Three-enzyme-inorganic hybrid nanoflower as well as preparation method and application method thereof
By immobilizing three-enzyme-inorganic hybrid nanoflowers, the problem of poor enzyme stability has been solved, enabling rapid and accurate detection of creatinine, which is applicable to the field of biosensing.
Patent Information
- Application Number
- CN202511509432.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-22
- Publication Date
- 2026-01-23
AI Technical Summary
The poor stability and difficulty in recovery of free enzymes limit the large-scale application of the three-enzyme system in creatinine detection. Traditional detection methods cannot achieve rapid, accurate, and convenient home testing.
The three-enzyme-inorganic hybrid nanoflower immobilization technology was adopted. Nanoflowers were formed by the self-assembly of mixed enzymes and calcium phosphate and immobilized in the working electrode area for electrochemical detection.
It improves the stability and activity of enzymes, enabling rapid, convenient, continuous, real-time, and accurate detection of creatinine. The operation is simple and environmentally friendly.
Smart Images
Figure CN121380035A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of enzyme immobilization, and particularly relates to a three-enzyme-inorganic hybrid nanoflower and a preparation and application method thereof. BACKGROUND
[0002] Enzymes are widely used in the fields of medicine, sensing, etc. due to their excellent catalytic activity and reaction specificity. However, the poor stability and difficulty in recycling of free enzymes limit their industrial application. In order to improve the stability and catalytic performance of enzymes, various enzyme immobilization technologies have emerged. The organic-inorganic hybrid nanoflower immobilization technology is a widely studied enzyme immobilization method in recent years. It forms a crystalline complex with different morphologies similar to natural petals through the self-assembly of enzyme proteins, metal cations and phosphate anions. In the immobilization process, enzyme proteins and metal ions are used as organic components and inorganic components, respectively. Therefore, this hybrid material is called an organic-inorganic hybrid nanomaterial or an enzyme-inorganic hybrid nanomaterial. This material increases the specific surface area and reduces the mass transfer resistance, and can usually improve the enzyme activity by several times and improve the stability and other enzymatic properties. When immobilizing enzymes, the selection of metal cations is crucial. Metal cations that do not destroy the enzyme and affect its enzyme activity need to be selected, and the synthesized immobilized enzyme should have the effect of improving enzyme activity.
[0003] Creatinine is the most commonly used and most core indicator for evaluating kidney function. Many early kidney diseases have no obvious symptoms, but the increase of serum creatinine can provide objective early evidence. By monitoring the changes in creatinine levels, a more intuitive way is provided for the prevention and evaluation of the development process of kidney disease. The traditional creatinine detection method relies on large instruments, cannot obtain feedback in time, and needs to frequently go to the hospital. Therefore, it is necessary to develop a creatinine detection method that can be used at home, quickly and accurately. Among them, the creatinine detection method based on the three-enzyme system has been widely concerned due to its rapidity, reversibility, and reusability. However, the three-enzyme method is difficult to achieve large-scale application due to the variety of enzymes, complex system, and low activity.
[0004] Therefore, it is very important to use a new enzyme immobilization method to realize the rapid, accurate and convenient detection of creatinine. SUMMARY
[0005] The purpose of the present application is to overcome the defects in the prior art and provide a three-enzyme-inorganic hybrid nanoflower and a preparation and application method thereof.
[0006] The specific technical solutions adopted by the present application are as follows: In a first aspect, the present application provides a preparation method of a three-enzyme-inorganic hybrid nanoflower, which is as follows: S1: adding a mixed enzyme aqueous solution into a phosphate buffer solution to obtain a mixed enzyme-phosphate buffer solution with a total enzyme concentration of 0.1-1 mg / mL; the mixed enzyme aqueous solution contains creatininase, creatine kinase and sarcosine oxidase; S2: adding a divalent calcium ion salt solution into the mixed enzyme-phosphate buffer solution under stirring to obtain a precursor solution of a three-enzyme-inorganic nanoflower; S3: ultrasonicating the precursor solution and then standing at room temperature or 4°C, and the precipitate obtained after centrifugal washing is a three-enzyme-inorganic hybrid nanoflower.
[0007] Preferably, in the mixed enzyme aqueous solution, the concentration of creatininase is 40-80 U / uL, the concentration of creatine kinase is 4-8 U / uL, and the concentration of sarcosine oxidase is 2-5 U / uL, and the three enzymes are mixed in equal volume ratio.
[0008] Preferably, the concentration of the phosphate buffer solution is 3-20 mM, and the pH value is 6.0-8.0.
[0009] Preferably, the divalent calcium ion salt solution is prepared by dissolving one or more of calcium chloride, calcium nitrate and calcium formate in water, and the concentration is 120 mM-375 mM.
[0010] Preferably, in the precursor solution, the volume ratio of the phosphate buffer solution to the divalent calcium ion salt solution is (50-150):1.
[0011] Preferably, the ultrasonicating time is 0-10 min, and the standing time is 2-24 hours; the three-enzyme-inorganic hybrid nanoflower is resuspended in ultrapure water and stored at 4°C for standby.
[0012] In a second aspect, the present application provides a three-enzyme-inorganic hybrid nanoflower prepared by the preparation method of any one of the first aspect.
[0013] In a third aspect, the present application provides a method for detecting creatinine based on the three-enzyme-inorganic hybrid nanoflower of the second aspect, which comprises fixing the three-enzyme-inorganic hybrid nanoflower in a working electrode region by in-situ growth or direct dropping, and then using a three-electrode system including the working electrode to electrochemically detect creatinine.
[0014] Preferably, the in-situ growth is performed as follows: The original working electrode is placed in the precursor solution of the three-enzyme-inorganic nanoflower, and stirring is sequentially performed for 2-10 min, ultrasonic is performed for 0-10 min, and standing is performed for 2-24 hours, so that the three-enzyme-inorganic nanoflower is fixed on the original working electrode, and finally the working electrode is taken out, washed with deionized water and dried. Then 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise, dried and stored at 4 DEG C for detection.
[0015] As preferred, the direct dropwise adding mode is specifically as follows: The three-enzyme-inorganic hybrid nanoflower is resuspended in ultrapure water to obtain a suspension, 8 uL of the suspension is added dropwise on the surface of the original working electrode, then 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise, dried and stored at 4 DEG C for detection.
[0016] Compared with the prior art, the present application has the following beneficial effects: 1) The present application is simple to operate, has mild preparation conditions, low raw material prices and is green and environmentally friendly.
[0017] 2) The three-enzyme-inorganic hybrid nanoflower prepared by the present application improves the stability and activity of the complex enzyme for creatinine detection, and the nanoflower is small with a size of 500 nm-1 um, which is convenient for fixing on the electrode surface.
[0018] 3) The present application has a wide application prospect in the field of creatinine detection, and can realize rapid, convenient, continuous, real-time and accurate detection of creatinine. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 It is a scanning electron microscope image of the shape of the three-enzyme-inorganic hybrid nanoflower prepared in the example with different PBS concentrations, the ultrasonic time is controlled to be 0 min, and the standing time is controlled to be 21 hours; Figure 2 It is a scanning electron microscope image of the shape of the three-enzyme-inorganic hybrid nanoflower prepared in the example with different ultrasonic times, the PBS concentration is controlled to be 8 mM, and the standing time is controlled to be 21 hours; Figure 3 It is a scanning electron microscope image of the shape of the three-enzyme-inorganic hybrid nanoflower prepared in the example with different standing times, the PBS concentration is controlled to be 8 mM, and the ultrasonic time is controlled to be 7 min; Figure 4 It is a chronoamperometric graph of the electrochemical creatinine detection using the three-enzyme-inorganic hybrid nanoflower in the example. DETAILED DESCRIPTION
[0020] The present application will be further described and explained in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment in the present application can be combined accordingly without conflict.
[0021] The present application provides a three-enzyme-inorganic hybrid nanoflower, wherein the three enzymes are creatininase, creatinase and sarcosine oxidase, the carrier is calcium phosphate, and the preparation raw materials are the three enzymes, phosphate and divalent calcium ion salt. The preparation method of the material is specifically as follows: S1: adding a mixed enzyme aqueous solution into a phosphate buffer solution to obtain a mixed enzyme-phosphate buffer solution with a total enzyme concentration of 0.1-1 mg / mL. The mixed enzyme aqueous solution contains creatininase, creatinase and sarcosine oxidase.
[0022] As a more preferred embodiment of the present application, the concentration of creatininase in the mixed enzyme aqueous solution is 40-80 U / uL, the concentration of creatinase is 4-8 U / uL, and the concentration of sarcosine oxidase is 2-5 U / uL, and the three enzymes are mixed in equal volume ratio.
[0023] As a more preferred embodiment of the present application, the concentration of the phosphate buffer solution is 3-20 mM, and the pH value is 6.0-8.0. The pH value of the mixed enzyme-phosphate buffer solution is also 6.0-8.0.
[0024] S2: adding a divalent calcium ion salt solution into the mixed enzyme-phosphate buffer solution obtained in S1, stirring while adding, to obtain a precursor solution of the three-enzyme-inorganic nanoflower.
[0025] As a more preferred embodiment of the present application, the divalent calcium ion salt solution is prepared by dissolving one or more of calcium chloride, calcium nitrate and calcium formate in water, and the concentration is 120 mM-375 mM.
[0026] As a more preferred embodiment of the present application, in the precursor solution, the volume ratio of the phosphate buffer solution to the divalent calcium ion salt solution is (50-150):1.
[0027] S3: ultrasonicating the precursor solution obtained in S2, and then standing at room temperature or 4°C, and the precipitate obtained after centrifugal washing is the three-enzyme-inorganic hybrid nanoflower.
[0028] As a more preferred embodiment of the present application, the ultrasonicating time is 0-10 min, and the standing time is 2-24 hours.
[0029] As a more preferred embodiment of the present application, the prepared three-enzyme-inorganic hybrid nanoflower can be resuspended in ultrapure water and stored at 4°C for standby.
[0030] The present application also provides an application method of the three-enzyme-inorganic hybrid nanoflower in creatinine detection, and the application method is specifically as follows: The three-enzyme-inorganic hybrid nanoflower is fixed on the working electrode area by in-situ growth or direct dropping, and then the three-enzyme-inorganic hybrid nanoflower is used for electrochemical detection of creatinine by using a three-electrode system including the working electrode.
[0031] As a preferred embodiment of the present application, the in-situ growth method is as follows: The original working electrode is placed in the three-enzyme-inorganic nanoflower precursor solution, and then stirring is performed for 2-10 min, ultrasonic treatment is performed for 0-10 min, and standing is performed for 2-24 hours, so that the three-enzyme-inorganic nanoflower is fixed on the original working electrode. Finally, the working electrode is taken out, washed with deionized water, and then dried. Then, 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise, and the mixture is dried and stored at 4°C for detection.
[0032] As a preferred embodiment of the present application, the direct dropping method is as follows: The three-enzyme-inorganic hybrid nanoflower is resuspended in ultrapure water to obtain a suspension, 8 uL of the suspension is added dropwise on the surface of the original working electrode, and then 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise. The mixture is dried and stored at 4°C for detection.
[0033] The material performance, preparation and application method of the present application will be described in detail through the following examples.
[0034] Example In this embodiment, a three-enzyme-inorganic hybrid nanoflower is prepared, and the preparation method is as follows: (1) 36 mL of phosphate buffer solution with different concentrations (3, 5, 8, and 10 mM) and pH of 7.2 is prepared. 720 uL (concentration of 200 mM) of calcium chloride aqueous solution is prepared. Creatinine enzyme (60 U / uL), creatine enzyme (6 U / uL), and sarcosine oxidase (5 U / uL) are mixed in a volume ratio of 1:1:1 to obtain a mixed enzyme aqueous solution, i.e., a mixed enzyme aqueous solution.
[0035] (2) The mixed enzyme aqueous solution in step (1) is added to the phosphate buffer solution to obtain a mixed enzyme-phosphate buffer solution with a total enzyme concentration of 0.5 mg / mL.
[0036] (3) The calcium chloride aqueous solution in step (1) is added to the mixed enzyme-phosphate buffer solution in step (2) while stirring to obtain a three-enzyme-inorganic nanoflower precursor solution.
[0037] (4) The precursor solution of step (3) is ultrasonicated for 0.7 min, and then left to stand at room temperature for 2, 4, 6, 8, 10, 12, 15, 18, 21, 24 hours, and finally centrifuged to obtain a precipitate, which is washed with deionized water, resuspended in 50 uL of ultrapure water after washing, and stored at 4°C for standby, to obtain a three-enzyme-inorganic hybrid nanoflower suspension.
[0038] In this example, nanoflowers formed under different conditions are prepared to obtain the optimal preparation conditions of nanoflowers. As shown in Figure 1 , first, the morphology of three-enzyme-inorganic hybrid nanoflowers prepared at different PBS concentrations (ultrasonic time is controlled at 0 min, and standing time is controlled at 21 hours) is explored. It can be found from the electron microscope images that as the PBS concentration increases, the shape of the nanoflowers becomes more and more complete, but when the PBS concentration is greater than 8 mM, the nanoflowers gradually grow into a spherical dense structure, which is not conducive to the cascade reaction of enzymes. Therefore, 8 mM is selected as the optimal PBS concentration. Next, by controlling the PBS concentration at 8 mM, the effects of ultrasonic time and standing time on the formation process of nanoflowers are studied in order to explore the optimal ultrasonic time and standing time. The results are shown in Figure 2 and Figure 3 . It can be seen from the figures that the ultrasonic time and standing time significantly affect the formation of nanoflowers. The longer the ultrasonic time and standing time, the faster the growth of nanoflowers, but too long ultrasonic time and standing time will lead to the formation of a dense spherical structure of nanoflowers, which is not conducive to the cascade reaction of enzymes. In order to reduce the overall reaction time, 7 min of ultrasonic time and 8 hours of standing time are selected as the optimal choice. Finally, the three-enzyme-inorganic hybrid nanoflowers prepared under the conditions of PBS concentration of 8 mM, ultrasonic time of 7 min and standing time of 8 hours are resuspended in 50 uL of ultrapure water and used for electrochemical creatinine detection in a three-electrode system.
[0039] The above three-enzyme-inorganic hybrid nanoflower suspension prepared in this example is added dropwise on the surface of the working electrode to prepare a sensing electrode for electrochemical detection of creatinine. The specific preparation steps are as follows: The three-enzyme-inorganic hybrid nanoflower suspension is mixed with an aqueous gelatin solution with a mass fraction of 5% at an equal volume ratio, and then 16 uL is added dropwise on the surface of the working electrode (Prussian blue is used as a sensitive material on the working electrode for detection of hydrogen peroxide, the substrate after enzyme catalysis). After drying at room temperature, the electrochemical workstation is used for testing. The test results are shown in Figure 4 . With the increase of creatinine concentration, the current response increases continuously, which further proves the successful preparation of the three-enzyme-inorganic hybrid nanoflowers in this example.
[0040] The present application has the advantages of simple operation, green and environmentally friendly, etc. The three-enzyme-inorganic nanoflower material prepared has high stability and rapid detection, and has good application prospect in the field of biosensing.
[0041] The above-described embodiments are merely preferred embodiments of the present application, but are not intended to limit the present application. Those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Therefore, any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the scope of the present application.
Claims
1. A method for preparing a tri-enzyme-inorganic hybrid nanoflower, characterized in that, Specifically as follows: S1: adding a mixed enzyme aqueous solution into a phosphate buffer solution to obtain a mixed enzyme-phosphate buffer solution with a total enzyme concentration of 0.1-1 mg / mL; the mixed enzyme aqueous solution contains creatininase, creatine kinase and sarcosine oxidase; S2: under stirring, adding a divalent calcium ion salt solution into the mixed enzyme-phosphate buffer solution to obtain a precursor solution of a three-enzyme-inorganic nanoflower; S3: after ultrasonic treatment of the precursor solution, standing at room temperature or 4°C, and centrifugal washing, the obtained precipitate is a three-enzyme-inorganic hybrid nanoflower.
2. The method according to claim 1, wherein the method is characterized by, In the mixed enzyme aqueous solution, the concentration of creatininase is 40-80 U / uL, the concentration of creatine kinase is 4-8 U / uL, and the concentration of sarcosine oxidase is 2-5 U / uL, and the three enzymes are mixed in equal volume ratio.
3. The method according to claim 1, wherein the method is characterized by, The concentration of the phosphate buffer solution is 3-20 mM, and the pH value is 6.0-8.
0.
4. The method according to claim 1, wherein the method is characterized by, The divalent calcium ion salt solution is prepared by dissolving one or more of calcium chloride, calcium nitrate and calcium formate in water, and the concentration is 120 mM-375 mM.
5. The method according to claim 1, wherein the method is characterized by, In the precursor solution, the volume ratio of the phosphate buffer solution to the divalent calcium ion salt solution is (50-150):
1.
6. The method according to claim 1, wherein the method is characterized by, The ultrasonic treatment time is 0-10 min, and the standing time is 2-24 hours; the three-enzyme-inorganic hybrid nanoflower is resuspended in ultrapure water to obtain a three-enzyme-inorganic hybrid nanoflower suspension, which is stored at 4°C for standby.
7. A three-enzyme-inorganic hybrid nanoflower prepared by the preparation method of any one of claims 1-6.
8. The application method of the three-enzyme-inorganic hybrid nanoflower based on claim 7 in the detection of creatinine, characterized by, The three-enzyme-inorganic hybrid nanoflower is fixed on the working electrode area by in-situ growth or direct dropping, and then a three-electrode system including the working electrode is used for electrochemical detection of creatinine.
9. The method for use according to claim 8 in the detection of creatinine, characterized in that, The in-situ growth method is specifically as follows: The original working electrode is placed in the precursor solution of the three-enzyme-inorganic nanoflower, and stirring is performed for 2-10 min, ultrasonic treatment is performed for 0-10 min, and standing is performed for 2-24 hours, so that the three-enzyme-inorganic nanoflower is fixed on the original working electrode, and finally the working electrode is taken out, washed with water and dried; then 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise, dried and stored at 4°C for detection.
10. The method of claim 8, wherein the method is used in the detection of creatinine. The direct dropping method is specifically as follows: The three-enzyme-inorganic hybrid nanoflower is resuspended in ultrapure water to obtain a suspension, 8 uL of the suspension is added dropwise on the surface of the original working electrode, then 3-8 uL of a 5% gelatin aqueous solution or a 0.5% chitosan solution is added dropwise, dried and stored at 4°C for detection.