A method for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozymes

Abstract

The present invention relates to a method for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozymes, comprising: (1) using silica nanoparticles as a template and adding one or more of iron salts, manganese salts, copper salts, zinc salts, nickel salts, cerium salts, and cobalt salts to incorporate one or more of iron, manganese, copper, zinc, nickel, cerium, and cobalt ions to obtain metal-based silicate nanozymes; (2) the metal-based silicate nanozymes exhibit peroxidase-like activity and catalyze the oxidation and color development of a chromogenic substrate in the presence of H2O2; (3) in the presence of pyrophosphate (PPi), PPi will complex with the metal ions in the metal-based silicate nanozymes, thereby inhibiting their peroxidase-like activity, and the colorless chromogenic substrate cannot be oxidized and developed; (4) alkaline phosphatase (ALP) can hydrolyze PPi to restore the peroxidase-like activity of the metal-based silicate nanozymes, and the colorless chromogenic substrate develops color; thus, metal-based silicate nanozymes with high specificity, stability, and sensitivity are prepared to achieve rapid and simple detection of PPi and ALP.

Description

Technical Field

[0001] The present invention discloses a new method for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozymes. Specifically, it relates to the preparation of nanozymes with peroxidase-like catalytic activity and the development of a new technology for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozymes. Background Art

[0002] Pyrophosphate (P2O7 4- , PPi) is an emerging biomarker for physiological functions and disease monitoring, playing an important role in physiological processes. Therefore, the detection of PPi has been an important research hotspot in recent years. Since PPi participates in the regulation of various physiological functions, the differences in PPi concentrations in various biological environments can also be used to monitor or diagnose various diseases such as arthritis, chondrocalcinosis, and hypophosphatemia. For example, excessive PPi content in synovial fluid is closely related to the accumulation of calcium pyrophosphate dihydrate crystals and the pathogenesis of arthritis-like diseases; high extracellular PPi concentrations can cause bone mineralization defects, i.e., hypophosphatemia, by inhibiting hydroxyapatite deposition. In addition, the activity of alkaline pyrophosphatase (ALP) can be detected using PPi as a mediator; and as a by-product of DNA polymerase chain reaction (PCR), PPi concentration can also be used for real-time monitoring of DNA sequencing, with potential applications in the detection of microbial pathogens.

[0003] Currently, the most common methods for detecting the presence and content changes of PPi include fluorescence method, enzymatic method, capillary electrophoresis method, ion chromatography method, and electrochemical analysis method, etc. Common PPi detection kits, such as the fluorescence-based pyrophosphate detection kit using a fluorescence probe, detect PPi by using a common ultraviolet-visible absorption spectrometer or microplate reader, and the PPi concentration can be quantified by fluorescence intensity. It has good stability, is fast and convenient, and is conducive to high-throughput detection, but has relatively low sensitivity, poor specificity, and the preparation of the probe is complex, difficult to store, and expensive; while the enzymatic method has high sensitivity, but has problems such as easy inactivation of the enzyme, high requirements for the catalytic environment, and high cost; other traditional methods also have problems such as long detection time, complex and time-consuming sample processing, and inapplicability to on-site detection. Therefore, it is extremely important to develop a simple and sensitive PPi detection method.

[0004] ALP is widely present in human tissues and organs. It participates in multiple physiological processes such as cell growth, apoptosis, and signal transduction. It is an indispensable biological enzyme in the human body. The activity of ALP is related to the development of many diseases, such as diabetes, hepatobiliary system diseases, bone diseases, prostate cancer, etc. Therefore, its activity is an important indicator for diagnosing normal body. At present, the most common methods for detecting the presence and activity changes of ALP include colorimetry, fluorescence, enzyme labeling, electrochemical method, electrophoresis, etc. Compared with other methods, colorimetry has the advantages of simple operation, low price, fast detection speed, and naked eye detection. However, the current traditional colorimetry also has problems such as low sensitivity and poor specificity. Therefore, it is urgent to develop a highly specific, highly sensitive, simple to operate, and low-cost detection method to detect ALP activity.

[0005] With the rapid development of nanotechnology, nanomaterials have also been widely used in research in various fields. Among them, nanozymes have been a research hotspot in the detection field in recent years because of their excellent enzyme-like activity, high catalytic efficiency, low requirements for the catalytic environment, good stability, simple preparation, reusability, low price, and convenient storage. Some metal-based silicate nanozymes, such as iron-based nanozymes and copper-based nanozymes, have excellent peroxidase-like activity and can catalyze the color-developing substrate to undergo a visible and obvious color change in the presence of hydrogen peroxide (H2O2). In addition, studies have shown that by doping different types of metal ions, the activity and selectivity of nanozymes can be directed and designed, or a synergistic catalytic effect can be achieved. There is a strong coordination effect between metal ions such as iron, copper, zinc, manganese, and cerium and PPi, which will significantly affect the enzymatic activity of metal-based silicate nanozymes, thereby inhibiting the color change of the color-developing substrate. In summary, by utilizing the excellent peroxidase-like activity of metal-based silicate nanozymes to amplify enzyme catalytic reactions, through the color development reaction of the enzyme substrate, not only can PPi and ALP be detected quickly, but also the dynamic changes of PPi and ALP can be monitored through the color development of the enzyme substrate. In recent years, metal-based silicate nanozymes have developed rapidly in the biomedical field. Compared with traditional natural enzymes, metal-based silicate nanozymes have higher catalytic activity and stability, making them promising new means for the development of fast and efficient PPi and ALP detection technologies. Summary of the invention

[0006] In view of the problems existing in the current detection methods for pyrophosphate and alkaline phosphatase, the present invention proposes a preparation method of metal-based silicate nanozyme and studies its application in the 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 hydrothermal method to obtain a metal-based silicate nanozyme with peroxidase-like activity. Then, a new detection technology for pyrophosphate and alkaline phosphatase based on the prepared metal-based silicate nanozyme is developed.

[0007] The technical solution of the present invention is as follows:

[0008] 1. A detection method for pyrophosphate and alkaline phosphatase based on metal-based silicate nanozyme, characterized by comprising the following steps:

[0009] I. Synthesis of metal-based silicate nanozyme

[0010] Using silica nanoparticles as a template, one or more of iron, manganese, copper, zinc, nickel, cerium, and cobalt ions are incorporated under hydrothermal conditions to prepare a metal-based silicate nanozyme with peroxidase-like activity;

[0011] II. Detection of pyrophosphate by metal-based silicate nanozyme

[0012] Under acidic conditions, the color reaction of catalyzing hydrogen peroxide (H2O2) to oxidize the chromogenic substrate by the metal-based silicate nanozyme and the rapid detection of PPi by the inhibition of the nanozyme activity by pyrophosphate (PPi) are utilized.

[0013] III. Detection of alkaline phosphatase by metal-based silicate nanozyme

[0014] After incubating alkaline phosphatase (ALP) with PPi for 10 - 90 min, it will hydrolyze PPi to relieve the inhibition of the enzyme activity of the metal-based silicate nanozyme by PPi, and catalyze the oxidation of the chromogenic substrate to rapidly detect the activity of ALP.

[0015] 2. Further, the preparation of the metal-based silicate nanozyme involved in step I

[0016] Disperse silica nanoparticles in an aqueous solution, add an aqueous solution containing one or more metal salts of iron / manganese / copper / zinc / nickel / cerium / cobalt, NH4Cl, and ammonia water. The molar ratio of silica nanoparticles, metal salt, NH4Cl, ammonia water (25 - 28 wt%), and water is 1:0.2 - 1.5:3 - 54:7 - 30:2000 - 7000. Stir the above solution for 3 - 10 min and then transfer it to a hydrothermal reaction kettle. Carry out hydrothermal reaction at 120 - 160 °C for 8 - 24 h, centrifuge, wash, and dry to obtain the metal-based silicate nanozyme.

[0017] 3. Further, the metal-based silicate nanozyme may be a silicate nanozyme containing one or more metal elements among iron, manganese, copper, zinc, nickel, cerium, and cobalt.

[0018] 4. Further, the metal salt is a mixture of one or more metal salts among ferric nitrate, ferrous nitrate, ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, iron(III) acetylacetonate, iron(II) acetylacetonate, manganese chloride, manganese acetate, manganese(III) acetylacetonate, manganese sulfate, copper chloride, copper nitrate, copper acetate, copper sulfate, copper(II) acetylacetonate, zinc chloride, zinc nitrate, zinc sulfate, zinc acetate, zinc(II) acetylacetonate, nickel chloride, nickel nitrate, nickel sulfate, nickel acetate, nickel(II) acetylacetonate, cerium chloride, cerium nitrate, cerium sulfate, cerium acetate, cerium(III) acetylacetonate, cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt acetate, cobalt(II) acetylacetonate and the corresponding metal salt hydrates.

[0019] 5. Further, the acidic environment is pH 2.0 - 7.0.

[0020] 6. Further, the metal-based silicate nanozyme exhibits peroxidase-like activity and shows a visible color change signal after contacting with a chromogenic substrate in the presence of H2O2.

[0021] 7. Further, in step II, a chromogenic solution containing H2O2 is added to the mixture of the sample to be tested and the metal-based silicate nanozyme, and the final concentration of H2O2 is 0.05 - 50 mM.

[0022] 8. Further, the chromogenic substrate is any one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD). The final concentration of TMB is 208 - 1664 μM, the final concentration of ABTS is 0.1 - 6 mM, and the final concentration of OPD is 0.1 - 3 mM.

[0023] 9. Further, for PPi detection, if the solution shows color, it indicates negative (PPi does not exist or is present in a small amount in the sample); if the color development of the solution is inhibited, it indicates positive (PPi exists in the sample); and the concentration of PPi can be quantitatively detected by an enzyme-linked immunosorbent assay (ELISA) reader or an ultraviolet spectrophotometer.

[0024] 10. Further, for ALP detection, if the solution shows color, it indicates positive (ALP exists in the sample); if the color development of the solution is inhibited, it indicates negative (ALP does not exist or has low activity in the sample); and the activity of ALP can be quantitatively detected by an ELISA reader or an ultraviolet spectrophotometer.

[0025] Under acidic conditions, a test sample containing PPi is thoroughly mixed with a metal-based silicate nanozyme, and then added to an acetate buffer containing a chromogenic substrate and H2O2. Based on the color development, it is determined whether PPi exists in the sample, and thus the positive and negative results are judged (the more PPi there is, the stronger the inhibitory effect on the enzyme-like activity of the nanozyme, and the stronger the inhibitory effect on its color development. If the solution system has no color, it indicates a positive result; 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 an enzyme-linked immunosorbent assay (ELISA) reader and an ultraviolet-visible absorption spectrometer to further accurately quantify the content of PPi. And ALP can hydrolyze PPi, thereby restoring the enzyme-like POD enzyme activity of the nanozyme inhibited by PPi, causing the chromogenic substrate to develop color and then detecting its content. Under acidic conditions, a test sample containing ALP is fully incubated with a fixed concentration of PPi, then thoroughly mixed with a metal-based silicate nanozyme, and then added to an acetate buffer containing a chromogenic substrate and H2O2. Based on the intensity of color development, the activity of ALP in the sample is judged, and thus the positive and negative results are judged (the stronger the ALP activity, the weaker the inhibitory effect of PPi on the enzyme-like activity of the nanozyme. If the solution system develops color, it indicates a positive result; vice versa). 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 also be detected by instruments such as an ELISA reader and an ultraviolet-visible absorption spectrometer to further accurately quantify the activity of ALP. The metal-based silicate nanozyme prepared by the present invention has good morphology, uniform particles, a simple synthesis method with good repeatability, is non-toxic, harmless, and environmentally friendly; the detection methods for pyrophosphate and alkaline phosphatase based on the metal-based silicate nanozyme are rapid, convenient, and highly sensitive, and have broad application prospects in the diagnosis of various diseases such as virus detection, arthritis, diabetes, and bone cancer, as well as other biomedical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the transmission electron microscopy image of the iron silicate nanozyme prepared in the present invention.

[0027] Figure 2 is the transmission electron microscopy image of the iron manganese silicate nanozyme prepared in the present invention.

[0028] Figure 3 is the transmission electron microscopy image of the iron copper silicate nanozyme prepared in the present invention.

[0029] Figure 4 is the schematic diagram of the detection of PPi and ALP by the metal-based silicate nanozyme.

[0030] Figure 5 is the detection sensitivity of the iron manganese silicate nanozyme for PPi.

[0031] Figure 6 is the detection specificity of the iron manganese silicate nanozyme for PPi.

[0032] Figure 7 It is the detection effect of iron-manganese silicate nanozyme on PPi and ALP. Specific implementation manners

[0033] The following further details the application of the present invention in combination with specific implementation manners and the drawings, but the protection scope of the present invention is not limited to the following implementation manners.

[0034] <Testing method>

[0035] 1. Morphology testing

[0036] The morphology of the metal-based silicate nanozyme was determined by a JEOL JEM-1011 field emission transmission electron microscope (TEM) made in Japan.

[0037] 2. Determination of the detection effect on pyrophosphate

[0038] Using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate, the specific detection of PPi by the metal-based silicate nanozyme was achieved by adding H2O2, the material and the sample to react in an acidic environment.

[0039] 3. Determination of the detection effect on alkaline phosphatase

[0040] Using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate, the specific detection of ALP by the metal-based silicate nanozyme was achieved by adding H2O2, the material and the sample incubated with PPi to react in an acidic environment.

[0041] Example 1 (Detection of pyrophosphate and alkaline phosphatase based on iron silicate nanozyme)

[0042] Weigh 30 mg of silica nanoparticles and disperse them evenly in 15 mL of ultrapure water, and place them on a magnetic stirrer and stir at a stirring rate of 300 r / min; successively weigh 962.82 mg of NH4Cl (18 mmol) and 125.10 mg of FeSO4·7H2O (0.45 mmol) and disperse them in 15 mL of ultrapure water, and stir rapidly on a magnetic stirrer. At the same time, add 700 μL of ammonia water (25 wt%), and after stirring evenly, immediately pour it into the silica nanoparticle aqueous solution. After stirring for 5 min, transfer it to a 50 mL hydrothermal reaction kettle, and carry out hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge and wash, and wash alternately with water and ethanol 5 times. After drying in a constant temperature drying oven at 60 °C, the iron silicate nanozyme is obtained. As Figure 4As shown, the test sample containing pyrophosphate (PPi) is fully mixed with iron silicate nanozyme (final concentration: 50 μg / mL) under the condition of pH 4.0, and then added to the acetate buffer solution (HAc / NaAc buffer solution, 0.1 M, pH 4.0) containing TMB (final concentration: 832 μM) and H2O2 (final concentration: 0.1 mM). When the test sample contains PPi, PPi will chelate with the iron in the iron silicate nanozyme, thereby inhibiting the peroxidase-like activity of the iron silicate nanozyme, and the TMB color development is inhibited; if the test sample does not contain PPi, in the presence of H2O2, the iron silicate nanozyme with peroxidase-like activity catalyzes the oxidation and color development of TMB, and the solution turns blue. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development situation. In addition to qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately quantitatively analyzed by combining an enzyme-linked immunosorbent assay (ELISA) reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve. The solution containing ALP (Tris-HCl buffer solution, 0.1 M, pH 8.5) and the PPi solution (final concentration: 300 μM) are incubated at 37 °C for 60 min. Then the above solution is fully mixed with the iron silicate nanozyme (final concentration: 50 μg / mL), and then added to the acetate buffer solution (HAc / NaAc buffer solution, 0.1 M, pH 4.0) containing TMB and H2O2. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development situation. In the presence of ALP, the TMB color development is restored; when ALP is absent or the activity is too low to hydrolyze PPi, the TMB color development is inhibited. Taking PPi as a medium, in addition to qualitatively analyzing the ALP activity according to the color depth, the ALP activity can be accurately quantitatively analyzed by combining an ELISA reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve.

[0043] Example 2 (Detection of Pyrophosphate and Alkaline Phosphatase Based on Iron Manganese Silicate Nanozyme)

[0044] Weigh 30 mg of silicon dioxide nanoparticles and disperse them evenly in 15 mL of ultrapure water, and place them on a magnetic stirrer to stir at a stirring rate of 300 r / min; weigh 18.88 mg of MnCl2 (0.15 mmol), 962.82 mg of NH4Cl (18 mmol), and 83.40 mg of FeSO4·7H2O (0.3 mmol) in sequence and disperse them in 15 mL of ultrapure water, and stir rapidly on a magnetic stirrer. At the same time, add 700 μL of ammonia water (25 wt%), and immediately pour it into the aqueous solution of silicon dioxide nanoparticles after stirring evenly. After stirring for 5 min, transfer it to a 50 mL hydrothermal reaction kettle, and carry out hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge and wash, and wash alternately with water and ethanol for 5 times. After drying in a constant temperature drying oven at 60 °C, the iron manganese silicate nanozyme is obtained. As Figure 4As shown, the test sample containing PPi was fully mixed with iron-manganese silicate nanozyme (final concentration: 50 μg / mL) under the condition of pH 4.0, and then added to the acetate buffer (HAc / NaAc buffer, 0.1 M, pH 4.0) containing TMB (final concentration: 832 μM) and H2O2 (final concentration: 0.1 mM). When the test sample contains PPi, PPi will complex with iron and manganese in the iron-manganese silicate nanozyme, thus inhibiting the peroxidase-like activity of the iron-manganese silicate nanozyme, and the TMB color development is inhibited; if the test sample does not contain PPi, in the presence of H2O2, the iron-manganese silicate nanozyme with peroxidase-like activity catalyzes the oxidation and color development of TMB, and the solution turns blue. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development situation. In addition to qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately quantitatively analyzed by combining an enzyme-linked immunosorbent assay (ELISA) reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve. Among them, as Figure 5 shown, the linear range of PPi detection is 0.6 - 4.5 μM (y = 0.1195x + 0.002624, R 2 = 0.9927). Under the condition of 3 times the signal-to-noise ratio, the detection limit is 205.47 nM; the solution containing ALP (Tris-HCl buffer, 0.1 M, pH 8.5) and the PPi solution (final concentration: 300 μM) were incubated at 37 °C for 60 min. Then the above solution was fully mixed with the iron-manganese silicate nanozyme (final concentration: 50 μg / mL), and then added to the acetate buffer (HAc / NaAc buffer, 0.1 M, pH 4.0) containing TMB and H2O2. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development situation. In the presence of ALP, the TMB color development is restored; when ALP is absent or the activity is too low to hydrolyze PPi, the TMB color development is inhibited. Taking PPi as a medium, in addition to qualitatively analyzing the ALP activity according to the color depth, the ALP activity can be accurately quantitatively analyzed by combining an ELISA reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve.

[0045] Example 3 (Detection of Pyrophosphate and Alkaline Phosphatase Based on Iron-Copper Silicate Nanozyme)

[0046] Weigh 30 mg of silica nanoparticles and disperse them evenly in 15 mL of ultrapure water, and place them on a magnetic stirrer and stir at a stirring rate of 300 r / min; weigh 962.82 mg NH4Cl (18 mmol), 83.40 mg FeSO4·7H2O (0.3 mmol), and 36.24 mg Cu(NO3)2·3H2O (0.15 mmol) in turn and disperse them in 15 mL of ultrapure water, and stir them quickly on a magnetic stirrer. At the same time, add 700 μL of ammonia water (25 wt%), and immediately pour it into the silica nanoparticle aqueous solution after stirring evenly. After stirring for 5 minutes, transfer it to a 50 mL hydrothermal reactor and perform a hydrothermal reaction at 140°C for 12 hours. After the reaction is completed, wash it by centrifugation, and wash it alternately with water and ethanol for 5 times. Dry it in a constant temperature drying oven at 60°C to obtain the iron-copper silicate nanozyme. Figure 4 As shown, the sample containing PPi is fully mixed with the iron copper silicate nanozyme (final concentration: 50 μg / mL) under pH 4.0, and then added to an acetate buffer (HAc / NaAc buffer, 0.1 μM, pH 4.0) containing TMB (final concentration: 832 μM) and H2O2 (final concentration: 0.1 mM). When the sample contains PPi, PPi will complex with the iron and copper in the iron copper silicate nanozyme to inhibit the peroxidase-like activity of the iron copper silicate nanozyme, and the TMB color development will be inhibited; if the sample does not contain PPi, in the presence of H2O2, the iron copper silicate nanozyme with peroxidase-like activity catalyzes the oxidation of TMB and the solution turns blue. After 15 minutes of reaction, the negative or positive result can be judged by the naked eye according to the color development. In addition to qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately quantitatively analyzed according to the absorbance corresponding to the standard curve in combination with an enzyme reader or ultraviolet visible spectrophotometer. A solution containing ALP (Tris-HCl buffer, 0.1M, pH 8.5) and PPi (final concentration: 300μM) were incubated at 37°C for 60 min. The above solution was then fully mixed with iron copper silicate nanozyme (final concentration: 50μg / mL) and added to an acetate buffer (HAc / NaAc buffer, 0.1M, pH 4.0) containing TMB and H2O2. After 15 minutes of reaction, the negative or positive result can be judged by the naked eye according to the color development. In the presence of ALP, TMB color development is restored; when ALP is absent or the activity is too low to hydrolyze PPi, TMB color development is inhibited. In addition to qualitatively analyzing ALP activity based on the depth of color development using PPi as a medium, ALP activity can be accurately quantitatively analyzed based on the absorbance corresponding to the standard curve using an ELISA reader or UV-visible spectrophotometer.

[0047] Example 4 (Detection of PPi content and ALP activity in real clinical samples based on metal-based silicate nanozymes)

[0048] As Figure 4 shown, at pH 4.0, a clinical sample of 1% diluted human serum was thoroughly mixed with a metal-based silicate nanozyme (final concentration: 50 μg / mL), and then added to an acetate buffer (HAc / NaAc buffer, 0.1 M, pH 4.0) containing TMB (final concentration: 832 μM) and H2O2 (final concentration: 0.1 mM). When the sample to be tested contains PPi, PPi will complex with the metal ions in the metal-based silicate nanozyme, thus inhibiting the peroxidase-like activity of the metal-based silicate nanozyme, and the TMB color development is inhibited; if the sample to be tested does not contain PPi, in the presence of H2O2, the metal-based silicate nanozyme with peroxidase-like activity catalyzes the oxidation and color development of TMB, and the solution turns blue. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development. In addition to qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately quantitatively analyzed by combining an enzyme-linked immunosorbent assay (ELISA) reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve. A buffer solution (Tris-HCl buffer, 0.1 M, pH 8.5) containing a 1% diluted human serum clinical sample and PPi (final concentration: 300 μM) was incubated at 37 °C for 60 min. Then the above solution was thoroughly mixed with a metal-based silicate nanozyme (final concentration: 50 μg / mL), and then added to an acetate buffer (HAc / NaAc buffer, 0.1 M, pH 4.0) containing TMB and H2O2. After a 15-minute reaction, the negative or positive result can be visually judged according to the color development. In the presence of ALP, the TMB color development is restored; when ALP is absent or the activity is too low to hydrolyze PPi, the TMB color development is inhibited. Using PPi as a medium, in addition to qualitatively analyzing the ALP activity according to the color depth, the ALP activity can be accurately quantitatively analyzed by combining an ELISA reader or ultraviolet-visible spectrophotometry according to the absorbance corresponding to the standard curve.

Claims

1. A method for detecting pyrophosphate and alkaline phosphatase based on metal-based silicate nanozyme, characterized in that, It includes the following steps: I Synthesis of metal-based silicate nanozyme Using silica nanoparticles as a template, incorporating one or more of iron, manganese, copper, zinc, nickel, cerium, and cobalt ions under hydrothermal conditions to prepare a metal-based silicate nanozyme with peroxidase-like activity; II Detection of pyrophosphate by metal-based silicate nanozyme Under acidic conditions, using the color reaction of the metal-based silicate nanozyme catalyzing the oxidation of hydrogen peroxide to a chromogenic substrate and the inhibition of the nanozyme activity by pyrophosphate (PPi) to rapidly detect PPi; III Detection of alkaline phosphatase by metal-based silicate nanozyme After incubating alkaline phosphatase (ALP) and PPi for 10 - 90 min, ALP will hydrolyze PPi to relieve the inhibition of the enzyme activity of the metal-based silicate nanozyme, and catalyze the oxidation of the chromogenic substrate to rapidly detect the ALP activity.

2. The method according to claim 1, characterized in that The preparation steps of the metal-based silicate nanozyme involved in step I are as follows: Disperse silica nanoparticles in an aqueous solution, add an aqueous solution containing one or more metal salts of iron / manganese / copper / zinc / nickel / cerium / cobalt, NH4Cl, and ammonia water. The molar ratio of silica nanoparticles, metal salts, NH4Cl, ammonia water with a concentration of 25 - 28 wt%, and water is 1:0.2 - 1.5:3 - 54:7 - 30:2000 - 7000; stir the above solution for 3 - 10 min and then transfer it to a hydrothermal reaction kettle, carry out hydrothermal reaction at 120 - 160 °C for 8 - 24 h, centrifuge, wash, and dry to obtain the metal-based silicate nanozyme.

3. The method according to claim 1, wherein The metal-based silicate nanozyme is a silicate nanozyme containing one or more metal elements of iron, manganese, copper, zinc, nickel, cerium, and cobalt.

4. The method according to claim 2, characterized in that The metal salts are one or a mixture of one or more metal salts among ferric nitrate, ferrous nitrate, ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, iron 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 the corresponding metal salt hydrates.

5. The method according to claim 1, characterized in that The acidic environment is pH 2.0 - 7.

0.

6. The method according to claim 1, wherein The metal-based silicate nanozyme exhibits peroxidase-like activity and shows a visible color change signal after contacting with the chromogenic substrate in the presence of H2O2.

7. The method according to claim 1, wherein In step II, a chromogenic solution containing H2O2 is added to the mixture of the sample to be measured and the metal-based silicate nanozyme, and the final concentration of H2O2 is 0.05 - 50 mM.

8. The method according to claim 1, characterized in that The chromogenic substrate is any one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD). The final concentration of TMB is 208 - 1664 μM, the final concentration of ABTS is 0.1 - 6 mM, and the final concentration of OPD is 0.1 - 3 mM.

9. The method according to claim 1, characterized in that For the detection of PPi, if the solution shows color, it indicates a negative result; if the color development of the solution is inhibited, it indicates a positive result; and the concentration of PPi is quantitatively detected by an enzyme-linked immunosorbent assay (ELISA) reader or a UV-visible spectrophotometer.

10. The method according to claim 1, wherein For the detection of ALP, if the solution shows color, it indicates a positive result; if the color development of the solution is inhibited, it indicates a negative result; and the activity of ALP is quantitatively detected by an ELISA reader or a UV-visible spectrophotometer.

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