A method for preparing CoP / RuP nanozymes and its application in the detection of alanine aminotransferase (ALT).

Abstract

This invention discloses a method for preparing CoP / RuP nanozymes and their application in the detection of alanine aminotransferase (ALT). Belonging to the fields of nanozyme preparation and medical detection technology, the CoP / RuP nanozyme is prepared by grinding ruthenium salt, cobalt salt, fumaric acid, phytic acid, and sodium chloride, followed by Joule heating. This nanozyme preparation process is simple, efficient, and has promising prospects for industrial application. The ALT colorimetric sensing method constructed based on this nanozyme exhibits excellent performance, with a detection limit as low as 1.22 U / L, demonstrating significant clinical application value and industrialization potential.

Description

Technical Field

[0001] This invention relates to the fields of nanozyme preparation and medical detection technology, and particularly to a method for preparing CoP / RuP nanozymes and its application in the detection of alanine aminotransferase (ALT). Background Technology

[0002] Alanine aminotransferase (ALT) is a key biomarker for liver function testing. Abnormally elevated ALT levels typically indicate hepatocellular damage or disease, such as hepatitis, cirrhosis, and drug-induced liver injury. Currently, clinical ALT detection mainly relies on traditional biochemical analysis methods. These methods suffer from drawbacks such as reliance on expensive reagents, complex procedures, stringent reaction conditions (e.g., requiring precise pH and temperature control), and high instrument dependence, limiting their application in primary healthcare and point-of-care testing (POCT). Therefore, developing novel, low-cost, highly stable, and easy-to-use ALT detection technologies is of significant clinical importance.

[0003] In recent years, colorimetric sensing methods based on peroxidase-hydrogen peroxide have been widely used for highly sensitive detection of biomarkers due to their simplicity, high sensitivity, and low cost. This method is highly resistant to interference, easy to operate, and has low dependence on specialized equipment. Based on this, ALT can be rapidly detected by constructing a colorimetric sensor using peroxidase to produce hydrogen peroxide via enzymatic catalysis. However, the peroxidases used in this method, such as horseradish peroxidase, are expensive to prepare and prone to inactivation, limiting their large-scale application. Nanozymes, as a novel type of artificial enzyme, possess the catalytic properties of traditional enzymes and have unique advantages, such as strong thermal stability, long shelf life, and tunable catalytic activity. Therefore, developing efficient peroxidase-like nanozymes to replace natural enzymes for ALT detection is of great significance.

[0004] Metal phosphides possess advantages such as rich crystal structure composition, varied coordination ratios between metal and phosphorus atoms, easily modifiable surface chemical states, and structural stability, showing great potential to replace natural peroxide enzymes. However, the activity of current phosphide nanozymes remains low, and the sensitivity of constructed colorimetric sensors for detecting biomarkers is also low. The activity of nanozymes is closely related to their morphology and structure. Generally, low-dimensionality (e.g., two-dimensional or one-dimensional) and porosity are key factors for achieving high catalytic activity in phosphide nanozymes. Low-dimensionality endows nanozymes with a larger specific surface area and shorter electron transport pathways, further enhancing catalytic kinetics, while porous structures facilitate reactant diffusion and product release. Currently, the synthesis of metal phosphides can be mainly divided into liquid-phase and solid-phase methods. Although the liquid-phase method can directly control the morphology and structure of transition metal phosphides, it primarily uses organophosphorus compounds (such as tri-n-octylphosphine or triphenylphosphine) as phosphorus sources, requiring expensive and toxic organic solvents, high reaction temperatures, and complete avoidance of oxygen during the reaction process. These characteristics limit the application of liquid-phase methods in the synthesis of transition metal phosphides. For example, Chinese patent document CN111668501B discloses an invention patent entitled "Anode Catalyst for Fuel Cells and its Preparation Method and Application." This patent first involves heating and stirring ruthenium acetylacetone and the reaction solvent trioctylphosphine oxide under vacuum conditions at a reaction temperature of 90–150 °C, and then reacting it with the phosphorus source tri-n-octylphosphine at 180–400 °C under an inert atmosphere to obtain ruthenium phosphide. Solid-phase methods are more convenient than liquid-phase methods for synthesizing transition metal phosphides, avoiding the use of surfactants and high-boiling-point organic solvents. For example, a Chinese invention patent titled "Preparation of a Noble Metal-Doped Porous Iron-Nickel Phosphate" (publication number CN114408886A) utilizes NaCl and noble metal chlorides to corrode foamed nickel-iron to synthesize a metal hydroxide precursor, which is then calcined at low temperature with sodium hypophosphite to obtain noble metal-doped porous iron-nickel phosphate nanomaterials. Another Chinese invention patent, publication number CN112960706A, discloses a flower-like nickel cobalt phosphate material, its preparation method, and its application. This patent first prepares flower-like nickel cobalt phosphate material via a hydrothermal method, and then calcines it with sodium hypophosphite to prepare the flower-like nickel cobalt phosphate material. However, this method is difficult to directly synthesize low-dimensional porous metal phosphides. It often requires the pre-synthesis of a low-dimensional precursor as a self-template, followed by high-temperature phosphating to form low-dimensional porous phosphides. This preparation process has high requirements for the precursor and is complex, lacking versatility.

[0005] Therefore, it is of great significance to develop a nanozyme with simple process, high catalyst preparation efficiency, and high sensitivity. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a CoP / RuP nanozyme with high peroxidase nanozyme activity, its preparation method, and its application in alanine aminotransferase (ALT) detection, which can solve the problem of low sensor sensitivity for biomarker detection caused by the low activity of traditional phosphide nanozymes. The CoP / RuP nanozyme preparation method provided by this invention is simple, has high catalyst preparation efficiency, and has promising prospects for industrial application; it can effectively solve the problem that it is difficult to directly synthesize low-dimensional porous metal phosphides using solid-phase methods.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, this invention provides a method for preparing CoP / RuP nanozymes, comprising the following steps:

[0009] (1) Dissolve ruthenium salt, cobalt salt, and fumaric acid in ethanol to obtain the first solution;

[0010] (2) Dissolve phytic acid in ethanol to obtain a second solution;

[0011] (3) Place sodium chloride in a mortar, and then add the first solution and the second solution in sequence during the grinding process. Grind until the ethanol is completely evaporated to obtain the first solid.

[0012] (4) The first solid was placed on the sample stage of the Joule heating device for Joule heating calcination. The calcined solid product was then rinsed with deionized water and dried to obtain CoP / RuP nanozyme.

[0013] Further, in step (1), the ruthenium salt is one of ruthenium chloride and ruthenium acetate; the cobalt salt is one of cobalt sulfate, cobalt nitrate, cobalt chloride, and cobalt acetate.

[0014] Furthermore, in step (1), the ratio of ruthenium salt, cobalt salt, fumaric acid, and ethanol is 1 mmol: 0.1~0.3 mmol: 2.5~3.5 mmol: 15~25 ml.

[0015] Furthermore, in step (3), the ratio of sodium chloride, the first solution, and the second solution is 1g: 0.2~0.3ml: 0.04~0.06ml.

[0016] Further, in step (4), the Joule heat calcination conditions are: the Joule heat shock temperature is 500~650℃, the heating rate is 2000~2500℃ / s, and the number of Joule heat shocks is 6~12 times; each Joule heat shock is to first heat the room temperature to 500~650℃, and then cool it naturally to room temperature.

[0017] In a second aspect, the present invention provides the application of CoP / RuP nanozymes prepared by any of the above preparation methods in the detection of alanine aminotransferase.

[0018] The beneficial effects of this invention include at least the following:

[0019] (1) This invention uses phytic acid, fumaric acid, cobalt ions, and ruthenium ions to form a uniformly distributed metal chelate, which is adsorbed onto the surface of sodium chloride crystals. During the Joule heating process, phytate ions and metal ions undergo an in-situ phosphating reaction on the surface of the sodium chloride template to form a two-dimensional CoP and RuP heterojunction. Simultaneously, phytate and fumarate ions generate a large amount of gas during pyrolysis, which can act as a foaming agent to form a porous structure inside the formed two-dimensional phosphide. This porous structure endows the two-component heterojunction with a rich interfacial structure, resulting in abundant phosphorus vacancies at the P atoms at the interface due to interfacial stress and other factors. During the cooling stage, due to the rapid cooling characteristic of Joule heating, it can work with the sodium chloride template to inhibit the sintering of the two-dimensional porous sheet, prevent the collapse of the two-dimensional porous structure, and stabilize the existence of P vacancies. This method has a simple preparation process, high efficiency, and promising prospects for industrial application.

[0020] (2) The CoP / RuP heterojunction nanozyme prepared in this invention achieves a significant improvement in catalytic performance through unique component design (CoP / RuP bimetallic synergy), structural regulation (two-dimensional porous structure), and defect engineering (high-density P vacancies): P vacancies can not only serve as active sites but also optimize the electronic structure of metal active sites, enhancing substrate adsorption and activation capabilities; the heterojunction interface synergistically enhances the catalytic activity of the nanozyme; and the porous structure significantly increases the exposure of active sites and accelerates the catalytic transformation of substrate molecules. Based on this, the ALT colorimetric sensing method exhibits excellent performance with a detection limit as low as 1.22 U / L, demonstrating significant clinical application value and industrialization prospects. Attached Figure Description

[0021] Figure 1 The image shows the X-ray powder diffraction (XRD) pattern of the nanozyme in Example 1.

[0022] Figure 2 The images show scanning electron microscope (SEM) images and elemental distribution maps (EDS Mapping) of the nanozyme in Example 1.

[0023] Figure 3 The image shows the nitrogen adsorption-desorption (N2-BET) diagram of the nanozyme in Example 1.

[0024] Figure 4 The image shows SEM images of the 1-3 nanozymes, which are comparative examples.

[0025] Figure 5 The electron paramagnetic resonance (EPR) spectra of the nanozymes of Example 1 and Comparative Examples 1-3 are shown.

[0026] Figure 6 The absorbance test results are shown for the nanozyme of Example 1 and the samples of Comparative Examples 1-5 when the ALT addition amount is 100 μL (2415 U / L).

[0027] Figure 7 The UV-Vis absorption spectra of the nanozyme of Example 1 in ALT solutions of different concentrations from 6 U / L to 210 U / L are shown.

[0028] Figure 8 The curve showing the relationship between the UV absorbance of the nanozyme in Example 1 and the ALT concentration is shown. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0031] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available materials; unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art.

[0032] The following specific embodiments illustrate the solution proposed in this invention:

[0033] Example 1

[0034] (1) Dissolve 0.25 mmol ruthenium chloride, 0.05 mmol cobalt chloride and 0.75 mmol fumaric acid in 5 mL ethanol to obtain the first solution;

[0035] (2) Dissolve 0.25 mmol of phytic acid in 1 mL of ethanol to obtain a second solution;

[0036] (3) Put 20g of sodium chloride into an agate mortar, and then add the first solution and the second solution in sequence during the grinding process. Grind until the ethanol is completely evaporated to obtain the first solid substance.

[0037] (4) The first solid material was placed in a Joule-heated sample stage and subjected to thermal shock calcination under an inert atmosphere. The Joule thermal shock temperature was 600℃, the heating rate was 2200℃ / s, and the number of Joule thermal shocks was 10 (one Joule thermal shock is achieved by first heating from room temperature to 600℃ and then naturally cooling to room temperature). The product was washed with deionized water by centrifugation and dried to obtain CoP / RuP nanozyme.

[0038] Example 2

[0039] (1) Dissolve 0.25 mmol ruthenium acetate, 0.025 mmol cobalt acetate and 0.625 mmol fumaric acid in 3.75 mL ethanol to obtain the first solution;

[0040] (2) Dissolve 0.188 mmol of phytic acid in 0.75 mL of ethanol to obtain a second solution;

[0041] (3) Place 18.75g of sodium chloride into a mortar, and then add the first solution and the second solution in sequence during the grinding process. Grind until the ethanol is completely evaporated to obtain the first solid substance.

[0042] (4) The first solid was placed in a Joule-heated sample stage and subjected to thermal shock calcination under an inert atmosphere. The Joule thermal shock temperature was 500℃, the heating rate was 2000℃ / s, and the number of Joule thermal shocks was 12. The product was washed with deionized water by centrifugation and dried to obtain CoP / RuP nanozyme.

[0043] Example 3

[0044] (1) Dissolve 0.25 mmol ruthenium chloride, 0.075 mmol cobalt chloride, and 0.875 mmol fumaric acid in 6.25 mL ethanol to obtain the first solution;

[0045] (2) Dissolve 0.313 mmol of phytic acid in 1.25 mL of ethanol to obtain a second solution;

[0046] (3) Place 20.83g of sodium chloride into a mortar, and then add the first solution and the second solution in sequence during the grinding process. Grind until the ethanol is completely evaporated to obtain the first solid substance.

[0047] (4) The first solid was placed in a Joule-heated sample stage and subjected to thermal shock calcination under an inert atmosphere. The Joule thermal shock temperature was 650℃, the heating rate was 2500℃ / s, and the number of Joule thermal shocks was 6. The product was washed with deionized water by centrifugation and dried to obtain CoP / RuP nanozyme.

[0048] Comparative Example 1:

[0049] The difference between Comparative Example 1 and Example 1 is that sodium chloride was not used in step (3), while the other methods were the same as in Example 1.

[0050] Comparative Example 2:

[0051] The difference between Comparative Example 2 and Example 1 is that in step (4), the Joule heating calcination method was changed to tube furnace calcination, and the calcination conditions were 650℃ for 1 hour and the heating rate was 5℃ / min. The product was washed with deionized water by centrifugation and dried.

[0052] Comparative Example 3:

[0053] The difference between Comparative Example 3 and Example 1 is that fumaric acid was not used in step (1), while the other methods were the same as in Example 1.

[0054] Comparative Example 4:

[0055] The difference between Comparative Example 4 and Example 1 is that ruthenium chloride was not used in step (1), while the other methods were the same as in Example 1.

[0056] Comparative Example 5:

[0057] The difference between Comparative Example 5 and Example 1 is that cobalt chloride was not used in step (1), while the other methods were the same as in Example 1.

[0058] Application Example 1:

[0059] The nanozymes prepared in the examples and comparative examples were applied to the detection of alanine aminotransferase (ALT), including the following application steps:

[0060] 50 μL of 600 U / L pyruvate oxidase, 150 μL of 600 mM L-alanine, 150 μL of 15 mM α-ketoglutarate, and 100 μL of ALT substrate solutions at different concentrations (69, 230, 460, 805, 1035, 1610, 2070, and 2415 U / L) were incubated together for 10 min. Then, 50 μL of the prepared nanozyme (1 mg / mL), 50 μL of 10 mM 2,2'-azido-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 600 μL of acetate buffer (pH=4.0) were added. After 30 min, the UV-Vis absorption spectrum was collected at 425 nm to evaluate the sensitivity of the colorimetric sensing method based on this nanozyme for ALT detection.

[0061] Figure 1 The image shows the XRD pattern of the nanozyme of Example 1. As can be seen from the image, the characteristic peaks of the XRD pattern of the nanozyme of Example 1 are consistent with the standard cards RuP PDF#97-064-8015 and CoP PDF#97-004-3249, indicating that the nanozyme of Example 1 is a heterojunction material composed of RuP and CoP.

[0062] Figure 2 SEM and EDS mapping images of the nanozyme of Example 1 are shown. The images reveal that Example 1 exhibits a two-dimensional porous nanosheet structure. Furthermore, Ru, Co, and P are uniformly distributed on the nanosheets, indicating that RuP and CoP are evenly dispersed. This good dispersion allows for the full utilization of the synergistic effect of the RuP and CoP components, which is beneficial for promoting the catalytic reaction and improving the sensitivity of the nanozyme sensor.

[0063] Figure 3 The N2-BET plot of the nanozyme of Example 1 is shown. As can be seen from the figure, the nanozyme of Example 1 is a mesoporous material with a pore size distribution mainly ranging from 2-30 nm and a specific surface area of ​​78.32 m². 2 / g. Example 1: The abundant mesopores and large specific surface area of ​​nanozymes can facilitate the exposure of nanozyme active sites, while promoting the adsorption, activation and desorption of substrate molecules, thereby significantly improving the sensitivity and lifespan of nanozyme-based ALT sensors.

[0064] Figure 4 SEM images of the nanozymes in Comparative Examples 1-3 are shown. As can be seen from the images, the nanozymes in Comparative Examples 1-2 all exhibit irregular blocky structures, while the nanozyme in Comparative Example 3 has a sintered sheet-like structure, rather than the two-dimensional porous nanosheet structure of Example 1. This is mainly because, during the preparation process of the examples, fumaric acid first forms metal clusters with ruthenium and cobalt ions, and then, in the presence of sodium chloride, forms a new metal chelate with phytic acid. This metal chelate is mainly formed by the chelation of phytate, fumarate, ruthenium, and cobalt ions, and is adsorbed onto the surface of sodium chloride crystals through grinding. During the Joule heating process, phytate ions provide a phosphorus source through pyrolysis and react with metal ions to form CoP and RuP, which nucleate and grow on the outer surface of the sodium chloride template, forming a two-dimensional sheet-like structure. Simultaneously, phytate and fumarate ions generate a large amount of gas during pyrolysis, which acts as a foaming agent, resulting in a porous structure within the formed two-dimensional phosphide. In addition, the uniform distribution of cobalt and ruthenium ions in the complex results in a uniform distribution of CoP and RuP within the two-dimensional porous phosphide produced by pyrolysis, which is beneficial for leveraging the synergistic effect of the two components. During the cooling stage, the rapid cooling characteristic of Joule heating, combined with the sodium chloride template, inhibits the sintering of the two-dimensional porous sheets, preventing the collapse of the two-dimensional porous structure. In contrast, Comparative Examples 1-3, compared to the examples, lacked the key factors of sodium chloride, Joule heating, and fumaric acid in their preparation processes, resulting in products exhibiting irregular blocky structures and sintered sheet-like structures, respectively.

[0065] Figure 5The EPR spectra of nanozymes from Example 1 and Comparative Examples 1-3 are shown. An EPR signal for a P vacancy appears at g=2.006, with the intensity order being: Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3. This indicates that the nanozyme of Example 1 has abundant P vacancies compared to Comparative Examples 1-3. The formation of these phosphorus vacancies is mainly due to the porous structure endowing the binary heterojunction with abundant interfacial structures. During high-temperature pyrolysis, the P atoms at the interface are in two coordination environments, making them susceptible to defects caused by factors such as lattice stress. The rapid cooling characteristic of Joule heating can stabilize these defects and prevent the phosphorus vacancies from being refilled. These P vacancies can serve as catalytic active sites in the nanozyme colorimetric reaction, and can also optimize the electronic structure of the metal active sites, promoting the catalytic transformation of the reaction substrate and improving the sensitivity of the nanozyme sensor.

[0066] Figure 6 The nanozyme activities of Example 1 and Comparative Examples 1-5 were compared when the ALT addition was 100 μL (2415 U / L). The figures show that Example 1 exhibited the strongest absorption peak at 425 nm, which was sequentially stronger than that of Comparative Examples 2, 1, 5, 3, and 4. This indicates that the order of nanozyme activity is Example 1 > Comparative Example 2 > Comparative Example 1 > Comparative Example 5 > Comparative Example 3 > Comparative Example 4. The performance comparison between Example 1 and Comparative Examples 4-5 shows that the synergistic effect between CoP and RuP enables Example 1 to exhibit superior nanozyme catalytic activity compared to single-component CoP (Comparative Example 4) and RuP (Comparative Example 5). The comparison between Example 1 and Comparative Examples 1-3 shows that the nanozyme of Example 1, due to its two-dimensional porous structure and abundant P vacancies, exhibits significantly higher nanozyme catalytic activity than Comparative Examples 1-3.

[0067] Figure 7 The UV-Vis absorption spectra of ALT solutions ranging from 6 U / L to 210 U / L were displayed. As the ALT concentration increased, the color of the sample solution gradually deepened, and the absorbance at 425 nm also gradually increased.

[0068] Figure 8 The graph shows the relationship between UV absorbance and ALT concentration. As can be seen from the figure, within the concentration range of 0 to 210 U / L, the UV absorbance exhibits a good linear relationship with ALT concentration, with the linear equation: y = 0.0053x + 0.067 (R² = 0.998). The detection limit of ALT is approximately 1.22 U / L, indicating that the colorimetric sensing method constructed using CoP / RuP nanozymes demonstrates excellent ALT detection sensitivity and possesses high scientific value and clinical application potential.

[0069] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0070] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing CoP / RuP nanozymes, characterized in that, Includes the following steps: (1) Dissolve ruthenium salt, cobalt salt, and fumaric acid in ethanol to obtain the first solution; (2) Dissolve phytic acid in ethanol to obtain a second solution; (3) Place sodium chloride in a mortar, and then add the first solution and the second solution in sequence during the grinding process. Grind until the ethanol is completely evaporated to obtain the first solid. (4) The first solid was placed on the sample stage of the Joule heating device for Joule heating calcination. The calcined solid product was then rinsed with deionized water and dried to obtain CoP / RuP nanozyme.

2. The preparation method according to claim 1, characterized in that, In step (1), the ruthenium salt is one of ruthenium chloride and ruthenium acetate; the cobalt salt is one of cobalt sulfate, cobalt nitrate, cobalt chloride, and cobalt acetate.

3. The preparation method according to claim 1, characterized in that, In step (1), the ratio of ruthenium salt, cobalt salt, fumaric acid, and ethanol is 1 mmol: 0.1~0.3 mmol: 2.5~3.5 mmol: 15~25 ml.

4. The preparation method according to claim 1, characterized in that, In step (3), the ratio of sodium chloride, the first solution, and the second solution is 1g: 0.2~0.3ml: 0.04~0.06ml.

5. The preparation method according to claim 1, characterized in that, In step (4), the Joule heat calcination conditions are: Joule heat shock temperature of 500~650℃, heating rate of 2000~2500℃ / s, and number of Joule heat shocks of 6~12 times; each Joule heat shock is to first heat the room temperature to 500~650℃, and then cool it naturally to room temperature.

6. The application of the CoP / RuP nanozyme prepared by the preparation method according to any one of claims 1 to 5 in the detection of alanine aminotransferase.

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