CN115629063A - Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method - Google Patents

Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method Download PDF

Info

Publication number
CN115629063A
CN115629063A CN202211223318.7A CN202211223318A CN115629063A CN 115629063 A CN115629063 A CN 115629063A CN 202211223318 A CN202211223318 A CN 202211223318A CN 115629063 A CN115629063 A CN 115629063A
Authority
CN
China
Prior art keywords
ppi
metal silicate
nanoenzyme
activity
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211223318.7A
Other languages
Chinese (zh)
Inventor
刘惠玉
苏昕
许溪璨
何梦雅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing University of Chemical Technology
Original Assignee
Beijing University of Chemical Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing University of Chemical Technology filed Critical Beijing University of Chemical Technology
Priority to CN202211223318.7A priority Critical patent/CN115629063A/en
Publication of CN115629063A publication Critical patent/CN115629063A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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

Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method
Technical Field
The invention discloses a novel method for detecting pyrophosphate and alkaline phosphatase based on metal silicate nano enzyme. In particular to preparation of peroxidase-like catalytic activity nano-enzyme and development of a new pyrophosphate and alkaline phosphatase detection technology based on metal silicate nano-enzyme.
Background
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 detection of PPi is an important research hotspot in recent years. Because PPi is involved in the regulation of various physiological functions, differences in PPi concentration in various biological environments can also be used to monitor or diagnose a variety of diseases such as arthritis, chondrocaliosis, and hypophosphatemia. For example, PPi with too high content in synovial fluid is closely related to the accumulation of calcium pyrophosphate dihydrate crystals and the pathogenesis of arthritis diseases; high levels of extracellular PPi concentration can cause bone mineralization defects, i.e. hypophosphatemia, by inhibiting hydroxyapatite deposition. In addition, the activity of alkaline pyrophosphatase (ALP) can be detected by taking PPi as a medium; as a byproduct of DNA Polymerase Chain Reaction (PCR), the PPi concentration can also be used for monitoring DNA sequencing in real time, and is expected to be applied to detection of microbial pathogens.
Currently, the most common methods for detecting the presence and amount change of PPi include fluorescence, enzymatic, capillary electrophoresis, ion chromatography, electrochemical analysis, and the like. A common PPi detection kit such as a fluorescence method pyrophosphoric acid detection kit based on a fluorescent probe detects PPi by using a common ultraviolet-visible absorption spectrometer or an enzyme-linked immunosorbent assay, can quantify the concentration of the PPi through fluorescence intensity, has good stability, is fast and convenient, is beneficial to high-throughput detection, but has relatively low sensitivity, poor specificity and complex preparation, difficult storage and high price of the probe; the enzymatic method has the defects of easy enzyme inactivation, high catalytic environment requirement, high cost and the like although the enzymatic method has high sensitivity; other conventional methods also have problems of long detection time, complex and time-consuming sample processing process, and unsuitability for field detection. Therefore, it is very important to develop a convenient and sensitive method for detecting PPi.
ALP is widely present in tissues and organs of the human body, is involved in various physiological processes such as cell growth, apoptosis, signal transduction, etc., and is an indispensable biological enzyme in the human body. The activity of ALP is associated with the development of various diseases such as diabetes, diseases of liver and gall system, bone diseases, prostate cancer, etc., and thus its activity is an important index for diagnosing the normal state of the body. Currently, the most common methods for detecting the presence and activity change of ALP include colorimetry, fluorescence, enzyme labeling, electrochemical methods, electrophoresis, and the like. Compared with other methods, the colorimetric method has the advantages of simple operation, low price, high detection speed, capability of realizing naked eye detection and the like, but the traditional colorimetric method at present also has the problems of low sensitivity, poor specificity and the like, so that the development of a detection method with high specificity, high sensitivity, simple operation and low price for detecting the ALP activity is urgently needed.
With the rapid development of nanotechnology, nanomaterials are also widely applied to research in various fields, wherein nanoenzymes have the advantages of excellent enzyme-like activity, high catalytic efficiency, low requirement on catalytic environment, good stability, simple preparation, reusability, low price, convenience for storage and the like, and are a research hotspot in the detection field in recent years. Some metallosilicate nanoenzymes such as iron-based nanoenzymes, copper-based nanoenzymes and the like have excellent peroxidase-like activity and can be applied to hydrogen peroxide (H) 2 O 2 ) In the presence of the catalytic chromogenic substrate, a macroscopic, distinct color change occurs. In addition, researches show that the activity and selectivity of the nano enzyme can be directionally designed or a synergistic catalytic effect can be realized by doping different types of metal ions. And strong coordination exists between metal ions such as iron, copper, zinc, manganese, cerium and the like and PPi, so that the enzyme activity of the metal silicate nanoenzyme can be obviously influenced, and the color change of a chromogenic substrate is inhibited. In conclusion, the excellent peroxidase-like activity of the metallosilicate nanoenzyme is utilized to amplify the enzyme catalytic reaction, and the rapid detection of PPi and ALP can be realized through the chromogenic reaction of the enzyme substrate, and the dynamic change monitoring of PPi and ALP can also be realized through the chromogenic degree of the enzyme substrate. In recent years, the metal silicate nano enzyme is rapidly developed in the field of biomedicine, and has higher catalytic activity and stability compared with the traditional natural enzyme, so that the metal silicate nano enzyme is expected to provide a new means for developing rapid and efficient PPi and ALP detection technologies.
Disclosure of Invention
The invention provides a preparation method of metal silicate nano enzyme and researches the application of the metal silicate nano enzyme in the detection of pyrophosphate and alkaline phosphatase aiming at the problems of the current detection method of pyrophosphate and alkaline phosphatase. Firstly, taking silicon dioxide nanoparticles as a template, doping one or more of iron, manganese, copper, zinc, nickel, cerium and cobalt ions by using a hydrothermal method to obtain the metal silicate nanoenzyme with peroxidase-like activity, and then developing a new pyrophosphate and alkaline phosphatase detection technology based on the metal silicate nanoenzyme by using the prepared metal silicate nanoenzyme.
The technical scheme of the invention is as follows:
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
Silicon dioxide nano-particles are used as a template, and one or more of iron, manganese, copper, zinc, nickel, cerium and cobalt ions are doped in a hydrothermal environment to prepare metal silicate nano-enzyme with peroxidase-like activity;
II detection of pyrophosphate by metal silicate nano enzyme
In an acidic environment, hydrogen peroxide (H) is catalyzed by using metal silicate nano enzyme 2 O 2 ) And (3) oxidizing the chromogenic reaction of a chromogenic substrate and quickly detecting PPi by inhibiting the activity of the nano enzyme 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, the PPi is hydrolyzed to remove the enzyme activity inhibition of the PPi on the metallosilicate nanoenzyme, and the ALP activity is rapidly detected by catalyzing, oxidizing and developing a substrate.
2. Further, the step I relates to the preparation of metal silicate nano enzyme
Dispersing silicon dioxide nano particles in an aqueous solution, adding one or more metal salts containing iron/manganese/copper/zinc/nickel/cerium/cobalt 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 (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. Further, the metal silicate nano enzyme can be silicate nano enzyme containing one or more metal elements of iron, manganese, copper, zinc, nickel, cerium and cobalt.
4. Further, the metal salt is one or a mixture of more metal salts 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 corresponding metal salts.
5. Further, the pH value of the acid environment is 2.0-7.0.
6. Further, the metal silicate nano enzyme shows 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. Further, step II involves adding H into the mixture of the sample to be measured 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 CM.
8. Further, the chromogenic substrate is any one of 3,3', 5' -Tetramethylbenzidine (TMB), 2' -diazobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and o-phenylenediamine (OPD), TMB final concentration is 208-1664 μm, ABTS final concentration is 0.1-6 m, and OPD final concentration is 0.1-3 m.
9. Further, for PPi detection, a color development of the solution indicates a negative result (no or low PPi content in the sample); a positive (presence of PPi in the sample) is indicated if the solution color development is inhibited; and the PPi concentration can be quantitatively detected by a microplate reader or an ultraviolet spectrophotometer.
10. Further, in the case of ALP detection, the solution develops color and shows positive (ALP exists in the sample); when the color development of the solution is inhibited, it indicates negative (ALP is absent or the activity is low in the sample); and ALP activity can be quantitatively detected by a microplate reader or an ultraviolet spectrophotometer.
Under the acidic condition, a sample to be detected containing PPi is fully mixed with metal silicate nano enzyme, and then the mixture is added into a sample containing a chromogenic substrate and H 2 O 2 According to the color development condition, judging whether PPi exists in the sample, and further judging the negative and positive results (the more PPi, the stronger the effect of inhibiting the enzyme activity of the nano enzymes, the stronger the inhibition effect of the color development, no color in the solution system and positive, or vice versa). Meanwhile, the existence of the PPi is directly observed by naked eyes and the content is qualitatively analyzed, and the absorbance can be detected by an enzyme-labeling instrument, an ultraviolet-visible absorption spectrometer and other instruments so as to further accurately and quantitatively analyze the content of the PPi. And the ALP can hydrolyze PPi, so that the activity of the nanometer enzyme POD enzyme inhibited by the PPi is recovered, and a chromogenic substrate is developed to detect the content of the chromogenic substrate. Under the acidic condition, a sample to be detected containing ALP is fully incubated with PPi with fixed concentration, then fully mixed with metal silicate nano enzyme, and then added into a solution containing a chromogenic substrate and H 2 O 2 In the acetate buffer solution, the activity of ALP in the sample is judged according to the intensity of the developed color, and then a positive result and a negative result are judged (the stronger the ALP activity is, the weaker the PPi effect of inhibiting the enzymatic activity of the nanoenzymes is, the solution system develops color, and the result is positive, and vice versa). Meanwhile, in addition to direct observation of the presence or absence of ALP by naked eyes and qualitative analysis of activity, absorbance may be detected by an microplate reader, an ultraviolet-visible absorption spectrometer or the like to further accurately and quantitatively analyze the activity of ALP. The metal silicate nano enzyme prepared by the invention has good appearance, uniform particles, simple synthetic method steps, good repeatability, no toxicity, no harm and environmental friendliness; pyrophosphate and alkaline phosphatase detection method based on metal silicate nano enzymeThe kit has high sensitivity, and has wide application prospect in virus detection, diagnosis of various diseases such as arthritis, diabetes, bone cancer and the like, and other biomedical fields.
Drawings
FIG. 1 is a transmission electron microscope image of the ferric silicate nanoenzyme prepared in the present invention.
FIG. 2 is a transmission electron microscope image of the ferrimanganic silicate nano enzyme prepared in the invention.
FIG. 3 is a transmission electron microscope image of iron copper silicate nanoenzyme prepared in the present invention.
FIG. 4 is a schematic diagram of detection of PPi and ALP by using metallosilicate nanoenzyme.
FIG. 5 shows the detection sensitivity of ferrimanganite nanoenzyme to PPi.
FIG. 6 shows the specificity of detection of PPi by ferrimanganite nanoenzyme.
FIG. 7 shows the effect of femtosilicate nanoenzyme on PPi and ALP detection.
Detailed Description
The present invention will be described in more detail with reference to the following embodiments and the accompanying drawings, but the scope of the present invention is not limited to the following embodiments.
< test method >
1. Topography testing
The morphology of the metal silicate nano-enzyme is determined by a Japanese electron JEM-1011 field emission Transmission Electron Microscope (TEM).
2. Determination of detection Effect of pyrophosphate
3,3', 5' -tetramethyl benzidine (TMB) is taken as a substrate, and H is added in an acidic environment 2 O 2 The material and the sample react to realize the specific detection of the metal silicate nano enzyme on the PPi.
3. Determination of alkaline phosphatase detection Effect
3,3', 5' -tetramethyl benzidine (TMB) is taken as a substrate, and H is added in an acidic environment 2 O 2 The material and the sample incubated with PPi react to realize the specific detection of the metal silicate nano enzyme on ALP.
Example 1 (detection of pyrophosphate and alkaline phosphatase based on iron silicate Nanolase)
Weighing 30mg of silicon dioxide nano particles, uniformly dispersing the silicon dioxide nano particles in 15mL of ultrapure water, and placing the silicon dioxide nano particles on a magnetic stirrer to stir at a stirring speed of 300 r/min; 962.82mg NH were weighed in turn 4 Cl(18mmol)、125.10mg FeSO 4 ·7H 2 Dispersing O (0.45 mmol) in 15mL of ultrapure water, rapidly stirring on a magnetic stirrer, simultaneously adding 700 mu L of ammonia water (25 wt%), stirring uniformly, immediately pouring into the aqueous solution of the silicon dioxide nanoparticles, stirring for 5min, transferring into a 50mL hydrothermal reaction kettle, carrying out hydrothermal reaction at 140 ℃ for 12h, after the reaction is finished, carrying out centrifugal washing, washing with water and ethanol alternately for 5 times, and drying in a constant-temperature drying oven at 60 ℃ to obtain the iron silicate nanoenzyme. As shown in FIG. 4, a sample to be tested containing pyrophosphate (PPi) was well mixed with ferric silicate nanoenzyme (final concentration: 50. Mu.g/mL) under pH 4.0, and then added with a solution containing TMB (final concentration: 832. Mu.M) and H 2 O 2 In acetate buffer (HAc/NaAc buffer, 0.1 CM, pH 4.0) (final concentration: 0.1M), when the sample to be tested contains PPi, PPi will complex with iron in the ferric silicate nanoenzyme to inhibit the peroxidase-like activity of the ferric silicate nanoenzyme, and TMB color development is inhibited; if the sample to be tested does not contain PPi, at H 2 O 2 In the presence of the enzyme, the ferric silicate nano-enzyme with peroxidase-like activity catalyzes TMB oxidation and color development, and the solution turns into blue. After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. Except for qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately and quantitatively analyzed according to the corresponding absorbance of a standard curve by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry. A solution containing ALP (Tris-HCl buffer, 0.1 CM, pH 8.5) and a PPi solution (final concentration: 300. Mu.M) were incubated at 37 ℃ for 60min. Then, the above solution was mixed well with ferric silicate nanoenzyme (final concentration: 50. Mu.g/mL), and added to a solution containing TMB and H 2 O 2 Acetate buffer (HAc/NaAc buffer, 0.1M, pH 4.0). After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. In the presence of ALP, TMB color development is restored; whereas in the absence of ALP or in activities low enough to hydrolyze PPi, TMB color development was inhibited. With PPi asExcept for qualitatively analyzing the ALP activity according to the color depth, the medium can accurately and quantitatively analyze the ALP activity by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry according to the corresponding absorbance of a standard curve.
Example 2 (detection of pyrophosphate and alkaline phosphatase based on Ferro-manganese silicate Nanolase)
Weighing 30mg of silicon dioxide nano particles, uniformly dispersing the silicon dioxide nano particles in 15mL of ultrapure water, and placing the silicon dioxide nano particles on a magnetic stirrer to stir at a stirring speed of 300 r/min; 18.88mg of MnCl are weighed in turn 2 (0.15mmol)、962.82mg NH 4 Cl(18mmol)、83.40mg FeSO 4 ·7H 2 Dispersing O (0.3 mmol) in 15mL of ultrapure water, rapidly stirring on a magnetic stirrer, simultaneously adding 700 mu L of ammonia water (25 wt%), stirring uniformly, immediately pouring into the aqueous solution of the silicon dioxide nanoparticles, stirring for 5min, transferring into a 50mL hydrothermal reaction kettle, carrying out hydrothermal reaction at 140 ℃ for 12h, after the reaction is finished, carrying out centrifugal washing, washing with water and ethanol alternately for 5 times, and drying in a constant-temperature drying oven at 60 ℃ to obtain the iron-manganese silicate nanoenzyme. As shown in FIG. 4, a sample to be tested containing PPi was mixed well with a ferromanganese silicate nanoenzyme (final concentration: 50. Mu.g/mL) under pH 4.0, and then added to a mixture containing TMB (final concentration: 832. Mu.M) and H 2 O 2 In acetate buffer solution (HAc/NaAc buffer solution, 0.1 CM, pH 4.0) (final concentration: 0.1 CM), when the sample to be detected contains PPi, PPi can be complexed with iron and manganese in the ferro-manganese silicate nanoenzyme so as to inhibit the peroxidase-like activity of the ferro-manganese silicate nanoenzyme, and TMB color development is inhibited; if the sample to be tested does not contain PPi, at H 2 O 2 Under the existence condition, the ferrimanganic silicate nano-enzyme with the peroxidase-like activity catalyzes TMB to carry out oxidation color development, and the solution turns into blue. After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. Except for qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately and quantitatively analyzed according to the corresponding absorbance of a standard curve by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry. Wherein, as shown in FIG. 5, the linear range of PPi detection is 0.6-4.5 μ M (y =0.1195x +0.002624, R) 2 = 0.9927), with a 3-fold signal-to-noise ratio, a detection limit of 205.47n cm; a solution containing ALP (Tris-HCl buffer, 0.1 CM, pH 8) was added.5) And PPi solution (final concentration: 300 μ M) was incubated at 37 ℃ for 60min. Then, the solution is fully mixed with ferrimanganic silicate nano enzyme (final concentration: 50 mu g/mL), and then is added into a mixture containing TMB and H 2 O 2 Acetate buffer (HAc/NaAc buffer, 0.1 CM, pH 4.0). After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. In the presence of ALP, TMB color development is restored; whereas TMB color development was inhibited when ALP was absent or not active enough to hydrolyze PPi. Except for qualitatively analyzing the ALP activity according to the color depth by taking the PPi as a medium, the ALP activity can be accurately and quantitatively analyzed by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry according to the corresponding absorbance of a standard curve.
Example 3 (detection of pyrophosphate and alkaline phosphatase based on iron copper silicate Nanolase)
Weighing 30mg of silicon dioxide nano particles, uniformly dispersing the silicon dioxide nano particles in 15mL of ultrapure water, and placing the silicon dioxide nano particles on a magnetic stirrer to stir at a stirring speed of 300 r/min; 962.82mg NH were weighed in turn 4 Cl(18mmol)、83.40mg FeSO 4 ·7H 2 O(0.3mmol)、36.24mg Cu(NO 3 ) 2 ·3H 2 Dispersing O (0.15 mmol) in 15mL of ultrapure water, rapidly stirring on a magnetic stirrer, simultaneously adding 700 mu L of ammonia water (25 wt%), stirring uniformly, immediately pouring into the aqueous solution of the silicon dioxide nanoparticles, stirring for 5min, transferring into a 50mL hydrothermal reaction kettle, carrying out hydrothermal reaction at 140 ℃ for 12h, after the reaction is finished, carrying out centrifugal washing, washing with water and ethanol alternately for 5 times, and drying in a constant-temperature drying oven at 60 ℃ to obtain the iron-copper silicate nanoenzyme. As shown in FIG. 4, a test sample containing PPi was thoroughly mixed with iron-copper silicate nanoenzyme (final concentration: 50. Mu.g/mL) at pH 4.0, and then added to a solution containing TMB (final concentration: 832. Mu.M) and H 2 O 2 In acetate buffer solution (HAc/NaAc buffer solution, 0.1 CM, pH 4.0) (final concentration: 0.1 CM), when the sample to be detected contains PPi, PPi can be complexed with iron and copper in the iron-copper silicate nanoenzyme so as to inhibit the peroxidase-like activity of the iron-copper silicate nanoenzyme, and TMB color development is inhibited; if the sample to be tested does not contain PPi, at H 2 O 2 In the presence of the enzyme, the iron-copper silicate nano-enzyme with peroxidase-like activity catalyzes TMB oxidationColor, solution turned blue. After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. Except for qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately and quantitatively analyzed according to the corresponding absorbance of a standard curve by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry. A solution containing ALP (Tris-HCl buffer, 0.1 CM, pH 8.5) and PPi (final concentration: 300. Mu.M) were incubated at 37 ℃ for 60min. Then, the solution was mixed well with iron-copper silicate nanoenzyme (final concentration: 50. Mu.g/mL), and added to a solution containing TMB and H 2 O 2 Acetate buffer (HAc/NaAc buffer, 0.1 CM, pH 4.0). After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. In the presence of ALP, TMB color development recovered; whereas TMB color development was inhibited when ALP was absent or not active enough to hydrolyze PPi. Except for qualitatively analyzing the ALP activity according to the color depth by taking the PPi as a medium, the ALP activity can be accurately and quantitatively analyzed by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry according to the corresponding absorbance of a standard curve.
Example 4 (detection of PPi content and ALP Activity in clinical authentic sample based on metallosilicate nanoenzyme)
As shown in FIG. 4, a clinical specimen containing 1% diluted human serum was well mixed with a metallosilicate nanoenzyme (final concentration: 50. Mu.g/mL) at pH 4.0, and then added to a sample containing TMB (final concentration: 832. Mu.M) and H 2 O 2 In acetate buffer (HAc/NaAc buffer, 0.1M, pH 4.0) of (final concentration: 0.1M), when the sample to be detected contains PPi, PPi can be complexed with metal ions in the metal silicate nanoenzyme to inhibit the peroxidase-like activity of the metal silicate nanoenzyme, and TMB color development is inhibited; if the sample to be tested does not contain PPi, at H 2 O 2 Under the existence condition, the metal silicate nano-enzyme with the peroxidase-like activity catalyzes TMB oxidation and color development, and the solution turns into blue. After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. Except for qualitatively analyzing the PPi content according to the color depth, the PPi concentration can be accurately and quantitatively analyzed according to the corresponding absorbance of a standard curve by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry. Clinical application of human serum containing 1% dilutionThe samples were incubated with buffer (Tris-HCl buffer, 0.1 CM, pH 8.5) and PPi (final concentration: 300. Mu.M) at 37 ℃ for 60min. Then, the above solution was sufficiently mixed with metallosilicate nanoenzyme (final concentration: 50. Mu.g/mL), and added to a solution containing TMB and H 2 O 2 Acetate buffer (HAc/NaAc buffer, 0.1 CM, pH 4.0). After 15min reaction, the negative or positive result can be judged by naked eyes according to the color development condition. In the presence of ALP, TMB color development recovered; whereas in the absence of ALP or in activities low enough to hydrolyze PPi, TMB color development was inhibited. Except for qualitatively analyzing the ALP activity according to the color depth by taking PPi as a medium, the ALP activity can be accurately and quantitatively analyzed by combining an enzyme-labeling instrument or ultraviolet-visible spectrophotometry according to the corresponding absorbance of a 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.
CN202211223318.7A 2022-10-09 2022-10-09 Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method Pending CN115629063A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211223318.7A CN115629063A (en) 2022-10-09 2022-10-09 Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211223318.7A CN115629063A (en) 2022-10-09 2022-10-09 Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method

Publications (1)

Publication Number Publication Date
CN115629063A true CN115629063A (en) 2023-01-20

Family

ID=84905074

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211223318.7A Pending CN115629063A (en) 2022-10-09 2022-10-09 Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method

Country Status (1)

Country Link
CN (1) CN115629063A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116735514A (en) * 2023-08-11 2023-09-12 昆明理工大学 Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116735514A (en) * 2023-08-11 2023-09-12 昆明理工大学 Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction
CN116735514B (en) * 2023-08-11 2023-11-03 昆明理工大学 Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction

Similar Documents

Publication Publication Date Title
AU2020100704A4 (en) A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2
Gao et al. Fluorometric method for the determination of hydrogen peroxide and glucose with Fe3O4 as catalyst
Yang et al. Cobalt oxyhydroxide nanoflakes with oxidase-mimicking activity induced chemiluminescence of luminol for glutathione detection
CN105891189B (en) A kind of copper ion detection kit and its application
CN109266332A (en) A kind of preparation method for the Ratiometric fluorescent probe of AChE and BChE in quantitative detection blood
CN107356591A (en) It is a kind of based on imitative enzyme nano material without one pot of glucose color developing detection method of enzyme
CN104267026A (en) Mercury-ion detection method simulating peroxidase based on nano platinum and kit
CZ972183A3 (en) Process of determining nad(p)h or substrates or enzymes reacting under the formation or consumption of nad(p)h and a testing system for making the same
CN108801998B (en) A method of the ratio fluorescent probe in detecting choline based on copper nano-cluster compound
Khachornsakkul et al. Development of an ultrasound-enhanced smartphone colorimetric biosensor for ultrasensitive hydrogen peroxide detection and its applications
Zhu et al. Application of thiamine as a fluorogenic substrate in the determination of hydrogen peroxide based on the catalytic effect of hemin
CN115629063A (en) Pyrophosphate based on metal silicate nano enzyme and alkaline phosphatase detection method
Wu et al. The screening of metal ion inhibitors for glucose oxidase based on the peroxidase-like activity of nano-Fe 3 O 4
Chen et al. Visual and dual-fluorescence homogeneous sensor for the detection of pyrophosphatase in clinical hyperthyroidism samples based on selective recognition of CdTe QDs and coordination polymerization of Ce 3+
Qu et al. Determination of butyrylcholinesterase activity based on thiamine luminescence modulated by MnO2 nanosheets
Al-mashriqi et al. Gold nanoclusters reversible switches based on aluminum ions-triggered for detection of pyrophosphate and acid phosphatase activity
Niu et al. Pyrophosphate-Mediated On–Off–On Oxidase-Like Activity Switching of Nanosized MnFe 2 O 4 for Alkaline Phosphatase Sensing
Zhang et al. Colorimetric copper (Ⅱ) ions detection in aqueous solution based on the system of 3′ 3′ 5′ 5′-tetramethylbenzidine and AgNPs in the presence of Na2S2O3
CN114275806A (en) Cadmium-zinc-selenium quantum dot, preparation method and application thereof, and ALP detection method
Zhao et al. The influence of substrates addition order on colorimetric assay based on MnO2 nanocubes: A novel turn-off H2O2 assay strategy in water-soak foods
Rahimi et al. MnO2 nanosheet-assisted ratiometric fluorescence probe for the detection of sulfide based on silicon nanoparticles and o-phenylenediamine
Li et al. MnO2 nanosheet-assisted ratiometric fluorescent sensor for ascorbic acid based on Pyronin Y and thiamine
Yao et al. Modulation of inner filter effect between persistent luminescent particles and 2, 3-diaminophenazine for ratiometric fluorescent assay of ascorbic acid and ascorbate oxidase activity
Wang et al. A reusable ratiometric fluorescent biosensor with simple operation for cysteine detection in biological sample
CN108613972B (en) Colorimetric sensing method for generating inorganic nanoparticles based on enzyme catalysis induction

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination