3, 4, 5, and 6, add reaction buffer in sequence, and then add PBNPs and TMB respectively to ensure that the final concentrations are 2.13 μM and 1.67 mM respectively. According to the label, add different volumes of H 2 O 2 , so that the final concentrations are 0mM, 1.18mM, 2.35mM, 3.53mM, 4.71mM, 5.88mM, and 7.06mM. The reaction solution was quickly transferred to a 96-well plate, and the absorbance at 625nm was measured every 30s using an ELISA reader. Figure 7 As shown, according to the reaction rate and H 2 O 2 The double reciprocal curve of concentration can be obtained using H 2 O 2 V max 2.33×10 -7 M.s-1 , K m 2.37mM. TMB was used as the substrate. Take 11 600μL centrifuge tubes and label them as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, add reaction buffer in sequence, and then add PBNPs to ensure that the final concentration is 2.13μM. According to the label, add different volumes of TMB to make the final concentrations 0mM, 0.17mM, 0.33mM, 0.50mM, 0.67mM, 0.83mM, 1.00mM, 1.17mM, 1.33mM, 1.5mM, 1.67mM. Finally, add H 2 O 2 , so that the final concentration is 11.8mM. The reaction solution was quickly transferred to a 96-well plate, and the absorbance at 625nm was measured every 30s using an ELISA reader. Figure 8 As shown in the figure, according to the double reciprocal curve of reaction rate and TMB concentration, it can be concluded that V max 1.51×10 -6 M.s -1 , K m It is 0.84mM.

[0080] Example 3: Preparation of dry chemical test strips

[0081] (1) Accurately weigh 38 mg of potassium dihydrogen phosphate, 102 mg of disodium hydrogen phosphate dihydrate, 1 mg of ethylenediaminetetraacetic acid, 1 mg of sodium benzoate, and 25 mg of bovine serum albumin and dissolve them in 10 mL of water. Then add 0.1% (v / v) Triton-X-100 and 5% (v / v) poly(methyl vinyl ether-alt-maleic anhydride) to prepare a reaction mother solution.

[0082] (2) PBNPs, LPL, GPO, GK, 4-AAP and MAOS were dissolved in the solution prepared in step (1) in sequence to ensure that their final concentrations were 1.4 mg / mL, 55 U / mL, 30 U / mL, 30 U / mL, 2 mg / mL and 4 mg / mL, respectively.

[0083] (3) The reaction solution of step (2) was sprayed onto the reaction layer at a spray volume of 7 μL / cm.

[0084] (4) The reaction layer obtained in step (3) was placed in an oven at 37° C. and dried for 60 min before being taken out.

[0085] (5) Fig. 9 As shown, the reaction layer obtained in step (4) is sequentially pasted onto the bottom plate together with the filter layer and the diffusion layer, and is cut into 50*6 mm test strips (from bottom to top, the bottom plate, the reaction layer, the filter layer and the diffusion layer) using a programmable strip cutter.

[0086] Example 4: Detection of triglycerides

[0087] (1) Specificity test. Blank, uric acid (UA), glucose (Glu), cholesterol (CHO), urea (Urea), glycine (Gly) and ascorbic acid (AA) were used as interferences to test the specificity of the test strip. The results are as follows: Fig.10 As shown. This proves that the test strip has good specificity.

[0088] (2) Preparation of standard curve. Prepare TG simulation samples of different concentrations (0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM), draw 15μL of sample and press Fig. 9 The RGB value was read by the smartphone through the APP, and the test results were linearly fitted by plotting B / (R+G+B) and TG concentration. Fig.11 As shown, the standard curve y=0.003x+0.339 was obtained, the standard deviation was 0.996, and the detection limit was 0.425mM.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the invention. Any modification, equivalent substitution or improvement made under the guidance of the basic principles and spirit of the present invention shall be regarded as part of the protection scope of the present invention.

[0090] After reading this specification and practicing the disclosed contents, it is not difficult for those skilled in the art to deduce other possible implementation methods of the present application. The present application is intended to cover all variants, applications or adaptive adjustments that follow the basic principles of the present application, including but not limited to common sense or standard technical means known in the art that are not explicitly mentioned in the present application. This specification and the embodiments provided are only for demonstration and are not intended to limit the scope of protection of the present application.

[0091] It should be clear that the present application is not limited to the specific structures described above and shown in the drawings, and various adjustments and improvements are allowed without deviating from its core principles.

Claims

1. A method for preparing Prussian blue nanozyme, characterized in that: include: Potassium hexacyanoferrate (III) is dissolved in water to prepare solution A; Dissolve ferric chloride in water to make solution B; Dilute hydrogen peroxide and ethylene glycol with water to form solution C and solution D respectively; Solutions A, B, and C are added to solution D in sequence, so that solutions A and B continue to react under constant temperature and constant speed stirring; the reaction solution is dialyzed, ultrafiltered, and then freeze-dried to obtain the Prussian blue nanozyme product.

2. The preparation method according to claim 1, characterized in that: The solutions A, B and C are added to the solution D in sequence, the final concentration of potassium hexacyanoferrate (III) is 0.5-1.0 M, the final concentration of ferric chloride is 0.5-1.0 M, the final concentration of hydrogen peroxide is 1.5-2.0 M, and the final concentration of ethylene glycol is 75-80% (v / v).

3. The preparation method according to claim 2, characterized in that: The solutions A, B and C are added to the solution D in sequence, the final concentration of potassium hexacyanoferrate (III) is 0.444 M, the final concentration of ferric chloride is 0.444 M, the final concentration of hydrogen peroxide is 1.46 M, and the final concentration of ethylene glycol is 79.4% (v / v).

4. The preparation method according to claim 1, characterized in that: The reaction is continuously carried out under constant temperature and constant speed stirring, specifically: the constant temperature is 30-40° C., the constant speed is 800-1000 rpm, and the reaction time is 60-80 min.

5. The preparation method according to claim 4, characterized in that: The constant temperature was 35°C, the constant speed was 1000 rpm, and the reaction time was 60 min.

6. The Prussian blue nanozyme prepared according to the preparation method according to any one of claims 1 to 5.

7. The Prussian blue nanozyme according to claim 6, characterized in that The average particle size of Prussian blue nanozyme is 15nm, showing the same crystal structure and absorption characteristics as Prussian blue. Prussian blue nanozyme exhibits significant peroxidase-like activity. Under the condition of pH 4, its activity remains at a high level in the temperature range of 15-45°C. When hydrogen peroxide is used as the substrate, the maximum reaction rate is 2.33×10 -7 M.s -1 , the Michaelis constant is 2.37 mM; when tetramethylbenzidine is used as the substrate, the maximum reaction rate is 1.51×10 -6 M.s -1 , the Michaelis constant is 0.84mM.

8. Use of the Prussian blue nanozyme prepared according to the preparation method according to any one of claims 1 to 5 or the Prussian blue nanozyme according to claim 6 or 7 in a dry chemical test paper; the dry chemical test paper is used to detect triglyceride concentration.

9. The use according to claim 8, characterized in that: The production method of dry chemical test paper is as follows: (1) dissolving potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, ethylenediaminetetraacetic acid, sodium benzoate and bovine serum albumin in water to ensure that their final concentrations are 25-30 mM, 55-60 mM, 3-4 mM, 6-7 mM and 35-40 μM, respectively, and then adding poly(methyl vinyl ether-alt-maleic anhydride) to a final concentration of 5% (v / v), and mixing well; (2) dissolving Prussian blue nanozyme, lipoprotein lipase, glycerol kinase, phosphoglycerol oxidase, 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate in the solution prepared in step (1) in sequence to ensure that their final concentrations are 1-1.8 mg / mL, 45-65 U / mL, 20-60 U / mL, 20-60 U / mL, 0.5-6 mg / mL and 1-8 mg / mL, respectively; (3) spraying the reaction solution of step (2) onto the reaction layer at a spray rate of 5 to 9 μL / cm; (4) placing the reaction layer obtained in step (3) in an oven at 37° C. for 60 min and then taking it out; (5) The reaction layer obtained in step (4) is sequentially pasted onto a bottom plate together with the filter layer and the diffusion layer to obtain a dry chemical test paper.

10. The use according to claim 8, characterized in that: The method for detecting triglyceride concentration is: Several triglyceride simulation samples of different concentrations were prepared, 15 μL of sample was aspirated and dripped vertically, and the RGB value was read through an APP on a smartphone. The test results were linearly fitted by plotting B / (R+G+B) and triglyceride concentration, and the standard curve y=0.003x+0.339 was obtained, with a standard deviation of 0.996 and a detection limit of 0.425 mM.

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