CN115629063A - A detection method for pyrophosphate and alkaline phosphatase based on metallosilicate nanozyme - Google Patents

Abstract

The invention relates to a pyrophosphate and alkaline phosphatase detection method based on metal silicate nano enzyme, which comprises the following steps: (1) Adding one or more of iron salt, manganese salt, copper salt, zinc salt, nickel salt, cerium salt and cobalt salt into the silicon dioxide nanoparticles serving as a template to obtain metal silicate nano enzyme by doping one or more of iron, manganese, copper, zinc, nickel, cerium and cobalt ions; (2) The metallosilicate nanoenzyme shows peroxidase-like activity in H ₂ O ₂ Catalyzing the chromogenic substrate to oxidize and develop color in the presence of the catalyst; (3) In the presence of PPi pyrophosphate, PPi can be complexed with metal ions in the metal silicate nanoenzyme to inhibit the activity of peroxidase-like enzyme, and a colorless chromogenic substrate cannot be oxidized to develop color; (4) Alkaline phosphatase ALP can hydrolyze PPi to recover peroxidase-like activity of metallosilicate nanoenzyme, and colorless chromogenic substrate is developed; thereby preparing high specificityThe metal silicate nano enzyme with stability and sensitivity is used for realizing the rapid and simple detection of PPi and ALP.

Description

Detection of pyrophosphate and alkaline phosphatase based on metallosilicate nanozyme method

technical field

The invention discloses a new method for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozyme. It specifically relates to the preparation of a nanozyme with peroxidase-like catalytic activity and the development of a new technology for the detection of pyrophosphate and alkaline phosphatase based on metal-based silicate nanozyme.

Background technique

Pyrophosphate (P ₂ O ₇ ^4- , PPi) is an emerging biomarker for physiological function and disease monitoring, and plays an important role in physiological processes, so the detection of PPi has become an important research hotspot in recent years. Since PPi is involved in the regulation of multiple physiological functions, the difference in PPi concentration in various biological environments can also be used to monitor or diagnose various diseases such as arthritis, cartilage calcification, and hypophosphatemia. For example, excessive PPi in synovial fluid is closely related to the accumulation of calcium pyrophosphate dihydrate crystals and the pathogenesis of arthritis-like diseases; high levels of extracellular PPi can cause defects in bone mineralization by inhibiting hydroxyapatite deposition , namely hypophosphatemia. In addition, using PPi as a medium can also detect the activity of alkaline pyrophosphatase (ALP); and as a by-product of DNA polymerase chain reaction (PCR), PPi concentration can also be used for real-time monitoring of DNA sequencing, which is expected to be applied to the detection of microbial pathogens. detection.

Currently, the most common methods for detecting the presence and content of PPi include fluorescence, enzymatic, capillary electrophoresis, ion chromatography, and electrochemical analysis. Common PPi detection kits, such as fluorescent probe-based fluorescent pyrophosphate detection kits, detect PPi by using ordinary UV-visible absorption spectrometers or microplate readers, and can quantify PPi concentrations through fluorescence intensity, which is stable, fast and convenient, and facilitates the detection of PPi. High-throughput detection, but the sensitivity is relatively low, the specificity is poor, and the preparation of the probe is more complicated, difficult to store, and expensive; while the enzymatic method has high sensitivity, but the enzyme is easily inactivated, the catalytic environment is high, High cost and other disadvantages; other traditional methods also have problems such as long detection time, complicated and time-consuming sample processing process, and inapplicability to on-site detection. Therefore, it is extremely important to develop a simple and sensitive method for PPi detection.

ALP widely exists in the tissues and organs of the human body. It participates in multiple physiological processes such as cell growth, apoptosis, and signal transduction, and is an indispensable biological enzyme in the human body. The activity of ALP is related to the development of various diseases, such as diabetes, hepatobiliary system diseases, bone diseases, prostate cancer, etc., so its activity is an important indicator for diagnosing the normal state of the body. At present, the most common methods for detecting the presence and activity changes of ALP include colorimetric methods, fluorescence methods, enzyme-labeled methods, electrochemical methods, and electrophoresis methods. Compared with other methods, the colorimetric method has the advantages of simple operation, low price, fast detection speed, and naked-eye detection. However, the current traditional colorimetric method also has problems such as low sensitivity and poor specificity. Therefore, it is urgent to develop a A detection method with high specificity, high sensitivity, simple operation and low price to detect ALP activity.

With the rapid development of nanotechnology, nanomaterials are also widely used in research in various fields, among which nanozymes have excellent enzyme-like activity, high catalytic efficiency, low requirements for catalytic environment, good stability, and easy preparation. The advantages of simplicity, reusability, low price, and good storage have become research hotspots in the detection field in recent years. Some metallosilicate nanozymes such as iron-based nanozymes and copper-based nanozymes have excellent peroxidase-like activity, and can catalyze chromogenic substrates in the presence of hydrogen peroxide (H ₂ O ₂ ). Visible, noticeable color change occurs. In addition, studies have shown that by doping different types of metal ions, the activity and selectivity of nanozymes can be directional designed, or a synergistic catalytic effect can be achieved. However, there is a strong coordination between iron, copper, zinc, manganese, cerium and other metal ions and PPi, which will significantly affect the enzymatic activity of metallosilicate nanozymes, thereby inhibiting the color change of the chromogenic substrate. In conclusion, using the excellent peroxidase-like activity of metallosilicate nanozymes to amplify the enzyme-catalyzed reaction, through the color reaction of the enzyme substrate, not only the rapid detection of PPi and ALP can be realized, but also the rapid detection of PPi and ALP can be realized through the enzyme substrate The degree of color rendering realizes the dynamic change monitoring of PPi and ALP. In recent years, metallosilicate nanozymes have developed rapidly in the field of biomedicine. Compared with traditional natural enzymes, metallosilicate nanozymes have higher catalytic activity and stability, making metallosilicate nanozymes promising for the development of rapid and efficient detection of PPi and ALP. Technology provides new means.

Contents of the invention

Aiming at the problems existing in the current detection methods of pyrophosphate and alkaline phosphatase, the invention proposes a preparation method of metal-based silicate nanozyme and studies its application in the direction of detection of pyrophosphate and alkaline phosphatase. First, using silica nanoparticles as a template, one or more of iron, manganese, copper, zinc, nickel, cerium, and cobalt ions are doped by a hydrothermal method to obtain a metal substrate with peroxidase-like activity. Silicate nanozyme, and then use the prepared metal-based silicate nanozyme to develop a new technology for the detection of pyrophosphate and alkaline phosphatase based on metal-based silicate nanozyme.

Technical scheme of the present invention is as follows:

1. a pyrophosphate radical and alkaline phosphatase detection method based on metallosilicate nanozyme, is characterized in that, comprises the following steps:

Synthesis of I metallosilicate nanozymes

Using silica nanoparticles as a template, doping one or more of iron, manganese, copper, zinc, nickel, cerium, and cobalt ions in a hydrothermal environment to prepare metals with peroxidase-like activity base silicate nanozyme;

II Metal-based silicate nanozyme detection of pyrophosphate

In an acidic environment, metallosilicate nanozymes were used to catalyze the chromogenic reaction of hydrogen peroxide (H ₂ O ₂ ) oxidation of chromogenic substrates and pyrophosphate (PPi) inhibited nanozyme activity to rapidly detect PPi.

III Metallosilicate Nanozyme Detection of Alkaline Phosphatase

After incubation with alkaline phosphatase (ALP) and PPi for 10-90 minutes, it will hydrolyze PPi to release the inhibitory effect of PPi on the enzyme activity of metal-based silicate nanozyme, and catalyze the oxidation of chromogenic substrates to quickly detect ALP activity.

2. further, in step 1, relate to the preparation of metallosilicate nanozyme

Disperse the silica nanoparticles in the aqueous solution, add one or more metal salts containing iron/manganese/copper/zinc/nickel/cerium/cobalt, NH ₄ Cl aqueous solution and ammonia water, wherein the silica nanoparticles , metal salt, NH ₄ Cl, ammonia water (25-28wt%), and water in a molar ratio of 1:0.2-1.5:3-54:7-30:2000-7000. The above solution is stirred for 3-10 minutes, then transferred to a hydrothermal reaction kettle, subjected to a hydrothermal reaction at 120-160° C. for 8-24 hours, centrifuged, washed and then dried to obtain the metallosilicate nanozyme.

3. Further, the metal-based silicate nanozyme may be a silicate nanozyme containing one or more metal elements of iron, manganese, copper, zinc, nickel, cerium, and cobalt.

4. Further, the metal salt is ferric nitrate, ferrous nitrate, ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, ferric acetylacetonate, ferrous acetylacetonate, manganese chloride, manganese acetate, acetyl Manganese acetonate, manganese sulfate, copper chloride, copper nitrate, copper acetate, copper sulfate, copper acetylacetonate, zinc chloride, zinc nitrate, zinc sulfate, zinc acetate, zinc acetylacetonate, nickel chloride, nickel nitrate, nickel sulfate, One of nickel acetate, nickel acetylacetonate, cerium chloride, cerium nitrate, cerium sulfate, cerium acetate, cerium acetylacetonate, cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt acetate, cobalt acetylacetonate and the corresponding metal salt hydrate or a mixture of metal salts.

5. Further, the acidic environment is pH 2.0-7.0.

6. Further, the metallosilicate nanozyme exhibits peroxidase-like activity, and exhibits a visible color change signal after being contacted with a chromogenic substrate in the presence of H ₂ O ₂ .

7. Further, in step II, it involves adding a chromogenic solution containing H ₂ O ₂ into the mixture of the sample to be tested and the metallosilicate nanozyme, wherein the final concentration of H ₂ O ₂ is 0.05-50 mм.

8. Further, the chromogenic substrates are 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis(3-ethylbenzothiazoline-6 -Any one of diammonium sulfonic acid (ABTS) and o-phenylenediamine (OPD), the final concentration of TMB is 208-1664 μм, the final concentration of ABTS is 0.1-6 mм, and the final concentration of OPD is 0.1-3 mм.

9. Further, for PPi detection, if the solution develops color, it means negative (there is no PPi or less content in the sample); if the solution color is suppressed, it means positive (PPi exists in the sample); A photometer can quantitatively detect PPi concentration.

10. Further, for ALP detection, if the solution develops color, it means positive (there is ALP in the sample); if the solution color is inhibited, it means negative (there is no ALP or low activity in the sample); A photometer can quantitatively detect ALP activity.

Under acidic conditions, fully mix the test sample containing PPi with the metallosilicate nanozyme, and then add it to the acetate buffer solution containing the chromogenic substrate and H ₂ O ₂ , and judge according to the color development Whether there is PPi in the sample, and then judge the negative and positive results (the more PPi, the stronger the effect of inhibiting the activity of nanozymes, and the stronger the effect of inhibiting the color development, and the solution system has no color, which means it is positive; and vice versa). At the same time, in addition to directly observing the presence or absence of PPi with the naked eye and qualitatively analyzing the content, the absorbance can also be detected by instruments such as a microplate reader and an ultraviolet-visible absorption spectrometer to further accurately and quantitatively analyze the content of PPi. ALP can hydrolyze PPi, thereby restoring the activity of nanozyme POD enzymes inhibited by PPi, allowing the chromogenic substrate to develop color and then detect its content. Under acidic conditions, fully incubate the test sample containing ALP with a fixed concentration of PPi, then fully mix with _{metallosilicate nanozyme} , and then add to the acetate containing chromogenic substrate and H2O2 In the buffer solution, judge the activity of ALP in the sample according to the strength of the color development, and then judge the negative and positive results (the stronger the ALP activity, the weaker the effect of PPi on inhibiting the activity of nanozyme enzymes, and the color development of the solution system indicates positive; otherwise as well). At the same time, in addition to directly observing the presence or absence of ALP with the naked eye and qualitatively analyzing the activity, the absorbance can be detected by microplate reader, ultraviolet-visible absorption spectrometer and other instruments to further accurately and quantitatively analyze the activity of ALP. The metal-based silicate nanozyme prepared by the invention has good appearance, uniform particles, simple synthesis method steps, good repeatability, non-toxic and harmless, and environment-friendly; The phosphatase detection method is fast, convenient and highly sensitive, and has broad application prospects in virus detection, arthritis, diagnosis of various diseases such as diabetes and bone cancer, and other biomedical fields.

Description of drawings

Fig. 1 is the transmission electron micrograph of ferrosilicate nanozyme prepared in the present invention.

Fig. 2 is a transmission electron micrograph of the ferromanganese silicate nanozyme prepared in the present invention.

Fig. 3 is a transmission electron micrograph of the iron-copper silicate nanozyme prepared in the present invention.

Fig. 4 is a schematic diagram of detection of PPi and ALP by metallosilicate nanozymes.

Fig. 5 is the detection sensitivity of iron manganese silicate nanozyme for PPi.

Fig. 6 is the detection specificity of iron manganese silicate nanozyme for PPi.

Fig. 7 is the detection effect of iron manganese silicate nanozyme on PPi and ALP.

Detailed ways

The application of the present invention will be further described below in conjunction with specific embodiments and drawings, but the scope of protection of the present invention is not limited to the following embodiments.

<test method>

1. Shape test

The morphology of metallosilicate nanozymes was determined by JEOL JEM-1011 field emission transmission electron microscope (TEM).

2. Determination of pyrophosphate detection effect

Using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, the reaction of metallosilicate nanozymes to PPi was achieved by adding H ₂ O ₂ , materials and samples in an acidic environment Specific detection.

3. Determination of alkaline phosphatase detection effect

Using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, metallosilicates were realized by adding H ₂ O ₂ , materials, and samples incubated with PPi in an acidic environment Nanozyme specific detection of ALP.

Embodiment 1 (detection of pyrophosphate and alkaline phosphatase based on iron silicate nanozyme)

Weigh 30mg of silicon dioxide nanoparticles and evenly disperse in 15mL of ultrapure water, and place on a magnetic stirrer to stir at a stirring rate of 300r/min; successively weigh 962.82mg of NH ₄ Cl (18mmol), 125.10mg of FeSO ₄ · 7H ₂ O (0.45mmol) was dispersed in 15mL of ultrapure water, and stirred rapidly on a magnetic stirrer, while adding 700 μL of ammonia water (25wt%), stirred evenly and immediately poured into the aqueous solution of silica nanoparticles, stirred for 5min Then transfer to a 50mL hydrothermal reaction kettle, and conduct a hydrothermal reaction at 140°C for 12 hours. After the reaction, centrifuge and wash 5 times alternately with water and ethanol, and dry in a constant temperature oven at 60°C to obtain the ferrosilicate nanozyme. As shown in Figure 4, under the condition of pH 4.0, the sample to be tested containing pyrophosphate (PPi) was fully mixed with iron silicate nanozyme (final concentration: 50 μg/mL), and then added to the solution containing TMB (final concentration: 832 μм ) and H ₂ O ₂ (final concentration: 0.1mм) in acetate buffer (HAc/NaAc buffer, 0.1м, pH 4.0), when the sample to be tested contains PPi, PPi will combine with iron silicate nanozyme The iron complex in the iron complexes to inhibit the peroxidase-like activity of iron silicate nanozyme, and the TMB color development is inhibited; if the sample to be tested does not contain PPi, in the presence of H ₂ O ₂ , it has a peroxidase-like The active ferrosilicate nanozyme catalyzes the oxidation of TMB, and the solution turns blue. After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In addition to the qualitative analysis of PPi content based on the color depth, the PPi concentration can be accurately quantitatively analyzed according to the absorbance corresponding to the standard curve in combination with a microplate reader or UV-visible spectrophotometry. ALP-containing solution (Tris-HCl buffer, 0.1 м, pH 8.5) and PPi solution (final concentration: 300 μм) were incubated at 37° C. for 60 min. Then the above solution was thoroughly mixed with iron silicate _nanozyme (final concentration: 50 μg/mL), and then added to acetate buffer (HAc/NaAc buffer, _0.1м , pH 4.0) containing TMB and H2O2 middle. After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In the presence of ALP, TMB color development was restored; however, TMB color development was inhibited in the absence or low activity of ALP to hydrolyze PPi. Using PPi as the medium, in addition to the qualitative analysis of ALP activity based on the depth of color development, combined with a microplate reader or UV-visible spectrophotometry, the ALP activity can be accurately and quantitatively analyzed according to the absorbance corresponding to the standard curve.

Embodiment 2 (detection of pyrophosphate and alkaline phosphatase based on ferromanganese silicate nanozyme)

Weigh 30mg of silica nanoparticles and evenly disperse in 15mL of ultrapure water, and place on a magnetic stirrer to stir at a stirring rate of 300r/min; weigh 18.88mg of MnCl ₂ (0.15mmol), 962.82mg of NH ₄ Cl in turn (18mmol), 83.40mg FeSO ₄ ·7H ₂ O (0.3mmol) were dispersed in 15mL ultrapure water, and stirred quickly on a magnetic stirrer, and 700μL ammonia water (25wt%) was added at the same time, and immediately poured into two In the aqueous solution of silicon oxide nanoparticles, stir for 5 minutes, transfer to a 50mL hydrothermal reaction kettle, and conduct a hydrothermal reaction at 140°C for 12h. After the reaction, wash by centrifugation, and alternately wash with water and ethanol for 5 times, and dry in a constant temperature drying oven at 60°C The ferromanganese silicate nanozyme is obtained. As shown in Figure 4, under the condition of pH 4.0, the sample to be tested containing PPi was fully mixed with ferromanganese silicate nanozyme (final concentration: 50 μg/mL), and then added to the solution containing TMB (final concentration: 832 μм) and H In acetate buffer (HAc/NaAc buffer, 0.1м, pH 4.0) of ₂ O ₂ (final concentration: 0.1mм), when the sample to be tested contains PPi, PPi will combine with ferromanganese silicate nanozyme The complexation of iron and manganese inhibits the peroxidase-like activity of iron _- manganese silicate _nanozyme , and the color development of TMB is inhibited; if the sample to be tested does not contain PPi, it has a peroxidase-like The iron-manganese-silicate nanozyme with oxidase activity catalyzes the oxidation of TMB to develop color, and the solution turns blue. After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In addition to the qualitative analysis of PPi content based on the color depth, the PPi concentration can be accurately quantitatively analyzed according to the absorbance corresponding to the standard curve in combination with a microplate reader or UV-visible spectrophotometry. Among them, as shown in Figure 5, the linear range of PPi detection is 0.6～4.5μм (y=0.1195x+0.002624, R ² =0.9927), and in the case of 3 times the signal-to-noise ratio, the detection limit is 205.47nм; it will contain ALP solution (Tris-HCl buffer, 0.1м, pH 8.5) and PPi solution (final concentration: 300μм) were incubated at 37°C for 60min. Then the above solution was thoroughly mixed with iron-manganese _- silicate _nanozyme (final concentration: 50 μg/mL), and then added to acetate buffer (HAc/NaAc buffer, 0.1 м, pH 4.0). After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In the presence of ALP, TMB color development was restored; however, TMB color development was inhibited in the absence or low activity of ALP to hydrolyze PPi. Using PPi as the medium, in addition to the qualitative analysis of ALP activity based on the depth of color development, combined with a microplate reader or UV-visible spectrophotometry, the ALP activity can be accurately and quantitatively analyzed according to the absorbance corresponding to the standard curve.

Embodiment 3 (detection of pyrophosphate and alkaline phosphatase based on iron-copper silicate nanozyme)

Weigh 30mg of silica nanoparticles and evenly disperse them in 15mL of ultrapure water, and place on a magnetic stirrer to stir at a stirring rate of 300r/min; successively weigh 962.82mg of NH ₄ Cl (18mmol), 83.40mg of FeSO ₄ · 7H ₂ O (0.3mmol), 36.24mg Cu(NO ₃ ) ₂ 3H ₂ O (0.15mmol) were dispersed in 15mL of ultrapure water, and stirred rapidly on a magnetic stirrer, while adding 700μL of ammonia water (25wt%), After stirring evenly, pour it into the aqueous solution of silica nanoparticles, stir for 5 minutes, transfer it to a 50mL hydrothermal reaction kettle, and conduct a hydrothermal reaction at 140°C for 12 hours. After drying in a constant temperature drying oven at 60°C, the iron-copper silicate nanozyme can be obtained. As shown in Figure 4, under the condition of pH 4.0, the sample to be tested containing PPi was fully mixed with iron-copper silicate nanozyme (final concentration: 50 μg/mL), and then added to the solution containing TMB (final concentration: 832 μм) and H In acetate buffer (HAc/NaAc buffer, 0.1m, pH 4.0) of ₂ O ₂ (final concentration: 0.1mм), when the sample to be tested contains PPi, PPi will combine with iron-copper silicate nanozyme The complexation of iron and copper inhibits the peroxidase-like activity of iron _- copper silicate _nanozyme , and the color development of TMB is inhibited; if the sample to be tested does not contain PPi, it has a peroxidase-like The iron-copper silicate nanozyme with oxidase activity catalyzes the oxidation of TMB to develop color, and the solution turns blue. After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In addition to the qualitative analysis of PPi content based on the color depth, the PPi concentration can be accurately quantitatively analyzed according to the absorbance corresponding to the standard curve in combination with a microplate reader or UV-visible spectrophotometry. A solution containing ALP (Tris-HCl buffer, 0.1 м, pH 8.5) and PPi (final concentration: 300 μм) was incubated at 37° C. for 60 min. Then the above solution was thoroughly mixed with iron _- copper silicate _nanozyme (final concentration: 50 μg/mL), and then added to acetate buffer (HAc/NaAc buffer, 0.1 м, pH 4.0). After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In the presence of ALP, TMB color development was restored; however, TMB color development was inhibited in the absence or low activity of ALP to hydrolyze PPi. Using PPi as the medium, in addition to the qualitative analysis of ALP activity based on the depth of color development, combined with a microplate reader or UV-visible spectrophotometry, the ALP activity can be accurately and quantitatively analyzed according to the absorbance corresponding to the standard curve.

Embodiment 4 (based on the detection of PPi content and ALP activity in clinical real samples by metallosilicate nanozyme)

As shown in Figure 4, under the condition of pH 4.0, the clinical sample containing 1% diluted human serum was fully mixed with metallosilicate nanozyme (final concentration: 50 μg/mL), and then added to the solution containing TMB (final concentration: 832 μм ) and H ₂ O ₂ (final concentration: 0.1mм) in acetate buffer (HAc/NaAc buffer, 0.1м, pH 4.0), when the sample to be tested contains PPi, PPi will combine with metal silicate The metal ions in the nanozyme are complexed to inhibit the peroxidase _- like activity of the metallosilicate _nanozyme , and the color development of TMB is inhibited; if the sample to be tested does not contain PPi, in the presence of H2O2, it has The metallosilicate nanozyme with peroxidase-like activity catalyzes the oxidation of TMB to develop color, and the solution turns blue. After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In addition to the qualitative analysis of PPi content based on the color depth, the PPi concentration can be accurately quantitatively analyzed according to the absorbance corresponding to the standard curve in combination with a microplate reader or UV-visible spectrophotometry. Buffer containing 1% diluted human serum clinical samples (Tris-HCl buffer, 0.1 м, pH 8.5) and PPi (final concentration: 300 μм) were incubated at 37° C. for 60 min. Then the above solution was thoroughly mixed with metallosilicate _{nanozymes (} final concentration: 50 μg/mL), and then added to acetate buffer (HAc/NaAc buffer, 0.1 м, pH 4.0). After 15 minutes of reaction, the negative or positive result can be judged by naked eyes according to the color development. In the presence of ALP, TMB color development was restored; however, TMB color development was inhibited in the absence or low activity of ALP to hydrolyze PPi. Using PPi as the medium, in addition to the qualitative analysis of ALP activity based on the depth of color development, combined with a microplate reader or UV-visible spectrophotometry, the ALP activity can be accurately and quantitatively analyzed according to the absorbance corresponding to the standard curve.

Claims (10)

1. A pyrophosphate and alkaline phosphatase detection method based on metal silicate nanoenzyme is characterized by comprising the following steps:

i Synthesis of metallosilicate nanoenzymes

The method comprises the following steps of (1) doping one or more of iron, manganese, copper, zinc, nickel, cerium and cobalt ions into silicon dioxide nanoparticles serving as a template in a hydrothermal environment to prepare the metal silicate nanoenzyme with peroxidase-like activity;

II detection of pyrophosphate by metal silicate nano enzyme

Under an acidic environment, catalyzing a color reaction of hydrogen peroxide oxidation chromogenic substrate by using metal silicate nano enzyme and rapidly detecting PPi by inhibiting nano enzyme activity by pyrophosphate PPi;

III detection of alkaline phosphatase by using metal silicate nano enzyme

After alkaline phosphatase ALP and PPi are incubated for 10-90 min, PPi is hydrolyzed to remove the enzyme activity inhibition effect of PPi on the metal silicate nano enzyme, and ALP activity is rapidly detected by catalyzing, oxidizing and developing a substrate.

2. The method according to claim 1, wherein the preparation steps involving metallosilicate nanoenzymes in step I are as follows:

adding sodium silicateDispersing rice particles in water solution, adding one or more metal salts containing Fe/Mn/Cu/Zn/Ni/Ce/Co, and NH ₄ Aqueous solution of Cl and ammonia water, wherein silicon dioxide nanoparticles, metal salt, NH ₄ The feeding molar ratio of Cl, ammonia water with the concentration of 25-28 wt% and water is 1; stirring the solution for 3-10 min, transferring the solution into a hydrothermal reaction kettle, carrying out hydrothermal reaction for 8-24 h at 120-160 ℃, centrifugally washing and drying to obtain the metal silicate nano enzyme.

3. The method according to claim 1, wherein the metallosilicate nanoenzyme is a silicate nanoenzyme containing one or more metal elements selected from iron, manganese, copper, zinc, nickel, cerium and cobalt.

4. The method of claim 1, wherein the metal salt is a mixture of one or more metal salts selected from the group consisting of ferric nitrate, ferrous nitrate, ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, ferric acetylacetonate, ferrous acetylacetonate, manganese chloride, manganese acetate, manganese acetylacetonate, manganese sulfate, copper chloride, copper nitrate, copper acetate, copper sulfate, copper acetylacetonate, zinc chloride, zinc nitrate, zinc sulfate, zinc acetate, zinc acetylacetonate, nickel chloride, nickel nitrate, nickel sulfate, nickel acetate, nickel acetylacetonate, cerium chloride, cerium nitrate, cerium sulfate, cerium acetate, cerium acetylacetonate, cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt acetate, cobalt acetylacetonate, and hydrates of the corresponding metal salts.

5. The method of claim 1, wherein the acidic environment is at a ph of 2.0 to 7.0.

6. The method of claim 1, wherein the metallosilicate nanoenzyme exhibits peroxidase-like activity in H ₂ O ₂ And exhibits a macroscopic discoloration signal upon contact with a chromogenic substrate in the presence of a light.

7. The method of claim 1, wherein step II involves adding H to the mixture of the sample to be tested and the metal silicate nanoenzyme ₂ O ₂ The color developing solution of (1), wherein H ₂ O ₂ The final concentration is 0.05-50 m.

8. The method according to claim 1, wherein the chromogenic substrate is any one of 3,3', 5' -Tetramethylbenzidine (TMB), 2' -diaza-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD), and the final concentration of TMB is 208 to 1664 μm, the final concentration of ABTS is 0.1 to 6m, and the final concentration of OPD is 0.1 to 3 m.

9. The method of claim 1, wherein for PPi detection, a solution color development indicates negative; if the solution color development is inhibited, the solution is positive; and the PPi concentration is quantitatively detected by a microplate reader or an ultraviolet spectrophotometer.

10. The method of claim 1, wherein for ALP detection, a solution that is colored indicates positive; if the solution color development is inhibited, the solution is negative; whereas ALP activity was quantitatively determined by a microplate reader or an ultraviolet spectrophotometer.

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